**Stoichiometric Ratio in Calixarene Complexes**

Flor de María Ramírez and Irma García-Sosa *Instituto Nacional de Investigaciones Nucleares* 

*México* 

### **1. Introduction**

Stoichiometry is a fundamental concept in chemistry that refers to the ratios of products and reactants in any chemical reaction. It is an important concept in both chemical reactions and biochemical processes. A stoichiometric ratio represents the relationship among the elements or molecules present in an equation. Using the correct stoichiometric amount of reactant will yield the maximum amount of product under the proper thermodynamic and kinetic conditions.

This chapter will be focused on the latest research, from our and other labs, on the determination of stoichiometric ratios for complexed species formed between substrates and calixarene receptors, the influence of size, conformation and functionalization site of calixarenes on the stoichiometry, as well as solvent effects and the stability of the complexes in the liquid and solid states. No formation (stability) constants will be discussed, although in some cases these could be mentioned.

Particular attention will be paid to the experimental methods used for stoichiometry determination. Furthermore, complexed calixarene molecules in 1:1, 1:2 and 2:1 (Substrate:Calixarene) stoichiometries calculated by Augmented MM3 and CONFLEX semiempirical procedures, and other computational calculations will be included.

The enormous number of sophisticated functionalized calixarenes published so far, forced us to be selective. Therefore, our attention will be devoted to the stoichiometry of complexes formed with parent calixarenes (Gutsche, 1989, 1998, Mandolini & Ungaro 2000) functionalized at the lower and upper rims, with linear arms, that were first reported between 1999 and 2011.

### **2. A brief overview of calixarenes**

Calixarenes are an important group of macrocycles, considered the third best host (receptors) molecules after cyclodextrins and crown ethers (Shinkai, 1993). They are prepared by condensation reactions between para-substituted phenols and formaldehyde (Gutsche, 1989). Here, we focus on conventional *endo*-calixarenes, which have a lipophilic cavity and two rims: the polar lower rim and the apolar upper rim that provides an unusual flexibility to the calixarene that can be modulated by adjusting cavity size and the rim substituents size. Its basket-like structure, with a lipophilic cavity made up of aromatic

Stoichiometric Ratio in Calixarene Complexes 5

Fig. 2. Some interesting calixarene molecules built with ChemDraw Ultra 10.0 software. a) Zheng et al. (2010), b) Liang et al. (2007), c) Leydier et al. (2008), d) Bew et al. (2010), e) Snejdarkova et al. (2010), f) Sliwa& Deska, (2008), g) Sliwka-Kaszynska et al. (2009), articles

The method of continuous variation involves a series of isomolar solutions of two reactants. It is one commonly used experimental mixing technique for determining formula and formation constants of complexes. The method is also known as Job's method, for his general development of spectrophotometric measurement techniques (Gil & Oliveira, 1990). However, it was Ostromisslensky in 1911 who (Hill & MacCarthy, 1986; Likussar & Boltz, 1971) first employed the method to establish the 1:1 stoichiometry of the adduct formed between nitrobenzene and aniline. Other methods, such as molar ratios and titration in a

Job's method is carried out in a batch mode (series of solutions) by mixing aliquots of two equimolecular stock solutions of two components (metal and ligand, or organic substrate and organic receptor) and diluting to a constant volume to get solutions with identical total molar concentrations but different mole fractions. The sum of the two components analytical

single flask, are also currently used to investigate the stoichiometry of complexes.

cited by Mokhtari et al., 2011b.

**3.1 Job's method** 

**3. Finding the stoichiometric ratio** 

nuclei and easily modified rims, has attracted the attention of many theoretical and experimental researchers.

With exceptions, macrocycle flexibility, i.e. conformational movement, increases with size. Calix[*n*]arenes (where *n* stands for the number of aryl groups in the macrocycle) with *n* = 4 to 20 have been synthesized (Gutsche, 1998), but only the smaller cycles (*n* = 4, 6 and 8) have been thoroughly studied. Calixarenes are versatile macrocycles with almost unlimited properties (Böhmer, 1995); they are excellent scaffolds since their conformation can be adapted to potential guests, and they can be selectively functionalized at three sites (Asfari et al., 2001; Mandolini & Ungaro, 2000). Most commonly, specific groups or substituents called pendant arms are added to the rims to impart a specific function.

Calixarenes are very attractive to researchers from numerous science and technology fields (Fig. 1). A vast array of functionalized calixarenes have been reported over the last decade (Alexandratos and Natesan, 2000; Gutsche, 1998; Lumetta et al., 2000; Mandolini and Ungaro, 2000; Mokhtari et al., 2011a, 2011b; Sliwa, 2002; Sliwa & Girck, 2010; Talanova, 2000). The challenge is to quickly synthesize suitable calixarenes on a large scale with minimal workup.

Fig. 1. Applications for calixarenes in science and technology.

In Fig. 2, a selection of recently reported calixarenes are presented.

nuclei and easily modified rims, has attracted the attention of many theoretical and

With exceptions, macrocycle flexibility, i.e. conformational movement, increases with size. Calix[*n*]arenes (where *n* stands for the number of aryl groups in the macrocycle) with *n* = 4 to 20 have been synthesized (Gutsche, 1998), but only the smaller cycles (*n* = 4, 6 and 8) have been thoroughly studied. Calixarenes are versatile macrocycles with almost unlimited properties (Böhmer, 1995); they are excellent scaffolds since their conformation can be adapted to potential guests, and they can be selectively functionalized at three sites (Asfari et al., 2001; Mandolini & Ungaro, 2000). Most commonly, specific groups or substituents called pendant arms are added to the rims to

Calixarenes are very attractive to researchers from numerous science and technology fields (Fig. 1). A vast array of functionalized calixarenes have been reported over the last decade (Alexandratos and Natesan, 2000; Gutsche, 1998; Lumetta et al., 2000; Mandolini and Ungaro, 2000; Mokhtari et al., 2011a, 2011b; Sliwa, 2002; Sliwa & Girck, 2010; Talanova, 2000). The challenge is to quickly synthesize suitable calixarenes on a large scale with

Fig. 1. Applications for calixarenes in science and technology.

In Fig. 2, a selection of recently reported calixarenes are presented.

experimental researchers.

impart a specific function.

minimal workup.

Fig. 2. Some interesting calixarene molecules built with ChemDraw Ultra 10.0 software. a) Zheng et al. (2010), b) Liang et al. (2007), c) Leydier et al. (2008), d) Bew et al. (2010), e) Snejdarkova et al. (2010), f) Sliwa& Deska, (2008), g) Sliwka-Kaszynska et al. (2009), articles cited by Mokhtari et al., 2011b.

### **3. Finding the stoichiometric ratio**

The method of continuous variation involves a series of isomolar solutions of two reactants. It is one commonly used experimental mixing technique for determining formula and formation constants of complexes. The method is also known as Job's method, for his general development of spectrophotometric measurement techniques (Gil & Oliveira, 1990). However, it was Ostromisslensky in 1911 who (Hill & MacCarthy, 1986; Likussar & Boltz, 1971) first employed the method to establish the 1:1 stoichiometry of the adduct formed between nitrobenzene and aniline. Other methods, such as molar ratios and titration in a single flask, are also currently used to investigate the stoichiometry of complexes.

### **3.1 Job's method**

Job's method is carried out in a batch mode (series of solutions) by mixing aliquots of two equimolecular stock solutions of two components (metal and ligand, or organic substrate and organic receptor) and diluting to a constant volume to get solutions with identical total molar concentrations but different mole fractions. The sum of the two components analytical

Stoichiometric Ratio in Calixarene Complexes 7

added until the absorbance at the analyzed wavelength does not change, or until a new band appears. Approximately 30 spectra are required, or enough data collected until a molar ratio [S]/[R]≥ 4 is reached. Specfit or Hyperquad software is used for stoichiometry

Spectra, solution concentrations, initial volume, aliquot number and volumes, and the total final volume are fed into the Specfit software. Factor analysis reveals the number of absorbing species, and the data are fitted to models to obtain the stoichiometry of the species and their stability constants. The same steps are followed if a fixed volume of the

In the case of calixarene complexes, the titration can be followed by analyzing changes in the physicochemical properties of the receptor or the substrate as the complex is formed (e.g.

UV/Vis, 1H'NMR, MS (mass spectrometry) and luminescence are the spectroscopic techniques that are most used for the elucidation of stoichiometry in calixarene complexes with organic or metallic substrates. In general, spectroscopic techniques are complementary, but if the solution contains only one complex species data from a single technique analyzed by Job or/and molar ratio methods may be sufficient. In these cases sometimes only 8 experimental points are required for a Job plot. However, if the solution contains more than two complex species, at least two different techniques and software packages are needed; in some cases the evaluation of stability constants is required to confirm the existence of a

The stoichiometry of isolated calixarene complexes in the solid state can be elucidated by microelemental analysis or, if possible, by its X-ray structure. If the complexes dissolve without decomposition, NMR and MS measurements are extremely useful for confirming

Calixarenes can interact with neutral, cationic and anionic organic substrates as well as metal ions; they are sometimes called "molecular baskets" because of this diverse capacity. Calixarenes are neutral but can act as cationic and anionic receptors if adequately functionalized (Asfari et al., 2001; Lumetta et al., 2000; Mandolini & Ungaro, 2000; Mokhtari et al., 2011a, 2011b; Sliwa & Girck, 2010). The calixarene-substrate interaction is illustrated in Fig. 3. Molecular recognition, the strength of the interaction between calixarenes and their substrates and thermodynamic and kinetic factors rule the complexes formation. It is important to keep in mind that the donating ability of solvents plays an import role (Asfari

In organic-calixarene complexes (Fig. 3, top right ), noncovalent interactions such as hydrogen bonds, π- hydrogen bonds, hydrophobic interactions, and cation-π and CH-π interactions are the main driving forces that allow stable complexes to form in the liquid and solid states (Asfari et al., 2001; García-Sosa & Ramírez, 2010; Mandolini & Ungaro, 2000).

substrate solution is titrated with a variable volume of the receptor solution.

absorbance, luminescence, NMR chemical shifts, calorimetric changes).

evaluation.

stoichiometry.

**4. Spectroscopic techniques** 

**5. Calixarene complexation** 

complex species in the determined stoichiometry.

et al., 2001; Mandolini & Ungaro, 2000) in complex stability.

concentration is constant while the Component 1:Component 2 ratio varies from flask to flask. Job's method is based on plotting measured absorbance (corrected for reactants absorbance) against the mole fraction of one single component. For stable complexes, the plot is a triangle, with the apex indicating the complex composition. For a moderately stable complex, the stoichiometry can be obtained from the intersection point of the curve tangents, this approximation being valid only for symmetrical plots (e.g. 1:1; 2:2). Weak complexes generally show a very curved plot.

This method works well for reactions where a single equilibrium, or simple equilibriums exist uninfluenced by ionic strength; no more than three species (two reactants and the product) can be present in solution and their complexes have to be very stable. Job's method may be applied to stoichiometric determination in two ways. The Standard Method was described above. The modified Limiting Reagent Method uses a series of solutions with a fixed "moles A" and a varying "moles B," such that the ratio moles B:moles A, goes from 0 to a value known to be larger than x (x equal to moles of B at intersection/(total moles of A plus B minus moles of B at intersection). The amount of product in each solution is then measured. Once the amount of B exceeds the stoichiometrically required amount, A becomes the limiting reagent and the amount of the formed product remains constant.

According to stoichiometric theory, the limiting reagent controls the yield, and the greatest yield is always produced at the stoichiometric equivalence point. The maximum amount of product should occur at the stoichiometric ratio that can be justified both intuitively and mathematically. For both methods, 8-15 experimental data points are required to determine the stoichiometry. The method can be applied to parameters obtained from techniques other than typical spectrophotometry, e.g. luminescence, nuclear magnetic resonance, calorimetry, conductometry.

Job's method has serious limitations for complex reactions systems where the number of species is larger than three. In these cases, two or more complexed species with different stoichiometries are formed, and/or the stability of the complexes is moderate. Moreover, the method fails when the formed complexes are only weakly stable. A simulation program (Gil & Oliveira, 1990) for reaction equilibriums has improved application of Job's method to more complicated systems.

### **3.2 Spectrophotometric titration**

There are several experimental methods to determine stoichiometries for systems with multiple complex species in solution. Spectrophotometric titration monitored by UV/Vis is largely used because UV/Vis spectrophotometers are widely available and inexpensive. The radiant energy absorption of a solution is measured spectrophotometrically after each increment of titrant, but the evaluation is somewhat limited by the solubility of the reactants (but not the products) in water and/or organic solvent.

In macrocyclic coordination chemistry equimolar solutions of the reactants in the same solvent with the same ionic strength are prepared, and two titrations are carried out, titrating the receptor with the substrate and the substrate with the receptor, respectively. In the former, a fixed volume of the receptor solution is titrated by adding varying volumes of the substrate solution. After each addition the UV/Vis spectrum is recorded. The titrant is

concentration is constant while the Component 1:Component 2 ratio varies from flask to flask. Job's method is based on plotting measured absorbance (corrected for reactants absorbance) against the mole fraction of one single component. For stable complexes, the plot is a triangle, with the apex indicating the complex composition. For a moderately stable complex, the stoichiometry can be obtained from the intersection point of the curve tangents, this approximation being valid only for symmetrical plots (e.g. 1:1; 2:2). Weak

This method works well for reactions where a single equilibrium, or simple equilibriums exist uninfluenced by ionic strength; no more than three species (two reactants and the product) can be present in solution and their complexes have to be very stable. Job's method may be applied to stoichiometric determination in two ways. The Standard Method was described above. The modified Limiting Reagent Method uses a series of solutions with a fixed "moles A" and a varying "moles B," such that the ratio moles B:moles A, goes from 0 to a value known to be larger than x (x equal to moles of B at intersection/(total moles of A plus B minus moles of B at intersection). The amount of product in each solution is then measured. Once the amount of B exceeds the stoichiometrically required amount, A becomes the limiting reagent and the amount of

According to stoichiometric theory, the limiting reagent controls the yield, and the greatest yield is always produced at the stoichiometric equivalence point. The maximum amount of product should occur at the stoichiometric ratio that can be justified both intuitively and mathematically. For both methods, 8-15 experimental data points are required to determine the stoichiometry. The method can be applied to parameters obtained from techniques other than typical spectrophotometry, e.g. luminescence, nuclear magnetic resonance, calorimetry,

Job's method has serious limitations for complex reactions systems where the number of species is larger than three. In these cases, two or more complexed species with different stoichiometries are formed, and/or the stability of the complexes is moderate. Moreover, the method fails when the formed complexes are only weakly stable. A simulation program (Gil & Oliveira, 1990) for reaction equilibriums has improved application of Job's method to

There are several experimental methods to determine stoichiometries for systems with multiple complex species in solution. Spectrophotometric titration monitored by UV/Vis is largely used because UV/Vis spectrophotometers are widely available and inexpensive. The radiant energy absorption of a solution is measured spectrophotometrically after each increment of titrant, but the evaluation is somewhat limited by the solubility of the reactants

In macrocyclic coordination chemistry equimolar solutions of the reactants in the same solvent with the same ionic strength are prepared, and two titrations are carried out, titrating the receptor with the substrate and the substrate with the receptor, respectively. In the former, a fixed volume of the receptor solution is titrated by adding varying volumes of the substrate solution. After each addition the UV/Vis spectrum is recorded. The titrant is

complexes generally show a very curved plot.

the formed product remains constant.

conductometry.

more complicated systems.

**3.2 Spectrophotometric titration** 

(but not the products) in water and/or organic solvent.

added until the absorbance at the analyzed wavelength does not change, or until a new band appears. Approximately 30 spectra are required, or enough data collected until a molar ratio [S]/[R]≥ 4 is reached. Specfit or Hyperquad software is used for stoichiometry evaluation.

Spectra, solution concentrations, initial volume, aliquot number and volumes, and the total final volume are fed into the Specfit software. Factor analysis reveals the number of absorbing species, and the data are fitted to models to obtain the stoichiometry of the species and their stability constants. The same steps are followed if a fixed volume of the substrate solution is titrated with a variable volume of the receptor solution.

In the case of calixarene complexes, the titration can be followed by analyzing changes in the physicochemical properties of the receptor or the substrate as the complex is formed (e.g. absorbance, luminescence, NMR chemical shifts, calorimetric changes).

### **4. Spectroscopic techniques**

UV/Vis, 1H'NMR, MS (mass spectrometry) and luminescence are the spectroscopic techniques that are most used for the elucidation of stoichiometry in calixarene complexes with organic or metallic substrates. In general, spectroscopic techniques are complementary, but if the solution contains only one complex species data from a single technique analyzed by Job or/and molar ratio methods may be sufficient. In these cases sometimes only 8 experimental points are required for a Job plot. However, if the solution contains more than two complex species, at least two different techniques and software packages are needed; in some cases the evaluation of stability constants is required to confirm the existence of a complex species in the determined stoichiometry.

The stoichiometry of isolated calixarene complexes in the solid state can be elucidated by microelemental analysis or, if possible, by its X-ray structure. If the complexes dissolve without decomposition, NMR and MS measurements are extremely useful for confirming stoichiometry.

### **5. Calixarene complexation**

Calixarenes can interact with neutral, cationic and anionic organic substrates as well as metal ions; they are sometimes called "molecular baskets" because of this diverse capacity. Calixarenes are neutral but can act as cationic and anionic receptors if adequately functionalized (Asfari et al., 2001; Lumetta et al., 2000; Mandolini & Ungaro, 2000; Mokhtari et al., 2011a, 2011b; Sliwa & Girck, 2010). The calixarene-substrate interaction is illustrated in Fig. 3. Molecular recognition, the strength of the interaction between calixarenes and their substrates and thermodynamic and kinetic factors rule the complexes formation. It is important to keep in mind that the donating ability of solvents plays an import role (Asfari et al., 2001; Mandolini & Ungaro, 2000) in complex stability.

In organic-calixarene complexes (Fig. 3, top right ), noncovalent interactions such as hydrogen bonds, π- hydrogen bonds, hydrophobic interactions, and cation-π and CH-π interactions are the main driving forces that allow stable complexes to form in the liquid and solid states (Asfari et al., 2001; García-Sosa & Ramírez, 2010; Mandolini & Ungaro, 2000).

Stoichiometric Ratio in Calixarene Complexes 9

applications. However, to the best of our knowledge, little work has been done related to

Mohammed-Ziegler et al. (2003) investigated complex formation between chromogenic capped calix[4]arene derivatives comprising indophenol indicator group(s) and aliphatic amines in ethanol using UV/Vis spectroscopy. Job's method was applied to quantify the spectral changes using various molar amine/calixarene ligand ratios by maintaining a constant ligand concentration (λmax = 520–534 nm) and varying the amine concentration. A new band emerged at 652–667 nm indicating complexation and adducts with a 1:1 stoichiometry were determined. The study indicated the formation of strong polar supramolecular complexes of calix[4]arenes capped by diamide bridges with the amines,

Zielenkiewicz et al. (2005) have thoroughly studied the complexation of isoleucine by phosphorylated calix[4]arene in methanol. Calorimetry, NMR and UV/Vis (Job's method) spectroscopy, as well as molecular modeling methods, were used. In methanol, amino acids occur primarily in the form of zwitterions [H3N+CH(R)CO2-], with a protonated amino acid and a dissociated carboxylic group. The association constants determined by spectroscopy agree with data obtained by calorimetry: 25,000 for a 1:1 complex and 1700 for 1:2 isoleucine:calixarene complexes, respectively. The formation of the former is driven by favorable changes both in enthalpy and entropy during complexation while the 1:2 complex

The 1:2 complex is the result of inclusion of the amino acid's alkyl chain into the cavity of one calixarene molecule and interaction of the amino acid amino group with the phosphoryl group of a second calixarene. The more stable 1:1 complex points to electrostatic interactions between the positively charged ammonium cations of the amino acid and the phosphoryl groups of the calixarene. The phosphoryl groups appear to serve as the anchoring points for the positively charged ammonium cations of the amino acid, thus leading to a more stable inclusion complex. Complexation of isoleucine also takes place through insertion of the aliphatic moiety of the substrate molecule into the calixarene cavity. Electrostatic interactions are the dominant forces in the complexation process. Molecular modeling results fit extraordinary well with experimental values, corroborating the 1:1 and 1:2:

Halder et al. (2010) investigated the effective and selective noncovalent interactions between fullerenes (C60 and C70) and para-tert-butylcalix[6]arene in toluene by UV/Vis and NMR methods. Both C60 and C70 form ground state noncovalent complexes with the calixarene; according to UV/Vis measurements, the complexation process is initiated by charge transfer transition. From Job's method, it was observed that both C60 and C70 form stable complexes with the calixarene ligand in a 1:1 stoichiometry. According to the binding constants, the calixarene bound strongly and selectively to C70, compared with C60, K = 110,000 and 32,400 dm3 mol-1, respectively. Proton NMR measurements support a strong complexation between

stoichiometric studies of calixarene complexes for biomedical applications.

stabilized by various types of host–guest interactions and by steric effects.

Here, we discuss some illustrative investigations.

is of entropic origin.

isoleucine:calixarene(s) stoichiometries.

C70 and the calixarene ligand.

**5.1.1 Stoichiometric ratio with neutral calixarenes** 

In metal-calixarene complexes (Fig. 3, bottom right), the coordination ability of the calixarene toward the metal ion determines complex formation. Ionic and covalent interactions dominate complex formation and define calixarene complex properties including stoichiometry.

Fig. 3. Schematic representation of calixarene complexation with neutral and cationic substrates.

### **5.1 Stoichiometry in calixarene complexes with organic substrates in solution**

There are a great number of calixarene complexes that are formed from functionalized calixarenes and organic substrates. Investigations into receptor-substrate recognition in solution afford fundamental knowledge and describe environmental, biological and medical

In metal-calixarene complexes (Fig. 3, bottom right), the coordination ability of the calixarene toward the metal ion determines complex formation. Ionic and covalent interactions dominate complex formation and define calixarene complex properties

Fig. 3. Schematic representation of calixarene complexation with neutral and cationic

**5.1 Stoichiometry in calixarene complexes with organic substrates in solution** 

There are a great number of calixarene complexes that are formed from functionalized calixarenes and organic substrates. Investigations into receptor-substrate recognition in solution afford fundamental knowledge and describe environmental, biological and medical

including stoichiometry.

substrates.

applications. However, to the best of our knowledge, little work has been done related to stoichiometric studies of calixarene complexes for biomedical applications.

Here, we discuss some illustrative investigations.

### **5.1.1 Stoichiometric ratio with neutral calixarenes**

Mohammed-Ziegler et al. (2003) investigated complex formation between chromogenic capped calix[4]arene derivatives comprising indophenol indicator group(s) and aliphatic amines in ethanol using UV/Vis spectroscopy. Job's method was applied to quantify the spectral changes using various molar amine/calixarene ligand ratios by maintaining a constant ligand concentration (λmax = 520–534 nm) and varying the amine concentration. A new band emerged at 652–667 nm indicating complexation and adducts with a 1:1 stoichiometry were determined. The study indicated the formation of strong polar supramolecular complexes of calix[4]arenes capped by diamide bridges with the amines, stabilized by various types of host–guest interactions and by steric effects.

Zielenkiewicz et al. (2005) have thoroughly studied the complexation of isoleucine by phosphorylated calix[4]arene in methanol. Calorimetry, NMR and UV/Vis (Job's method) spectroscopy, as well as molecular modeling methods, were used. In methanol, amino acids occur primarily in the form of zwitterions [H3N+CH(R)CO2-], with a protonated amino acid and a dissociated carboxylic group. The association constants determined by spectroscopy agree with data obtained by calorimetry: 25,000 for a 1:1 complex and 1700 for 1:2 isoleucine:calixarene complexes, respectively. The formation of the former is driven by favorable changes both in enthalpy and entropy during complexation while the 1:2 complex is of entropic origin.

The 1:2 complex is the result of inclusion of the amino acid's alkyl chain into the cavity of one calixarene molecule and interaction of the amino acid amino group with the phosphoryl group of a second calixarene. The more stable 1:1 complex points to electrostatic interactions between the positively charged ammonium cations of the amino acid and the phosphoryl groups of the calixarene. The phosphoryl groups appear to serve as the anchoring points for the positively charged ammonium cations of the amino acid, thus leading to a more stable inclusion complex. Complexation of isoleucine also takes place through insertion of the aliphatic moiety of the substrate molecule into the calixarene cavity. Electrostatic interactions are the dominant forces in the complexation process. Molecular modeling results fit extraordinary well with experimental values, corroborating the 1:1 and 1:2: isoleucine:calixarene(s) stoichiometries.

Halder et al. (2010) investigated the effective and selective noncovalent interactions between fullerenes (C60 and C70) and para-tert-butylcalix[6]arene in toluene by UV/Vis and NMR methods. Both C60 and C70 form ground state noncovalent complexes with the calixarene; according to UV/Vis measurements, the complexation process is initiated by charge transfer transition. From Job's method, it was observed that both C60 and C70 form stable complexes with the calixarene ligand in a 1:1 stoichiometry. According to the binding constants, the calixarene bound strongly and selectively to C70, compared with C60, K = 110,000 and 32,400 dm3 mol-1, respectively. Proton NMR measurements support a strong complexation between C70 and the calixarene ligand.

Stoichiometric Ratio in Calixarene Complexes 11

structure of the free calixarene switches to an opposite C2v pinched-cone conformation, with the two carboxylato-bearing rings pointing inward the calixarene cavity to maximize electrostatic and van der Waals interactions with the cationic paraquat. These adaptive conformational changes were fully confirmed by molecular modeling. The complexation of paraquat with tetrametoxi- para-carboxylatocalix[4]arene was also monitored with Diffusion-Ordered Spectroscopy (DOSY) NMR. DOSY has been particularly used in the characterization of host–guest systems in solution. The results indicated the formation of a complex in a 1:1 stoichiometry; in this complex, the calixarene was in the cone conformation

Methiocarb [3,5-dimethyl-4-(methylthio) phenyl methylcarbamate] is one of the mostly important N-methylcarbamate pesticides, used worldwide in agriculture and health programs. Ding et al. (2011) investigated the complexation between tetrabutyl ether derivatives of p-sulfonatocalix[4]arene (SC4Bu) and methiocarb by fluorescence spectrometry in a mixture of water and DMSO. It was observed that upon the addition of methiocarb, the fluorescence intensity of SC4Bu was quenched and a slight red shift was observed for the maximum emission peak, which is indicative of a calixarene-methiocarb interaction. The results indicated that the SC4Bu-methiocarb complex was formed in a 1:1 mole ratio and that the electrostatic effect is not the main driving force. Using a modeling package it was proposed that complexation was an "external" inclusion process and that the hydrogen bonding between the methyl H atom of methiocarb and the sulfonate of SC4Bu facilitates the formation of this SC4Bu–methiocarb complex in a 1:1 stoichiometry. This

while the free calixarene was predominantly in the partial-cone conformation.

study provides useful information for applying calixarenes to pesticide detection.

occur under desirable conditions close to standard temperature and pressure.

**state** 

complexes seems to be 1:1.

solution and in the solid state.

**5.2 Stoichiometry in calixarene complexes formed with organic substrates in solid** 

For years, organic frameworks have been suggested as promising gas storage substrates, but purely organic molecular crystals have received little attention. Atwood et al. (2005) studied the absorption of methane in p-tert-butylcalix[4]arene at room temperature and pressures of one atmosphere and lower. The results, supported by purely size–shape considerations, suggest that it is possible to accommodate at least two CH4 molecules within each dimeric capsule (i.e., calixarene:substrate = 1:1). On the basis of this assumption, preliminary results indicate that on average, at 0.54 atm, 14% of capsules are occupied by two molecules of methane. The results demonstrate that low-density organic systems do indeed deserve consideration as potential sorbants for volatile gases, and that such sorption processes can

Ferreira et al. (2010) studied the inclusion of biacetyl within p-tert-butylcalix[n=4,6]arenes in powdered solid samples using luminescence and diffuse reflectance. Lifetime distribution analysis of the phosphorescence of both complexes suggested *endo-*inclusion calixarene complexes with p-tert-butylcalix[n=4]arene, and *exo*-inclusion calixarene complexes with ptert-butylcalix[n=6]arene. Although it is not explicit, the stoichiometry of the inclusion

Table 1 summarizes the stoichiometric ratio of some of the discussed organic complexes in

### **5.1.2 Stoichiometric ratio with charged calixarenes**

Kunsági-Máté et al. (2004) investigated complex formation between C60 fullerene (dissolved in toluene) and water-soluble hexasulfonated calix[6]arenes functionalized with sulfonates in the upper rim. Photoluminescence (PL) measurements and Job's method were used for this investigation. The stoichiometry of the complex was 1:1, and related quantum chemical calculations demonstrated that the C60 fullerene is located deep within the cavity of the calixarene. This observation makes this calixarene a promising candidate for overcoming the natural water-repulsive character of C60 fullerene and could improve the application of fullerenes to biochemical processes.

Zhou et al. (2008) used spectrofluorometric titrations to investigate the inclusion behavior of *p*-(*p*-carboxyl benzeneazo) calix[4]arene with norfloxacin (fungicide) in sodium acetateacetic acid buffer solution. The fluorescence results indicated a 1:1 complex stoichiometry. Stoichiometry was also evaluated by applying Job's method to fluorescence measurements to verify the 1:1 inclusion complex. Hydrogen bonding and structural matching effects were proposed to play important roles in the formation of the calixarene–norfloxacin complex in water. Furthermore, various factors affecting the inclusion process, such as pH value, ionic strength and surfactants, were examined in detail; the results corroborated the formation of this inclusion complex. The nature of the fungicide-calixarene interaction was mainly electrostatic. This investigation is crucial, since norfloxacin is a third-generation synthetic antibacterial fluoroquinolone agent that is used in the treatment of urinary and respiratory tract infections and gastro-intestinal and sexually transmitted diseases. Therefore, azocalix[*n*]arenes and its inclusion compounds may have potential biological and medical applications.

The effect of adding a macrocycle such as para-sulfonatocalix[6]arene on the fluorescence of benzoimidazolic fungicides such as Benomyl (BY) and Carbendazim (CZ) has been studied by Pacioni et al. (2008) using spectrofluorimetric titrations. Benomyl (BY) and Carbendazim (CZ) interact with para-sulfonatocalix[6]arene to form complexes. Calixarene enhanced the fluorescence of BY in water at pH 1.000 and 25o C. An inclusion complex with 1:1 stoichiometry was formed with the pesticide. The nature of the interactions was mainly electrostatic, i.e. cation–π and ion–ion interactions between the cationic BY and sulfonate groups. The use of macrocyclic nanocavities, compared with other methods, is a very good alternative to determine BY residues in water and fruit samples at low levels, with better or in the same order. The complexation of the neutral CZ occurs at pH 6.994. Two complexes with 1:1 and 1:2 stoichiometries were formed; the latter complex was less fluorescent than the free CZ. π-π stacking and hydrogen bonding are the main driving forces for the 1:1 and 1:2 complexes.

Organic quaternary ammonium ions have been of great interest in molecular recognition studies; paraquat (1,1' -dimethyl-4,4'-bipyridinium dichloride) is an example. Pierro et al. (2009) investigated the conformation fitting of tetramethoxy-para-carboxylatocalix[4]arene and tetrapropiloxi-para-carboxylatocalix[4]arene to paraquat in a CDCl3/CD3OD mixture using NMR measurements. This study demonstrated that tetrapropiloxi-paracarboxylatocalix[4]arene and paraquat formed a stable complex with a 1:1 stoichiometry, as estimated by a Job plot. NMR measurements indicated that upon complexation the C2v

Kunsági-Máté et al. (2004) investigated complex formation between C60 fullerene (dissolved in toluene) and water-soluble hexasulfonated calix[6]arenes functionalized with sulfonates in the upper rim. Photoluminescence (PL) measurements and Job's method were used for this investigation. The stoichiometry of the complex was 1:1, and related quantum chemical calculations demonstrated that the C60 fullerene is located deep within the cavity of the calixarene. This observation makes this calixarene a promising candidate for overcoming the natural water-repulsive character of C60 fullerene and could improve the application of

Zhou et al. (2008) used spectrofluorometric titrations to investigate the inclusion behavior of *p*-(*p*-carboxyl benzeneazo) calix[4]arene with norfloxacin (fungicide) in sodium acetateacetic acid buffer solution. The fluorescence results indicated a 1:1 complex stoichiometry. Stoichiometry was also evaluated by applying Job's method to fluorescence measurements to verify the 1:1 inclusion complex. Hydrogen bonding and structural matching effects were proposed to play important roles in the formation of the calixarene–norfloxacin complex in water. Furthermore, various factors affecting the inclusion process, such as pH value, ionic strength and surfactants, were examined in detail; the results corroborated the formation of this inclusion complex. The nature of the fungicide-calixarene interaction was mainly electrostatic. This investigation is crucial, since norfloxacin is a third-generation synthetic antibacterial fluoroquinolone agent that is used in the treatment of urinary and respiratory tract infections and gastro-intestinal and sexually transmitted diseases. Therefore, azocalix[*n*]arenes and its inclusion compounds may have potential biological and medical

The effect of adding a macrocycle such as para-sulfonatocalix[6]arene on the fluorescence of benzoimidazolic fungicides such as Benomyl (BY) and Carbendazim (CZ) has been studied by Pacioni et al. (2008) using spectrofluorimetric titrations. Benomyl (BY) and Carbendazim (CZ) interact with para-sulfonatocalix[6]arene to form complexes. Calixarene enhanced the fluorescence of BY in water at pH 1.000 and 25o C. An inclusion complex with 1:1 stoichiometry was formed with the pesticide. The nature of the interactions was mainly electrostatic, i.e. cation–π and ion–ion interactions between the cationic BY and sulfonate groups. The use of macrocyclic nanocavities, compared with other methods, is a very good alternative to determine BY residues in water and fruit samples at low levels, with better or in the same order. The complexation of the neutral CZ occurs at pH 6.994. Two complexes with 1:1 and 1:2 stoichiometries were formed; the latter complex was less fluorescent than the free CZ. π-π stacking and hydrogen bonding are the main driving forces for the 1:1 and

Organic quaternary ammonium ions have been of great interest in molecular recognition

(2009) investigated the conformation fitting of tetramethoxy-para-carboxylatocalix[4]arene and tetrapropiloxi-para-carboxylatocalix[4]arene to paraquat in a CDCl3/CD3OD mixture using NMR measurements. This study demonstrated that tetrapropiloxi-paracarboxylatocalix[4]arene and paraquat formed a stable complex with a 1:1 stoichiometry, as estimated by a Job plot. NMR measurements indicated that upon complexation the C2v


**5.1.2 Stoichiometric ratio with charged calixarenes** 

fullerenes to biochemical processes.

applications.

1:2 complexes.

studies; paraquat (1,1'

structure of the free calixarene switches to an opposite C2v pinched-cone conformation, with the two carboxylato-bearing rings pointing inward the calixarene cavity to maximize electrostatic and van der Waals interactions with the cationic paraquat. These adaptive conformational changes were fully confirmed by molecular modeling. The complexation of paraquat with tetrametoxi- para-carboxylatocalix[4]arene was also monitored with Diffusion-Ordered Spectroscopy (DOSY) NMR. DOSY has been particularly used in the characterization of host–guest systems in solution. The results indicated the formation of a complex in a 1:1 stoichiometry; in this complex, the calixarene was in the cone conformation while the free calixarene was predominantly in the partial-cone conformation.

Methiocarb [3,5-dimethyl-4-(methylthio) phenyl methylcarbamate] is one of the mostly important N-methylcarbamate pesticides, used worldwide in agriculture and health programs. Ding et al. (2011) investigated the complexation between tetrabutyl ether derivatives of p-sulfonatocalix[4]arene (SC4Bu) and methiocarb by fluorescence spectrometry in a mixture of water and DMSO. It was observed that upon the addition of methiocarb, the fluorescence intensity of SC4Bu was quenched and a slight red shift was observed for the maximum emission peak, which is indicative of a calixarene-methiocarb interaction. The results indicated that the SC4Bu-methiocarb complex was formed in a 1:1 mole ratio and that the electrostatic effect is not the main driving force. Using a modeling package it was proposed that complexation was an "external" inclusion process and that the hydrogen bonding between the methyl H atom of methiocarb and the sulfonate of SC4Bu facilitates the formation of this SC4Bu–methiocarb complex in a 1:1 stoichiometry. This study provides useful information for applying calixarenes to pesticide detection.

### **5.2 Stoichiometry in calixarene complexes formed with organic substrates in solid state**

For years, organic frameworks have been suggested as promising gas storage substrates, but purely organic molecular crystals have received little attention. Atwood et al. (2005) studied the absorption of methane in p-tert-butylcalix[4]arene at room temperature and pressures of one atmosphere and lower. The results, supported by purely size–shape considerations, suggest that it is possible to accommodate at least two CH4 molecules within each dimeric capsule (i.e., calixarene:substrate = 1:1). On the basis of this assumption, preliminary results indicate that on average, at 0.54 atm, 14% of capsules are occupied by two molecules of methane. The results demonstrate that low-density organic systems do indeed deserve consideration as potential sorbants for volatile gases, and that such sorption processes can occur under desirable conditions close to standard temperature and pressure.

Ferreira et al. (2010) studied the inclusion of biacetyl within p-tert-butylcalix[n=4,6]arenes in powdered solid samples using luminescence and diffuse reflectance. Lifetime distribution analysis of the phosphorescence of both complexes suggested *endo-*inclusion calixarene complexes with p-tert-butylcalix[n=4]arene, and *exo*-inclusion calixarene complexes with ptert-butylcalix[n=6]arene. Although it is not explicit, the stoichiometry of the inclusion complexes seems to be 1:1.

Table 1 summarizes the stoichiometric ratio of some of the discussed organic complexes in solution and in the solid state.

Stoichiometric Ratio in Calixarene Complexes 13

The preliminary test in water suggests that the stability of the PQ-para-tertbutylcalix[8]arene complex does not depend on the pH while that of PQ-para-tertbutylcalix[6]arene does. Being the former complex much more stable than the latter in aqueous media, we envision that para-tert-butylcalix[8]arene could be useful in stabilizing paraquat dichloride in solid, organic or water solutions to deposit/de-activate it as waste.

Calixarenes have been used to separate organic pesticides, pharmaceuticals and dyes that pollute water. However, little attention has been dedicated to the stoichiometric ratio of the

Shimojo & Goto (2005) studied the synergistic extraction of nucleobases by a combination of calixarene and D2EHPA ligands. Liquid-liquid extraction of various nucleobases with a para-tert-octylcalix[6]arene carboxylic acid derivative was carried out to elucidate its molecular recognition properties. Bis-(2-ethylhexyl)phosphoric acid (D2EHPA) was tested as a synergistic reagent to improve the extraction capability of the calixarene toward nucleobases (in buffered water). The efficiency of adenine and cytosine extraction increased drastically in the presence of both extractant ligands dissolved in isooctane. Neither calix[6]arene nor D2EHPA were very effective at nucleobase extraction. According to Job's method, the stoichiometry of the extracted stable species in the case of the better-extracted adenine was 1 adenine:1 calix[6]arene:2 D2EHPA. The authors proposed that D2EHPA is involved in the secondary coordination sphere of the calixarene-adenine complex and that its highly hydrophobic nature enhances the distribution of the complex into isooctane. Recovery of adenine from the organic phase to the aqueous receiving phase is readily achieved under acidic conditions. These results highlight the great potential of macrocyclic

As seen above, calixarenes are relevant to the removal of biological or pharmaceutical compounds from wastewater. Elsellami et al. (2009) implemented a coupling process between solid–liquid extraction and photocatalytic degradation for the selective separation of amino acids from water by calix[*n*]arene carboxylic acid derivatives and degradation by activation of a photocatalyst (TiO2) under UV light. The advantage of this liquid-solid extraction is that it selectively preconcentrates pollutants (tryptophan, phenyalanine and histidine). Apparently, the extracted complex was stabilized in a stoichiometric ratio of 1 calixarene:1 amino acid. The photodegradation followed a first-order kinetic, and the rate constant increased with amino acid concentration. Clearly, solid–liquid extraction is a simple, useful method, and the reagents are recyclable. Although these results are only preliminary, they suggest further possibilities for optimal extraction of amino acids and

**5.4 Stoichiometry in calixarene complexes formed with metallic substrates in solution**  Several books and reviews on calixarenes have been dedicated to the coordination chemistry of calixarenes with most of the metal elements of the periodic table (Alexandratos & Natesan, 2000; Lumetta et al., 2000; Mandolini and Ungaro, 2000; Mokhtari et al., 2011a, 2011b; Sliwa, 2002; Sliwa & Girck, 2010; Talanova, 2000). The most important feature of

**5.3 Stoichiometry of extracted species formed with organic substrates and** 

extracted species. Here, two examples are presented.

ligand calixarenes as extractants for bioproducts.

other pharmaceuticals.

**calixarenes** 


Table 1. Stoichiometry of organic calixarene species, in solution and in the solid state, and the used techniques for their identification and determination.

Garcia-Sosa & Ramírez (2010) found that para-tert-butylcalix[6]arene and para-tertbutylcalix[8]arene form stable 1:1 complexes with paraquat dichloride (PQ) in the solid state (the complexes were studied in liquid and solid states). The biexponential decay of luminescence and lifetimes proved that the quaternary ammoniums (quats) of the paraquat were not in the same environment for both complexes. This fact correlated with molecular models, since two de-excitation pathways were present with very different lifetimes; the longer lifetime was associated with one methyl-pyridinium head closer to the aryl rings, and the shorter lifetime was associated with the methyl-pyridinium head far from the aryl ring. The former ring is more shielded than the latter. Solution studies (UV/Vis, luminescence) and molecular modeling suggested that the calixarenes interact with the herbicide through cation-π interactions. Paraquat is included in the para-tert-butylcalix[8]arene cavity, but only partially included in the para-tert-butylcalix[6]arene cavity. The theoretical results, in particular using MOPAC procedures, were in good agreement with experimental findings.

UV/Vis titration in ethanol

Insight II package

Calorimetry; NMR titration Methanol

Fluorescence titration

Fluorescence titration

Liquid-liquid extraction

UV/Vis titration

Methane/calixarene X-ray diffraction 1:1 also modeled Atwood et al.

Table 1. Stoichiometry of organic calixarene species, in solution and in the solid state, and

Garcia-Sosa & Ramírez (2010) found that para-tert-butylcalix[6]arene and para-tertbutylcalix[8]arene form stable 1:1 complexes with paraquat dichloride (PQ) in the solid state (the complexes were studied in liquid and solid states). The biexponential decay of luminescence and lifetimes proved that the quaternary ammoniums (quats) of the paraquat were not in the same environment for both complexes. This fact correlated with molecular models, since two de-excitation pathways were present with very different lifetimes; the longer lifetime was associated with one methyl-pyridinium head closer to the aryl rings, and the shorter lifetime was associated with the methyl-pyridinium head far from the aryl ring. The former ring is more shielded than the latter. Solution studies (UV/Vis, luminescence) and molecular modeling suggested that the calixarenes interact with the herbicide through cation-π interactions. Paraquat is included in the para-tert-butylcalix[8]arene cavity, but only partially included in the para-tert-butylcalix[6]arene cavity. The theoretical results, in particular using MOPAC procedures, were in good agreement with experimental findings.

toluene

fluorescence titration

NMR titration/ CDCl3/CD3OD

Fluorescence titration/ H2O-DMSO

the used techniques for their identification and determination.

stoichiometry (M:L)

Molecular Modeling /(1:1)

Job's Method/ (1:1, 1:2 2:1; 1:1) /Modeling

Job's Method / BY(1:1), CZ(1:1, 1:2)

Molecular Modeling (1:1)

Job's Method/(1:1) Mohammed-

Job's Method /(1:1) Zhou et al.

Job's Method /(1:1) Kunsági-

Job's Method/(1:1) Shimojo &

Job's Method (1:1) Halder et al.

Job's Method(1:1) Pierro et al.

References

Ziegler, et al. (2003)

Zielenkiewicz et al. (2005)

(2008).

Máté et al. (2004)

Goto (2005).

Pacioni, et al. (2008).

(2010)

(2005**)** 

(2009)

Ding et al. (2011)

Organic Substrate/calixarene Technique Method/

Aliphatic amines /chromogenic capped calix[4]arene derivatives

Isoleucine(peptide)/

C60 fullerene / sulfonated calixarene

D2EHPA

phosporylated calix[4]arene.

Norfloxacin*/p*-(*p*-carboxyl benzene-azo) calix[4]arene

Adenine, guanine, cytosine, thymine /calixarene-

para-sulfonatocalix[6]arene.

paraquat / tetraalquiloxipara-carboxylatocalix[4]arene

N-methylcarbamate/parasulfonatocalix[4]arene

fullerenes (C60 and C70) /para-tert-butylcalix[6]arene

Benomyl (BY) and Carbendazim (CZ)**/**  The preliminary test in water suggests that the stability of the PQ-para-tertbutylcalix[8]arene complex does not depend on the pH while that of PQ-para-tertbutylcalix[6]arene does. Being the former complex much more stable than the latter in aqueous media, we envision that para-tert-butylcalix[8]arene could be useful in stabilizing paraquat dichloride in solid, organic or water solutions to deposit/de-activate it as waste.

### **5.3 Stoichiometry of extracted species formed with organic substrates and calixarenes**

Calixarenes have been used to separate organic pesticides, pharmaceuticals and dyes that pollute water. However, little attention has been dedicated to the stoichiometric ratio of the extracted species. Here, two examples are presented.

Shimojo & Goto (2005) studied the synergistic extraction of nucleobases by a combination of calixarene and D2EHPA ligands. Liquid-liquid extraction of various nucleobases with a para-tert-octylcalix[6]arene carboxylic acid derivative was carried out to elucidate its molecular recognition properties. Bis-(2-ethylhexyl)phosphoric acid (D2EHPA) was tested as a synergistic reagent to improve the extraction capability of the calixarene toward nucleobases (in buffered water). The efficiency of adenine and cytosine extraction increased drastically in the presence of both extractant ligands dissolved in isooctane. Neither calix[6]arene nor D2EHPA were very effective at nucleobase extraction. According to Job's method, the stoichiometry of the extracted stable species in the case of the better-extracted adenine was 1 adenine:1 calix[6]arene:2 D2EHPA. The authors proposed that D2EHPA is involved in the secondary coordination sphere of the calixarene-adenine complex and that its highly hydrophobic nature enhances the distribution of the complex into isooctane. Recovery of adenine from the organic phase to the aqueous receiving phase is readily achieved under acidic conditions. These results highlight the great potential of macrocyclic ligand calixarenes as extractants for bioproducts.

As seen above, calixarenes are relevant to the removal of biological or pharmaceutical compounds from wastewater. Elsellami et al. (2009) implemented a coupling process between solid–liquid extraction and photocatalytic degradation for the selective separation of amino acids from water by calix[*n*]arene carboxylic acid derivatives and degradation by activation of a photocatalyst (TiO2) under UV light. The advantage of this liquid-solid extraction is that it selectively preconcentrates pollutants (tryptophan, phenyalanine and histidine). Apparently, the extracted complex was stabilized in a stoichiometric ratio of 1 calixarene:1 amino acid. The photodegradation followed a first-order kinetic, and the rate constant increased with amino acid concentration. Clearly, solid–liquid extraction is a simple, useful method, and the reagents are recyclable. Although these results are only preliminary, they suggest further possibilities for optimal extraction of amino acids and other pharmaceuticals.

### **5.4 Stoichiometry in calixarene complexes formed with metallic substrates in solution**

Several books and reviews on calixarenes have been dedicated to the coordination chemistry of calixarenes with most of the metal elements of the periodic table (Alexandratos & Natesan, 2000; Lumetta et al., 2000; Mandolini and Ungaro, 2000; Mokhtari et al., 2011a, 2011b; Sliwa, 2002; Sliwa & Girck, 2010; Talanova, 2000). The most important feature of

Stoichiometric Ratio in Calixarene Complexes 15

and excimer decreased in a ratiometric manner. This ratiometric change is attributable to a combination of heavy metal ion effects, reverse-PET (photoinduced electron transfer) and conformational changes of the pyrene during the chelation of Cu2+and Pb2+ to form the 1:1 complex. The authors conclude that this calixarene acts as a selective sensor of Cu2+ and Pb2+ ions. Cu2+ is both a pollutant and an essential trace element, while elevated levels of Pb2+ in the environment cause anemia, kidney damage, blood disorders, memory loss, muscle

Arena et al. (2003) functionalized the 1- and 3- positions of a calix[4]arene with two dipyridyl pendants to create a ligand that complexes Cu(2+) and Co(2+). The new ligand, fixed in its 1,3-alternate conformation, forms stable complexes with both Co(2+) and Cu(2+), as shown by UV/Vis titrations carried out in acetonitrile. The stoichiometry of the main Co and Cu -calixarene species was determined by molar ratio and Job plot methods. Both methods indicated a single complex species with a 1:1 stoichiometry for Co2+, and two complex species for Cu2+ in 1:1 and 2:1 (metal:ligand) stoichiometry. In the case of Cu2+, speciation was confirmed by the multivariate and multiwavelength treatment of the data (60-70 points) using two different software packages (Specfit and Hyperquad). The existence of two complexes with the above-mentioned stoichiometry was confirmed. The authors conclude that the new ligand efficiently targets ions, and proposed that the cobalt complex is a good candidate as di-oxygen carrier, while the two copper complexes are good low molecular weight model systems for the study of copper enzyme catalytic activity in nonaqueous environments. The extracted complex species with 1:1 stoichiometry is a good candidate for nanoswitches; cyclic voltammetry studies showed reversible

Kumar et al. (2010) reported fluorescent sensors based on (N-(pyrenyl-1-methylimine) derivatized calix[4]arenes and investigated their metal-ion binding (Li+, Na+, K+, Pb2+, Zn2+, Hg2+ and Ag+) by UV and fluorescence spectroscopy in CH2Cl2/CH3CN. Two of these receptors in a cone conformation showed ratiometric sensing while the third receptor in a 1,3 alternate conformation showed 'On–Off' signaling for Pb2+. The stoichiometry of Pb2+ with the three ligands was 1:1 as established by a Job's plot of fluorescence titrations. The cation binding properties of the ligands were examined by 1H' NMR for Pb2+ in CDCl3 /CD3CN (1:9 v/v). The significant downfield shift of the imino protons (>1.5 ppm) indicated strong complexation between imino nitrogen atoms and Pb2+ ions. Fitting the changes to the ligands fluorescence spectra with other metal ions using the nonlinear regression analysis program SPECFIT gave good fit with a 1:1 (metal:ligand) stoichiometry. The stability constant data indicated that these ligands bind strongly to Pb2+ ions. Ag+ is

Bayrakc et al. (2009) synthesized several dinitro-substituted calix[4]arene-based receptors for extracting chromate and arsenate anions. Chromate and arsenate anions are important because of their high toxicity and presence in soil and water. Humans are sensitive to arsenic carcinogenesis; prolonged exposure to arsenic damages the central nervous system

to oxidize biological molecules. Therefore, the treatment of waste water containing Cr(6+) prior to discharge is essential. The upper and lower rims of para-tert-butylcalix[4]arene (L) were modified in order to create binding sites for the recognition of arsenate and dichromate


bound to ligands more weakly than Pb2+, but more strongly than Li+.

Chromium(6+) can be toxic, as it can diffuse as Cr2O72- or HCr2O7

and results in liver, lung, bladder, and skin cancers.

paralysis and mental retardation by lead poisoning.

oxidation/reduction behavior.

functionalized calixarenes for coordination with metal ions is that metal complexation properties depend not only on the nature of the binding groups grafted to the calixarene platform but also on their stereochemical arrangement, determined by the calixarene conformation and regulated by its size. The coordination ability of the calixarene can be completely changed, when functionalized by the same groups, by simply changing the rim these groups are attached to; the stability, selectivity and stoichiometric ratio of the metal calixarene complexes will be consequently altered. Metal ions can be highly toxic pollutants, pesticides, radionuclides, materials, nanomaterials, oligoelements, and motivate the current interest in synthesizing calixarenes with enhanced selectivity in the liquid and solid states.

### **5.4.1 Stoichiometric ratio with alkali and earth alkaline and metal transition ions**

Joseph et al. (2009) synthesized an amide-linked lower rim 1,3-bis(2-picolyl)amine calix[4]arene derivative. Binding properties of this ligand toward ten different biologically relevant metal ions (Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cu2+, Na+, K+, Ca2+, and Mg2+) have been studied by fluorescence and absorption spectroscopy in methanol and aqueous methanol. This ligand is a highly discriminating fluorescence sensor that selectively detects Cu2+ down to a concentration of 196 and 341 ppb in methanol and 1:1 aqueous methanol, respectively, even if other metal ions are present. Based on competitive metal ion titration studies, Cu2+ can be sensed even in the presence of other biologically relevant ions in aqueous solution. Both the calix[4]arene platform and the pyridyl binding core are required for selective recognition of Cu2+, as established by comparison of results obtained with the relevant control molecules e.g. the upper-rim-based quinoline derivative. The stoichiometry of the complex was 1:1, as calculated by a Job plot and confirmed by ESI MS. The computationally obtained structure for the Cu2+ complex exhibited a tetracoordinate geometry that is also seen in the blue copper protein, i.e. plastocyanin.

Dendrimers and hyperbranched molecules special properties come from their very peculiar molecular structures. Their structures have been accurately defined, and are prepared by established reactions and chosen pathways. In preliminary work, Mahouachi et al. (2006) investigated the extraction of solid zinc(2+) picrate hydrate into CDCl3 solutions (10-3 M) by a linear dendrimer composed of six para-tertbutylcalix[4]arene linked together by amideethylene-amine and amide-ethylene chains. 1H NMR spectra of the resulting solutions remained unchanged after 24 hours, indicating a stable complex. The integration ratio between the singlet of the picrate at 8.52 ppm and the aromatic protons of the linear dendrimer indicates that the stoichiometry of the complex is 2:1 (metal:ligand). However, the spectrum of the complex was broad, and the signal patterns could not be interpreted. The authors proposed that this broadening was due to metal coordination site exchange, since the ligand has three potential Zn(2+)-coordination sites, delineated by the amide functions and/or from a mixture of different arrangements of trinuclear complexes.

Sahin & Yilmaz (2011) synthesized a new fluorogenic calixarene bearing two pyrene amine groups; the ligand shows selectivity for Cu2+ and Pb2+ due to a conformational change upon chelation of the ions. Absorption (1x10-4 M) and fluorescence (1x10-6 M) spectra of ligands in CH3CN/CH2Cl2 solutions containing 10 mol equivalents of the appropriate metal perchlorate salt were recorded. The Job method was applied to determine the stoichiometry of the complexes, and the stability constants and quenching constants were determined by fluorimetric titration. When Cu2+and Pb2+ are bound to the calixarene, the pyrene monomer

functionalized calixarenes for coordination with metal ions is that metal complexation properties depend not only on the nature of the binding groups grafted to the calixarene platform but also on their stereochemical arrangement, determined by the calixarene conformation and regulated by its size. The coordination ability of the calixarene can be completely changed, when functionalized by the same groups, by simply changing the rim these groups are attached to; the stability, selectivity and stoichiometric ratio of the metal calixarene complexes will be consequently altered. Metal ions can be highly toxic pollutants, pesticides, radionuclides, materials, nanomaterials, oligoelements, and motivate the current interest in synthesizing calixarenes with enhanced selectivity in the liquid and solid states.

**5.4.1 Stoichiometric ratio with alkali and earth alkaline and metal transition ions** 

seen in the blue copper protein, i.e. plastocyanin.

Joseph et al. (2009) synthesized an amide-linked lower rim 1,3-bis(2-picolyl)amine calix[4]arene derivative. Binding properties of this ligand toward ten different biologically relevant metal ions (Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cu2+, Na+, K+, Ca2+, and Mg2+) have been studied by fluorescence and absorption spectroscopy in methanol and aqueous methanol. This ligand is a highly discriminating fluorescence sensor that selectively detects Cu2+ down to a concentration of 196 and 341 ppb in methanol and 1:1 aqueous methanol, respectively, even if other metal ions are present. Based on competitive metal ion titration studies, Cu2+ can be sensed even in the presence of other biologically relevant ions in aqueous solution. Both the calix[4]arene platform and the pyridyl binding core are required for selective recognition of Cu2+, as established by comparison of results obtained with the relevant control molecules e.g. the upper-rim-based quinoline derivative. The stoichiometry of the complex was 1:1, as calculated by a Job plot and confirmed by ESI MS. The computationally obtained structure for the Cu2+ complex exhibited a tetracoordinate geometry that is also

Dendrimers and hyperbranched molecules special properties come from their very peculiar molecular structures. Their structures have been accurately defined, and are prepared by established reactions and chosen pathways. In preliminary work, Mahouachi et al. (2006) investigated the extraction of solid zinc(2+) picrate hydrate into CDCl3 solutions (10-3 M) by a linear dendrimer composed of six para-tertbutylcalix[4]arene linked together by amideethylene-amine and amide-ethylene chains. 1H NMR spectra of the resulting solutions remained unchanged after 24 hours, indicating a stable complex. The integration ratio between the singlet of the picrate at 8.52 ppm and the aromatic protons of the linear dendrimer indicates that the stoichiometry of the complex is 2:1 (metal:ligand). However, the spectrum of the complex was broad, and the signal patterns could not be interpreted. The authors proposed that this broadening was due to metal coordination site exchange, since the ligand has three potential Zn(2+)-coordination sites, delineated by the amide

functions and/or from a mixture of different arrangements of trinuclear complexes.

Sahin & Yilmaz (2011) synthesized a new fluorogenic calixarene bearing two pyrene amine groups; the ligand shows selectivity for Cu2+ and Pb2+ due to a conformational change upon chelation of the ions. Absorption (1x10-4 M) and fluorescence (1x10-6 M) spectra of ligands in CH3CN/CH2Cl2 solutions containing 10 mol equivalents of the appropriate metal perchlorate salt were recorded. The Job method was applied to determine the stoichiometry of the complexes, and the stability constants and quenching constants were determined by fluorimetric titration. When Cu2+and Pb2+ are bound to the calixarene, the pyrene monomer and excimer decreased in a ratiometric manner. This ratiometric change is attributable to a combination of heavy metal ion effects, reverse-PET (photoinduced electron transfer) and conformational changes of the pyrene during the chelation of Cu2+and Pb2+ to form the 1:1 complex. The authors conclude that this calixarene acts as a selective sensor of Cu2+ and Pb2+ ions. Cu2+ is both a pollutant and an essential trace element, while elevated levels of Pb2+ in the environment cause anemia, kidney damage, blood disorders, memory loss, muscle paralysis and mental retardation by lead poisoning.

Arena et al. (2003) functionalized the 1- and 3- positions of a calix[4]arene with two dipyridyl pendants to create a ligand that complexes Cu(2+) and Co(2+). The new ligand, fixed in its 1,3-alternate conformation, forms stable complexes with both Co(2+) and Cu(2+), as shown by UV/Vis titrations carried out in acetonitrile. The stoichiometry of the main Co and Cu -calixarene species was determined by molar ratio and Job plot methods. Both methods indicated a single complex species with a 1:1 stoichiometry for Co2+, and two complex species for Cu2+ in 1:1 and 2:1 (metal:ligand) stoichiometry. In the case of Cu2+, speciation was confirmed by the multivariate and multiwavelength treatment of the data (60-70 points) using two different software packages (Specfit and Hyperquad). The existence of two complexes with the above-mentioned stoichiometry was confirmed. The authors conclude that the new ligand efficiently targets ions, and proposed that the cobalt complex is a good candidate as di-oxygen carrier, while the two copper complexes are good low molecular weight model systems for the study of copper enzyme catalytic activity in nonaqueous environments. The extracted complex species with 1:1 stoichiometry is a good candidate for nanoswitches; cyclic voltammetry studies showed reversible oxidation/reduction behavior.

Kumar et al. (2010) reported fluorescent sensors based on (N-(pyrenyl-1-methylimine) derivatized calix[4]arenes and investigated their metal-ion binding (Li+, Na+, K+, Pb2+, Zn2+, Hg2+ and Ag+) by UV and fluorescence spectroscopy in CH2Cl2/CH3CN. Two of these receptors in a cone conformation showed ratiometric sensing while the third receptor in a 1,3 alternate conformation showed 'On–Off' signaling for Pb2+. The stoichiometry of Pb2+ with the three ligands was 1:1 as established by a Job's plot of fluorescence titrations. The cation binding properties of the ligands were examined by 1H' NMR for Pb2+ in CDCl3 /CD3CN (1:9 v/v). The significant downfield shift of the imino protons (>1.5 ppm) indicated strong complexation between imino nitrogen atoms and Pb2+ ions. Fitting the changes to the ligands fluorescence spectra with other metal ions using the nonlinear regression analysis program SPECFIT gave good fit with a 1:1 (metal:ligand) stoichiometry. The stability constant data indicated that these ligands bind strongly to Pb2+ ions. Ag+ is bound to ligands more weakly than Pb2+, but more strongly than Li+.

Bayrakc et al. (2009) synthesized several dinitro-substituted calix[4]arene-based receptors for extracting chromate and arsenate anions. Chromate and arsenate anions are important because of their high toxicity and presence in soil and water. Humans are sensitive to arsenic carcinogenesis; prolonged exposure to arsenic damages the central nervous system and results in liver, lung, bladder, and skin cancers.

Chromium(6+) can be toxic, as it can diffuse as Cr2O7 2- or HCr2O7 - through cell membranes to oxidize biological molecules. Therefore, the treatment of waste water containing Cr(6+) prior to discharge is essential. The upper and lower rims of para-tert-butylcalix[4]arene (L) were modified in order to create binding sites for the recognition of arsenate and dichromate

Stoichiometric Ratio in Calixarene Complexes 17

**5.4.2 Stoichiometric ratio with lanthanide and actinide ions in liquid and solid states**  In the last decade, many functionalized calixarenes have been used as lanthanide ion (Ln(3+)) receptors, either to improve their photophysical properties by the antenna effect of

Le Saulnier et al. (1999) investigated the coordination chemistry of a tetraphosphinoylated para-tertbutylcalix[4]arene (B4bL4) with lanthanides, Ln, (Ln(3+) = La, Eu and Tb) and their luminescence. The stoichiometry of the complexes, both in solution and in the solid state, was 1:1 and 1:2 (M: B4bL4), as demonstrated by UV/Vis, NMR and ES-MS titrations and by applying Specfit software to UV/Vis data to determine speciation and stability constants in acetonitrile. Although the isolated complexes were very stable, the calixarene did not

Ramírez et al. (2001) prepared a tetra-ether-amide-para-tertbutylcalix[4]arene (L= A4bL4) and studied its coordination ability toward Ln(3+) = Eu, Gd, Tb, and Lu. The stoichiometry of the complex species in acetonitrile solution was demonstrated to be 1:1 (M:L) by 1H'- 13C-NMR and ES-MS titrations. The stability constants of the 1:1 species were estimated using the MINEQL+ program. A**4**bL**4** reacted with Ln(3+) in acetonitrile to yield a 1:1 complex. The crystal structure of the lutetium complex [Lu(A**4**bL**4**)(H**2**O)](CF**3**SO**3**)**3** 2Et**2**O corroborated the 1:1 stoichiometry and showed the metal ion encapsulated in the cavity formed by the four arms. Lu was 9-coordinate, bound to the four ether and four carbonyl functions and a water molecule that was itself H-bonded to the phenolic ether functions, rigidifying the cavity formed by the pendant arms. Additionally, an ether molecule is inserted into the hydrophobic cavity defined by the aromatic rings. Both NMR (La, Lu) and luminescence (Eu, Tb) data pointed to high local symmetry at the metal center while lifetime determinations were consistent with the coordination of an inner-sphere water molecule.

Although the calixarene sensitized the luminescence of the Tb ion, the quantum yield measured in acetonitrile was relatively low (*Q***abs** = 5.8%, *τ***<sup>F</sup>** = 1.42 ms), particularly for Eu (*Q***abs** = 2.0%, *τ***<sup>F</sup>** = 0.73 ms). This is most likely due to the presence of a ligand metal charge transfer (LMCT) state that severely limits such a process. This study demonstrated once more the calixarene platform potentiality to simultaneously complex inorganic and organic guests. This finding might be helpful for modeling and designing extraction processes.

A hexaphosphinoylated para-tertbutyl calix[6]arene (B**6**bL**6**) was synthesized by Ramírez et al. (2002). Temperature-dependent **1**H and **31**P NMR studies indicate a mixture of conformers with a time-averaged *C*6v symmetry at 405 K in dmso-d**6**; Δ*G*<sup>≠</sup> values for conformational inter conversion processes were equal to 68(1) and 75(2) kJ mol-1 and reveal a semi-flexible macrocycle with alternate in-out cone conformation in DMSO and CHCl**<sup>3</sup>** solutions, confirmed by molecular mechanics and dynamics calculations. B**6**bL**<sup>6</sup>** crystallized as a dimer, where the two calixarenes are linked through hydrogen bonding and surrounded by water and toluene molecules in the lattice. UV/Vis spectrophotometric titration of B**6**bL**6** with La(3+) in acetonitrile yielded stability constants of log *β***<sup>1</sup>** = 9.8 and log *β***<sup>2</sup>** = 19.6 for the 1 : 1

Complexes with La, Eu, Gd and Tb in 1:1 and 1:2 (M:calixarene) stoichiometries were isolated and characterized. Lifetime determinations of the Eu(3+) and Tb(3+) complexes in acetonitrile solution were consistent with no, or little, interaction of water molecules in the inner coordination sphere. The B**6**bL**<sup>6</sup>** sensitized the luminescence of Tb(3+) (*Q***abs** = 4.8%, *τ***f** = 2.1 ms, 1 :

the calixarene, or as selective extractants of Ln and actinides (An).

sensitize the Eu and Tb luminescence of the complexes.

and 1 : 2 (Ln : B**6**bL**6**) species, respectively.

anions. Protonated alkylammonium forms of the ionophores showed high affinity toward dichromate and arsenate anions. Both oxyanions were extracted; extraction of the dichromate ions from water into dichloromethane follows a linear relationship between log D versus log [L] at different concentrations of L, with the slope ≈ 1 at pH=1.5, suggesting that two calixarenes form 1:1 complexes with the extracted dichromate anion.

Table 2 summarizes the stoichiometric ratio of some of the discussed metal complexes in solution.


Table 2. Stoichiometry of metal calixarene species, in solution, and the used techniques for their identification and determination.

anions. Protonated alkylammonium forms of the ionophores showed high affinity toward dichromate and arsenate anions. Both oxyanions were extracted; extraction of the dichromate ions from water into dichloromethane follows a linear relationship between log D versus log [L] at different concentrations of L, with the slope ≈ 1 at pH=1.5, suggesting

Table 2 summarizes the stoichiometric ratio of some of the discussed metal complexes in

Stoichiometry(M:L)

Job plot/ (1:1) Sahin &

Specfit/ (1:1, 1:2) Le Saulnier

Specfit/ (1:1, 1:2) Ramirez et

Specfit/ (1:1, 2:1) Puntus et al.,

Job plot/ ESI-MS

Job plot/ Specfit / Hyperquad/Co2+1 (1:1)Cu2+(1:1, 2:1)

Job plot/Specfit/

Liquid-liquid extraction / (1:1)

MINEQL+ program/(1:1)

Specfit/ (1:1, 1:2) Liquid-liquid extraction/(1:1)

(1:1, 1:2) (1:1, 1:2 or 2:1)

Liquid-liquid extraction (1:1, 1:2)

/(1:1)

(1:1)

References

Joseph et al.,

Yilmaz, 2011

Arena et al., 2003

Kumar et al.,

Bayrakc et al., 2009

et al., 1999

Ramirez et al., 2001

al., 2002

Ramírez et al., 2008

Karavan et al., 2010

2007

2010

2009

that two calixarenes form 1:1 complexes with the extracted dichromate anion.

Fluorescence, UV/Vis; CH3OH; CH3OH-H2O

Fluorescence titration /CH2Cl2/CH3CN

Fluorescence titration /CH2Cl2/CH3CN

UV/Vis, NMR,ES-MS titrations/CH3CN

UV/Vis titration/

UV/Vis , Atomic absorption

NMR titration/

UV/Vis titration/

MS,UV/Vis, NMR titrations/CH3CN

UV/Vis titration/

Microcalorimetric, UV/Vis titrations/ CH3CN, CH3OH

Table 2. Stoichiometry of metal calixarene species, in solution, and the used techniques for

CH3CN

CH3CN

CH3CN UV/Vis

UV/Vis

CH3CN

Substrate Techniques/Solvent Methods/

solution.

calix[4]

derivatives

Cu2+/amide linked lower rim 1,3-bis(2-picolyl)amine

Cu2+ and Pb2+/ pyrenearmed calix[4]arene

Li1+,Na1+,K1+,Pb2+,Zn2+, Hg2+,Ag2+/(N-(pyrenyl methylimine)calix[4]

Ln/tetraphosphinoylated paratertbutylcalix[4]arene

Ln/ hexaphosphinoylated paratertbutylcalix[6]arene

Ln/octaphosphinoylated paratertbutylcalix[8]arene

An/hexaphosphinoylated paratertbutylcalix[6]arene

An, Ln/ phosphorylated calixarenes (upper rim) Ln,An/ phosphorylated calixarenes (lower rim)

An, Ln/ phosphorylated calixarenes (lower and

their identification and determination.

An, Ln/ B6bL6

upper rims)

Ln/ tetra-ether-amideparatertbutylcalix[4]arene

Co2+and Cu2+/ 1,3 calix[4]arene with two dipyridyl pendants

Cromate; Arsenate anions/ dinitro – substituted calix[4]arene

### **5.4.2 Stoichiometric ratio with lanthanide and actinide ions in liquid and solid states**

In the last decade, many functionalized calixarenes have been used as lanthanide ion (Ln(3+)) receptors, either to improve their photophysical properties by the antenna effect of the calixarene, or as selective extractants of Ln and actinides (An).

Le Saulnier et al. (1999) investigated the coordination chemistry of a tetraphosphinoylated para-tertbutylcalix[4]arene (B4bL4) with lanthanides, Ln, (Ln(3+) = La, Eu and Tb) and their luminescence. The stoichiometry of the complexes, both in solution and in the solid state, was 1:1 and 1:2 (M: B4bL4), as demonstrated by UV/Vis, NMR and ES-MS titrations and by applying Specfit software to UV/Vis data to determine speciation and stability constants in acetonitrile. Although the isolated complexes were very stable, the calixarene did not sensitize the Eu and Tb luminescence of the complexes.

Ramírez et al. (2001) prepared a tetra-ether-amide-para-tertbutylcalix[4]arene (L= A4bL4) and studied its coordination ability toward Ln(3+) = Eu, Gd, Tb, and Lu. The stoichiometry of the complex species in acetonitrile solution was demonstrated to be 1:1 (M:L) by 1H'- 13C-NMR and ES-MS titrations. The stability constants of the 1:1 species were estimated using the MINEQL+ program. A**4**bL**4** reacted with Ln(3+) in acetonitrile to yield a 1:1 complex. The crystal structure of the lutetium complex [Lu(A**4**bL**4**)(H**2**O)](CF**3**SO**3**)**3** 2Et**2**O corroborated the 1:1 stoichiometry and showed the metal ion encapsulated in the cavity formed by the four arms. Lu was 9-coordinate, bound to the four ether and four carbonyl functions and a water molecule that was itself H-bonded to the phenolic ether functions, rigidifying the cavity formed by the pendant arms. Additionally, an ether molecule is inserted into the hydrophobic cavity defined by the aromatic rings. Both NMR (La, Lu) and luminescence (Eu, Tb) data pointed to high local symmetry at the metal center while lifetime determinations were consistent with the coordination of an inner-sphere water molecule.

Although the calixarene sensitized the luminescence of the Tb ion, the quantum yield measured in acetonitrile was relatively low (*Q***abs** = 5.8%, *τ***<sup>F</sup>** = 1.42 ms), particularly for Eu (*Q***abs** = 2.0%, *τ***<sup>F</sup>** = 0.73 ms). This is most likely due to the presence of a ligand metal charge transfer (LMCT) state that severely limits such a process. This study demonstrated once more the calixarene platform potentiality to simultaneously complex inorganic and organic guests. This finding might be helpful for modeling and designing extraction processes.

A hexaphosphinoylated para-tertbutyl calix[6]arene (B**6**bL**6**) was synthesized by Ramírez et al. (2002). Temperature-dependent **1**H and **31**P NMR studies indicate a mixture of conformers with a time-averaged *C*6v symmetry at 405 K in dmso-d**6**; Δ*G*<sup>≠</sup> values for conformational inter conversion processes were equal to 68(1) and 75(2) kJ mol-1 and reveal a semi-flexible macrocycle with alternate in-out cone conformation in DMSO and CHCl**<sup>3</sup>** solutions, confirmed by molecular mechanics and dynamics calculations. B**6**bL**<sup>6</sup>** crystallized as a dimer, where the two calixarenes are linked through hydrogen bonding and surrounded by water and toluene molecules in the lattice. UV/Vis spectrophotometric titration of B**6**bL**6** with La(3+) in acetonitrile yielded stability constants of log *β***<sup>1</sup>** = 9.8 and log *β***<sup>2</sup>** = 19.6 for the 1 : 1 and 1 : 2 (Ln : B**6**bL**6**) species, respectively.

Complexes with La, Eu, Gd and Tb in 1:1 and 1:2 (M:calixarene) stoichiometries were isolated and characterized. Lifetime determinations of the Eu(3+) and Tb(3+) complexes in acetonitrile solution were consistent with no, or little, interaction of water molecules in the inner coordination sphere. The B**6**bL**<sup>6</sup>** sensitized the luminescence of Tb(3+) (*Q***abs** = 4.8%, *τ***f** = 2.1 ms, 1 :

Stoichiometric Ratio in Calixarene Complexes 19

pathways that facilitate 2:1 complex formation. This work demonstrated that the stoichiometry of lanthanide complexes with calixarenes can be tuned by a narrow and/or

Puntus et al. (2007) synthesized an octa-phosphinoylated para-tertbutylcalix[8]arene (B8bL8), and its structure was studied in solution. According to temperature-dependent 1H and 31P NMR spectroscopic data, the calix[8]arene adopts a so-called in–out cone conformation. Its coordination ability toward Ln (Ln (3+) = La, Eu, Tb, Lu) was probed by MS, UV/Vis and NMR spectroscopic titrations. Although both 1:1 (in the presence of triflate) and 2:1 (in the presence of nitrate) Ln:B8bL8 complexes could be isolated in the solid state, it was clear from the titration results that the major species present in methanol (solubility problems prevented the study in acetonitrile) had a 1:1 stoichiometry (irrespective of the anion), and the minor species a 2:1 stoichiometry. Observation of the 2:1 species was consistent with the bimetallic complexes usually isolated with calix[8]arenes, but only in the presence of a

NMR spectroscopic data indicated a common conformation for the 1:1 complexes in solution. Ln ions were coordinated by four of the eight phosphinoyl arms, with a coordination sphere completed by methanol molecules or nitrate ions, as ascertained by IR and MS spectra. B8bL8 displayed a weak absorption at 360 nm that can be assigned to an intraligand charge-transfer (ILCT) state that is very sensitive to coordination. Photophysical data for the Eu 2:1 complex pointed to similar chemical environments provided by the metal ion sites and no coordinated water, contrary to what is observed in the 1:1 complex. In this work, optical electronegativity predicted the energy of the charge-transfer states in the lanthanide systems with inequivalent ligands, and extensive analysis of the vibronic satellites of the Eu(5D0→7FJ) transitions allowed

Karavan et al. (2010) investigated the binding properties of three series of phosphorylated calixarene derivatives (bearing phosphine oxide or phosphonate groups either at the wide or the narrow rims) toward some representative lanthanide and actinide ions in solution. Complexation was studied in single media (methanol and acetonitrile) followed by UV spectrophotometric and isoperibolic (micro)calorimetric titrations (ITC). Using upper rim phosphorylated calixarenes, it was found that the solvating ability of methanol and acetonitrile influences stoichiometry, number and constant stability of the europium complex species (1:1 and/or 2:1 M: L species). In a single solvent, the major influence on the

stoichiometry of a complex is the length of the substituents bound to the OP groups.

No influence of calixarene size or substituent type in the upper rim was observed in the stoichiometry of uranyl calixarene species (1:1) when lower-rim-phosphorylated calixarenes were used in methanol, while in acetonitrile, a 2:1 species was found with the detertbutylated tetramer derivative. Similar stoichiometries were determined for europium complex species using the tertbutylated tetramer in methanol, where a 1:2 complex species

Calorimetry was very useful for the determination of stoichiometries, particularly when complexation did not induce significant spectral changes. It also provided full thermodynamic characterization of the complex species in organic solution. The influence of some structural features of the ligands on the nature of the substituents as well as the condensation degree of the calixarene moiety on the complexation thermodynamic parameters were thus established. It is clear that wide rim and narrow rim phosphine oxide

wide rim substituents suitable choice.

nitrate counterion as a result of its bidentate chelating mode.

the authors to draw conclusions about Eu(III) coordination.

formation was also observed.

1 complex) and Eu(3+) (*Q***abs** = 2.5%, *τ* = 2.0 ms, 1 : 2 complex) reasonably well in comparison with B4bL4. Molecular modeling calculations confirmed that the structure observed in the solid state, with phosphoryl groups interacting with water molecules, is a good model for the solution structure. The stability constants for the complexes with La(3+) were either smaller (1:1 complex) or equal (1:2) to the ones found for the corresponding B4bL4, in view of the larger flexibility of the calix[6]arene macrocycle. Photophysical properties were enhanced with respect to the smaller calix[4]arene, which opens the way for sensitive luminescence detection of these complexes, a definite advantage for quantifying extraction processes.

The coordination ability of the B6bL6 calixarene toward actinides was established by Ramírez et al. (2008). Spectrophotometric titration of uranyl with B6bL6 in CH3CN yielded log *β***11** = 7.1 and log *β***12** = 12.5 for the 1:1 and 1:2 (UO2 2+: B6bL6) species, respectively. UO2 2+ and Th(IV) complexes with 1:1 and 1:2 (M:L) stoichiometries were isolated and characterized. Uranyl compounds only fulfilled their CN=8 with B6bL6, while thorium compounds required coordinated nitrates and/or water molecules. The luminescence spectra, photophysical parameters and luminescence lifetimes of the uranyl complexes permitted an understanding of the coordination chemistry of these actinide calixarene complexes.

Energy transfer from the B6bL6 ligand to the uranyl ion was relevant in the 1:1 complex, with *Q*abs = 2.0%. The uranyl complex emission revealed biexponential decay for both complexes. The conclusion that we drew from this luminescence study and comparison with the emission spectra of uranyl nitrate recorded under various experimental conditions is that coordination of uranyl to the calixarene results in stabilization of its triplet state (heavy atom effect). This coordination promotes efficient energy transfer, although incomplete in the case of the 1:2 complex. Additionally, the macrocyclic molecule(s) provide(s) a protective environment, minimizing nonradiative deactivation processes.

A de-tert-butylated calix[6]arene (A6L6) fitted with six ether-amide pendant arms in the lower rim was synthesized and characterized in solution (Ramírez et al., 2004). NMR spectroscopic data point to the six phenoxide units adopting an average *D*6*<sup>h</sup>* conformation on the NMR time scale (1,2,3-alternate conformation). According to Augmented MM3 molecular mechanics and MOPAC quantum mechanical calculations, A6L6 is a ditopic ligand featuring two nonadentate coordination sites, each built from three pendant arms, and extending in opposite directions, with one arm above and the other below the main ring. A6L6 reacted with Ln ions (Ln(3+) = La, Eu) in acetonitrile to successively form 1:1 and 2:1 complexes. The isolated Eu 2:1 complex was luminescent (*Q***abs** = 2.5% in acetonitrile, upon ligand excitation), with bi-exponential luminescent decay, pointing to the presence of two differently coordinated metal ions, one with no bound water molecules, and the other one with two molecules bound.

According to molecular mechanics calculations, the more stable isomer was indeed asymmetric, with two nine-coordinate metal ions. Both Eu ions are bound to three bidentate arms and one monodentate triflate anion, but one metal ion completes its coordination sphere with two phenoxide oxygen atoms while the other uses two water molecules, which is consistent with IR spectroscopic and luminescence data. The two metal ion sites became equivalent in acetonitrile, and the relatively long lifetime (1.35 ms) indicates a coordination environment free of water molecules. The absence of substituents on the narrower rim of H6L6 (calix[6]arene) allows easy interconversion between different conformers, through either the ''tert-butyl through the annulus'' or ''narrower rim through the annulus''

1 complex) and Eu(3+) (*Q***abs** = 2.5%, *τ* = 2.0 ms, 1 : 2 complex) reasonably well in comparison with B4bL4. Molecular modeling calculations confirmed that the structure observed in the solid state, with phosphoryl groups interacting with water molecules, is a good model for the solution structure. The stability constants for the complexes with La(3+) were either smaller (1:1 complex) or equal (1:2) to the ones found for the corresponding B4bL4, in view of the larger flexibility of the calix[6]arene macrocycle. Photophysical properties were enhanced with respect to the smaller calix[4]arene, which opens the way for sensitive luminescence detection

The coordination ability of the B6bL6 calixarene toward actinides was established by Ramírez et al. (2008). Spectrophotometric titration of uranyl with B6bL6 in CH3CN yielded log *β***11** = 7.1 and log *β***12** = 12.5 for the 1:1 and 1:2 (UO22+: B6bL6) species, respectively. UO22+ and Th(IV) complexes with 1:1 and 1:2 (M:L) stoichiometries were isolated and characterized. Uranyl compounds only fulfilled their CN=8 with B6bL6, while thorium compounds required coordinated nitrates and/or water molecules. The luminescence spectra, photophysical parameters and luminescence lifetimes of the uranyl complexes permitted an

Energy transfer from the B6bL6 ligand to the uranyl ion was relevant in the 1:1 complex, with *Q*abs = 2.0%. The uranyl complex emission revealed biexponential decay for both complexes. The conclusion that we drew from this luminescence study and comparison with the emission spectra of uranyl nitrate recorded under various experimental conditions is that coordination of uranyl to the calixarene results in stabilization of its triplet state (heavy atom effect). This coordination promotes efficient energy transfer, although incomplete in the case of the 1:2 complex. Additionally, the macrocyclic molecule(s) provide(s) a protective

A de-tert-butylated calix[6]arene (A6L6) fitted with six ether-amide pendant arms in the lower rim was synthesized and characterized in solution (Ramírez et al., 2004). NMR spectroscopic data point to the six phenoxide units adopting an average *D*6*<sup>h</sup>* conformation on the NMR time scale (1,2,3-alternate conformation). According to Augmented MM3 molecular mechanics and MOPAC quantum mechanical calculations, A6L6 is a ditopic ligand featuring two nonadentate coordination sites, each built from three pendant arms, and extending in opposite directions, with one arm above and the other below the main ring. A6L6 reacted with Ln ions (Ln(3+) = La, Eu) in acetonitrile to successively form 1:1 and 2:1 complexes. The isolated Eu 2:1 complex was luminescent (*Q***abs** = 2.5% in acetonitrile, upon ligand excitation), with bi-exponential luminescent decay, pointing to the presence of two differently coordinated metal ions, one with no bound water molecules, and the other

According to molecular mechanics calculations, the more stable isomer was indeed asymmetric, with two nine-coordinate metal ions. Both Eu ions are bound to three bidentate arms and one monodentate triflate anion, but one metal ion completes its coordination sphere with two phenoxide oxygen atoms while the other uses two water molecules, which is consistent with IR spectroscopic and luminescence data. The two metal ion sites became equivalent in acetonitrile, and the relatively long lifetime (1.35 ms) indicates a coordination environment free of water molecules. The absence of substituents on the narrower rim of H6L6 (calix[6]arene) allows easy interconversion between different conformers, through either the ''tert-butyl through the annulus'' or ''narrower rim through the annulus''

understanding of the coordination chemistry of these actinide calixarene complexes.

environment, minimizing nonradiative deactivation processes.

one with two molecules bound.

of these complexes, a definite advantage for quantifying extraction processes.

pathways that facilitate 2:1 complex formation. This work demonstrated that the stoichiometry of lanthanide complexes with calixarenes can be tuned by a narrow and/or wide rim substituents suitable choice.

Puntus et al. (2007) synthesized an octa-phosphinoylated para-tertbutylcalix[8]arene (B8bL8), and its structure was studied in solution. According to temperature-dependent 1H and 31P NMR spectroscopic data, the calix[8]arene adopts a so-called in–out cone conformation. Its coordination ability toward Ln (Ln (3+) = La, Eu, Tb, Lu) was probed by MS, UV/Vis and NMR spectroscopic titrations. Although both 1:1 (in the presence of triflate) and 2:1 (in the presence of nitrate) Ln:B8bL8 complexes could be isolated in the solid state, it was clear from the titration results that the major species present in methanol (solubility problems prevented the study in acetonitrile) had a 1:1 stoichiometry (irrespective of the anion), and the minor species a 2:1 stoichiometry. Observation of the 2:1 species was consistent with the bimetallic complexes usually isolated with calix[8]arenes, but only in the presence of a nitrate counterion as a result of its bidentate chelating mode.

NMR spectroscopic data indicated a common conformation for the 1:1 complexes in solution. Ln ions were coordinated by four of the eight phosphinoyl arms, with a coordination sphere completed by methanol molecules or nitrate ions, as ascertained by IR and MS spectra. B8bL8 displayed a weak absorption at 360 nm that can be assigned to an intraligand charge-transfer (ILCT) state that is very sensitive to coordination. Photophysical data for the Eu 2:1 complex pointed to similar chemical environments provided by the metal ion sites and no coordinated water, contrary to what is observed in the 1:1 complex. In this work, optical electronegativity predicted the energy of the charge-transfer states in the lanthanide systems with inequivalent ligands, and extensive analysis of the vibronic satellites of the Eu(5D0→7FJ) transitions allowed the authors to draw conclusions about Eu(III) coordination.

Karavan et al. (2010) investigated the binding properties of three series of phosphorylated calixarene derivatives (bearing phosphine oxide or phosphonate groups either at the wide or the narrow rims) toward some representative lanthanide and actinide ions in solution. Complexation was studied in single media (methanol and acetonitrile) followed by UV spectrophotometric and isoperibolic (micro)calorimetric titrations (ITC). Using upper rim phosphorylated calixarenes, it was found that the solvating ability of methanol and acetonitrile influences stoichiometry, number and constant stability of the europium complex species (1:1 and/or 2:1 M: L species). In a single solvent, the major influence on the stoichiometry of a complex is the length of the substituents bound to the OP groups.

No influence of calixarene size or substituent type in the upper rim was observed in the stoichiometry of uranyl calixarene species (1:1) when lower-rim-phosphorylated calixarenes were used in methanol, while in acetonitrile, a 2:1 species was found with the detertbutylated tetramer derivative. Similar stoichiometries were determined for europium complex species using the tertbutylated tetramer in methanol, where a 1:2 complex species formation was also observed.

Calorimetry was very useful for the determination of stoichiometries, particularly when complexation did not induce significant spectral changes. It also provided full thermodynamic characterization of the complex species in organic solution. The influence of some structural features of the ligands on the nature of the substituents as well as the condensation degree of the calixarene moiety on the complexation thermodynamic parameters were thus established. It is clear that wide rim and narrow rim phosphine oxide

Stoichiometric Ratio in Calixarene Complexes 21

of the aliphatic substituents, notwithstanding solvent effects, are all factors that define the

Jean Marie Lehn (Lehn, 1990) established that molecular recognition is key not only to biological macromolecules but also to macrocyclic complexation. Experimental results are sometimes not sufficient to elucidate how recognition occurs, which sites coordinate, how the stereochemical arrangement influences stabilization of the complex molecule, or why the substrate physicochemical properties drastically change after interaction with a certain macrocycle. Since suitable single crystals of macrocycle complexes are difficult to obtain, simulation of complexes with molecular modeling has become extremely useful. New molecular receptors can also be designed, built and optimized with the same software, although successful prediction depends on the molecule and the calculation approach used. Molecular modeling is mostly useful when the molecule is based on experimental data. It has to be used with a great care to avoid meaningless results. The more complicated the molecule, the less likely the model is to represent the real complex. Before 1999, only empirical calculations were used for calixarene complexes, but over the last decade, ab initio

Kunsági-Máté et al. (2004) used the HYPERCHEM Professional 7 and related quantumchemical calculations to elucidate the inclusion of C60 fullerene in the hexasulfonated calix[6]arene functionalized at the upper rim. The calculation showed that C60 lies deep in

Zielenkiewicz et al. (2005) used INSIGHT II to show that a phosphorylated calix[4]arene effectively bound for the amino acid isoleucine. Based on the calculation results for 1:1 and 1:2 (isoleucine: calixarene) complexes, the inclusion of the isoleucine into the calixarene cavity stabilizes the macrocyclic skeleton in the regular cone conformation. Strong electrostatic interactions between the phosphoryl group of the calixarene and the positively charged amino

Atwood et al. (2005) visualized purely size–shape considerations with X-SEED to determine whether two CH4 molecules could fit within each dimeric capsule. The calculation

To understand the structural features of the 1:1 complex formed between an amide-linked lower rim 1,3-bis(2-picolyl)amine derivative of calix[4]arene and Cu2+, Joseph et al. (2009) calculated the complex using GAUSSIAN 03 and DFT calculations. The calculated structure for the Cu2+ complex exhibited a tetracoordinate geometry, where all four pyridyl moieties were involved in binding and the coordination of Cu2+ center was a highly distorted

Ding et al. (2011) calculated the most stable structures of the complex (the lowest energy was 0 kcal/mol) formed between tetrabutyl ether derivatives of p-sulfonatocalix[4]arene (SC4Bu) and the methiocarb pesticide using GAUSSIAN 03. The complexation was an "external" inclusion process, and hydrogen bonding between the methyl H atom of methiocarb and the sulfonate of SC4Bu facilitated the formation of this SC4Bu–methiocarb

group of the amino acid played an important role in the complexation process.

illustrated the capsules' excellent size–shape compatibility.

and semi-empirical calculations have been successfully attempted.

stoichiometry of extracted species.

**6. Molecular modeling** 

the cavity of the calixarene.

tetrahedral.

complex.

derivatives formed 1:1 complexes with europium and uranyl, accompanied in some cases with 2:1 complexes or 1:2 species with europium.

The stabilization origin of the complexes is quite different for the two cations and depends on the solvent. Whereas entropy terms are generally favorable in methanol for europium complexes, the entropy contributions appear to be very negative and hence unfavorable in acetonitrile. This finding indicates the importance of solvation/desolvation in the complexation process. In contrast, the stabilization of the uranyl complexes is mostly enthalpy-driven in both solvents.

### **5.4.3 Stoichiometric ratio in extracted species formed with lanthanide and actinides ions**

Studies of metal ion separations using liquid-liquid and liquid-solid extraction systems and the evaluation of their experimental parameters—extraction percentage, distribution (coefficients) ratios, loading capacity and the stoichiometry of the extracted species among others— allow for gaining a complete physicochemical understanding of the extraction system and its applications.

Long-lived radionuclides, actinides in particular, are the most hazardous components of nuclear waste. The recovery of these elements from waste mass, alone or combined with other elements like lanthanides before disposal or reprocessing, would significantly enhance the ecological safety and efficiency of the nuclear fuel cycle. Phosphorylated calixarenes offer numerous possibilities for selective complexation of metal ions and will likely be essential in the treatment of nuclear waste.

Here, we focus on the Ln and An extracted species with phosphinoylated (phosphorylated) calixarenes from aqueous media to organic media. The usefulness of this type of calixarene for Ln/An separation with high efficiency was proved several years ago (Lumetta et al., 2000). The stoichiometry of extracted species with a certain calixarene is not necessarily in agreement with that of its complex species. Furthermore, a functionalized calixarene with the same phosphinoylated arms in the lower rim or in the upper rim does not extract a species with the same stoichiometry (Arnaud-Neu et al., 2000; Karavan et al., 2010).

Solvent can influence the conformational arrangement of the calixarene and thus the stabilization of certain complex species. It can also compete for metal ions, affecting the M:calixarene stoichiometric ratio. Experiments were conducted on the B6bL6 calixarene functionalized in the lower rim mentioned above (Ramírez et al., 2008) in a liquid-liquid extraction system of metal salt/1 M HNO3/3.5 M NaNO3-calixarene in chloroform. A 1:1 stoichiometry was found for the Eu(3+), UO2 2+ and Th(4+) extracted species while the complexation study in acetonitrile demonstrated 1:1 and 1:2 complexes in the solution and solid state.

Karavan et al. (2010) found that for liquid–liquid extraction from nitric acid to mnitrobenzotrifluoride uranyl extracted species were in a 1:2 stoichiometry using a pentamer functionalized on the lower rim while in methanol and in acetonitrile the uranyl complex species were in 1:1 stoichiometry. However, europium extracted species kept the same stoichiometry. In contrast, upper-rim functionalized calixarenes using the same extraction system formed 1:1 uranyl extracted species and europium in 1:1 and 1:2 stoichiometries. Thus, the size, conformation, choice of rim (upper or lower), and length and isomeric nature of the aliphatic substituents, notwithstanding solvent effects, are all factors that define the stoichiometry of extracted species.

### **6. Molecular modeling**

20 Stoichiometry and Research – The Importance of Quantity in Biomedicine

derivatives formed 1:1 complexes with europium and uranyl, accompanied in some cases

The stabilization origin of the complexes is quite different for the two cations and depends on the solvent. Whereas entropy terms are generally favorable in methanol for europium complexes, the entropy contributions appear to be very negative and hence unfavorable in acetonitrile. This finding indicates the importance of solvation/desolvation in the complexation process. In contrast, the stabilization of the uranyl complexes is mostly

**5.4.3 Stoichiometric ratio in extracted species formed with lanthanide and actinides** 

Studies of metal ion separations using liquid-liquid and liquid-solid extraction systems and the evaluation of their experimental parameters—extraction percentage, distribution (coefficients) ratios, loading capacity and the stoichiometry of the extracted species among others— allow for gaining a complete physicochemical understanding of the extraction

Long-lived radionuclides, actinides in particular, are the most hazardous components of nuclear waste. The recovery of these elements from waste mass, alone or combined with other elements like lanthanides before disposal or reprocessing, would significantly enhance the ecological safety and efficiency of the nuclear fuel cycle. Phosphorylated calixarenes offer numerous possibilities for selective complexation of metal ions and will likely be

Here, we focus on the Ln and An extracted species with phosphinoylated (phosphorylated) calixarenes from aqueous media to organic media. The usefulness of this type of calixarene for Ln/An separation with high efficiency was proved several years ago (Lumetta et al., 2000). The stoichiometry of extracted species with a certain calixarene is not necessarily in agreement with that of its complex species. Furthermore, a functionalized calixarene with the same phosphinoylated arms in the lower rim or in the upper rim does not extract a

Solvent can influence the conformational arrangement of the calixarene and thus the stabilization of certain complex species. It can also compete for metal ions, affecting the M:calixarene stoichiometric ratio. Experiments were conducted on the B6bL6 calixarene functionalized in the lower rim mentioned above (Ramírez et al., 2008) in a liquid-liquid extraction system of metal salt/1 M HNO3/3.5 M NaNO3-calixarene in chloroform. A 1:1 stoichiometry was found for the Eu(3+), UO22+ and Th(4+) extracted species while the complexation study in acetonitrile demonstrated 1:1 and 1:2 complexes in the solution and

Karavan et al. (2010) found that for liquid–liquid extraction from nitric acid to mnitrobenzotrifluoride uranyl extracted species were in a 1:2 stoichiometry using a pentamer functionalized on the lower rim while in methanol and in acetonitrile the uranyl complex species were in 1:1 stoichiometry. However, europium extracted species kept the same stoichiometry. In contrast, upper-rim functionalized calixarenes using the same extraction system formed 1:1 uranyl extracted species and europium in 1:1 and 1:2 stoichiometries. Thus, the size, conformation, choice of rim (upper or lower), and length and isomeric nature

species with the same stoichiometry (Arnaud-Neu et al., 2000; Karavan et al., 2010).

with 2:1 complexes or 1:2 species with europium.

enthalpy-driven in both solvents.

system and its applications.

essential in the treatment of nuclear waste.

**ions** 

solid state.

Jean Marie Lehn (Lehn, 1990) established that molecular recognition is key not only to biological macromolecules but also to macrocyclic complexation. Experimental results are sometimes not sufficient to elucidate how recognition occurs, which sites coordinate, how the stereochemical arrangement influences stabilization of the complex molecule, or why the substrate physicochemical properties drastically change after interaction with a certain macrocycle. Since suitable single crystals of macrocycle complexes are difficult to obtain, simulation of complexes with molecular modeling has become extremely useful. New molecular receptors can also be designed, built and optimized with the same software, although successful prediction depends on the molecule and the calculation approach used.

Molecular modeling is mostly useful when the molecule is based on experimental data. It has to be used with a great care to avoid meaningless results. The more complicated the molecule, the less likely the model is to represent the real complex. Before 1999, only empirical calculations were used for calixarene complexes, but over the last decade, ab initio and semi-empirical calculations have been successfully attempted.

Kunsági-Máté et al. (2004) used the HYPERCHEM Professional 7 and related quantumchemical calculations to elucidate the inclusion of C60 fullerene in the hexasulfonated calix[6]arene functionalized at the upper rim. The calculation showed that C60 lies deep in the cavity of the calixarene.

Zielenkiewicz et al. (2005) used INSIGHT II to show that a phosphorylated calix[4]arene effectively bound for the amino acid isoleucine. Based on the calculation results for 1:1 and 1:2 (isoleucine: calixarene) complexes, the inclusion of the isoleucine into the calixarene cavity stabilizes the macrocyclic skeleton in the regular cone conformation. Strong electrostatic interactions between the phosphoryl group of the calixarene and the positively charged amino group of the amino acid played an important role in the complexation process.

Atwood et al. (2005) visualized purely size–shape considerations with X-SEED to determine whether two CH4 molecules could fit within each dimeric capsule. The calculation illustrated the capsules' excellent size–shape compatibility.

To understand the structural features of the 1:1 complex formed between an amide-linked lower rim 1,3-bis(2-picolyl)amine derivative of calix[4]arene and Cu2+, Joseph et al. (2009) calculated the complex using GAUSSIAN 03 and DFT calculations. The calculated structure for the Cu2+ complex exhibited a tetracoordinate geometry, where all four pyridyl moieties were involved in binding and the coordination of Cu2+ center was a highly distorted tetrahedral.

Ding et al. (2011) calculated the most stable structures of the complex (the lowest energy was 0 kcal/mol) formed between tetrabutyl ether derivatives of p-sulfonatocalix[4]arene (SC4Bu) and the methiocarb pesticide using GAUSSIAN 03. The complexation was an "external" inclusion process, and hydrogen bonding between the methyl H atom of methiocarb and the sulfonate of SC4Bu facilitated the formation of this SC4Bu–methiocarb complex.

Stoichiometric Ratio in Calixarene Complexes 23

The aim of this chapter was to establish the relevance of stoichiometric studies in understanding complexes formed, either in solution or in the solid state, between calixarene receptors and organic or metal substrates. Spectroscopic techniques are highly useful in investigating the stoichiometry of calixarene complexes. Nevertheless, many stoichiometric studies require more than one technique, or one that affords thermodynamic parameters. Computational calculations have gained an important place in determining the stoichiometry of calixarene complexes. Visualizing the structural arrangement of a calixarene complex with a particular stoichiometry can help elucidate how the platform of a conformational functionalized calixarene at the upper or lower rim can drastically change the physical, chemical or physicochemical properties of the complexed substrate. Great care must be taken in experimentally determining stoichiometry and in correlating these ratios

In this chapter we have focused our attention on investigations linking basic research to real problems, from pollution and water treatment, to nuclear waste treatment, fuel storage and luminescent materials. The presented examples were carefully chosen to provide sufficient knowledge of stoichiometric studies from the experimental, theoretical and applications point of view. Furthermore, these examples afford an understanding of the physical, chemical and stereochemical parameters that are responsible for the stoichiometry and

The relevancy of stoichiometry and structure of the discussed examples for their future

The increasingly interest in calixarenes resides in their versatile intrinsic properties allowing them to be functionalized and to envision their potential applications in different fields of science and technology. Particular attention has to be paid to the use of functionalized calixarenes in biomedical studies, particularly on the stoichiometry and structure of organic

The authors thank CONACYT (México), project Nr. 36689-E and the Swiss National Science Foundation project SCOPES No 7BUPJ062293; their works cited in this chapter were developed with their support. They also thanks to Mr. Claudio Fernández from the Library

Alexandratos, S. D. & Natesan, S. (2000). Coordination Chemistry of Phosphorylated

Arena, G., Contino, A., Longo, E., Sciotto, D., Sgarlata C. & Spoto G. (2003). Synthesis,

Calixarenes and Their Application to Separations Science. *Industrial & Engineering Chemistry Research*, Vol. 39, No. 11, (Sept. 2000), pp.3998-4010, DOI: 10.1021

Characterization of a Novel Calixarene Having Dipyridyl Pendants and Study of its Complexes with Cu(II) and Co(II). *Tetrahedron Letters*, Vol. 44, No. 29, (July 2003),

structure of an organic calixarene complex or of a metal calixarene complex.

and/ or metal complexes with potential biomedical applications.

with molecular modeling to avoid incorrect conclusions.

**7. Conclusions** 

applications is undoubted.

**8. Acknowledgment**

**9. References** 

/ie000294x.

of ININ for his help anytime they needed it.

pp. 5415–5418, ISSN: 0040-4039

We have used molecular modeling in our current work with calixarenes and calixarene complexes. Structures have been built, and their minimum energies calculated, using the CAChe WorkSystem Pro 5.02 for Windows®. Free calixarenes (Ramírez et al., 2004; Ramírez et al., 2008; García-Sosa & Ramírez, 2010) and calixarenes complexed with organic substrates (García-Sosa & Ramírez, 2010) have been simulated by sequential application of Augmented MM3/CONFLEX/Augmented MM3/ MOPAC/PM5 or PM3/ MOPAC/PM5 or PM3/COSMO procedures.

MOPAC/PM5 or PM3/COSMO procedures calculate the most stable molecules under aqueous solvent effects and the heat formation of the most stable molecule (given in kcal.mol-1).

The calixarene complexes formed with lanthanide and actinide ions were calculated by sequential application of Augmented MM3/CONFLEX procedures (Ramírez et al., 2004; Ramírez et al., 2008). Augmented MM3 yielded the optimum structure, and CONFLEX yielded the most stable conformers (kcal.mol-1).

Fig. 4 shows the modeled structure of the de-tert-butylated calix[6]arene fitted with six ether amide pendant arms, A6L6 (Fig. 4, left) and its europium complex in a 2:1 (metal: ligand) stoichiometry (Fig. 4, right). The modeling shows that the free calixarene is a ditopic ligand featuring two nonadentate coordination sites; each is built from three pendant arms and extends in opposite directions, one site above, and the other under the main ring. The modeled structure of the dimetallic complex is a stable asymmetric isomer with two ninecoordinate metal ions.

Fig. 4. Molecular modeling of A6L6 calixarene (left) and optimized geometry of the Eu (3+) 2:1 complex with A6L6 (right), as determined by MM3 Augmented and Conflex CAChe procedures (Ramírez et al., 2004).

Both Eu (3+) ions are bound to three bidentate arms and one monodentate triflate anion, but one of the metal ion completes its coordination sphere with two phenoxide oxygen atoms whereas the other uses two water molecules. These computational results are consistent with IR and luminescence data (Ramírez et al., 2004) and elucidate the luminescence behavior of the complex in solid and in solution.

### **7. Conclusions**

22 Stoichiometry and Research – The Importance of Quantity in Biomedicine

We have used molecular modeling in our current work with calixarenes and calixarene complexes. Structures have been built, and their minimum energies calculated, using the CAChe WorkSystem Pro 5.02 for Windows®. Free calixarenes (Ramírez et al., 2004; Ramírez et al., 2008; García-Sosa & Ramírez, 2010) and calixarenes complexed with organic substrates (García-Sosa & Ramírez, 2010) have been simulated by sequential application of Augmented MM3/CONFLEX/Augmented MM3/ MOPAC/PM5 or PM3/

MOPAC/PM5 or PM3/COSMO procedures calculate the most stable molecules under aqueous solvent effects and the heat formation of the most stable molecule (given in kcal.mol-1). The calixarene complexes formed with lanthanide and actinide ions were calculated by sequential application of Augmented MM3/CONFLEX procedures (Ramírez et al., 2004; Ramírez et al., 2008). Augmented MM3 yielded the optimum structure, and CONFLEX

Fig. 4 shows the modeled structure of the de-tert-butylated calix[6]arene fitted with six ether amide pendant arms, A6L6 (Fig. 4, left) and its europium complex in a 2:1 (metal: ligand) stoichiometry (Fig. 4, right). The modeling shows that the free calixarene is a ditopic ligand featuring two nonadentate coordination sites; each is built from three pendant arms and extends in opposite directions, one site above, and the other under the main ring. The modeled structure of the dimetallic complex is a stable asymmetric isomer with two nine-

Fig. 4. Molecular modeling of A6L6 calixarene (left) and optimized geometry of the Eu (3+) 2:1 complex with A6L6 (right), as determined by MM3 Augmented and Conflex CAChe

Both Eu (3+) ions are bound to three bidentate arms and one monodentate triflate anion, but one of the metal ion completes its coordination sphere with two phenoxide oxygen atoms whereas the other uses two water molecules. These computational results are consistent with IR and luminescence data (Ramírez et al., 2004) and elucidate the luminescence

MOPAC/PM5 or PM3/COSMO procedures.

yielded the most stable conformers (kcal.mol-1).

coordinate metal ions.

procedures (Ramírez et al., 2004).

behavior of the complex in solid and in solution.

The aim of this chapter was to establish the relevance of stoichiometric studies in understanding complexes formed, either in solution or in the solid state, between calixarene receptors and organic or metal substrates. Spectroscopic techniques are highly useful in investigating the stoichiometry of calixarene complexes. Nevertheless, many stoichiometric studies require more than one technique, or one that affords thermodynamic parameters. Computational calculations have gained an important place in determining the stoichiometry of calixarene complexes. Visualizing the structural arrangement of a calixarene complex with a particular stoichiometry can help elucidate how the platform of a conformational functionalized calixarene at the upper or lower rim can drastically change the physical, chemical or physicochemical properties of the complexed substrate. Great care must be taken in experimentally determining stoichiometry and in correlating these ratios with molecular modeling to avoid incorrect conclusions.

In this chapter we have focused our attention on investigations linking basic research to real problems, from pollution and water treatment, to nuclear waste treatment, fuel storage and luminescent materials. The presented examples were carefully chosen to provide sufficient knowledge of stoichiometric studies from the experimental, theoretical and applications point of view. Furthermore, these examples afford an understanding of the physical, chemical and stereochemical parameters that are responsible for the stoichiometry and structure of an organic calixarene complex or of a metal calixarene complex.

The relevancy of stoichiometry and structure of the discussed examples for their future applications is undoubted.

The increasingly interest in calixarenes resides in their versatile intrinsic properties allowing them to be functionalized and to envision their potential applications in different fields of science and technology. Particular attention has to be paid to the use of functionalized calixarenes in biomedical studies, particularly on the stoichiometry and structure of organic and/ or metal complexes with potential biomedical applications.

### **8. Acknowledgment**

The authors thank CONACYT (México), project Nr. 36689-E and the Swiss National Science Foundation project SCOPES No 7BUPJ062293; their works cited in this chapter were developed with their support. They also thanks to Mr. Claudio Fernández from the Library of ININ for his help anytime they needed it.

### **9. References**


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**2** 

*Korea* 

**Water-Soluble Calix[4]arene Derivatives:** 

Satish Balasaheb Nimse1, Keum-Soo Song2 and Taisun Kim1\*

If hydrophobic molecules are inserted into an aqueous medium, the water molecules order around the hydrophobic ones to build a quasi crystalline surface. In this way, the hydrogen bonding of the water molecules around a hydrophobic surface is maximized. If two hydrophobic molecules meet they will associate with their hydrophobic surfaces towards each other. The water molecules, previously attached to these surfaces, will be distributed back into the bulk solvent resulting in favourable entropy. The entropic gain is responsible for almost all associations in the medium water and hence extremely important for life (e.g. formation of membranes, micelles, and for protein folding where folding starts often with tryptophan residues forming a hydrophobic core). The hydrophobic effect is shown below (**Figure 1**).

Similarly, in most cases the protein-substrate binding is a result of the hydrophobic effect. However, there are evidences suggesting that the water molecules play an important role in the protein-substrate binding. Water molecules could participate in hydrogen bonding networks that link side chain and main chain atoms with the functional groups on the bases,

Fig. 1. Host-guest binding mechanism in aqueous medium.

**1. Introduction** 

*1Institute for Applied Chemistry and Department of Chemistry,* 

**Evaluation of the Host-Guest** 

**Recognition Mechanism** 

*Hallym University, Chuncheon* 

*2Biometrix Technology Inc., Chuncheon,* 

**Binding Stoichiometry and Spectroscopic** 


## **Water-Soluble Calix[4]arene Derivatives: Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism**

Satish Balasaheb Nimse1, Keum-Soo Song2 and Taisun Kim1\* *1Institute for Applied Chemistry and Department of Chemistry,* 

> *Hallym University, Chuncheon 2Biometrix Technology Inc., Chuncheon, Korea*

### **1. Introduction**

26 Stoichiometry and Research – The Importance of Quantity in Biomedicine

Puntus, L. N., Chauvin, A-S., Varbanov, S. & Bünzli, J-C. G. (2007). Lanthanide Complexes

Ramírez, F. de M., Charbonnière, L., Muller, G., Scopelliti, R. & Bünzli, J.-C. G. (2001). A p-

Ramírez, F. M., Varbonov, S., Cécile, C., Muller, G., Fatin- Rouge, N., Scopelliti, R. & Bünzli,

Ramírez, F. M., Charbonnière, L., Muller, G. & Bünzli, J.-C. G. (2004). Tuning the

*Inorganic Chemistry,* (April 2004), pp.2348-2355, DOI: 10.1002/ejic.200300824 Ramírez, F. de M., Varbanov, S., Padilla, J. & Bünzli, J-C G. (2008) Physicochemical

Sahin, O. & Yilmaz, M., (2011). Synthesis and Fluorescence Sensing Properties of Novel

Shimojo, K. & Goto, M. (2005). Synergistic Extraction of Nucleobases by the Combination of

Shinkai, S. (1993). Calixarenes-The Third Generation of Supramolecules. *Tetrahedron*, Vol. 49,

Sliwa, W. & Girek, T. J. (2010). Calixarene Complexes with Metal Ions. *Journal of Inclusion* 

Sliwa, W. (2002). Calixarene Complexes with Transition Metal, Lanthanide and Actinide Ions*. Croatica Chemica Acta,* Vol. 75, No. 1, pp. 131-153, ISSN: 0011-1643 Talanova, G. G. (2000). Phosphorus-Containing Macrocyclic Ionophores in Metal Ion

Zhou, Y., Xu, H., Yu, H., Chun, L., Lu, Q. & Wang, L. (2008). Spectrofluorometric Study on

Zielenkiewicz, W., Marcinowicz, A., Poznanski, J., Cherenok, S. & Kalchenko, V. (2005).

*Transactions,* (Oct. 2001), pp. 3205–3213, DOI: 10.1039/b105513p

(Aug. 2008), pp. 10976–10988, DOI: 10.1021/jp710848m

1099-0682

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No. 40, pp. 8933-8968, ISSN: 0040-4020

2000), pp. 3550 -3565, DOI: 10.1021/ie0001467

with a Calix[8]arene Bearing Phosphinoyl Pendant Arms. *European. Journal of Inorganic. Chemistry*, No. 22, (Apr. 2007), pp. 2315-2326, ISSN: 1434-1948, ISSN:

tert-butylcalix[4]arene Functionalised at its Lower Rim with Ether-amide Pendant Arms Acts as an Inorganic–organic Receptor: Structural and Photophysical Properties of its Lanthanide Complexes. *Journal of the Chemical Society, Dalton* 

J.-C. G. (2002). A p-tert-butylcalix[6]arene Bearing Phosphinoyl Pendant Arms for the Complexation and Sensitisation of Lanthanide Ions. *Journal of the Chemical Society, Dalton Transactions*, No. 23, (Sept 2002), pp. 4505-4513, DOI:

Stoichiometry of Lanthanide Complexes with Calixarenes: Bimetallic Complexes with a Calix[6]arene Bearing Ether-amide Pendant Arms. *European Journal of* 

Properties and Theoretical Modeling of Actinide Complexes with a para-tertbutylcalix[6]arene Bearing Phosphinoyl Pendants. Extraction Capability of the Calixarene toward f Elements. *The Journal of Physical Chemistry B*, Vol. 112, No. 35,

Pyrene-armed Calix[4]arene Derivatives. *Tetrahedron,* Vol. 67, No. 19, ( May 2011),

Calixarene and D2EHPA. *Separation and Purification Technology,* Vol. 44, No. 2, (July

*Phenomena and Macrocyclic Chemistry*, Vol. 66, pp. 15–41, DOI: 10.1007/s10847-009-

Separations*. Industrial & Engineering Chemistry Research,* Vol. 39, No. 10, (Sept.

The Inclusion Behavior of p-(p-carboxylbenzeneazo) calix[4]arene with Norfloxacin. *Spectrochimica Acta Part A,* Vol. 70, No. 2, (July 2008), pp. 411–415,

Complexation of Isoleucine by Phosphorylated Calix[4]arene in Methanol Followed by Calorimetry, NMR and UV–VIS Spectroscopies, and Molecular Modeling Methods. *Journal of Molecular Liquids,* Vol. 121, No.1, (July 2005), pp. 8 –14, ISSN: If hydrophobic molecules are inserted into an aqueous medium, the water molecules order around the hydrophobic ones to build a quasi crystalline surface. In this way, the hydrogen bonding of the water molecules around a hydrophobic surface is maximized. If two hydrophobic molecules meet they will associate with their hydrophobic surfaces towards each other. The water molecules, previously attached to these surfaces, will be distributed back into the bulk solvent resulting in favourable entropy. The entropic gain is responsible for almost all associations in the medium water and hence extremely important for life (e.g. formation of membranes, micelles, and for protein folding where folding starts often with tryptophan residues forming a hydrophobic core). The hydrophobic effect is shown below (**Figure 1**).

Fig. 1. Host-guest binding mechanism in aqueous medium.

Similarly, in most cases the protein-substrate binding is a result of the hydrophobic effect. However, there are evidences suggesting that the water molecules play an important role in the protein-substrate binding. Water molecules could participate in hydrogen bonding networks that link side chain and main chain atoms with the functional groups on the bases,

Water-Soluble Calix[4]arene Derivatives:

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 29

The protein enzymatic activity, being a surface function, depends on the recognition efficiency of negatively charged and polar amino acids of the substrate peptide.7 The bulklike environment in the close vicinity of the surface would enhance the interaction with the substrate (**Figure 3**). On the other hand, the structured water molecules are needed around the protein surface to be part of an efficient chemistry and possibly maintain a three dimensional structure.8 From these observations, it is clear that the water molecules play a crucial role in the receptor-substrate binding, probably be due to the hydration effect or hydrophobic effect. Scientist has always tried to find the answers related to the biological receptor-substrate interactions using the host-guest chemistry of synthetic counterparts.

Fig. 3. High-resolution X-ray structure of the Subtilisin *Carlsberg* (SC) protein. This structure was downloaded from the Protein Data Bank and processed with WEBLAB-VIEWERLITE, Accelrys, San Diego, CA. (*Top*) Position of the protein single Trp residue. Note the bound water molecules around this residue. (*Middle*) Two of the nine potential binding sites for DC labeling are shown. (*Bottom*) Illustration of a micelle with a NATA molecule included. Molecular structures of the probes are presented on the right of each illustration. (*Reproduced* 

*from Proc. Nat. Acad. Sci.* 2002, 99(4): 1763–1768)

and the phosphodiester backbone anionic oxygens.1 Macromolecular crystallography provided the necessary supportive view, that water molecules act as major contributors to stability and specificity.2,3,4

Thermodynamic analyses of protein-DNA binding suggest that water released from protein-DNA interfaces is favourable to binding. Structural analyses of the remaining water at the interface in protein-DNA complexes indicate that a majority of these water molecules promote binding by screening protein and DNA electrostatic repulsions between electronegative atoms/like charges. A small fraction of the observed interfacial waters act as linkers to form extended hydrogen bonds between the protein and the DNA, compensating for the lack of a direct hydrogen bond.5

Is it by design or by default that water molecules are observed at the interfaces of some protein-DNA complexes? Both experimental and theoretical studies on the thermodynamics of protein-DNA binding overwhelmingly support the extended hydrophobic view that water release from interfaces supports binding. Structural and energy analyses indicate that the remaining waters at the protein-DNA complexes interfaces ensure liquid-state packing densities, screen the electrostatic repulsions between like charges (which seems to be by design), and in a few cases act as linkers between complementary charges on the biomolecules (which may well be by default). Protein-drug binding and DNA-small molecule binding also revealed the possibility of the role played by the water molecules in the receptors binding pockets. The binding of the cardiac toponin-I (cTnI) with the small molecule (Fluorescent probe) revealed the enzyme hydrophobic binding region as shown in **Figure 2**.6

Fig. 2. Binding mode of cardiac toponin-I (cTnI) with the fluorescent probe. (*Reproduced from J. Am. Chem Soc.*133(38):14972-14974).

and the phosphodiester backbone anionic oxygens.1 Macromolecular crystallography provided the necessary supportive view, that water molecules act as major contributors to

Thermodynamic analyses of protein-DNA binding suggest that water released from protein-DNA interfaces is favourable to binding. Structural analyses of the remaining water at the interface in protein-DNA complexes indicate that a majority of these water molecules promote binding by screening protein and DNA electrostatic repulsions between electronegative atoms/like charges. A small fraction of the observed interfacial waters act as linkers to form extended hydrogen bonds between the protein and the DNA, compensating

Is it by design or by default that water molecules are observed at the interfaces of some protein-DNA complexes? Both experimental and theoretical studies on the thermodynamics of protein-DNA binding overwhelmingly support the extended hydrophobic view that water release from interfaces supports binding. Structural and energy analyses indicate that the remaining waters at the protein-DNA complexes interfaces ensure liquid-state packing densities, screen the electrostatic repulsions between like charges (which seems to be by design), and in a few cases act as linkers between complementary charges on the biomolecules (which may well be by default). Protein-drug binding and DNA-small molecule binding also revealed the possibility of the role played by the water molecules in the receptors binding pockets. The binding of the cardiac toponin-I (cTnI) with the small molecule (Fluorescent

Fig. 2. Binding mode of cardiac toponin-I (cTnI) with the fluorescent probe. (*Reproduced from* 

probe) revealed the enzyme hydrophobic binding region as shown in **Figure 2**.6

stability and specificity.2,3,4

for the lack of a direct hydrogen bond.5

*J. Am. Chem Soc.*133(38):14972-14974).

The protein enzymatic activity, being a surface function, depends on the recognition efficiency of negatively charged and polar amino acids of the substrate peptide.7 The bulklike environment in the close vicinity of the surface would enhance the interaction with the substrate (**Figure 3**). On the other hand, the structured water molecules are needed around the protein surface to be part of an efficient chemistry and possibly maintain a three dimensional structure.8 From these observations, it is clear that the water molecules play a crucial role in the receptor-substrate binding, probably be due to the hydration effect or hydrophobic effect. Scientist has always tried to find the answers related to the biological receptor-substrate interactions using the host-guest chemistry of synthetic counterparts.

Fig. 3. High-resolution X-ray structure of the Subtilisin *Carlsberg* (SC) protein. This structure was downloaded from the Protein Data Bank and processed with WEBLAB-VIEWERLITE, Accelrys, San Diego, CA. (*Top*) Position of the protein single Trp residue. Note the bound water molecules around this residue. (*Middle*) Two of the nine potential binding sites for DC labeling are shown. (*Bottom*) Illustration of a micelle with a NATA molecule included. Molecular structures of the probes are presented on the right of each illustration. (*Reproduced from Proc. Nat. Acad. Sci.* 2002, 99(4): 1763–1768)

Water-Soluble Calix[4]arene Derivatives:

**2.2 Job plot (method of continuous variation)** 

adopted here to determine the stoichiometry.

**[Complex]**

Fig. 4. Job plot for a 1:1 host–guest complex.

usually directly proportional to the complex concentration.

**2.3 Methods for association constant measurements** 

to following equation

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 31

There are different methods to determine the stoichiometry, e.g. Continuous Variation Methods (**Figure 4**),9 the Slope Ratio Method,10 the Mole Ratio Method,11 and others. Being the Continuous Variation Method the most popular among these, this method has been

There is a strong bias in the host-guest chemistry literature towards the fitting of data to 1:1 stoichiometries, and it is a common mistake to neglect higher complexes. Binding stoichiometry may be confirmed in most kinds of titration experiments, allowing the complex concentration to be determined by making up a series of solutions with varying host-guest ratios such that the total concentration of host and guest remains constant. Monitoring the changing concentration of the host–guest complex in these samples allows a plot of [Complex] against ([Host]/([Host] + [Guest])) to be constructed (**Figure 4**). For a 1:1 complex, this kind of representation (referred to as a Job plot) should give a peak at 0.5 (**Figure 4**), a peak at 0.66 would correspond to a 2:1 stoichiometry and so on. The complex concentration is generally taken to be related to an observable quantity such as Δ*δ* according

**0 0.2 0.4 0.6 0.8 1 [Host] / [Host] + [Guest]** 

[Complex]��������mole�������o��o���o�� In a spectrophotometric experiment, absorbance, at a properly chosen wavelength, is

Generally, the complex formation mechanism between a host and a guest is a basic and important process in supramolecular chemistry. Selectivity in the complexation is a crucial property in determining the molecular recognition ability of the host molecule, which discriminates among different guest species. The association constant's ratio of the corresponding complexation is usually treated as a measure of the selectivity. However,

This chapter presents a survey of the current literature on water-soluble calix[4]arenes (hosts) complexes with various substrates (guests), elaborating the water-soluble calix[4]arenes applications. Moreover, a critique of various data interpretations, in the context of the water molecules role in host-guest binding and in general of the water-soluble calix[4]arene guest recognition principles, is also provided.

### **2. Stoichiometry of the water-soluble calix[4]arene complexes: The methods**

### **2.1 Binding constant**

The thermodynamic stability of a host-guest (e.g. metal–macrocycle) complex, in a given solvent (often water or methanol) at a given temperature, is gauged by the binding constant, *K*, measurement. The binding constant is the most widely used method for host-guest affinity assessment in solution, and it is of fundamental importance in supramolecular chemistry. The binding constant is merely the equilibrium constant for the reaction between a Host, H, and Guest, G, in water, described in the following equation:

$$H(H\_2O)n + G(H\_2O)n \rightleftharpoons [H.G] + n(H\_2O)$$

$$K = \frac{[H.G]}{[H(H\_2O)n][G(H\_2O)n]}$$

Thus a large binding constant corresponds to a high equilibrium concentration of bound guest, and hence to a more stable host–guest complex.

If a sequential process of more than one guest is involved in the binding process, then two *K*  values may be measured for the 1:1 and 1:2 complexes, respectively: *K*1 and *K*2.

$$H(H\_2O)n + G(H\_2O)n \xrightleftharpoons \xrightleftharpoons[H.G.O]{K\_1} + n(H\_2O)$$

$$[H.G](H\_2O)n + G(H\_2O)n \xrightleftharpoons[H.G\_2]{K\_2}[H.G\_2] + n(H\_2O)n$$

$$K\_2 = \frac{[H.G\_2]}{[[H.G](H\_2O)n][G(H\_2O)n]}$$

In these circumstances, an overall binding constant, *β*, may be defined for the complete process, with the individual *K* values known as the stepwise binding constants:

$$
\mathcal{B} = K\_1 \,\, X \,\, K\_2,
$$

Magnitudes of binding constants can widely change, so they are often reported as log *K*, hence:

$$
\log \beta = \log(K\_1 \, X \, K\_2) = \log K\_1 + \log K\_2
$$

The host-guest complex binding constant depends on the complex stoichiometry. As shown in the equations above, a key aspect of such calculations is the use of the correct stoichiometry model (i.e. the ratio of host to guest, which must be assumed or determined), so it is worthy spending some time in understanding the method to determine it.

### **2.2 Job plot (method of continuous variation)**

30 Stoichiometry and Research – The Importance of Quantity in Biomedicine

This chapter presents a survey of the current literature on water-soluble calix[4]arenes (hosts) complexes with various substrates (guests), elaborating the water-soluble calix[4]arenes applications. Moreover, a critique of various data interpretations, in the context of the water molecules role in host-guest binding and in general of the water-soluble

**2. Stoichiometry of the water-soluble calix[4]arene complexes: The methods** 

The thermodynamic stability of a host-guest (e.g. metal–macrocycle) complex, in a given solvent (often water or methanol) at a given temperature, is gauged by the binding constant, *K*, measurement. The binding constant is the most widely used method for host-guest affinity assessment in solution, and it is of fundamental importance in supramolecular chemistry. The binding constant is merely the equilibrium constant for the reaction between

�(���)���(���)� � [�� �] � �(���)

[�(���)�][�(���)�] Thus a large binding constant corresponds to a high equilibrium concentration of bound

If a sequential process of more than one guest is involved in the binding process, then two *K* 

*<sup>K</sup>*1 [�� �] � �(���)

*<sup>K</sup>*2[�� ��] � �(���)

� = [�� �]

values may be measured for the 1:1 and 1:2 complexes, respectively: *K*1 and *K*2.

�(���)���(���)� 

[�� �](���)���(���)� 

�� <sup>=</sup> [�� ��]

process, with the individual *K* values known as the stepwise binding constants:

so it is worthy spending some time in understanding the method to determine it.

[[�� �](���)�][�(���)�] In these circumstances, an overall binding constant, *β*, may be defined for the complete

�=�� � �� Magnitudes of binding constants can widely change, so they are often reported as log *K*,

���� = ���(�� � ��) = ����� � �����) The host-guest complex binding constant depends on the complex stoichiometry. As shown in the equations above, a key aspect of such calculations is the use of the correct stoichiometry model (i.e. the ratio of host to guest, which must be assumed or determined),

calix[4]arene guest recognition principles, is also provided.

a Host, H, and Guest, G, in water, described in the following equation:

guest, and hence to a more stable host–guest complex.

**2.1 Binding constant** 

hence:

There are different methods to determine the stoichiometry, e.g. Continuous Variation Methods (**Figure 4**),9 the Slope Ratio Method,10 the Mole Ratio Method,11 and others. Being the Continuous Variation Method the most popular among these, this method has been adopted here to determine the stoichiometry.

Fig. 4. Job plot for a 1:1 host–guest complex.

There is a strong bias in the host-guest chemistry literature towards the fitting of data to 1:1 stoichiometries, and it is a common mistake to neglect higher complexes. Binding stoichiometry may be confirmed in most kinds of titration experiments, allowing the complex concentration to be determined by making up a series of solutions with varying host-guest ratios such that the total concentration of host and guest remains constant. Monitoring the changing concentration of the host–guest complex in these samples allows a plot of [Complex] against ([Host]/([Host] + [Guest])) to be constructed (**Figure 4**). For a 1:1 complex, this kind of representation (referred to as a Job plot) should give a peak at 0.5 (**Figure 4**), a peak at 0.66 would correspond to a 2:1 stoichiometry and so on. The complex concentration is generally taken to be related to an observable quantity such as Δ*δ* according to following equation

### [Complex]��������mole�������o��o���o��

In a spectrophotometric experiment, absorbance, at a properly chosen wavelength, is usually directly proportional to the complex concentration.

### **2.3 Methods for association constant measurements**

Generally, the complex formation mechanism between a host and a guest is a basic and important process in supramolecular chemistry. Selectivity in the complexation is a crucial property in determining the molecular recognition ability of the host molecule, which discriminates among different guest species. The association constant's ratio of the corresponding complexation is usually treated as a measure of the selectivity. However,

Water-Soluble Calix[4]arene Derivatives:

association constants in many systems.18,19

**2.3.3 Fluorescence titration** 

*K*s = 0, we obtain:

should yield a straight line of slope *K*s.

**2.3.4 UV-Vis Spectrophotometric titration** 

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 33

method,15,16 and the non-linear curve fitting analysis (few examples of the software programs are EQNMR, AGRNMRL, GRAFIT, and GRAPHPAD PRISM)17, give quantitative information about the association constant. NMR spectroscopic methods are useful for binding constants in the range 10–104 M-1. Recently, the diffusion NMR spectroscopy has become popular in supramolecular chemistry, due to its application in determining

Fluorescence titration measurements are based on the proportion of fluorescence intensity to fluorophore concentration (i.e. concentration of fluorescent species in solution; this is often a fluorescent guest, G). For a 1:1 complex with host, H, with stability constant *Ks=*

*F* = *k*G [G] + *k*s [HG] Where, the *k*G and *k*s represent proportionality constants for the guest and the 1:1 host– guest complex respectively. In the absence of host the fluorescence intensity, *F*o, is given by:

*F0 = K* o Gtotal

Where Gtotal = [G] + [HG]. Combining these two relationships gives the following equation, which provides the basis

� ⁄ � � ��� ��

����� This equation is greatly simplified when either the guest or host–guest complex are nonfluorescent (i.e. the fluorescence is 'turned on' by complexation, or, in the case of quenching, by the host), in which case either *K*G or *K*s become zero. For example, for *K*G = *K0*G 0 and

� ⁄ ����

for almost all the fluorimetric methods for stability constant (*K*11) measurements:

<sup>=</sup> ��� ��

��

� =����� A simple plot of *F*o/*F* against [H] from the quenching host titration into a guest solution

UV-Vis spectroscopic titration (or Spectrophotometric titration) involves monitoring the intensity of an electronic absorption band at a particular wavelength, characteristic of either the complex or free host or guest, and it is closely related to the fluorescence titration method. An absorbance intensity *vs*. concentration plot is generated by adding a guest to a solution of constant host concentration.10,11,12,13,14 Software such as *Specfit*® can then be used, in association with an appropriate stoichiometry model, to evaluate the association constant. Both fluorescent and UV-Vis spectroscopic methods have the advantage over NMR methods of being more sensitive, hence lower concentrations of host and guest can be used. Unlike

� ��

[HG]/[H][G] the fluorescence intensity *F* is given by the following equation:

theoretically the association constants can be measured by any experimental technique yielding information about the concentration of a complex, [Host-Guest], as a function of the host or guest concentration changes. In practice, the methods described below are of common use. In every case a concentration range must be chosen to have equilibrium between significant amounts of complexed and free host and guest, limiting the range of binding constants that can be measured by a particular technique. If the binding by the target host is too strong, then a competing host is sometimes added, in order to reduce the apparent (measured) association constant according to the difference in guest affinity between the two hosts. The true affinity can then be extrapolated from the knowledge of the binding constant of the guest for the host with the lower affinity.

### **2.3.1 Potentiometric titrations**

If the host molecules are susceptible to protonation (e.g. the aminocalix[4]arenes with their basic tertiary amine nitrogen), the protonation constants, and consequently the p*K*a values, may be readily determined using pH electrodes to monitor a simple acid–base titration. Initially, this will give the acid dissociation constant (p*K*a) of the hosts conjugate acid, *H*.H+.12 Addition of a guest cation will perturb the hosts basicity by competition with H+ ions for the ligand lone pair(s) and hence will affect the titration curves shape. Analysis of the various equilibrium by a curve-fitting computer program (such as *sigmaplot* or *Hyperquad*), along with knowledge of the hosts p*K*a, allows the determination of the amount of uncomplexed host and subsequently the concentration of the complex and the stability constants for the host-guest complexation reaction, as shown in the following equation,

$$K = \frac{[H^+][H]}{[G.H^+]}$$

### **2.3.2 Nuclear magnetic resonance titration**

If the exchange of complexed and un-complexed guest is slow on the nuclear magnetic resonance (NMR) time scale, then the association constant may be approximately evaluated under the prevailing conditions of concentration, temperature solvent *etc.* by simple integration of the NMR signals for complexed and un-complexed host or guest. However, most host–guest equilibrium are fast on the (relatively slow) NMR spectroscopic time scale, and the chemical shift observed for a particular resonance (that is sensitive to the complexation reaction) is a weighted average between the chemical shift of the free and bound species.

In a typical NMR titration experiment, small aliquots of guest are added to a host solution of known concentration in a deuterated solvent, and the NMR spectrum of the sample monitored as a function of guest concentration, or host:guest ratio. Commonly, changes in chemical shift (Δ*δ*) are noted for various atomic nuclei present (e.g. 1H in 1H NMR) as a function of the guest binding influence on their magnetic environment. As a result, two kinds of information are gained. Firstly, the location of the most affected nuclei may give qualitative information about the guest binding regioselectivity (e.g. is the guest inside the host cavity?). More importantly, the treatment of the titration curve data (a plot of Δ*δ* against added guest concentration, e.g. Figure 1.4) by different methods such as the Benesi-Hildebrand (Hanna-Ashbaugh) treatment,13 the Rose-Drago,14 the Scatchard (Foster-Fyfe) method,15,16 and the non-linear curve fitting analysis (few examples of the software programs are EQNMR, AGRNMRL, GRAFIT, and GRAPHPAD PRISM)17, give quantitative information about the association constant. NMR spectroscopic methods are useful for binding constants in the range 10–104 M-1. Recently, the diffusion NMR spectroscopy has become popular in supramolecular chemistry, due to its application in determining association constants in many systems.18,19

### **2.3.3 Fluorescence titration**

32 Stoichiometry and Research – The Importance of Quantity in Biomedicine

theoretically the association constants can be measured by any experimental technique yielding information about the concentration of a complex, [Host-Guest], as a function of the host or guest concentration changes. In practice, the methods described below are of common use. In every case a concentration range must be chosen to have equilibrium between significant amounts of complexed and free host and guest, limiting the range of binding constants that can be measured by a particular technique. If the binding by the target host is too strong, then a competing host is sometimes added, in order to reduce the apparent (measured) association constant according to the difference in guest affinity between the two hosts. The true affinity can then be extrapolated from the knowledge of the

If the host molecules are susceptible to protonation (e.g. the aminocalix[4]arenes with their basic tertiary amine nitrogen), the protonation constants, and consequently the p*K*a values, may be readily determined using pH electrodes to monitor a simple acid–base titration. Initially, this will give the acid dissociation constant (p*K*a) of the hosts conjugate acid, *H*.H+.12 Addition of a guest cation will perturb the hosts basicity by competition with H+ ions for the ligand lone pair(s) and hence will affect the titration curves shape. Analysis of the various equilibrium by a curve-fitting computer program (such as *sigmaplot* or *Hyperquad*), along with knowledge of the hosts p*K*a, allows the determination of the amount of uncomplexed host and subsequently the concentration of the complex and the stability constants for the host-guest complexation reaction, as shown in the following equation,

> � � [H�][�] [�� ��]

If the exchange of complexed and un-complexed guest is slow on the nuclear magnetic resonance (NMR) time scale, then the association constant may be approximately evaluated under the prevailing conditions of concentration, temperature solvent *etc.* by simple integration of the NMR signals for complexed and un-complexed host or guest. However, most host–guest equilibrium are fast on the (relatively slow) NMR spectroscopic time scale, and the chemical shift observed for a particular resonance (that is sensitive to the complexation reaction) is a weighted average between the chemical shift of the free and

In a typical NMR titration experiment, small aliquots of guest are added to a host solution of known concentration in a deuterated solvent, and the NMR spectrum of the sample monitored as a function of guest concentration, or host:guest ratio. Commonly, changes in chemical shift (Δ*δ*) are noted for various atomic nuclei present (e.g. 1H in 1H NMR) as a function of the guest binding influence on their magnetic environment. As a result, two kinds of information are gained. Firstly, the location of the most affected nuclei may give qualitative information about the guest binding regioselectivity (e.g. is the guest inside the host cavity?). More importantly, the treatment of the titration curve data (a plot of Δ*δ* against added guest concentration, e.g. Figure 1.4) by different methods such as the Benesi-Hildebrand (Hanna-Ashbaugh) treatment,13 the Rose-Drago,14 the Scatchard (Foster-Fyfe)

binding constant of the guest for the host with the lower affinity.

**2.3.1 Potentiometric titrations** 

**2.3.2 Nuclear magnetic resonance titration** 

bound species.

Fluorescence titration measurements are based on the proportion of fluorescence intensity to fluorophore concentration (i.e. concentration of fluorescent species in solution; this is often a fluorescent guest, G). For a 1:1 complex with host, H, with stability constant *Ks=* [HG]/[H][G] the fluorescence intensity *F* is given by the following equation:

$$F = kG\text{ [G] + ks\ [HG]}$$

Where, the *k*G and *k*s represent proportionality constants for the guest and the 1:1 host– guest complex respectively. In the absence of host the fluorescence intensity, *F*o, is given by:

$$F\_0 = K \circ \mathbf{G}\_{\text{total}}$$

$$\text{Where } \mathbf{G}\_{\text{total}} = [\mathbf{G}] + [\mathbf{H}\mathbf{G}].$$

Combining these two relationships gives the following equation, which provides the basis for almost all the fluorimetric methods for stability constant (*K*11) measurements:

$$\frac{F}{F\_0} = \frac{(K\_G/K\_G^0) + (K\_s/K\_G^0)K\_sH}{1 + K\_SH}$$

This equation is greatly simplified when either the guest or host–guest complex are nonfluorescent (i.e. the fluorescence is 'turned on' by complexation, or, in the case of quenching, by the host), in which case either *K*G or *K*s become zero. For example, for *K*G = *K0* G 0 and *K*s = 0, we obtain:

$$\frac{F\_0}{F} = 1 + K\_s H$$

A simple plot of *F*o/*F* against [H] from the quenching host titration into a guest solution should yield a straight line of slope *K*s.

### **2.3.4 UV-Vis Spectrophotometric titration**

UV-Vis spectroscopic titration (or Spectrophotometric titration) involves monitoring the intensity of an electronic absorption band at a particular wavelength, characteristic of either the complex or free host or guest, and it is closely related to the fluorescence titration method. An absorbance intensity *vs*. concentration plot is generated by adding a guest to a solution of constant host concentration.10,11,12,13,14 Software such as *Specfit*® can then be used, in association with an appropriate stoichiometry model, to evaluate the association constant. Both fluorescent and UV-Vis spectroscopic methods have the advantage over NMR methods of being more sensitive, hence lower concentrations of host and guest can be used. Unlike

Water-Soluble Calix[4]arene Derivatives:

Fig. 5. water-soluble calix[4]arene derivatives **1 - 6**

on the complexation with water-soluble hosts.

pH= 8.2.28,29

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 35

Several other calixarenes for complexation of nuclides have also garnered attention (**Figure 5**), including a water-soluble calix[4]arene-bis-benzocrown-6 (**1)** for selective Cs+ complexation (1:1) in moderately salted media.26 The water-soluble calix[4]arene-bisbenzocrown-6 (**1)** derivatives are also reported to separate the caesium–sodium by nanofiltration–complexation.27 The voltammetric study on a water-soluble calix[4]arene (calix[4]arene-triacid-monoquinone (**2)**, CTA, and calix[4]arene-triacid-diquinone (**3)**), which bind with the Ca2+, Sr2+, Ba2+ in basic aqueous solution, provided important information about the unique electrochemical behaviour of Ca2+–CTA 1:1 complex at

The 1:1 stoichiometric complexation of lanthanoid(III) nitrates (La-Gd, Tb) with watersoluble calix[4]arenesulfonate (**4)**, and its structurally similar derivatives (**5)** and (**6)** is reported (**Table 1**).30 The water-soluble calix[4]arenesulfonates (**5)** possessing four carboxylic groups at the lower rim of parent calix[4]arenesulfonate (**4)**, displayed the enhanced binding abilities for Sm3+. As compared with (**4)** and (**5)**, *p*sulfonatothiacalix[4]arene (**6)** gives not only the lower binding constants for all of lanthanoid(III) ions but also lower cations selectivity. Thermodynamically, the resulting complexes of lanthanoid(III) ions with (**4)** and its derivatives (**5)** and (**6)** are entirely entropydriven in aqueous solution, typically showing larger positive entropy changes. These changes (*T∆S*◦), and somewhat smaller positive enthalpy changes (*∆H*◦), are directly

It is interesting to notice that in all cases the solvated metal ions are the guests which form respective complexes with the water-soluble calix[4]arene derivatives (hosts) in the aqueous medium. Therefore, further studies are needed to evaluate the cations hydration shell effect

contributed to the stability of the complexes as a compensative consequence.

fluorescence methods, the observation of one or more clear isosbestic points is common in absorption spectroscopic titrations. An isosbestic point is reached when the observed absorption intensity remains constant throughout the titration. Furthermore, the observation of an isosbestic point is a good evidence for the free host conversion into a complex without any other significant intermediate species involved. The understanding of the statistical treatment of the obtained data to determine the association constants with the knowledge of primary statistics is the main feature of this method. When the complex stoichiometry is not 1 to 1, or when other premises are not satisfied, the data treatment should be changed or modified. Nonlinear least square data manipulation is one of the best approximations.

### **2.3.5 Calorimetric titration**

Calorimetric titration, also known as isothermal titration calorimetry (ITC), involves accurate measurement of the heat (enthalpy) evolved from a carefully insulated sample as a function of added guest or host concentration.20,21,22 The gradient of the ITC curve can be fitted to determine the binding constant and Δ*G*complex. Integration of the total area under the ITC plot gives the complexation enthalpy (Δ*H*complex)which allows for all the system thermodynamic parameters evaluation, being Δ*G*complex = Δ*H*complex - TΔ*S*complex. ITC is useful for determination of binding constants in range from *ca.*102 - 107 M-1.

### **2.3.6 Mass spectrometry**

Several electrospray-mass spectrometry (ESI-MS)-based methods are available for association constants (*K*S) determination between a protein and a small substrate. Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer, it allows the investigation of the molecular composition of liquid samples. A large variety of chemical substances can be ionized with electrospray. Moreover, there is no limitation in mass which thus enables even the investigation of large non-covalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion.23,24,25

### **3. Molecular recognition by water-soluble calix[4]arenes**

### **3.1 Cation recognition**

Non-covalent interactions play a dominant role in many forefront areas of modern chemistry, from materials design to molecular biology. A detailed understanding of the physical origin and scope of such interactions has become a major goal of physical organic chemistry. The cation-*π* interaction is an important non-covalent kind of interaction, including hydrogen bonds, ion pairs (salt bridges), and the hydrophobic interaction.

### **3.1.1 Metal ion recognition**

The first patent explicitly describing a calixarene for a practical application of p-tertbutylcalix[8]arene for the recovery of cesium from nuclear wastes, came in 1984. Numerous papers relating to the complexation of cesium by modified calixarenes have appeared since then.

Several other calixarenes for complexation of nuclides have also garnered attention (**Figure 5**), including a water-soluble calix[4]arene-bis-benzocrown-6 (**1)** for selective Cs+ complexation (1:1) in moderately salted media.26 The water-soluble calix[4]arene-bisbenzocrown-6 (**1)** derivatives are also reported to separate the caesium–sodium by nanofiltration–complexation.27 The voltammetric study on a water-soluble calix[4]arene (calix[4]arene-triacid-monoquinone (**2)**, CTA, and calix[4]arene-triacid-diquinone (**3)**), which bind with the Ca2+, Sr2+, Ba2+ in basic aqueous solution, provided important information about the unique electrochemical behaviour of Ca2+–CTA 1:1 complex at pH= 8.2.28,29

Fig. 5. water-soluble calix[4]arene derivatives **1 - 6**

34 Stoichiometry and Research – The Importance of Quantity in Biomedicine

fluorescence methods, the observation of one or more clear isosbestic points is common in absorption spectroscopic titrations. An isosbestic point is reached when the observed absorption intensity remains constant throughout the titration. Furthermore, the observation of an isosbestic point is a good evidence for the free host conversion into a complex without any other significant intermediate species involved. The understanding of the statistical treatment of the obtained data to determine the association constants with the knowledge of primary statistics is the main feature of this method. When the complex stoichiometry is not 1 to 1, or when other premises are not satisfied, the data treatment should be changed or modified. Nonlinear least square data manipulation is one of the best approximations.

Calorimetric titration, also known as isothermal titration calorimetry (ITC), involves accurate measurement of the heat (enthalpy) evolved from a carefully insulated sample as a function of added guest or host concentration.20,21,22 The gradient of the ITC curve can be fitted to determine the binding constant and Δ*G*complex. Integration of the total area under the ITC plot gives the complexation enthalpy (Δ*H*complex)which allows for all the system thermodynamic parameters evaluation, being Δ*G*complex = Δ*H*complex - TΔ*S*complex. ITC is useful

Several electrospray-mass spectrometry (ESI-MS)-based methods are available for association constants (*K*S) determination between a protein and a small substrate. Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer, it allows the investigation of the molecular composition of liquid samples. A large variety of chemical substances can be ionized with electrospray. Moreover, there is no limitation in mass which thus enables even the investigation of large non-covalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and

Non-covalent interactions play a dominant role in many forefront areas of modern chemistry, from materials design to molecular biology. A detailed understanding of the physical origin and scope of such interactions has become a major goal of physical organic chemistry. The cation-*π* interaction is an important non-covalent kind of interaction,

The first patent explicitly describing a calixarene for a practical application of p-tertbutylcalix[8]arene for the recovery of cesium from nuclear wastes, came in 1984. Numerous papers relating to the complexation of cesium by modified calixarenes have appeared since

including hydrogen bonds, ion pairs (salt bridges), and the hydrophobic interaction.

for determination of binding constants in range from *ca.*102 - 107 M-1.

quantified on trace amounts in a high throughput fashion.23,24,25

**3. Molecular recognition by water-soluble calix[4]arenes** 

**2.3.5 Calorimetric titration** 

**2.3.6 Mass spectrometry** 

**3.1 Cation recognition** 

**3.1.1 Metal ion recognition** 

then.

The 1:1 stoichiometric complexation of lanthanoid(III) nitrates (La-Gd, Tb) with watersoluble calix[4]arenesulfonate (**4)**, and its structurally similar derivatives (**5)** and (**6)** is reported (**Table 1**).30 The water-soluble calix[4]arenesulfonates (**5)** possessing four carboxylic groups at the lower rim of parent calix[4]arenesulfonate (**4)**, displayed the enhanced binding abilities for Sm3+. As compared with (**4)** and (**5)**, *p*sulfonatothiacalix[4]arene (**6)** gives not only the lower binding constants for all of lanthanoid(III) ions but also lower cations selectivity. Thermodynamically, the resulting complexes of lanthanoid(III) ions with (**4)** and its derivatives (**5)** and (**6)** are entirely entropydriven in aqueous solution, typically showing larger positive entropy changes. These changes (*T∆S*◦), and somewhat smaller positive enthalpy changes (*∆H*◦), are directly contributed to the stability of the complexes as a compensative consequence.

It is interesting to notice that in all cases the solvated metal ions are the guests which form respective complexes with the water-soluble calix[4]arene derivatives (hosts) in the aqueous medium. Therefore, further studies are needed to evaluate the cations hydration shell effect on the complexation with water-soluble hosts.

Water-Soluble Calix[4]arene Derivatives:

Fig. 6. Water-soluble calix[4]arene host(s) **7** and guests **8, 9.** 

Fig. 7. Hosts with deep hydrophobic cavities (**10 - 12**), and guests **14** 

(Phenyltrimethylammonium chloride), **15** (Benzyltrimethylammonium chloride).

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 37

study37 involving a variety of ammonium guests, including acetylcholine and Nmethylquinuclidinium, reached the same conclusion that the guest is in the cavity and that its ammonium portion is closely associated with the calixarene aromatic rings. This interaction was discussed as a π-cation interaction.38,39,40 The N,N,N,-trimethylanilinium (TMA) cation (**14)** orientation was reported to have a dual binding mode (charged group vs. aromatic moiety inclusion) which occurs in a nonselective fashion with flexible watersoluble calix[4]arene hosts (**7a**, n=4). The binding mode can, however, be effectively controlled and turned into a selective process by preorganization of the calixarene cavity into the cone structure (**7b**, n=4). The presence of sulfonate groups at the upper rim provides, anchoring points for the positively charged guests, the sulfonate groups significantly deepening the cavity of host (**7)**, thus improving its inclusion capability.


Values are the averages of more than three independent measurements in pH = 2 acidic aqueous solution

Table 1. Complex stability constants (log *Ka*) and thermodynamic parameters (kJ mol-1) for complexation of lanthanoid(III) nitrates with 4, 5, and 6 in acidic aqueous solution (pH = 2) at 25 ◦C.

### **3.1.2 Molecular cation recognition and hydrophobic cavity depth of water-soluble calix[4]arenes**

The complexation of molecular cations by the water-soluble calix[4]arenes is widely studied. Shinkai and coworkers31 were the first to investigate molecular cation complexation with psulfonatocalix[4]arenes (**7)** (**Figure 6**) as hosts and trimethylanilinium as a guest.

By measuring the 1H NMR shift values over a temperature range of 0–800C, they calculated ∆Go, ∆Ho and ∆So values and concluded that complexation with the cyclic tetramer (7.8, n=4) was driven by a favourable enthalpy change (stronger electrostatic interaction). It was emphasized that in studies with water soluble calixarenes an important feature that had to be taken into consideration was their aggregation properties.32 A calix[4]arene with anionic groups (SO3- and CO2-) on both exo and endo rims, forms fairly strong complexes with cations such as PhCH2NMe3+ (*K*s = 2500M-1) (**15)**.33 For cations derived from amines, the organic moiety introduces a significant steric factor, with the ammonium cation included in the host cavity.34,35,36 A closely related study37 involving a variety of ammonium guests, including acetylcholine and Nmethylquinuclidinium, reached the same conclusion that the guest is in the cavity and that its ammonium portion is closely associated with the calixarene aromatic rings. This interaction was discussed as a π-cation interaction.38,39,40 The N,N,N,-trimethylanilinium (TMA) cation (**14)** orientation was reported to have a dual binding mode (charged group vs. aromatic moiety inclusion) which occurs in a nonselective fashion with flexible watersoluble calix[4]arene hosts (**7a**, n=4). The binding mode can, however, be effectively controlled and turned into a selective process by preorganization of the calixarene cavity into the cone structure (**7b**, n=4). The presence of sulfonate groups at the upper rim provides, anchoring points for the positively charged guests, the sulfonate groups significantly deepening the cavity of host (**7)**, thus improving its inclusion capability.

Fig. 6. Water-soluble calix[4]arene host(s) **7** and guests **8, 9.** 

36 Stoichiometry and Research – The Importance of Quantity in Biomedicine

Nd3+ 4.08 23.3 ± 0.3 9.5 ± 0.2 32.8 ± 0.5 Sm3+ 3.82 21.8 ± 0.2 10.4 ± 0.2 32.2 ± 0.4 Eu3+ 3.83 21.9 ± 0.2 12.5 ± 0.2 34.4 ± 0.4 Gd3+ 3.94 22.5 ± 0.3 9.8 ± 0.3 32.2 ± 0.6

Ce3+ 3.82 ± 0.01 21.8 ± 0.1 5.1 ± 0.3 26.9 ± 0.4 Pr3+ 3.97 ± 0.04 22.7 ± 0.3 4.5 ± 0.4 27.2 ± 0.1 Nd3+ 4.09 ± 0.03 23.4 ± 0.6 4.0 ± 0.1 27.4 ± 0.2 Sm3+ 4.08 ± 0.02 23.3 ± 0.4 3.9 ± 0.1 27.2 ± 0.8 Eu3+ 3.51 ± 0.04 20.1 ± 0.1 7.3 ± 0.3 27.4 ± 0.1 Gd3+ 3.86 ± 0.05 22.0 ± 0.3 5.5 ± 0.2 27.5 ± 0.3 Tb3+ 3.63 ± 0.01 20.9 ± 0.2 6.8 ± 0.7 27.7 ± 0.5

Ce3+ 3.41 ± 0.02 19.4 ± 0.2 7.0 ± 0.1 26.5 ± 0.2 Pr3+ 3.42 ± 0.03 19.6 ± 0.3 6.9 ± 0.1 26.5 ± 0.3 Nd3+ 3.40 ± 0.01 19.4 ± 0.1 6.8 ± 0.3 26.2 ± 0.1 Sm3+ 3.37 ± 0.04 19.2 ± 0.2 7.2 ± 0.2 26.4 ± 0.4 Eu3+ 3.26 ± 0.03 18.6 ± 0.4 7.5 ± 0.3 26.0 ± 0.3 Gd3+ 3.30 ± 0.02 17.7 ± 0.6 9.0 ± 0.1 26.6 ± 0.1 Tb3+ 3.33 ± 0.02 19.0 ± 0.1 7.7 ± 0.1 26.7 ± 0.5

Host Guest (Cation) Log Ks *-∆G*◦ *∆H*◦ *T∆S*◦ **4** La3+ 4.23 24.1 ± 0.3 9.2 ± 0.1 33.3 ± 0.4

**5** La3+ 3.73 ± 0.03 21.3 ± 0.4 5.1 ± 0.5 26.5 ± 0.3

**6** La3+ 3.45 ± 0.02 19.7 ± 0.1 7.2 ± 0.2 26.8 ± 0.3

Values are the averages of more than three independent measurements in pH = 2 acidic aqueous

Table 1. Complex stability constants (log *Ka*) and thermodynamic parameters (kJ mol-1) for complexation of lanthanoid(III) nitrates with 4, 5, and 6 in acidic aqueous solution (pH = 2)

**3.1.2 Molecular cation recognition and hydrophobic cavity depth of water-soluble** 

sulfonatocalix[4]arenes (**7)** (**Figure 6**) as hosts and trimethylanilinium as a guest.

The complexation of molecular cations by the water-soluble calix[4]arenes is widely studied. Shinkai and coworkers31 were the first to investigate molecular cation complexation with p-

By measuring the 1H NMR shift values over a temperature range of 0–800C, they calculated ∆Go, ∆Ho and ∆So values and concluded that complexation with the cyclic tetramer (7.8, n=4) was driven by a favourable enthalpy change (stronger electrostatic interaction). It was emphasized that in studies with water soluble calixarenes an important feature that had to be taken into consideration was their aggregation properties.32 A calix[4]arene with anionic groups (SO3- and CO2-) on both exo and endo rims, forms fairly strong complexes with cations such as PhCH2NMe3+ (*K*s = 2500M-1) (**15)**.33 For cations derived from amines, the organic moiety introduces a significant steric factor, with the ammonium cation included in the host cavity.34,35,36 A closely related

solution

at 25 ◦C.

**calix[4]arenes** 

Fig. 7. Hosts with deep hydrophobic cavities (**10 - 12**), and guests **14**  (Phenyltrimethylammonium chloride), **15** (Benzyltrimethylammonium chloride).

Water-Soluble Calix[4]arene Derivatives:

**3.2 Anion recognition** 

**3.2.1 Inorganic anions** 

for such studies.

reactions in aqueous solution.47

Fig. 9. Water-soluble iminecalix[4]arene **16**, **28** and guests **17-27.**

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 39

Anion recognition (binding) plays an important role in a variety of chemical reactions and biochemical events as outlined in various reports.44 This molecular recognition process has

The hydrogen-bond dynamics of water molecules solvating a Cl-, Br-, or I- anion is slow compared with neat liquid water, indicating that the aqueous solvation shells of these ions are rigid. This rigidity can play an important role in the overall dynamics of chemical

Furthermore, the anions complexation can be more difficult than that of cations, and a variety of considerations come into play, including (a) the charge, (b) the size, which is often larger than the metal cation one, (c) the shape; whereas the metal cations are spherical, the anions frequently are not, (d) pH dependence, often more critical than in the case of metal cations and (e) solvation, which has a strong influence on the binding strength. There are enormous reports on the recognition of various anions (inorganic) by the calix[4]arene derivatives in the organic solvents but there are only few reports on the anion complexation by the water-soluble calix[4]arenes in the aqueous medium,48 which opens a new direction

Functionalisation of calix[4]arenes with carbohydrate moieties results in receptors which show considerable water solubility. A number of calixsugars have been developed49 and their binding characteristics studied. Neutral guests such as carbohydrates and N-protected

been the subject of numerous experimental and theoretical studies in recent years.45,46

The complexation of the water-soluble aminocalix[4]arenes containing deep hydrophobic cavities with cations have been reported.41 However, the guest recognition and the orientation in the cavity of the host were reported to be dependent on the depth of the host hydrophobic cavity. The host (**11**, **12)** interacts with both the cationic function and the aromatic moiety in the guests (**14**, **15**), but with a slight preference for the cationic functions. The host (**13)** selectively recognizes the trimethylammonium functions of the guests (**14** and **15)**.

Fig. 8. A) Inclusion mode of the guest **15** by hosts **11-13**; B) Inclusion mode of the guest **15** by host **10**

However, the host (**10)** selectively recognizes the aromatic moiety of the ditopic trimethylammonium guests (**14** and **15)**. These results suggest that the water molecules around the calix[4]arene nucleus in the hosts (**11 – 13)** may assist the hydrophilic trimethylammonium function in entering the cavity. Furthermore, in case of the host (**10),** possessing a deep hydrophobic cavity, the trimethylammonium function cannot deeply enter into the calix[4]arene nucleus, being solvated by the water. As the guest molecules trimethylammonium function is engaged on the mouth of the host **(10)** deep hydrophobic cavity, the guest aromatic moiety is selected by the host (**10)** to form the inclusion complexes. These results suggest that the guest recognition and orientation in the cavity of the host are directly dependent on the host hydrophobic cavity depth.

The water-soluble iminecalix[4]arene (**16, Figure 9**) with deep hydrophobic cavity was also recognized for its selective recognition of the guest.42,43 The negatively charged four carboxylate functions on the top of the deep hydrophobic cavity play a major role in the recognition of charged molecular species. The 1H NMR titration experiments revealed that host (**16)** binds with cationic (**15**, **21, 22**) and neutral guests (**17-20**) in water, with high binding constants in order of 104-105 M-1. Cationic guest (**15)** showed the highest binding constant of 2.81× 105 M-1 . These studies revealed that except for the -CH···π and π-π stacking interactions, the hydrophobic interactions proved to be crucial in the molecular recognition process in aqueous medium.

Fig. 9. Water-soluble iminecalix[4]arene **16**, **28** and guests **17-27.**

### **3.2 Anion recognition**

38 Stoichiometry and Research – The Importance of Quantity in Biomedicine

The complexation of the water-soluble aminocalix[4]arenes containing deep hydrophobic cavities with cations have been reported.41 However, the guest recognition and the orientation in the cavity of the host were reported to be dependent on the depth of the host hydrophobic cavity. The host (**11**, **12)** interacts with both the cationic function and the aromatic moiety in the guests (**14**, **15**), but with a slight preference for the cationic functions. The host (**13)** selectively

Fig. 8. A) Inclusion mode of the guest **15** by hosts **11-13**; B) Inclusion mode of the guest **15** by

However, the host (**10)** selectively recognizes the aromatic moiety of the ditopic trimethylammonium guests (**14** and **15)**. These results suggest that the water molecules around the calix[4]arene nucleus in the hosts (**11 – 13)** may assist the hydrophilic trimethylammonium function in entering the cavity. Furthermore, in case of the host (**10),** possessing a deep hydrophobic cavity, the trimethylammonium function cannot deeply enter into the calix[4]arene nucleus, being solvated by the water. As the guest molecules trimethylammonium function is engaged on the mouth of the host **(10)** deep hydrophobic cavity, the guest aromatic moiety is selected by the host (**10)** to form the inclusion complexes. These results suggest that the guest recognition and orientation in the cavity of

The water-soluble iminecalix[4]arene (**16, Figure 9**) with deep hydrophobic cavity was also recognized for its selective recognition of the guest.42,43 The negatively charged four carboxylate functions on the top of the deep hydrophobic cavity play a major role in the recognition of charged molecular species. The 1H NMR titration experiments revealed that host (**16)** binds with cationic (**15**, **21, 22**) and neutral guests (**17-20**) in water, with high binding constants in order of 104-105 M-1. Cationic guest (**15)** showed the highest binding constant of 2.81× 105 M-1 . These studies revealed that except for the -CH···π and π-π stacking interactions, the hydrophobic interactions proved to be crucial in the molecular

the host are directly dependent on the host hydrophobic cavity depth.

recognition process in aqueous medium.

recognizes the trimethylammonium functions of the guests (**14** and **15)**.

host **10**

Anion recognition (binding) plays an important role in a variety of chemical reactions and biochemical events as outlined in various reports.44 This molecular recognition process has been the subject of numerous experimental and theoretical studies in recent years.45,46

### **3.2.1 Inorganic anions**

The hydrogen-bond dynamics of water molecules solvating a Cl-, Br-, or I- anion is slow compared with neat liquid water, indicating that the aqueous solvation shells of these ions are rigid. This rigidity can play an important role in the overall dynamics of chemical reactions in aqueous solution.47

Furthermore, the anions complexation can be more difficult than that of cations, and a variety of considerations come into play, including (a) the charge, (b) the size, which is often larger than the metal cation one, (c) the shape; whereas the metal cations are spherical, the anions frequently are not, (d) pH dependence, often more critical than in the case of metal cations and (e) solvation, which has a strong influence on the binding strength. There are enormous reports on the recognition of various anions (inorganic) by the calix[4]arene derivatives in the organic solvents but there are only few reports on the anion complexation by the water-soluble calix[4]arenes in the aqueous medium,48 which opens a new direction for such studies.

Functionalisation of calix[4]arenes with carbohydrate moieties results in receptors which show considerable water solubility. A number of calixsugars have been developed49 and their binding characteristics studied. Neutral guests such as carbohydrates and N-protected

Water-Soluble Calix[4]arene Derivatives:

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 41

been recently reported.55 The pH of the solution shows a significant effect on the dynamics of the gate (formed by eight benzylic functions) and portal on the hydrophobic cavity of the water-soluble aminocalix[4]arene host (**10)**. At pH 5.8 the gate closes and prevents the entry of anionic guests. However, at pH 7.3 the gate opens and allows the entry of anionic guests (**23**, **24)** to the hydrophobic cavity. Host **10** not only shows a similar behaviour towards guests **23** and **24** but also shows a preference for sulfonate derivatives. This preference can be assigned to the tripodal symmetry of sulfonate function, instead of dipodal in carboxylate, and its electron withdrawing effect. The tripodal symmetry gives extra room for negative charges of guest molecules on the cavity of host **10** reducing the electrostatic repulsion. The electron withdrawing effect prevails and increases the π-π stacking interactions between the guest (**23)** and the host (**10)**. The deep hydrophobic cavity of the water-soluble aminocalix[4]arene role in the recognition of anionic guests cannot be neglected, despite the absence of favourable electrostatic interaction shown by host **30**

The complexation of neutral molecules by water-soluble calixarenes was carried out in the eighties and has been already critically reviewed.56,57,58 The pioneering work59 on the complexation of aromatic hydrocarbons by hosts **33** (n=4) has however to be mentioned, since it disclosed a rough correlation between binding constants and host-guest complementarily. Calix[4]arenes are too small to host durene or naphthalene, calix[5]- or -

Sciotto et al. have studied the interactions between alcohols, ketones, nitriles and psulfonatocalixarenes (**4)** and its derivatives by 1H NMR spectroscopy,60,61 proving that the apolar aliphatic portions of the guests were included into the host hydrophobic cavity with the terminal polar groups directed towards the polar sulfonate groups of the host and to the solvent. The two most important factors for the complexation of the investigated hosts and guests are conformational properties of the receptors and electrostatic effects. Methanol is not included by p-sulfonatocalixarenes at all, probably due to the fact that the small methyl group inclusion inside the hydrophobic cavity would lead to a partial inclusion of polar OH

The interactions of aromatic substrates (**34, 35, 36,** and **37**) (**Figure 12**) with **4** were studied by Schatz and co-workers via 1H NMR titration experiments and molecular modelling

towards guests **23** and **24**, host **10** showing strong binding with them**.**

Fig. 11. Water-soluble calix[4]arene derivatives **30** (cationic), **33.**

[6]arenes preferring naphthalene, anthracene and phenanthrene.

group, causing the polar hydroxyl group to be less exposed to polar solvent.

**3.3 Recognition of neutral molecules** 

amino acids failed to bind. However, 1:1 complexation of dihydrogen phosphate was seen for (**28),** offering opportunities for the binding of larger, phosphate containing biological substrates.

Fig. 10. Water-soluble π-metalated calix[n]arene **29** and complexes

A series of π-metalated calix[n]arenes were synthesised, among which compounds **29** are water-soluble due to the presence of six positive charges.50 The calixarenes cavities are therefore electron-poor and able to complex anions both in the solid state and in water. The X-ray crystal structures of compounds **29a, 29c** and **29d** showed that a BF4- , SO4 2-, and Ianion is complexed in the calixarene cavity, respectively, tetrafluoroborate being the most deeply included one. Acetate, phosphate and sulfate anions are not bound by host (**29b),**  due to their high hydrophilicity. An interesting inversion of the expected selectivity, on the basis of the free hydration energy order (Hofmeister series), is observed for halide ions due to size complementarity between the guest and the calixarene cavity.51

### **3.2.2 Molecular anion recognition and depth of hydrophobic cavity of water-soluble calix[4]arenes**

Very few examples of anion complexation by water-soluble calixarenes have been reported so far. This is probably due to the fact that anion recognition is a rather new field in supramolecular chemistry and that anions are more highly hydrated than cations of comparable size and, therefore, their complexation in water is a remarkably difficult task. In the case of l-anilino-8-naphthalenesulfonate (ANS)52 and 2-*p*-toluidino-6 naphthalenesulfonate (TNS) 53 the lipophilic residue of the guest is included inside the calixarene cavity.54

A cationic calix[4]arene derivative (**30)** binds both aliphatic (**31**, **32)** and aromatic, sulfonate (**23)** and carboxylate (**26)** anions in aqueous solution with a Log *K* of 1.50, 1.48, 2.44, 2.32, respectively, as a result of concerted electrostatic and hydrophobic interactions. The sulfonate ion in guest **23** may show good electrostatic interaction with the cations on the top of the cavity. However, the sulfonate guest inclusion is affected by the host different mobility caused by the pH change. An interesting example of the anionic host (**10)** complexation with the anionic sulfonate (**23)** (Log *K*= 4.3, pH=7.3; Log *K*= 0, pH=5.8) has been recently reported.55 The pH of the solution shows a significant effect on the dynamics of the gate (formed by eight benzylic functions) and portal on the hydrophobic cavity of the water-soluble aminocalix[4]arene host (**10)**. At pH 5.8 the gate closes and prevents the entry of anionic guests. However, at pH 7.3 the gate opens and allows the entry of anionic guests (**23**, **24)** to the hydrophobic cavity. Host **10** not only shows a similar behaviour towards guests **23** and **24** but also shows a preference for sulfonate derivatives. This preference can be assigned to the tripodal symmetry of sulfonate function, instead of dipodal in carboxylate, and its electron withdrawing effect. The tripodal symmetry gives extra room for negative charges of guest molecules on the cavity of host **10** reducing the electrostatic repulsion. The electron withdrawing effect prevails and increases the π-π stacking interactions between the guest (**23)** and the host (**10)**. The deep hydrophobic cavity of the water-soluble aminocalix[4]arene role in the recognition of anionic guests cannot be neglected, despite the absence of favourable electrostatic interaction shown by host **30** towards guests **23** and **24**, host **10** showing strong binding with them**.**

Fig. 11. Water-soluble calix[4]arene derivatives **30** (cationic), **33.**

### **3.3 Recognition of neutral molecules**

40 Stoichiometry and Research – The Importance of Quantity in Biomedicine

amino acids failed to bind. However, 1:1 complexation of dihydrogen phosphate was seen for (**28),** offering opportunities for the binding of larger, phosphate containing biological

A series of π-metalated calix[n]arenes were synthesised, among which compounds **29** are water-soluble due to the presence of six positive charges.50 The calixarenes cavities are therefore electron-poor and able to complex anions both in the solid state and in water. The

anion is complexed in the calixarene cavity, respectively, tetrafluoroborate being the most deeply included one. Acetate, phosphate and sulfate anions are not bound by host (**29b),**  due to their high hydrophilicity. An interesting inversion of the expected selectivity, on the basis of the free hydration energy order (Hofmeister series), is observed for halide ions due

**3.2.2 Molecular anion recognition and depth of hydrophobic cavity of water-soluble** 

Very few examples of anion complexation by water-soluble calixarenes have been reported so far. This is probably due to the fact that anion recognition is a rather new field in supramolecular chemistry and that anions are more highly hydrated than cations of comparable size and, therefore, their complexation in water is a remarkably difficult task. In the case of l-anilino-8-naphthalenesulfonate (ANS)52 and 2-*p*-toluidino-6 naphthalenesulfonate (TNS) 53 the lipophilic residue of the guest is included inside the

A cationic calix[4]arene derivative (**30)** binds both aliphatic (**31**, **32)** and aromatic, sulfonate (**23)** and carboxylate (**26)** anions in aqueous solution with a Log *K* of 1.50, 1.48, 2.44, 2.32, respectively, as a result of concerted electrostatic and hydrophobic interactions. The sulfonate ion in guest **23** may show good electrostatic interaction with the cations on the top of the cavity. However, the sulfonate guest inclusion is affected by the host different mobility caused by the pH change. An interesting example of the anionic host (**10)** complexation with the anionic sulfonate (**23)** (Log *K*= 4.3, pH=7.3; Log *K*= 0, pH=5.8) has

, SO42-, and I-

Fig. 10. Water-soluble π-metalated calix[n]arene **29** and complexes

X-ray crystal structures of compounds **29a, 29c** and **29d** showed that a BF4-

to size complementarity between the guest and the calixarene cavity.51

substrates.

**calix[4]arenes** 

calixarene cavity.54

The complexation of neutral molecules by water-soluble calixarenes was carried out in the eighties and has been already critically reviewed.56,57,58 The pioneering work59 on the complexation of aromatic hydrocarbons by hosts **33** (n=4) has however to be mentioned, since it disclosed a rough correlation between binding constants and host-guest complementarily. Calix[4]arenes are too small to host durene or naphthalene, calix[5]- or - [6]arenes preferring naphthalene, anthracene and phenanthrene.

Sciotto et al. have studied the interactions between alcohols, ketones, nitriles and psulfonatocalixarenes (**4)** and its derivatives by 1H NMR spectroscopy,60,61 proving that the apolar aliphatic portions of the guests were included into the host hydrophobic cavity with the terminal polar groups directed towards the polar sulfonate groups of the host and to the solvent. The two most important factors for the complexation of the investigated hosts and guests are conformational properties of the receptors and electrostatic effects. Methanol is not included by p-sulfonatocalixarenes at all, probably due to the fact that the small methyl group inclusion inside the hydrophobic cavity would lead to a partial inclusion of polar OH group, causing the polar hydroxyl group to be less exposed to polar solvent.

The interactions of aromatic substrates (**34, 35, 36,** and **37**) (**Figure 12**) with **4** were studied by Schatz and co-workers via 1H NMR titration experiments and molecular modelling

Water-Soluble Calix[4]arene Derivatives:

do not enter the hydrophobic cavity.

**4. Conclusion** 

**5. Acknowledgement** 

**6. References** 

77

7:126–34

Chuncheon, Korea for helpful discussions.

recognition. 1999. *Structure* 7:R277–79

Binding Stoichiometry and Spectroscopic Evaluation of the Host-Guest Recognition Mechanism 43

The NMR investigations indicate that host **11** and **12** can form 1:1 host–guest inclusion complexes with aromatic cationic guests and pyridine derivatives with high binding constants. Both hosts refused to recognize the hydrophilic anionic guests, possibly due to the electrostatic repulsion arising from carboxylate functions on the cavity of the host. The host **12**, with hydrophobic mouth, showed high binding constant for 4 methylbenzylammonium, as the carboxylate functions of the mouth showed strong electrostatic interactions with the ammonium function. However, the hydrophilic mouth of host **11** enhances the binding of 4-ethylpyridine. It is clear from the data that the cavity of both hosts has a preference for structurally flat guests containing methyl groups (either a CH3 in *para* position of an aromatic ring or a presence of trimethylammonium group) and a very poor one for smaller but more hydrophilic primary ammonium groups, which indeed

Mimicry of the molecular recognition features of naturally occurring proteins by synthetic receptors is one of the challenging research topics of supramolecular chemistry. The substrates and enzymes (host-guest) features can be studied by Potentiometry, NMR Spectroscopy, UV-Visible Spectroscopy, Fluorescence Spectroscopy, and Calorimetry. In some cases the ESI-MS can be employed to study the protein-protein, or protein-small molecule interactions. It is quite obvious that the exact host-guest complex stoichiometry is the most critical parameter in the evaluation of the host-guest interactions. The molecular recognition properties of the water-soluble calix[4]arene derivatives revealed that the hydrophobic cavity of these hosts play an important role in the guests recognition. Increasing the hydrophobic cavity depth, like in the water-soluble aminocalix[4]arene hosts, results in an increased binding of the guest into the hosts deep hydrophobic pockets. Synthetically tailored hosts based on the calix[4]arene framework can be used to probe the

naturally occurring biomolecular reactions based on the non-covalent interactions.

This work was supported by the Ministry of Knowledge and Economy of South Korea. We also acknowledge Song Keum-soo and Kim Junghoon of Biometrix Technology Inc.

[1] Seeman NC, Rosenberg JM, Rich A. Sequence specific recognition of double helical

[2] Janin J. Wet and dry interfaces: the role of solvent in protein-protein and protein-DNA

[3] Woda J, Schneider B, Patel K, Mistry K, Berman HM. 1998. An analysis of the

[4] Schwabe JW. The role of water in protein-DNA interactions. 1997. *Curr. Opin. Struct. Biol.* 

relationship between hydration and protein- DNA interactions. *Biophys. J.* 75:2170–

nucleic acids by proteins. 1976. *Proc. Natl. Acad. Sci. USA* 73:804–8

studies combined with abinitio NMR shift calculation at neutral aqueous solutions.62 All the guests are included into the hosts cavities, with a mechanism which is mainly driven by enthalpy term. In most cases, the five aromatic protons are pointing inside and the guest functional group is located outside the hosts cavities due to hydrophobic and π-π interactions. For (**34)**, the complex binding mode is different, probably because the methyl group is included into the host cavity, contributing to the favorable C–H-π interactions and hydrophobic interactions.63

Fig. 12. Aromatic neutral guests (**34**-**37**), and cationic guests **38**, **39.**

Fig. 13. Hosts **11** and **12,** A) host **12** (side view), B) host **11** (side view).

The new water-soluble aminocalix[4]arene hosts **11** and **12** with deep hydrophobic cavity facilitate hydrophilic mouth and hydrophobic mouth, respectively.64 The 1H NMR titrations revealed that host **12** shows high selectivity for neutral guests (**18** and **19)**, with log *K* of 4.2 and 4.6, respectively. The host **11** shows log *K* of 4.9 for binding with guest **39**. Moreover, the host **11** binding ability for guest **38** is stronger by a factor of 1000 than that of the host **12**.

The NMR investigations indicate that host **11** and **12** can form 1:1 host–guest inclusion complexes with aromatic cationic guests and pyridine derivatives with high binding constants. Both hosts refused to recognize the hydrophilic anionic guests, possibly due to the electrostatic repulsion arising from carboxylate functions on the cavity of the host. The host **12**, with hydrophobic mouth, showed high binding constant for 4 methylbenzylammonium, as the carboxylate functions of the mouth showed strong electrostatic interactions with the ammonium function. However, the hydrophilic mouth of host **11** enhances the binding of 4-ethylpyridine. It is clear from the data that the cavity of both hosts has a preference for structurally flat guests containing methyl groups (either a CH3 in *para* position of an aromatic ring or a presence of trimethylammonium group) and a very poor one for smaller but more hydrophilic primary ammonium groups, which indeed do not enter the hydrophobic cavity.

### **4. Conclusion**

42 Stoichiometry and Research – The Importance of Quantity in Biomedicine

studies combined with abinitio NMR shift calculation at neutral aqueous solutions.62 All the guests are included into the hosts cavities, with a mechanism which is mainly driven by enthalpy term. In most cases, the five aromatic protons are pointing inside and the guest functional group is located outside the hosts cavities due to hydrophobic and π-π interactions. For (**34)**, the complex binding mode is different, probably because the methyl group is included into the host cavity, contributing to the favorable C–H-π interactions and

Fig. 12. Aromatic neutral guests (**34**-**37**), and cationic guests **38**, **39.**

Fig. 13. Hosts **11** and **12,** A) host **12** (side view), B) host **11** (side view).

The new water-soluble aminocalix[4]arene hosts **11** and **12** with deep hydrophobic cavity facilitate hydrophilic mouth and hydrophobic mouth, respectively.64 The 1H NMR titrations revealed that host **12** shows high selectivity for neutral guests (**18** and **19)**, with log *K* of 4.2 and 4.6, respectively. The host **11** shows log *K* of 4.9 for binding with guest **39**. Moreover, the host **11** binding ability for guest **38** is stronger by a factor of 1000 than that of the host **12**.

hydrophobic interactions.63

Mimicry of the molecular recognition features of naturally occurring proteins by synthetic receptors is one of the challenging research topics of supramolecular chemistry. The substrates and enzymes (host-guest) features can be studied by Potentiometry, NMR Spectroscopy, UV-Visible Spectroscopy, Fluorescence Spectroscopy, and Calorimetry. In some cases the ESI-MS can be employed to study the protein-protein, or protein-small molecule interactions. It is quite obvious that the exact host-guest complex stoichiometry is the most critical parameter in the evaluation of the host-guest interactions. The molecular recognition properties of the water-soluble calix[4]arene derivatives revealed that the hydrophobic cavity of these hosts play an important role in the guests recognition. Increasing the hydrophobic cavity depth, like in the water-soluble aminocalix[4]arene hosts, results in an increased binding of the guest into the hosts deep hydrophobic pockets. Synthetically tailored hosts based on the calix[4]arene framework can be used to probe the naturally occurring biomolecular reactions based on the non-covalent interactions.

### **5. Acknowledgement**

This work was supported by the Ministry of Knowledge and Economy of South Korea. We also acknowledge Song Keum-soo and Kim Junghoon of Biometrix Technology Inc. Chuncheon, Korea for helpful discussions.

### **6. References**


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**3** 

*Romania* 

**The Determination of the Stoichiometry** 

**of Cyclodextrin Inclusion Complexes by** 

Cristina Tablet, Iulia Matei and Mihaela Hillebrand *Dept. Physical Chemistry, University of Bucharest, Bucharest,* 

**Spectral Methods: Possibilities and Limitations** 

The inclusion complexes of many organic ligands, drugs or metal ions, in cyclodextrins (CDs) represent a class of the simplest supramolecular systems widely studied for the last several decades. The increasing interest for their investigation arises from both a theoretical and an applicative points of view. Considering the first one, their study contributes to the understanding of the molecular recognition and molecular interactions, emphasizing the role of different structural factors. From a more practical purpose, the encapsulation of different drugs in the CD cavity produces an increase in the solubility (Brewster & Loftsson, 2007) and allows for a more controlled oral, parental, ocular, nasal or rectal drug release (Challa et al., 2005). A special interest resides in the CDs potential as alternatives for conventional anti-obesity medications (Grunberger et al., 2007). The same goal is also realized using the supramolecular systems, such as hydrogels, obtained by the selfassembling of CDs with some polymers (Li, 2010; Zhang & Ma, 2010). One of the CDs applications that deserves both an experimental and theoretical focus is based on their interaction with the cellular or model membranes, resulting in cholesterol extraction (Abi-Mosleh et al, 2009). It was shown that the cholesterol removal occurs via inclusion complex formation, for which the stoichiometry plays an important role. Thus, it was stated on both experimental grounds and molecular dynamics calculations that the efficient stoichiometry for the extraction is 1:2 guest:host, requiring, for a good effect, the presence of CD dimers oriented in an appropriate way on the membrane layer (Lopez et al., 2011). Besides these, a remarkable amount of studies were devoted to CDs inclusion complexes, covering an extended basis for the discussion of their properties and applications (Arunkumar et al., 2005; Challa et al., 2005; Li & Loh, 2008; Loftsson et al., 2004; Loftsson & Duchene, 2007;

Continuing our studies on the CDs inclusion process (Oana et al., 2002; Matei et al., 2007; Tablet & Hillebrand, 2008, Tintaru et al., 2003), we report here some cases encountered in the study of some potentially bioactive compounds like coumarin and phenoxathiin derivatives, and of two drugs, atenolol and indapamide, presenting some peculiar structural factors which make their characterization more difficult. We will focus on the possibilities and limitations of some spectroscopic methods for the estimation of the stoichiometry and

**1. Introduction** 

Martin Del Valle, 2004; Sjetli, 1982; Vyas et al., 2008).


## **The Determination of the Stoichiometry of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations**

Cristina Tablet, Iulia Matei and Mihaela Hillebrand *Dept. Physical Chemistry, University of Bucharest, Bucharest, Romania* 

### **1. Introduction**

46 Stoichiometry and Research – The Importance of Quantity in Biomedicine

[56] Gutsche CD. *Calixarenes,* The Royal Society of Chemistry (Ed.: J. F. Stoddart),

[57] Shinkai S. in *Calixarenes, a Versatile Class of Macrocyclic Compounds,* Kluwer Academic Publishers (Eds.: J. Vicens, V. Böhmer), Dordrecht, 1991**,** pp. 173-198. [58] Pochini A, Ungaro R. in *Comprehensive Supramolecular Chemistry, Vol. 2,* Pergamon Press

[60] Arena G, Casnati A, Contino A, Sciotto D, Ungaro R. Charge assisted hydrophobic

[61] Arena G, Contino A, Gulino FG, Magrí A, Sciotto D, Ungaro R. Complexation of small

[62] Baur M, Frank M, Schatz J, Schildbach F. Water-soluble calix[n]arenes as receptor

[63] Guo D-S, Wang K, Liu y. Selective binding behaviors of p-sulfonatocalixarenes in

[64] Nimse SB, Nguyen V, Kim J, Kim H, Song K, Eoum W, Jung C, Ta V, Seelam SR, Kim T.

aqueous solution. 2008. *Incl. Phenom. Macrocycl. Chem.* 62(1-2):1-21.

binding of ethanol into the cavity of calix[4]arene receptors in aqueous solution.

neutral organic molecules by water soluble calix[4]arenes. 2000. *Tetrahedron Lett*ers

molecules for non-polar substrates and inverse phase transfer catalysts. 2001.

Water-soluble aminocalix[4]arene receptors with hydrophobic and hydrophilic

Cambridge, 1989.

41: 9327–9330.

*Tetrahedron* 57:6985–6991.

(Ed.: F. Vögtle), Oxford, 1996**,** pp. 103-142. [59] Gutsche CD, Alam I. 1988. *Tetrahedron* 4689-4694.

1997. *Tetrahedron Letters* 38(26):4685–4688.

mouths. 2010. *Tetrahedron Letters* 51:2840-2845.

The inclusion complexes of many organic ligands, drugs or metal ions, in cyclodextrins (CDs) represent a class of the simplest supramolecular systems widely studied for the last several decades. The increasing interest for their investigation arises from both a theoretical and an applicative points of view. Considering the first one, their study contributes to the understanding of the molecular recognition and molecular interactions, emphasizing the role of different structural factors. From a more practical purpose, the encapsulation of different drugs in the CD cavity produces an increase in the solubility (Brewster & Loftsson, 2007) and allows for a more controlled oral, parental, ocular, nasal or rectal drug release (Challa et al., 2005). A special interest resides in the CDs potential as alternatives for conventional anti-obesity medications (Grunberger et al., 2007). The same goal is also realized using the supramolecular systems, such as hydrogels, obtained by the selfassembling of CDs with some polymers (Li, 2010; Zhang & Ma, 2010). One of the CDs applications that deserves both an experimental and theoretical focus is based on their interaction with the cellular or model membranes, resulting in cholesterol extraction (Abi-Mosleh et al, 2009). It was shown that the cholesterol removal occurs via inclusion complex formation, for which the stoichiometry plays an important role. Thus, it was stated on both experimental grounds and molecular dynamics calculations that the efficient stoichiometry for the extraction is 1:2 guest:host, requiring, for a good effect, the presence of CD dimers oriented in an appropriate way on the membrane layer (Lopez et al., 2011). Besides these, a remarkable amount of studies were devoted to CDs inclusion complexes, covering an extended basis for the discussion of their properties and applications (Arunkumar et al., 2005; Challa et al., 2005; Li & Loh, 2008; Loftsson et al., 2004; Loftsson & Duchene, 2007; Martin Del Valle, 2004; Sjetli, 1982; Vyas et al., 2008).

Continuing our studies on the CDs inclusion process (Oana et al., 2002; Matei et al., 2007; Tablet & Hillebrand, 2008, Tintaru et al., 2003), we report here some cases encountered in the study of some potentially bioactive compounds like coumarin and phenoxathiin derivatives, and of two drugs, atenolol and indapamide, presenting some peculiar structural factors which make their characterization more difficult. We will focus on the possibilities and limitations of some spectroscopic methods for the estimation of the stoichiometry and

The Determination of the Stoichiometry

involved in the electronic transition.

**1.1.2 The stoichiometry** 

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 49

in its intrinsic dichroic signal; b) for an achiral guest, CD inclusion may lead to the appearance of an induced dichroic signal of the guest, by chirality transfer from the CD. As the circular dichroism technique offers data on the steric match between the two interacting partners, i.e. on the "geometry" of the interaction, the dimensions of the CD cavity mainly determine the changes observed in the spectrum of the guest upon CD incorporation. The dichroic band position is the same as the absorption one corresponding to the chromophore

The sign of the dichroic band of a guest molecule included in the CD cavity depends on the angle between the electronic transition moment and the symmetry axis of the CD. The rules of Harata and Kodaka state that the dichroic signal is positive for an axial inclusion (parallel orientation of the transition moment with respect to the CD axis) and negative for an equatorial inclusion (perpendicular orientation) (Harata & Uedaira, 1975; Kodaka, 1993). Thus, it is possible to obtain valuable information on the stoichiometry and the orientation of the guest molecule in the complex. Moreover, the intensity of the dichroic signal gives an

The stoichiometry of the complex is given by the number of G and H molecules contained in the supramolecular complex, the general notation being GnHm; the most common stoichiometry is 1:1 (GH), implying the inclusion of a single guest molecule, but other stoichiometries like G1H2, G2H1, G2H2, G1H3, G3H1, etc., can be encountered as well (Baglole et al., 2005; De Azevedo et al., 2000; Ge et al., 2011; Sancho et al., 2011; Shen et al., 1998) and some of them will be further discussed from a structural point of view. As the formation of the G1H2 complex can be the result of two successive equilibriums, the simultaneous

Self-assembled, stable supramolecular systems can also be formed, containing a large number of H and G molecules. Such systems include the rotaxanes, catenanes and nanotubes (Li et al., 2011; Qu et al. 2005; Yui et al., 2009). In some cases, the obtained complexes form extended linear aggregates, revealed mostly from the unexpectedly large value of the fluorescence anisotropy, e.g. 2,5-diphenyl-oxazole with *γ*-CD (Agbaria & Gill, 1988), 2,5-diphenyl-1,3,4-oxadiazole, 2-phenyl-5-(4-biphenyl)-1,3,4-oxadiazole and 2,5 biphenyl-1,3,4-oxadiazole with *γ*-CD (Agbaria & Gill, 1994), 2,5-bis-(4-methylphenyl) oxazole with *β*-CD and *γ*-CD (Yarabe et al., 2002), 1,4-diphenyl-1,3-butadiene with *β*-CD and *γ*-CD (De La Pena et al., 1997), trans-1,6-diphenyl-1,3,5-hexatriene with *β*-CD and *γ*-CD (Li & McGown, 1994), 4,4′-bis(2-benzoxazolyl) stilbene with *β*-CD and *γ*-CD (Wu et al., 2006) and

The quantitative measure of the guest–host interaction at a given temperature is represented by the value of the equilibrium constant governing the formation of the inclusion complex. It is also referred to as binding or association constant. The mathematical models for processing the experimental data in order to estimate the association constants will be

indication on the magnitude of the interaction, namely on the induced asymmetry.

presence of 1:1 and 1:2 complexes is also frequently mentioned.

coumarin 153 with *γ*-CD (Mandal et al., 2011).

discussed further for some of the most common cases.

**1.1.3 The association constant** 

association constants for both cases, i.e. when the procedure is straightforward and when some problems were encountered. The guest molecules were chosen to elucidate some difficulties occurring during the analysis of the experimental data in establishing the real stoichiometry of the inclusion complexes.

This paper starts with a short description of the main features of CDs and their inclusion complexes. After a summary of the main mathematical models used for the estimation of the inclusion complexes properties, we will discuss our results on the specific aforementioned cases.

### **1.1 Cyclodextrins and the characterization of their inclusion complexes**

Native CDs are macrocyclic oligosaccharides formed by the *α*-1,4-linkage of 6–8 glucose residues and named *α*-, *β*- and *γ*-CD, respectively. They have a toroidal structure and, based on molecular recognition principles, are able to include in their cavity different organic molecules and ions. Therefore, they will be hereafter named as Host, H, while the ligands are generally labelled as Guests, G. Differing from the hydrophobic character of the cavity, the presence of the primary and secondary OH groups ensures an hydrophilic exterior. Besides these three CDs, a large number of modified CDs were synthesized and used for experimental studies, aiming to a better understanding of the driving forces of the inclusion process. Furthermore there is an increasing interest on CDs, due to their multiple applications, especially in pharmacology and biotechnology. The characterization of a CD inclusion complex can be performed using different experimental methods and implicates several factors briefly summarized below.

### **1.1.1 Methodology**

The spectral methods are based on monitoring the guest corresponding spectra, at a constant concentration, when increasing amounts of CDs are added. In absorption and fluorescence spectroscopy, the main effects in the guest spectra are changes in the intensity and/or band shifts. They may be accompanied by the appearance of isosbestic/isoemissive points, which indicate an equilibrium between the free and the complexed guest. Differently, the absence of isosbestic points can be indicative of the occurrence of high order associations in solution (Hamai et al., 1992). In the spectral range of the common guest molecules, there is no interference from the absorption bands of the CDs, located in the far UV region. Using NMR spectroscopy, the experimental data for characterizing the complexes are the variation in the chemical shifts of both guest and host protons (Tintaru et al., 2003). IR and Raman spectroscopies can be used as well for the characterization of the inclusion complexes.

A spectral method which brings about interesting information is the circular dichroism spectroscopy. Circular dichroism is essentially an absorption phenomenon occurring when an optically active molecule absorbs to different degrees the right and left components of a circularly polarized light beam. The magnitude of the dichroic signal depends on the difference between the molar extinction coefficients of the molecule for the right- and lefthanded components. The changes that inclusion to CDs, chiral environments themselves, may produce in the circular dichroism spectrum of a guest molecule are of two types, depending on the nature of the guest: a) for a chiral guest, CD inclusion may lead to changes in its intrinsic dichroic signal; b) for an achiral guest, CD inclusion may lead to the appearance of an induced dichroic signal of the guest, by chirality transfer from the CD. As the circular dichroism technique offers data on the steric match between the two interacting partners, i.e. on the "geometry" of the interaction, the dimensions of the CD cavity mainly determine the changes observed in the spectrum of the guest upon CD incorporation. The dichroic band position is the same as the absorption one corresponding to the chromophore involved in the electronic transition.

The sign of the dichroic band of a guest molecule included in the CD cavity depends on the angle between the electronic transition moment and the symmetry axis of the CD. The rules of Harata and Kodaka state that the dichroic signal is positive for an axial inclusion (parallel orientation of the transition moment with respect to the CD axis) and negative for an equatorial inclusion (perpendicular orientation) (Harata & Uedaira, 1975; Kodaka, 1993). Thus, it is possible to obtain valuable information on the stoichiometry and the orientation of the guest molecule in the complex. Moreover, the intensity of the dichroic signal gives an indication on the magnitude of the interaction, namely on the induced asymmetry.

### **1.1.2 The stoichiometry**

48 Stoichiometry and Research – The Importance of Quantity in Biomedicine

association constants for both cases, i.e. when the procedure is straightforward and when some problems were encountered. The guest molecules were chosen to elucidate some difficulties occurring during the analysis of the experimental data in establishing the real

This paper starts with a short description of the main features of CDs and their inclusion complexes. After a summary of the main mathematical models used for the estimation of the inclusion complexes properties, we will discuss our results on the specific aforementioned

Native CDs are macrocyclic oligosaccharides formed by the *α*-1,4-linkage of 6–8 glucose residues and named *α*-, *β*- and *γ*-CD, respectively. They have a toroidal structure and, based on molecular recognition principles, are able to include in their cavity different organic molecules and ions. Therefore, they will be hereafter named as Host, H, while the ligands are generally labelled as Guests, G. Differing from the hydrophobic character of the cavity, the presence of the primary and secondary OH groups ensures an hydrophilic exterior. Besides these three CDs, a large number of modified CDs were synthesized and used for experimental studies, aiming to a better understanding of the driving forces of the inclusion process. Furthermore there is an increasing interest on CDs, due to their multiple applications, especially in pharmacology and biotechnology. The characterization of a CD inclusion complex can be performed using different experimental methods and implicates

The spectral methods are based on monitoring the guest corresponding spectra, at a constant concentration, when increasing amounts of CDs are added. In absorption and fluorescence spectroscopy, the main effects in the guest spectra are changes in the intensity and/or band shifts. They may be accompanied by the appearance of isosbestic/isoemissive points, which indicate an equilibrium between the free and the complexed guest. Differently, the absence of isosbestic points can be indicative of the occurrence of high order associations in solution (Hamai et al., 1992). In the spectral range of the common guest molecules, there is no interference from the absorption bands of the CDs, located in the far UV region. Using NMR spectroscopy, the experimental data for characterizing the complexes are the variation in the chemical shifts of both guest and host protons (Tintaru et al., 2003). IR and Raman spectroscopies can be used as well for

A spectral method which brings about interesting information is the circular dichroism spectroscopy. Circular dichroism is essentially an absorption phenomenon occurring when an optically active molecule absorbs to different degrees the right and left components of a circularly polarized light beam. The magnitude of the dichroic signal depends on the difference between the molar extinction coefficients of the molecule for the right- and lefthanded components. The changes that inclusion to CDs, chiral environments themselves, may produce in the circular dichroism spectrum of a guest molecule are of two types, depending on the nature of the guest: a) for a chiral guest, CD inclusion may lead to changes

**1.1 Cyclodextrins and the characterization of their inclusion complexes** 

stoichiometry of the inclusion complexes.

several factors briefly summarized below.

the characterization of the inclusion complexes.

**1.1.1 Methodology** 

cases.

The stoichiometry of the complex is given by the number of G and H molecules contained in the supramolecular complex, the general notation being GnHm; the most common stoichiometry is 1:1 (GH), implying the inclusion of a single guest molecule, but other stoichiometries like G1H2, G2H1, G2H2, G1H3, G3H1, etc., can be encountered as well (Baglole et al., 2005; De Azevedo et al., 2000; Ge et al., 2011; Sancho et al., 2011; Shen et al., 1998) and some of them will be further discussed from a structural point of view. As the formation of the G1H2 complex can be the result of two successive equilibriums, the simultaneous presence of 1:1 and 1:2 complexes is also frequently mentioned.

Self-assembled, stable supramolecular systems can also be formed, containing a large number of H and G molecules. Such systems include the rotaxanes, catenanes and nanotubes (Li et al., 2011; Qu et al. 2005; Yui et al., 2009). In some cases, the obtained complexes form extended linear aggregates, revealed mostly from the unexpectedly large value of the fluorescence anisotropy, e.g. 2,5-diphenyl-oxazole with *γ*-CD (Agbaria & Gill, 1988), 2,5-diphenyl-1,3,4-oxadiazole, 2-phenyl-5-(4-biphenyl)-1,3,4-oxadiazole and 2,5 biphenyl-1,3,4-oxadiazole with *γ*-CD (Agbaria & Gill, 1994), 2,5-bis-(4-methylphenyl) oxazole with *β*-CD and *γ*-CD (Yarabe et al., 2002), 1,4-diphenyl-1,3-butadiene with *β*-CD and *γ*-CD (De La Pena et al., 1997), trans-1,6-diphenyl-1,3,5-hexatriene with *β*-CD and *γ*-CD (Li & McGown, 1994), 4,4′-bis(2-benzoxazolyl) stilbene with *β*-CD and *γ*-CD (Wu et al., 2006) and coumarin 153 with *γ*-CD (Mandal et al., 2011).

### **1.1.3 The association constant**

The quantitative measure of the guest–host interaction at a given temperature is represented by the value of the equilibrium constant governing the formation of the inclusion complex. It is also referred to as binding or association constant. The mathematical models for processing the experimental data in order to estimate the association constants will be discussed further for some of the most common cases.

The Determination of the Stoichiometry

experimental accessible range.

Table 1.

Fig. 1. Typical Job's plots for 1:1 and 1:2 complexes.

0.00

0.05

0.10

Δ**P (a.u.)**

0.15

0.20

0.25

**1.3 Determination of both the stoichiometry and the association constant** 

The determination of the stoichiometry in the host–guest interaction is strongly correlated with the estimation of the association constant. Excepting Job's method which gives indications only on the stoichiometry of the inclusion complexes, for all other methods the following procedure is applied. Several stoichiometries are assumed and the experimental data are fitted to the corresponding linear or nonlinear models. The description of all these models and their different applications are given in very well known books and reviews (Connors, 1987; Singh et al., 2010). Therefore, we will further present in Table 1 the main formula used in the analysis of the spectral experimental data, without the corresponding deductions. Starting with the equations of the assumed chemical equilibria, the general idea is to monitor the changes of an experimental property (Pobs) directly correlated with the concentration of the former or the new-formed species, at gradual host addition. The function Pobs = f(Ci, parameters), where Ci represents the equilibrium concentration of the species *i*, is called the binding isotherm. As for Job's plot, this property can be the absorbance (A), fluorescence quantum yield or fluorescence emission (Φ or F), ellipticity (θ) or NMR chemical shift (Δδ). Starting from the binding isotherm, the equations corresponding to some widely used linear (eqs. 1, 2, 5) (Benesi & Hildebrand, 1949; Scott, 1956) and nonlinear (eqs. 3, 4, 6–9) (Liu et al., 2001; Park et al., 2002) models are given in

0.0 0.2 0.4 0.6 0.8 1.0

**XH**

XH=0.37 **1:2** 

XH=0.5 **1:1** 

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 51

A development of Job's method was described by Landy *et al.* (Landy et al., 2007) for the determination of the stoichiometry of CD inclusion complexes and named Competitive Continuous Variation Plot. In fact, this new approach represents a coupling of Job's method with the competitive experiments, spectral displacements, well known in the study of biopolymer–ligand interactions. The basic idea was to monitor the changes of a given experimental property and to build a Job plot when a competitor ligand, for which the features of the inclusion complex were previously determined, is introduced in the system. The method is recommended for the cases in which either the low solubility prevents the usual experimental determinations or the spectral properties of the guest are not in the

### **1.1.4 The thermodynamic parameters**

Performing the experiments at different temperatures and using the estimated associated constants in a Van't Hoff treatment allow for the determination of the thermodynamic parameters of the inclusion process, ΔH and ΔS (Tablet & Hillebrand, 2008). The sign and the absolute relative values of these quantities are further used in the discussion of the main forces involved in the inclusion process, i.e. electrostatic, hydrophobic, hydrogen bonds, etc.

### **1.1.5 Structural aspects**

The structure of an inclusion complex in solution is difficult to be established. The guest can penetrate the CD cavity in several ways, through the wide or the narrow rim, in an axial or equatorial position with respect to the cavity long axis. Although for obtaining the previously mentioned features all the spectral methods (absorption, fluorescence, circular dichroism and NMR spectroscopy) can be used, information on the structure of the complexes can be experimentally obtained only from 2D-NMR and induced circular dichroism (ICD) spectra. The presence of the asymmetric environment of the cavity can induce a dichroic signal even for the achiral guests. According to the Harata-Kodaka rules, the positive/negative sign of the dichroic band indicates the axial/equatorial inclusion of the guest in respect to the cavity main axis. This information can be used as starting point to elaborate structural models that will be further optimized at different theoretical levels (molecular mechanics, semiempirical methods, DFT). The best test for judging the consistency of the model, although difficult to reach, is the agreement between the simulated ICD spectrum of the GnHm system and the experimental one.

### **1.2 Determination of the stoichiometry. Job's method**

One of the first methods used for the determination of the stoichiometry of inclusion complexes was Job's method, also known as the continuous variation method (Job, 1928). The experiments use stock solutions with equimolecular concentrations of H and G components. The samples are prepared by mixing different volumes of these two solutions in such a way that the total concentration [H]+[G] remains constant and the molar fraction of the guest, XG varies in the range 0–1. The variation of the experimental measured property, ΔP, in presence of the host in respect with the value for the free guest is plotted vs. XG or XH. The value of XG for which the plot presents the maximum deviation gives the stoichiometry of the inclusion complex (XG = 0.5 for 1:1 or 2:2 G:H complexes; XH = 0.33 for 1:2 G:H complexes). Although, in most cases, in a Job plot ΔP represents the change of the absorbance of the guest during addition of the host, ΔA, (Rajaram et al., 2011), other properties correlated with the concentration of the complex, like the change in the NMR chemical shifts (Δδ) or the enthalpy changes (ΔH) can be used as well (Chadha et al., 2011; Kacso et al., 2010; Thi et al., 2011).

Two typical schematic Job's plots for 1:1 and 1:2 inclusion complexes are given in Fig. 1, considering ΔP as the absorbance change, ΔA, for the HG complexes and the NMR proton shifts (Δδ) for the HG2 complexes. It can be seen that in the case of 1:1 complexes, the maximum deviation is obtained for XH = 0.5, while for the second type of complexes the maximum is reached for XH ~ 0.37. Literature data offers a lot of examples for the application of Job's method (Ge et al., 2011; Jadhav et al., 2007; Liu et al., 2001; Sainz-Rozas et al., 2005).

Performing the experiments at different temperatures and using the estimated associated constants in a Van't Hoff treatment allow for the determination of the thermodynamic parameters of the inclusion process, ΔH and ΔS (Tablet & Hillebrand, 2008). The sign and the absolute relative values of these quantities are further used in the discussion of the main forces involved in the inclusion process, i.e. electrostatic, hydrophobic, hydrogen bonds, etc.

The structure of an inclusion complex in solution is difficult to be established. The guest can penetrate the CD cavity in several ways, through the wide or the narrow rim, in an axial or equatorial position with respect to the cavity long axis. Although for obtaining the previously mentioned features all the spectral methods (absorption, fluorescence, circular dichroism and NMR spectroscopy) can be used, information on the structure of the complexes can be experimentally obtained only from 2D-NMR and induced circular dichroism (ICD) spectra. The presence of the asymmetric environment of the cavity can induce a dichroic signal even for the achiral guests. According to the Harata-Kodaka rules, the positive/negative sign of the dichroic band indicates the axial/equatorial inclusion of the guest in respect to the cavity main axis. This information can be used as starting point to elaborate structural models that will be further optimized at different theoretical levels (molecular mechanics, semiempirical methods, DFT). The best test for judging the consistency of the model, although difficult to reach, is the agreement between the

One of the first methods used for the determination of the stoichiometry of inclusion complexes was Job's method, also known as the continuous variation method (Job, 1928). The experiments use stock solutions with equimolecular concentrations of H and G components. The samples are prepared by mixing different volumes of these two solutions in such a way that the total concentration [H]+[G] remains constant and the molar fraction of the guest, XG varies in the range 0–1. The variation of the experimental measured property, ΔP, in presence of the host in respect with the value for the free guest is plotted vs. XG or XH. The value of XG for which the plot presents the maximum deviation gives the stoichiometry of the inclusion complex (XG = 0.5 for 1:1 or 2:2 G:H complexes; XH = 0.33 for 1:2 G:H complexes). Although, in most cases, in a Job plot ΔP represents the change of the absorbance of the guest during addition of the host, ΔA, (Rajaram et al., 2011), other properties correlated with the concentration of the complex, like the change in the NMR chemical shifts (Δδ) or the enthalpy changes (ΔH) can be used as well (Chadha et al., 2011;

Two typical schematic Job's plots for 1:1 and 1:2 inclusion complexes are given in Fig. 1, considering ΔP as the absorbance change, ΔA, for the HG complexes and the NMR proton shifts (Δδ) for the HG2 complexes. It can be seen that in the case of 1:1 complexes, the maximum deviation is obtained for XH = 0.5, while for the second type of complexes the maximum is reached for XH ~ 0.37. Literature data offers a lot of examples for the application of Job's method (Ge et al., 2011; Jadhav et al., 2007; Liu et al., 2001; Sainz-Rozas et al., 2005).

simulated ICD spectrum of the GnHm system and the experimental one.

**1.2 Determination of the stoichiometry. Job's method** 

Kacso et al., 2010; Thi et al., 2011).

**1.1.4 The thermodynamic parameters** 

**1.1.5 Structural aspects** 

A development of Job's method was described by Landy *et al.* (Landy et al., 2007) for the determination of the stoichiometry of CD inclusion complexes and named Competitive Continuous Variation Plot. In fact, this new approach represents a coupling of Job's method with the competitive experiments, spectral displacements, well known in the study of biopolymer–ligand interactions. The basic idea was to monitor the changes of a given experimental property and to build a Job plot when a competitor ligand, for which the features of the inclusion complex were previously determined, is introduced in the system. The method is recommended for the cases in which either the low solubility prevents the usual experimental determinations or the spectral properties of the guest are not in the experimental accessible range.

Fig. 1. Typical Job's plots for 1:1 and 1:2 complexes.

### **1.3 Determination of both the stoichiometry and the association constant**

The determination of the stoichiometry in the host–guest interaction is strongly correlated with the estimation of the association constant. Excepting Job's method which gives indications only on the stoichiometry of the inclusion complexes, for all other methods the following procedure is applied. Several stoichiometries are assumed and the experimental data are fitted to the corresponding linear or nonlinear models. The description of all these models and their different applications are given in very well known books and reviews (Connors, 1987; Singh et al., 2010). Therefore, we will further present in Table 1 the main formula used in the analysis of the spectral experimental data, without the corresponding deductions. Starting with the equations of the assumed chemical equilibria, the general idea is to monitor the changes of an experimental property (Pobs) directly correlated with the concentration of the former or the new-formed species, at gradual host addition. The function Pobs = f(Ci, parameters), where Ci represents the equilibrium concentration of the species *i*, is called the binding isotherm. As for Job's plot, this property can be the absorbance (A), fluorescence quantum yield or fluorescence emission (Φ or F), ellipticity (θ) or NMR chemical shift (Δδ). Starting from the binding isotherm, the equations corresponding to some widely used linear (eqs. 1, 2, 5) (Benesi & Hildebrand, 1949; Scott, 1956) and nonlinear (eqs. 3, 4, 6–9) (Liu et al., 2001; Park et al., 2002) models are given in Table 1.

The Determination of the Stoichiometry

equations and to obtain K11 and K21 or K22.

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 53

inclusion of a second guest molecule in a 1:1 complex already formed, or 2:2, resulted from the association of two 1:1 complexes. Over the time, Hamai has reported extensive studies on this regard (Hamai, 1990, 1999, 2005, 2010). Fluorescence is an excellent tool to reveal the formation of these complexes because the dimer, called excimer in excited state, has a distinct band localized at longer wavelength than the monomer. Monitoring the dependence of its fluorescence intensity *vs.* its concentration we can distinguish between the 2:1 and 2:2 stoichiometries using the eqs. 8 and 9 from Table 1. There are two ways to find the K21 and K22 values. The first one is to drive the experiment at small guest concentrations, when the dimer does not exist, and thus to obtain the K11 value. Then, with this value, we can simulate curves according to eqs. 8 and 9 for various values of K21 and K22. The best fit gives the value of the association constant. The second one is to fit the data directly with the said

When several complex types, i.e. complexes with different stoichiometry, are present in the system, the necessity to introduce several fitting parameters reduces the reliability of the fits. Therefore, for spectral measurements, one recommended method (Davies & Deary, 1999; Sainz-Rozas et al., 2005) is to work with sets of data read at different wavelengths and to perform a multivariable analysis of the whole set of data, imposing the condition that the

A special case when it is impossible to unambiguously differentiate by fitting procedures between the 1:1 and the mixture of 1:1+1:2 complexes was discussed by Pistolis and Malliaris (Pistolis & Malliaris, 1999). They found that this happens when the ratio of the corresponding equilibrium constants K11/K12 depends on the extinction coefficients of the species present in the system, the free guest, the 1:1 complex and the 1:2 complex, according

2

ε

 εε

The same relationship holds also for the analysis of fluorescence data, with the fluorescence quantum yields of the three species, Φ*<sup>G</sup>* , Φ*GH* , *GH*<sup>2</sup> Φ , replacing the extinction coefficients. The authors recommend that in such cases additional experiments must be carried out, for

The most used equations are the Benesi-Hildebrand linear or double reciprocal equations. However, their reliability was the subject of many discussions, especially to differentiate the formation of 1:1 and 1:2 complexes. Besides the usual approximations included in the deduction of the Benesi-Hildebrand equation, Wang (Wang & Yu, 2007) used computer simulations to establish some required experimental conditions for obtaining consistent results with the Benesi-Hildebrand equations, rules that can also be applied for the guest–

Shortly, they stated that the CD concentration should be at least an order of magnitude larger than the guest's, the minimum concentration ratio of the two partners should be sufficiently large and the ratio 1/(K[G]) should be large, around 20. Analyzing the electronic absorption data, they also emphasized the role of the differences in the extinction

( ) ( )( ) *GH G GH G GH GH*

 ε

2

2

 ε

<sup>−</sup> <sup>=</sup> − − (10)

association constants are the same, independent on the wavelength.

11 12

ε

*K K*

example fluorescence time-resolved or anisotropy experiments.

coefficients for the 1:1 and 1:2 complexes compared to the free guest.

CD systems.

to eq. 10, written in respect to the extinction coefficients:


Table 1. Nonlinear and linear fitting models for the determination of the stoichiometry and association constant of a CD inclusion complex.

An interesting case is the formation of inclusion complexes in which the guest is included as a dimer. There are two types of stoichiometry for such complexes: 2:1, obtained by the

G + H ←⎯⎯⎯→

Δ− − (1)

*obs GH GH*

<sup>0</sup> 11 11

*P PK H*

1 [] *G GH*

*K H* <sup>+</sup> <sup>=</sup> + (3)

11 11 11 1 ([ ] [ ] ) ([ ] [ ] ) 4[ ][ ])

⎯ GH2

0 0 2

> 12 2

2

(6)

2

2 11 11 11 21 0 11 21 ( [ ] 1) ( [ ] 1) 8 [ ][ ] [ ] 4 [] *K H K H KK HG*

⎯ GH2

⎯ G2H

⎯ G2H2

22 2

2

(7)

[ ]

*P P KP* = + <sup>Δ</sup> <sup>⋅</sup> (2)

*P H G H G HG obs K K* Δ= ++ − ++ − (4)

G + 2H ←⎯⎯⎯→

11 1 [ ] *P ( obs (P P )K H GH G PGH GP )* = +

12

0 2

+

0 2

<sup>2</sup> 0 11

− ++ + + <sup>=</sup>

2 2 0 11

− ++ + + <sup>=</sup>

*G GH GH*

Δ − <sup>−</sup> (5)

12

11 11 12

⎯ GH; GH + G ←⎯⎯⎯→

<sup>1</sup> ([ ] [ ] [ ][ ])

⎯ GH; GH + GH ←⎯⎯⎯→

<sup>1</sup> ([ ] [ ] [ ][ ])

*P P G G K HG* = −− *G H* (9)

*KK H*

11 11 11 22 0 2 2 11 22 ( [ ] 1) ( [ ] 1) 8 [ ] [ ] [ ] 4 [] *K H K H KK H G*

*KK H*

1 [] []

*P P G G K HG* = −− *G H* (8)

*+K H +K K H*

*P P K H +P K K H*

⎯ GH; GH + H ←⎯⎯⎯→

11 11 12

[] []

1 [] *G GH*

*+K H*

*P PKH*

11 1 [ ] *P (P P )K H (P P ) obs GH G GH G* = +

0 0

[ ][ ] [ ] 1

*HG G*

*obs*

*P*

*obs*

+

2

2

Table 1. Nonlinear and linear fitting models for the determination of the stoichiometry and

An interesting case is the formation of inclusion complexes in which the guest is included as a dimer. There are two types of stoichiometry for such complexes: 2:1, obtained by the

G + H ←⎯⎯⎯→

G + H ←⎯⎯⎯→

G + H ←⎯⎯⎯→

*obs*

where

where

association constant of a CD inclusion complex.

*G*

*G*

*P =*

*P =*

⎯ GH

0 0 11

[ ]

2

' '

**Stoichiometry Equations** 

2

1:1

1:2

1:1+1:2

2:1

2:2

inclusion of a second guest molecule in a 1:1 complex already formed, or 2:2, resulted from the association of two 1:1 complexes. Over the time, Hamai has reported extensive studies on this regard (Hamai, 1990, 1999, 2005, 2010). Fluorescence is an excellent tool to reveal the formation of these complexes because the dimer, called excimer in excited state, has a distinct band localized at longer wavelength than the monomer. Monitoring the dependence of its fluorescence intensity *vs.* its concentration we can distinguish between the 2:1 and 2:2 stoichiometries using the eqs. 8 and 9 from Table 1. There are two ways to find the K21 and K22 values. The first one is to drive the experiment at small guest concentrations, when the dimer does not exist, and thus to obtain the K11 value. Then, with this value, we can simulate curves according to eqs. 8 and 9 for various values of K21 and K22. The best fit gives the value of the association constant. The second one is to fit the data directly with the said equations and to obtain K11 and K21 or K22.

When several complex types, i.e. complexes with different stoichiometry, are present in the system, the necessity to introduce several fitting parameters reduces the reliability of the fits. Therefore, for spectral measurements, one recommended method (Davies & Deary, 1999; Sainz-Rozas et al., 2005) is to work with sets of data read at different wavelengths and to perform a multivariable analysis of the whole set of data, imposing the condition that the association constants are the same, independent on the wavelength.

A special case when it is impossible to unambiguously differentiate by fitting procedures between the 1:1 and the mixture of 1:1+1:2 complexes was discussed by Pistolis and Malliaris (Pistolis & Malliaris, 1999). They found that this happens when the ratio of the corresponding equilibrium constants K11/K12 depends on the extinction coefficients of the species present in the system, the free guest, the 1:1 complex and the 1:2 complex, according to eq. 10, written in respect to the extinction coefficients:

$$\frac{K\_{11}}{K\_{12}} = \frac{(\varepsilon\_{\rm GH\_2} - \varepsilon\_G)^2}{(\varepsilon\_{\rm GH} - \varepsilon\_G)(\varepsilon\_{\rm GH\_2} - \varepsilon\_{\rm GH})} \tag{10}$$

The same relationship holds also for the analysis of fluorescence data, with the fluorescence quantum yields of the three species, Φ*<sup>G</sup>* , Φ*GH* , *GH*<sup>2</sup> Φ , replacing the extinction coefficients. The authors recommend that in such cases additional experiments must be carried out, for example fluorescence time-resolved or anisotropy experiments.

The most used equations are the Benesi-Hildebrand linear or double reciprocal equations. However, their reliability was the subject of many discussions, especially to differentiate the formation of 1:1 and 1:2 complexes. Besides the usual approximations included in the deduction of the Benesi-Hildebrand equation, Wang (Wang & Yu, 2007) used computer simulations to establish some required experimental conditions for obtaining consistent results with the Benesi-Hildebrand equations, rules that can also be applied for the guest– CD systems.

Shortly, they stated that the CD concentration should be at least an order of magnitude larger than the guest's, the minimum concentration ratio of the two partners should be sufficiently large and the ratio 1/(K[G]) should be large, around 20. Analyzing the electronic absorption data, they also emphasized the role of the differences in the extinction coefficients for the 1:1 and 1:2 complexes compared to the free guest.

The Determination of the Stoichiometry

as compared to aqueous solution.

*β*-CD complex (Matei et al, 2007).

(6)

(1)

**3.2 7-Diethylamino-3-carboxycoumarin (DEAC)** 

450 500 550 600

S O

N N

λ **(nm)**

of the fit.

0

200

**F (a.u.)**

400

600

2009; Zhang, et al., 2008).

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 55

polarity and ability to form hydrogen bonds. In presence of 2-HP-*β*-CD, a decrease in the emission from QP is observed (Fig. 2A). The changes in the fluorescence intensity of guests upon CD inclusion are usually rationalized by comparing the emission properties of the complexed guest with those of the free guest in different solvents (Wagner et al., 2003). The fluorescence quantum yield of QP in propanol, solvent of polarity similar to that of the CD cavity, is smaller than in dimethylformamide–aqueous solution, the medium in which the experiment was performed. On this basis, the experimentally observed fluorescence decrease was ascribed as due to the lower polarity experienced by QP inside the CD cavity,

In this case, the fluorescence–concentration dependence reflects reliable experimental data; the entire binding isotherm was obtained even without using the maximum possible host concentration. Fitting the data with eq. 3 evidenced the 1:1 stoichiometry of the complex (Fig. 2B) and the value of its association constant (inset of Fig. 2B). The results obtained for the 2-HP-*β*-CD complex of QP are in accordance with our results previously obtained for its

Fig. 2. (A) The fluorescence spectrum of QP (10-5 M in dimethylformamide:water 1:9 v:v) in absence (1) and presence (2–6) of increasing amounts of 2-HP-*β*-CD (up to 3×10-3 M). (B) Determination of the 1:1 stoichiometry of the complex by means of eq. 3. Inset: parameters

300

400

**F (a.u.)**

500

0.000 0.001 0.002 0.003

**[2-HP-**β**-CD] (M)**

K11=11581±526 M-1

=0.995; F-stat=3665

R2

The CD inclusion complexes were often considered as a starting point for choosing good fluorophores to be used in protein studies (Abou-Zied & Al-Hinai, 2006). The changes in the photophysical properties of the guests upon inclusion in the CD cavity can be correlated to their sensitivity on the local polarity, possibility to be involved in hydrogen bonds, etc. Our previous studies on 3-carboxy-coumarin derivatives (Varlan & Hillebrand, 2011) showed that this class of compounds, present in aqueous solution of pH = 7.4 as the corresponding carboxylate ions, are suitable for protein studies. Among the carboxycoumarin derivatives, DEAC represents an interesting guest to be studied in the presence of CD, due to the existence of a second substituent with donor character (the diethylamino group), (Jung et al.,

A slight different methodology for the association constants estimation considers the timeresolved fluorescence experiments. The study of the decay fluorescence curves allows for the estimation of the stoichiometry, and, consequently of the association constants. In the case of the 1:1 complexes, the decay fit by two exponentials is expected, corresponding to the presence of two species in the system, namely the complexed and uncomplexed guest. Considering the ratio of the two pre-exponential coefficients (B), the following relation is obtained:

$$\frac{B\_{bound}}{B\_{free}} = \frac{\mathbf{C}\_{bound} k r\_{bound} \mathbf{c}\_{bound}}{\mathbf{C}\_{free} k r\_{free} \mathbf{c}\_{free}} \tag{11}$$

where C, kr and ε represent the concentration, the radiative rate constant and the extinction coefficient, respectively, for the bound and free species (Monti et al., 1993).

Since kr is constant and we can consider εbound = εfree, in excess of CD eq. 11 becomes:

$$\frac{B\_{bound}}{B\_{free}} = \text{K[CD]}\tag{12}$$

The linearity of the plot of *Bbound*/*Bfree vs*. the CD concentration confirms the formation of a complex with 1:1 stoichiometry and allows for the estimation of the association constant (Rajaram et al., 2011).

Some examples, emphasizing different situations encountered in the CD inclusion complexes study, starting with a guest for which the stoichiometry and the association constants were unambiguously established and continuing with cases of increasing complexity, are reported.

### **2. Experimental methods and computational details**

*α*-, *β*-, 2-HP-*β* and *γ*-CDs (Aldrich) were used as received. 2-[2'-quinoxalinyl]-phenoxathiin was synthesised as described in Ref. (Nicolae et al., 1998). Indapamide was purchased from Helcor (Romania). Two samples of atenolol were used, one purchased from Helcor and the other from IPCA (India). The guest–CD samples were prepared from stock solution of both components, keeping in all cases the guest concentration constant (10-6–10-5 M for fluorescence measurements and 10-5–10-4 M for absorption and circular dichroism measurements). The spectra were recorded on a Jasco FP-6300 spectrofluorimeter, Jasco J-815 CD spectropolarimeter and Jasco V-560 UV-VIS spectrophotometer.

All the necessary computational steps to find the most stable complexes in vacuo and in water were detailed in a previous paper (Matei et al., 2009).

### **3. Some particular cases encountered in the estimation of the stoichiometry of guest–cyclodextrin inclusion complexes**

### **3.1 2-[2'-quinoxalinyl]-phenoxathiin (QP)**

An unequivocal example for the determination of the stoichiometry is given by the inclusion complex formed by QP with 2-HP-*β*-CD. QP is a fluorescence probe sensitive to the medium

A slight different methodology for the association constants estimation considers the timeresolved fluorescence experiments. The study of the decay fluorescence curves allows for the estimation of the stoichiometry, and, consequently of the association constants. In the case of the 1:1 complexes, the decay fit by two exponentials is expected, corresponding to the presence of two species in the system, namely the complexed and uncomplexed guest. Considering the ratio of the two pre-exponential coefficients (B), the following relation is

> *bound bound bound bound free free free free*

where C, kr and ε represent the concentration, the radiative rate constant and the extinction

[ ] *bound*

The linearity of the plot of *Bbound*/*Bfree vs*. the CD concentration confirms the formation of a complex with 1:1 stoichiometry and allows for the estimation of the association constant

Some examples, emphasizing different situations encountered in the CD inclusion complexes study, starting with a guest for which the stoichiometry and the association constants were unambiguously established and continuing with cases of increasing

*α*-, *β*-, 2-HP-*β* and *γ*-CDs (Aldrich) were used as received. 2-[2'-quinoxalinyl]-phenoxathiin was synthesised as described in Ref. (Nicolae et al., 1998). Indapamide was purchased from Helcor (Romania). Two samples of atenolol were used, one purchased from Helcor and the other from IPCA (India). The guest–CD samples were prepared from stock solution of both components, keeping in all cases the guest concentration constant (10-6–10-5 M for fluorescence measurements and 10-5–10-4 M for absorption and circular dichroism measurements). The spectra were recorded on a Jasco FP-6300 spectrofluorimeter, Jasco J-

All the necessary computational steps to find the most stable complexes in vacuo and in

**3. Some particular cases encountered in the estimation of the stoichiometry** 

An unequivocal example for the determination of the stoichiometry is given by the inclusion complex formed by QP with 2-HP-*β*-CD. QP is a fluorescence probe sensitive to the medium

ε

<sup>=</sup> (11)

*<sup>B</sup>* <sup>=</sup> (12)

ε

*B C kr B C kr*

Since kr is constant and we can consider εbound = εfree, in excess of CD eq. 11 becomes:

*free <sup>B</sup> K CD*

coefficient, respectively, for the bound and free species (Monti et al., 1993).

**2. Experimental methods and computational details** 

water were detailed in a previous paper (Matei et al., 2009).

**of guest–cyclodextrin inclusion complexes** 

**3.1 2-[2'-quinoxalinyl]-phenoxathiin (QP)** 

815 CD spectropolarimeter and Jasco V-560 UV-VIS spectrophotometer.

obtained:

(Rajaram et al., 2011).

complexity, are reported.

polarity and ability to form hydrogen bonds. In presence of 2-HP-*β*-CD, a decrease in the emission from QP is observed (Fig. 2A). The changes in the fluorescence intensity of guests upon CD inclusion are usually rationalized by comparing the emission properties of the complexed guest with those of the free guest in different solvents (Wagner et al., 2003). The fluorescence quantum yield of QP in propanol, solvent of polarity similar to that of the CD cavity, is smaller than in dimethylformamide–aqueous solution, the medium in which the experiment was performed. On this basis, the experimentally observed fluorescence decrease was ascribed as due to the lower polarity experienced by QP inside the CD cavity, as compared to aqueous solution.

In this case, the fluorescence–concentration dependence reflects reliable experimental data; the entire binding isotherm was obtained even without using the maximum possible host concentration. Fitting the data with eq. 3 evidenced the 1:1 stoichiometry of the complex (Fig. 2B) and the value of its association constant (inset of Fig. 2B). The results obtained for the 2-HP-*β*-CD complex of QP are in accordance with our results previously obtained for its *β*-CD complex (Matei et al, 2007).

Fig. 2. (A) The fluorescence spectrum of QP (10-5 M in dimethylformamide:water 1:9 v:v) in absence (1) and presence (2–6) of increasing amounts of 2-HP-*β*-CD (up to 3×10-3 M). (B) Determination of the 1:1 stoichiometry of the complex by means of eq. 3. Inset: parameters of the fit.

### **3.2 7-Diethylamino-3-carboxycoumarin (DEAC)**

The CD inclusion complexes were often considered as a starting point for choosing good fluorophores to be used in protein studies (Abou-Zied & Al-Hinai, 2006). The changes in the photophysical properties of the guests upon inclusion in the CD cavity can be correlated to their sensitivity on the local polarity, possibility to be involved in hydrogen bonds, etc. Our previous studies on 3-carboxy-coumarin derivatives (Varlan & Hillebrand, 2011) showed that this class of compounds, present in aqueous solution of pH = 7.4 as the corresponding carboxylate ions, are suitable for protein studies. Among the carboxycoumarin derivatives, DEAC represents an interesting guest to be studied in the presence of CD, due to the existence of a second substituent with donor character (the diethylamino group), (Jung et al., 2009; Zhang, et al., 2008).

The Determination of the Stoichiometry

residuals for the (A) 1:1+1:2 and (B) 1:1 fits.

absorbance increase depends on the CD type.

0.0

0.5

**A**

1.0

1.5

**3.3 Indapamide** 

1.00

1.25

1.50

**F/F0**

1.75

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 57

Fig. 4. Normalized experimental data fits with eqs. 7 (solid line) and 3 (dotted line) for the determination of the stoichiometry of the DEAC-γ-CD complex. Inset: The distribution of the

1:1+1:2 1:1

**-0.018 -0.012 -0.006 0.000 0.006 0.012 0.018**

(B)

**-0.06 -0.04 -0.02 0.00 0.02 0.04 0.06**

Cl

S O <sup>O</sup> NH2

Residuals

(A)

**1:1+1:2**

**1:1**

Residuals

0.000 0.005 0.010 0.015

**[**γ−**CD] (M)**

Indapamide is an antihypertensive, diuretic drug of the sulphonamide class (Ciborowski et al., 2004; Ghugare et al., 2010; Radi & Eissa, 2011). We have studied its complexation with *α*-, *β*-, 2-HP- *β*- and *γ*-CD, by means of absorption spectroscopy. The isolated guest presents two absorption bands, located at 240 nm and 280 nm. In presence of CD, the absorbance of these bands increases, without any band shift or appearance of isosbestic points (Fig. 5). Absorbance increase by complexation has been reported by several authors (El-Kemary, 2002) and considered as evidence for the formation of an inclusion complex. The shape of the spectra of the complexes is similar, irrespective of the CD type, but the magnitude of the

Fig. 5. The absorption spectrum of indapamide (10-5 M in methanol:water 1:9 v:v) in absence

λ **(nm)**

N H N O

CH3

200 250 300

(3)

(1)

(1) and presence (2, 3) of increasing concentrations of 2-HP-*β*-CD (up to 2×10-2 M).

The fluorescence spectrum of DEAC in the presence of γ-CD, the largest native cyclodextrin, is given in Fig. 3. The increase of the γ-CD concentration induces an enhancement of the emission intensity accompanied by a blue shift of the band (~7 nm), without the appearance of an isoemissive point. All this data clearly indicate that the DEAC molecule has passed from the bulk solution to the less polar cavity of the CD, where it has a higher quantum yield and emits at shorter wavelength, as stated by Ramakrishna and Ghosh (Ramakrishna & Ghosh, 2002).

Fig. 3. The fluorescence spectrum of DEAC (2×10-6 M) in the presence of increasing amounts of γ-CD. λ*ex* = 410 nm.

In order to find the stoichiometry of the complexes, the dependence of the fluorescence intensity *vs.* the *γ*-CD concentration was analyzed. The experimental points could be fitted with good statistical parameters to both eqs. 3 and 7 corresponding to the formation of 1:1 and of a mixture of 1:1+1:2 complexes (Fig. 4), the parameters being given in Table 2. As it can be seen from the R2 values, the better fit corresponds to the mixture of 1:1+1:2 complexes. This is also supported by the lower and more random distribution of the fits residuals, included as insets in Fig. 4. Another point to be stressed is that when the ratio of intensities of the bound and free guest in a 1:1 complex is near 1, as in the case of DEAC (F11/F0=1.19), it is very important to have experimental data in the first domain of concentration, otherwise the complexation process will be misinterpreted as a solely 1:1 complex formation.


Table 2. Fitted parameters for the DEAC–γ-CD interaction: association constants (K), normalized fluorescence of the complex (F/F0); R2 – correlation coefficient; F-stat – Fisher statistic coefficient.

Fig. 4. Normalized experimental data fits with eqs. 7 (solid line) and 3 (dotted line) for the determination of the stoichiometry of the DEAC-γ-CD complex. Inset: The distribution of the residuals for the (A) 1:1+1:2 and (B) 1:1 fits.

### **3.3 Indapamide**

56 Stoichiometry and Research – The Importance of Quantity in Biomedicine

The fluorescence spectrum of DEAC in the presence of γ-CD, the largest native cyclodextrin, is given in Fig. 3. The increase of the γ-CD concentration induces an enhancement of the emission intensity accompanied by a blue shift of the band (~7 nm), without the appearance of an isoemissive point. All this data clearly indicate that the DEAC molecule has passed from the bulk solution to the less polar cavity of the CD, where it has a higher quantum yield and emits at shorter wavelength, as stated by Ramakrishna and Ghosh (Ramakrishna

(6)

(1)

Fig. 3. The fluorescence spectrum of DEAC (2×10-6 M) in the presence of increasing amounts

450 500 550 600

(C2H5)2N O

COOH

O

λ **(nm)**

In order to find the stoichiometry of the complexes, the dependence of the fluorescence intensity *vs.* the *γ*-CD concentration was analyzed. The experimental points could be fitted with good statistical parameters to both eqs. 3 and 7 corresponding to the formation of 1:1 and of a mixture of 1:1+1:2 complexes (Fig. 4), the parameters being given in Table 2. As it can be seen from the R2 values, the better fit corresponds to the mixture of 1:1+1:2 complexes. This is also supported by the lower and more random distribution of the fits residuals, included as insets in Fig. 4. Another point to be stressed is that when the ratio of intensities of the bound and free guest in a 1:1 complex is near 1, as in the case of DEAC (F11/F0=1.19), it is very important to have experimental data in the first domain of concentration, otherwise the complexation process will be misinterpreted as a solely 1:1

**Stoichiometry K11 (M-1) K12 (M-1) F11/F0 F12/F0 R2 F-stat** 

1:1 1256±112 – 1.64±0.01 – 0.971 831

1:1+1:2 15450±2987 421±36 1.19±0.01 1.72±0.08 0.998 5721


γ

normalized fluorescence of the complex (F/F0); R2 – correlation coefficient; F-stat – Fisher

& Ghosh, 2002).

of γ-CD.

λ

complex formation.

statistic coefficient.

Table 2. Fitted parameters for the DEAC–

*ex* = 410 nm.

0

60

120

**F (a.u.)**

180

240

Indapamide is an antihypertensive, diuretic drug of the sulphonamide class (Ciborowski et al., 2004; Ghugare et al., 2010; Radi & Eissa, 2011). We have studied its complexation with *α*-, *β*-, 2-HP- *β*- and *γ*-CD, by means of absorption spectroscopy. The isolated guest presents two absorption bands, located at 240 nm and 280 nm. In presence of CD, the absorbance of these bands increases, without any band shift or appearance of isosbestic points (Fig. 5). Absorbance increase by complexation has been reported by several authors (El-Kemary, 2002) and considered as evidence for the formation of an inclusion complex. The shape of the spectra of the complexes is similar, irrespective of the CD type, but the magnitude of the absorbance increase depends on the CD type.

Fig. 5. The absorption spectrum of indapamide (10-5 M in methanol:water 1:9 v:v) in absence (1) and presence (2, 3) of increasing concentrations of 2-HP-*β*-CD (up to 2×10-2 M).

The Determination of the Stoichiometry

points at high CD concentrations.

0.000 0.025 0.050 0.075 0.100 0.125

**[**α**-CD] (M)**

**?**

**3.4.1 Photophysical properties of atenolol** 

**3.4 Atenolol** 

1.0 1.5 2.0 2.5 3.0 3.5 4.0

**A/A0**

(A)

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 59

employed before, for the stoichiometries determination of some simvastatin–CD complexes (Matei et al., 2009). From Fig. 7A we can observe that up to a CD concentration of ~2.5×10-2 M, the two curves are identical, the difference between them appearing only at high CD concentrations. Therefore, in the absence of experimental points in this range, it is impossible to asses without a doubt the stoichiometry of the inclusion complexes. As these experimental data are of crucial importance for the determination of the stoichiometry, the working conditions must be selected in such a way to be as close as possible to the CD solubility limit,

We performed such an experiment, using indapamide and high concentrations of *α*-CD (Fig. 7B). This experiment was revelatory and allowed us to discard the formation of mixtures of 1:1+1:2 complexes. Thus, when the experimental points at high CD concentrations were added to the previous experimental data, they aligned on the generated curve corresponding to the

Fig. 7. Generation of the curves describing the formation of 1:2 (eq. 6) and 1:1+1:2 (eq. 7)

1.00

1.25

1.50

1.75

**A/A0**

2.00

2.25 (B)

0.000 0.025 0.050 0.075 0.100

**[**α**-CD] (M)**

1:2

Atenolol (inset of Fig. 8), a beta-blocker drug used in the treatment of cardiovascular diseases (Bontchev et al., 2000; Castro et al., 1998; Esteves de Castro et al., 2007; Moloney et al., 1998; Pandeeswaran & Elango, 1009; Pulgarin et al., 1998; Ranta et al., 2002), is a very flexible molecule, consisting of fragments with different features: an amido-substituted aromatic ring, a flexible three-carbon chain (Siqintuya et al., 2007) and a dimethyl-substituted amino group.

The photophysical properties of atenolol, measured prior to the study of its CD inclusion complexes, showed very interesting features. The absorption spectra of atenolol in water, methanol and acetonitrile are presented in Fig. 8A. An intense band (ε ~ 104 M-1cm-1) is recorded at 225 nm and assigned to a π-π\* transition correlated to the presence of the aromatic ring (Pillot et al., 1997). In the range of 265–290 nm, a second band is observed,

complexes on a greater domain of CD concentrations than the experimental one (experimental data as open circles), in the absence (A) and presence (B) of experimental

1:2

i.e. *α*: 1.5×10-1 M, *β*: 1.6×10-2 M, 2-HP-*β*: 3.3×10-1 M, *γ*: 1.8×10-1 M (Connors, 1987).

formation of 1:2 complexes, revealing that this is the real stoichiometry.

1:1+1:2

In the first attempt to determine the stoichiometry of the complexes, the Benesi-Hildebrand model was applied. As previously discussed, a linear dependency of the type 1/(*A-A0*) *vs.* 1/[CD]n, with n = 1 or 2, indicates the presence of complexes of 1:1 or 1:2 stoichiometry, respectively. For indapamide, the plot 1/(*A-A0*) *vs.* 1/[CD] shows a positive deviation, irrespective of the CD type (inset of Fig. 6A), which eliminates the hypothesis of the formation of solely 1:1 complexes. This is in accordance with the lack of isosbestic points. The plot 1/(*A*-*A0*) *vs.* 1/[CD]2, although characterized by a good correlation coefficient (R2 = 0.990), has unacceptably high standard errors and standard deviations of the fits (Fig. 6A). The Benesi-Hildebrand model cannot offer conclusive data on the presence of the 1:2 stoichiometry.

Applying the nonlinear model describing the formation of a 1:2 complex (eq. 6, dotted line), we obtained good results for all CDs, with small errors (<15%), good correlation coefficients (R2 > 0.990) and Fisher statistic coefficients (F ~ 1500–7000) (Fig. 6B). The predicted association constants are: *Kα*-CD = 2447±89 M-1, *Kβ*-CD = 25550±783 M-1, *K2-HP-β*-CD = 3330±370 M-1 and *Kγ-*CD = 3897±481 M-1.

Fig. 6. (A) Benesi-Hildebrand plots for the investigation of the formation of indapamide–α-CD complexes of 1:2 and 1:1 (inset) stoichiometry. (B) The dependence of the normalized absorption of indapamide on the CD concentration. Fits with eqs. 6 and 7.

However, the examination of the first segment of the curves, corresponding to low CD concentrations, reveals some deviations of the experimental points, reflected in the shape of the fit residuals. This prompted us to check for the presence of a mixture of 1:1+1:2 complexes (eq. 7, solid line). These fits are characterized by higher correlation coefficients (R2 > 0.995), as well as smaller and more randomly distributed residuals for the first segment of the curve. Still, in this case the standard errors have high values for K12 and A12, due to the uncertainty introduced in the fit by the lack of experimental points on the last segment of the curve, at high CD concentrations.

In the attempt to discern between the two cases, i.e. the 1:2 stoichiometry and the mixture of 1:1+1:2 complexes, we have generated fitting curves on a broader range of CD concentrations, using the values of the association constants and absorbances of the complexes obtained with eqs. 6 and 7, respectively (Fig. 7A). This technique has been employed before, for the stoichiometries determination of some simvastatin–CD complexes (Matei et al., 2009). From Fig. 7A we can observe that up to a CD concentration of ~2.5×10-2 M, the two curves are identical, the difference between them appearing only at high CD concentrations. Therefore, in the absence of experimental points in this range, it is impossible to asses without a doubt the stoichiometry of the inclusion complexes. As these experimental data are of crucial importance for the determination of the stoichiometry, the working conditions must be selected in such a way to be as close as possible to the CD solubility limit, i.e. *α*: 1.5×10-1 M, *β*: 1.6×10-2 M, 2-HP-*β*: 3.3×10-1 M, *γ*: 1.8×10-1 M (Connors, 1987).

We performed such an experiment, using indapamide and high concentrations of *α*-CD (Fig. 7B). This experiment was revelatory and allowed us to discard the formation of mixtures of 1:1+1:2 complexes. Thus, when the experimental points at high CD concentrations were added to the previous experimental data, they aligned on the generated curve corresponding to the formation of 1:2 complexes, revealing that this is the real stoichiometry.

Fig. 7. Generation of the curves describing the formation of 1:2 (eq. 6) and 1:1+1:2 (eq. 7) complexes on a greater domain of CD concentrations than the experimental one (experimental data as open circles), in the absence (A) and presence (B) of experimental points at high CD concentrations.

### **3.4 Atenolol**

58 Stoichiometry and Research – The Importance of Quantity in Biomedicine

In the first attempt to determine the stoichiometry of the complexes, the Benesi-Hildebrand model was applied. As previously discussed, a linear dependency of the type 1/(*A-A0*) *vs.* 1/[CD]n, with n = 1 or 2, indicates the presence of complexes of 1:1 or 1:2 stoichiometry, respectively. For indapamide, the plot 1/(*A-A0*) *vs.* 1/[CD] shows a positive deviation, irrespective of the CD type (inset of Fig. 6A), which eliminates the hypothesis of the formation of solely 1:1 complexes. This is in accordance with the lack of isosbestic points. The plot 1/(*A*-*A0*) *vs.* 1/[CD]2, although characterized by a good correlation coefficient (R2 = 0.990), has unacceptably high standard errors and standard deviations of the fits (Fig. 6A). The Benesi-Hildebrand model cannot offer conclusive data on the presence of the 1:2

Applying the nonlinear model describing the formation of a 1:2 complex (eq. 6, dotted line), we obtained good results for all CDs, with small errors (<15%), good correlation coefficients (R2 > 0.990) and Fisher statistic coefficients (F ~ 1500–7000) (Fig. 6B). The predicted association constants are: *Kα*-CD = 2447±89 M-1, *Kβ*-CD = 25550±783 M-1, *K2-HP-β*-CD = 3330±370

Fig. 6. (A) Benesi-Hildebrand plots for the investigation of the formation of indapamide–α-CD complexes of 1:2 and 1:1 (inset) stoichiometry. (B) The dependence of the normalized

1.2

1.6

2.0

**A/A0**

2.4

2.8

(B)

1.2 1.6

**A/A0**

0.000 0.005 0.010 0.015 0.020 0.025

0.00 0.01 0.02

**[CD] (M)**

 2-HP-β-CD γ-CD

2.0 <sup>β</sup>-CD

**[**α**-CD] (M)**

 1:2 1:1+1:2

However, the examination of the first segment of the curves, corresponding to low CD concentrations, reveals some deviations of the experimental points, reflected in the shape of the fit residuals. This prompted us to check for the presence of a mixture of 1:1+1:2 complexes (eq. 7, solid line). These fits are characterized by higher correlation coefficients (R2 > 0.995), as well as smaller and more randomly distributed residuals for the first segment of the curve. Still, in this case the standard errors have high values for K12 and A12, due to the uncertainty introduced in the fit by the lack of experimental points on the last

In the attempt to discern between the two cases, i.e. the 1:2 stoichiometry and the mixture of 1:1+1:2 complexes, we have generated fitting curves on a broader range of CD concentrations, using the values of the association constants and absorbances of the complexes obtained with eqs. 6 and 7, respectively (Fig. 7A). This technique has been

absorption of indapamide on the CD concentration. Fits with eqs. 6 and 7.

100 200 300 400

**1/[**α**-CD] (M-1 )**

1:1

segment of the curve, at high CD concentrations.

0 5x104 1x105 2x105 2x105

**1/(A-A0**

**)**

 **(M-2 )**

**1/[**α**-CD]2**

stoichiometry.

0

30

**1/(A-A0**

**)**

60

90

(A)

M-1 and *Kγ-*CD = 3897±481 M-1.

1:2

Atenolol (inset of Fig. 8), a beta-blocker drug used in the treatment of cardiovascular diseases (Bontchev et al., 2000; Castro et al., 1998; Esteves de Castro et al., 2007; Moloney et al., 1998; Pandeeswaran & Elango, 1009; Pulgarin et al., 1998; Ranta et al., 2002), is a very flexible molecule, consisting of fragments with different features: an amido-substituted aromatic ring, a flexible three-carbon chain (Siqintuya et al., 2007) and a dimethyl-substituted amino group.

### **3.4.1 Photophysical properties of atenolol**

The photophysical properties of atenolol, measured prior to the study of its CD inclusion complexes, showed very interesting features. The absorption spectra of atenolol in water, methanol and acetonitrile are presented in Fig. 8A. An intense band (ε ~ 104 M-1cm-1) is recorded at 225 nm and assigned to a π-π\* transition correlated to the presence of the aromatic ring (Pillot et al., 1997). In the range of 265–290 nm, a second band is observed,

The Determination of the Stoichiometry

(1)

(4)

protic solvents.

0

50

100

150

**F (a.u.)**

200

<sup>250</sup> (A)

accumulation of the species emitting at 350 nm.

spectroscopy is used as the method of investigation.

strongly dependent on the excitation wavelength (Fig. 11).

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 61

Fig. 9. Evolution of the B1 and B2 fluorescence bands of atenolol in methanol (A) upon dilution with methanol and (B) upon irradiation at 254 nm (three steps of 25 minutes each).

300 350 400 450

λ **(nm)**

All these data show that atenolol exists in solution as several conformers differently influenced by the solvent, the excitation energy and/or the temperature. Furthermore, considering the pKa value of 9.6 of atenolol (Kasim et al., 2004), we can not rule out the presence of different amounts of protonated/nonprotonated species, especially in water and

0

100

(4)

(1)

200

**F (a.u.)**

<sup>300</sup> (B)

300 350 400 450

λ **(nm)**

In order to obtain more information on the species present in solution, the fluorescence spectrum of atenolol was recorded after irradiation with a mercury lamp at 254 nm. The results are displayed in Fig. 9B and show unambiguously that the irradiation favours the

In conclusion, the experimental data on the emission process of atenolol showed the presence of two emitting species, influenced by the concentration, excitation wavelength, temperature and irradiation of the system. All these data must be considered in the further discussion of the atenolol–CD inclusion complexes, especially when the fluorescence

Several spectral methods (FTIR, DSC, SEM, etc*.*) have been employed for the characterization of the CD inclusion complexes of atenolol in solid state (Borodi et al., 2008; Ficarra et al., 2000a, 2000b). In the following, we will present our results on the interaction of atenolol with *α*-, *β*- and *γ*-CD in solution, studied by means of fluorescence, circular dichroism and absorption spectroscopies. Recording the fluorescence spectra of atenolol in the presence of CDs, we observed slight changes in the intensity of B1 and the strong increase of a band in the same spectral region and with the same shape as the B2 band of uncomplexed atenolol (Fig. 10). This increase indicates that the inclusion process favours the same process as previously discussed for uncomplexed atenolol, *i.e.* the emission correlated to the amino group evidenced in several experimental conditions. This could be due to the inclusion of the isopropylamine fragment in the CD cavity. The fluorescence spectra are

**3.4.2 Characterization of the atenolol-cyclodextrin inclusion complexes** 

characterized by a lower ε value (~103 M-1cm-1), at the limit of a weak π-π\* and a forbidden n-π\* transition. According to literature data, this band was considered a benzenic band bathochromically shifted due to the amide substitution (Gratzer, 1967).

Fig. 8. Absorption (A) and fluorescence (B) spectra of atenolol in different solvents. Inset: Deconvolution of the fluorescence spectrum of atenolol in dimethylformamide. *λex* = 275 nm.

The fluorescence spectrum of atenolol was recorded in nonpolar, polar aprotic and polar protic solvents considering several excitation wavelengths, in the range of the two aforementioned bands. An intense emission band, hereafter labelled B1, slightly influenced by the solvents, was obtained in the range 299–306 nm (Fig. 8B), characterized by a fluorescence quantum yield of 0.11 in water and 0.20 in acetonitrile. A careful examination of the band shape revealed an asymmetry at the longer wavelength range, better evidenced by a deconvolution process, as it can be seen from the inset of Fig. 8B. Besides the main band (300 nm), two other bands were found, one at 316 nm, very close to the first band, and another at 344 nm. This new band, located at 344 nm, much lower in intensity than the former one, will be further called B2. Performing a systematic scanning of the role of different experimental factors (concentration, excitation wavelength, etc.) on the shape of the fluorescence bands, we have found that B2 strongly increases in intensity by dilution, presents in fact two maxima (Fig. 9A) and that it is very sensitive to the excitation wavelength, being much enhanced for λ*ex* = 250 nm. The ratio of the two bands composing B2 remains constant upon dilution, suggesting a vibrational structure of the band. By comparison with the fluorescence spectrum of a related compound, 4-phenyl-1-N,N-dimethylaminobutane (Xie et al., 2004), B1 was assigned to the emission of the excited aromatic system and B2 to a species in which the excitation is localized on the amino chromophore.

For obtaining supplementary data on the emission process, the fluorescence spectra were recorded at several temperatures in the range 25–75°C using different excitation wavelengths. By cooling back to 25°C, we have observed a reversible decrease of the intensity of the B1 band, which allowed for the estimation of the activation energy of the nonradiative processes. In the limit of experimental errors, the obtained values showed that the process is not influenced by the excitation wavelength (Ea ~ 1.2 kcal/mol). Analyzing the behaviour of B2 in the same temperature range, a different result was obtained, *i.e.* a larger value of the activation energy of the process, 5.38 kcal/mol, for λ*ex* = 250 nm.

characterized by a lower ε value (~103 M-1cm-1), at the limit of a weak π-π\* and a forbidden n-π\* transition. According to literature data, this band was considered a benzenic band

Fig. 8. Absorption (A) and fluorescence (B) spectra of atenolol in different solvents. Inset: Deconvolution of the fluorescence spectrum of atenolol in dimethylformamide. *λex* = 275 nm.

being much enhanced for

**Anorm-275 nm**

(A) water

acetonitrile metanol

λ

220 240 260 280 300

λ **(nm)**

O NH2

which the excitation is localized on the amino chromophore.

value of the activation energy of the process, 5.38 kcal/mol, for

The fluorescence spectrum of atenolol was recorded in nonpolar, polar aprotic and polar protic solvents considering several excitation wavelengths, in the range of the two aforementioned bands. An intense emission band, hereafter labelled B1, slightly influenced by the solvents, was obtained in the range 299–306 nm (Fig. 8B), characterized by a fluorescence quantum yield of 0.11 in water and 0.20 in acetonitrile. A careful examination of the band shape revealed an asymmetry at the longer wavelength range, better evidenced by a deconvolution process, as it can be seen from the inset of Fig. 8B. Besides the main band (300 nm), two other bands were found, one at 316 nm, very close to the first band, and another at 344 nm. This new band, located at 344 nm, much lower in intensity than the former one, will be further called B2. Performing a systematic scanning of the role of different experimental factors (concentration, excitation wavelength, etc.) on the shape of the fluorescence bands, we have found that B2 strongly increases in intensity by dilution, presents in fact two maxima (Fig. 9A) and that it is very sensitive to the excitation wavelength,

0

dimethylsulfoxide water

20

40

**F (a.u.)**

25

50

**Fnorm-300 nm**

75

100

(B)

constant upon dilution, suggesting a vibrational structure of the band. By comparison with the fluorescence spectrum of a related compound, 4-phenyl-1-N,N-dimethylaminobutane (Xie et al., 2004), B1 was assigned to the emission of the excited aromatic system and B2 to a species in

For obtaining supplementary data on the emission process, the fluorescence spectra were recorded at several temperatures in the range 25–75°C using different excitation wavelengths. By cooling back to 25°C, we have observed a reversible decrease of the intensity of the B1 band, which allowed for the estimation of the activation energy of the nonradiative processes. In the limit of experimental errors, the obtained values showed that the process is not influenced by the excitation wavelength (Ea ~ 1.2 kcal/mol). Analyzing the behaviour of B2 in the same temperature range, a different result was obtained, *i.e.* a larger

*ex* = 250 nm. The ratio of the two bands composing B2 remains

λ

*ex* = 250 nm.

300 350 400 450

λ **(nm)**

dioxane

<sup>60</sup> R2

300 400 500 <sup>0</sup>

λ **(nm)**

=0.999 F-stat=32078 Predicted bands: 300 nm 316 nm 344 nm

bathochromically shifted due to the amide substitution (Gratzer, 1967).

OH NH CH3 CH3

O

Fig. 9. Evolution of the B1 and B2 fluorescence bands of atenolol in methanol (A) upon dilution with methanol and (B) upon irradiation at 254 nm (three steps of 25 minutes each).

All these data show that atenolol exists in solution as several conformers differently influenced by the solvent, the excitation energy and/or the temperature. Furthermore, considering the pKa value of 9.6 of atenolol (Kasim et al., 2004), we can not rule out the presence of different amounts of protonated/nonprotonated species, especially in water and protic solvents.

In order to obtain more information on the species present in solution, the fluorescence spectrum of atenolol was recorded after irradiation with a mercury lamp at 254 nm. The results are displayed in Fig. 9B and show unambiguously that the irradiation favours the accumulation of the species emitting at 350 nm.

In conclusion, the experimental data on the emission process of atenolol showed the presence of two emitting species, influenced by the concentration, excitation wavelength, temperature and irradiation of the system. All these data must be considered in the further discussion of the atenolol–CD inclusion complexes, especially when the fluorescence spectroscopy is used as the method of investigation.

### **3.4.2 Characterization of the atenolol-cyclodextrin inclusion complexes**

Several spectral methods (FTIR, DSC, SEM, etc*.*) have been employed for the characterization of the CD inclusion complexes of atenolol in solid state (Borodi et al., 2008; Ficarra et al., 2000a, 2000b). In the following, we will present our results on the interaction of atenolol with *α*-, *β*- and *γ*-CD in solution, studied by means of fluorescence, circular dichroism and absorption spectroscopies. Recording the fluorescence spectra of atenolol in the presence of CDs, we observed slight changes in the intensity of B1 and the strong increase of a band in the same spectral region and with the same shape as the B2 band of uncomplexed atenolol (Fig. 10). This increase indicates that the inclusion process favours the same process as previously discussed for uncomplexed atenolol, *i.e.* the emission correlated to the amino group evidenced in several experimental conditions. This could be due to the inclusion of the isopropylamine fragment in the CD cavity. The fluorescence spectra are strongly dependent on the excitation wavelength (Fig. 11).

The Determination of the Stoichiometry

superposition of several effects.

fluorescent conformation.

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 63

The 1:2 stoichiometry of the inclusion complexes in methanol:water has been determined by fitting the experimental data with eq. 6. In the case of the atenolol–*α*-CD complex, the readings have been made on both B1 and B2, at *λex* = 282 nm (Fig. 12). In an attempt to obtain a more consistent value of the association constant, for B2, the emission intensities were read at three wavelengths within the band (345, 358 and 380 nm). The obtained data for all CDs are summarized in Table 3. For *λex* = 225 and 275 nm, the fits resulted in high standard errors, while for *λex* = 250 nm, due to high data scattering, no reliable results could be obtained. Considering that this band is also evidenced in absence of CD and most prominently at *λex* = 250 nm, it must be stressed that the great deviations may be due to the

The influence of the temperature on the equilibriums present in solution is the same, irrespective of the CD type and *λex* (Fig. 13), and it is similar to that discussed for uncomplexed atenolol in different solvents. Upon raising the temperature, we observed an increase in the intensity of B1 and a decrease of B2. Similar data were obtained for all CDs. The trend of B1 is an usually encountered for fluorescent molecules one, and can be explained by an increased magnitude of the nonradiative deactivation processes. Differently, the behaviour of B2 could be rationalized in terms of a conformational change leading to an atenolol molecule in a highly fluorescent conformation. This process could be favoured by the decreased viscosity of the medium, leading to an increased mobility of the molecular fragments. Interesting information can be obtained from the behaviour of B1 and B2 upon cooling. While B1 temperature variation is reversible, the intensity of B2 doesn't return to its original value upon decreasing the temperature. This irreversibility could indicate that the amino group is included in the CD cavity and only successively fixed in a

Fig. 12. Plots for the determination of the stoichiometry and association constant of the atenolol–*α*-CD complex in methanol:water 1:9 v:v using fluorescence data read on B2 at

0.0000 0.0002 0.0004 0.0006 0.0008

0.0000 0.0003 0.0006 0.0009

**[**α**-CD] (M)**

M-1

**[**α**-CD] (M)**

K=5.14x10<sup>5</sup>

K=4.07x10<sup>5</sup>

K=4.32x10<sup>5</sup>

M-1

M-1

M-1

three excitation wavelengths (225, 275 and 282 nm) and B1 (inset) at 282 nm.

 225 nm 275 nm 282 nm

1.04 K=1.83x10<sup>6</sup>

1.00 1.01 1.02 1.03

**F**

**/300 nm**

**norm**

**F**

**/351 nm**

**norm**

Fig. 10. The fluorescence spectrum of atenolol (2×10-5 M in methanol:water 1:9 v:v) in absence (1) and presence (2–9) of increasing amounts of CD: (A) *β* (up to 5.5×10-4 M) and (B) *γ* (up to 7.3×10-4 M). *λex* = 250 nm.

Fig. 11. The fluorescence spectrum of atenolol (2×10-5 M in methanol:water 1:9 v:v) in absence (1) and presence (2–10) of increasing amounts of *α*-CD (up to 7.4×10-4 M), at various *λex* (nm): (A) 225, (B) 250, (C) 275 and (D) 282.

Fig. 10. The fluorescence spectrum of atenolol (2×10-5 M in methanol:water 1:9 v:v) in absence (1) and presence (2–9) of increasing amounts of CD: (A) *β* (up to 5.5×10-4 M) and (B)

0

0

(D)

(8)

(1)

0

50

**F (a.u.)**

100

50

100

**F (a.u.)**

<sup>150</sup> (B)

(10) (1)

50

**F (a.u.)**

100

(B)

(1)

(9)

250 300 350 400 450

(10)

250 300 350 400 450

(8)

(1)

λ **(nm)**

250 300 350 400 450 500

λ **(nm)**

(1)

λ **(nm)**

(1)

(9)

Fig. 11. The fluorescence spectrum of atenolol (2×10-5 M in methanol:water 1:9 v:v) in absence (1) and presence (2–10) of increasing amounts of *α*-CD (up to 7.4×10-4 M), at various

*λex* (nm): (A) 225, (B) 250, (C) 275 and (D) 282.

*γ* (up to 7.3×10-4 M). *λex* = 250 nm.

(3) (1)

(A) (7)

0

0

<sup>150</sup> (C)

(5) (1)

0

50

100

**F (a.u.)**

50

100

**F (a.u.)**

150

(A)

50

**F (a.u.)**

100

250 300 350 400 450

λ **(nm)**

250 300 350 400

λ **(nm)**

250 300 350 400 450 500

λ **(nm)**

(5) (1)

(3) (1)

(1)

The 1:2 stoichiometry of the inclusion complexes in methanol:water has been determined by fitting the experimental data with eq. 6. In the case of the atenolol–*α*-CD complex, the readings have been made on both B1 and B2, at *λex* = 282 nm (Fig. 12). In an attempt to obtain a more consistent value of the association constant, for B2, the emission intensities were read at three wavelengths within the band (345, 358 and 380 nm). The obtained data for all CDs are summarized in Table 3. For *λex* = 225 and 275 nm, the fits resulted in high standard errors, while for *λex* = 250 nm, due to high data scattering, no reliable results could be obtained. Considering that this band is also evidenced in absence of CD and most prominently at *λex* = 250 nm, it must be stressed that the great deviations may be due to the superposition of several effects.

The influence of the temperature on the equilibriums present in solution is the same, irrespective of the CD type and *λex* (Fig. 13), and it is similar to that discussed for uncomplexed atenolol in different solvents. Upon raising the temperature, we observed an increase in the intensity of B1 and a decrease of B2. Similar data were obtained for all CDs. The trend of B1 is an usually encountered for fluorescent molecules one, and can be explained by an increased magnitude of the nonradiative deactivation processes. Differently, the behaviour of B2 could be rationalized in terms of a conformational change leading to an atenolol molecule in a highly fluorescent conformation. This process could be favoured by the decreased viscosity of the medium, leading to an increased mobility of the molecular fragments. Interesting information can be obtained from the behaviour of B1 and B2 upon cooling. While B1 temperature variation is reversible, the intensity of B2 doesn't return to its original value upon decreasing the temperature. This irreversibility could indicate that the amino group is included in the CD cavity and only successively fixed in a fluorescent conformation.

Fig. 12. Plots for the determination of the stoichiometry and association constant of the atenolol–*α*-CD complex in methanol:water 1:9 v:v using fluorescence data read on B2 at three excitation wavelengths (225, 275 and 282 nm) and B1 (inset) at 282 nm.

The Determination of the Stoichiometry

to the symmetry axis of *β*-CD.

from UV-vis absorption data.

*vs*. the *β*-CD concentration.

0

200

**F (a.u.)**

400

(1)

(15)

(1)

600

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 65

To obtain more data on the atenolol complexation process, several other methods were used, including circular dichroism and absorption spectroscopies, together with molecular modeling. Therefore, the stoichiometry of the inclusion complex of atenolol with *β*-CD has been determined *via* circular dichroism spectroscopy. While in absence of CD no signal is recorded for atenolol, upon CD addition a negative dichroic signal appears at 275 nm (Fig. 15A). The corresponding calculated transition moment is located in the plane of the aromatic ring, perpendicular to the long molecular axis (*vide infra*). The appearance of an induced circular dichroism signal of atenolol confirms its inclusion into the asymmetric CD cavity and indicates that the aromatic ring is perturbed by CD incorporation. Moreover, its negative sign indicates the perpendicular orientation of the transition moment with respect

The data for the system atenolol–*β*-CD were fitted with eq. 3, revealing the formation of complexes of 1:1 stoichiometry (Fig. 15B). One can observe the greater scattering of the experimental points, characteristic to the measurements by circular dichroism. The data were also analyzed using the Scott model (eq. 2, inset of Fig. 15B), which yielded a K11 value in good accordance with the results of the nonlinear model, although the fit was of somewhat lower quality. The value of the association constant also correlates to that obtained by us (Fig. 16) and by Borodi *et al.* (Borodi et al., 2008) (110±22 M-1, Scott's model)

Fig. 14. The fluorescence spectrum of atenolol (2×10-5 M in dimethylformamide) in the absence (1) and presence (2–30) of increasing amounts of *β*-CD (up to 8×10-4 M). *λex* = 275 nm. The DMF spectrum has been subtracted. Inset: Plot of the fluorescence intensity of B2

300 350 400 450

0

100

200

**F (a.u.)**

**1:2**

K12=(8.76±3.12)10<sup>4</sup>

M-1

300

R2 =0.999 F-stat=21972

0.0000 0.0004 0.0008

**[**β**-CD] (M)**

λ **(nm)**

(15)


Table 3. Parameters of the 1:2 atenolol–CD inclusion complexes in methanol:water 1:9 v:v. *λex* = 282 nm.

Fig. 13. The temperature dependence of the fluorescence intensity of the atenolol–*γ*-CD system in methanol:water 1:9 v:v (A) and the activation energies of the respective processes (B). *λex* = 250 nm.

Differing from the spectra in methanol:water, using dimethylformamide as solvent we observed an enhanced decrease of the B1 band intensity, together with a significant increase of B2 (Fig. 14). Despite the significant intensity change of B1, the readings on this band could not be used for the stoichiometry determination. On band B2, the fits, according to eq. 6, indicated the presence of 1:2 complexes (inset of Fig. 14).

**Band K12×10-6 (M-1) F12/F0 R2; F-stat**  *α***-CD** 

> 15.22 29.13 24.71

3.47 5.67 6.48

5.63 10.44 9,19

0.997; 5291 0.998; 7786 0.997; 6133

0.991; 1056 0.988; 887 0.992; 1312

0.992; 2168 0.992; 2252 0,988; 1484

0.0030 0.0032 0.0034

**1/T (K-1 )**

B1, 300 nm 1.83±0.43 1.08 0.987; 739

*β***-CD** 

*γ***-CD** 

Table 3. Parameters of the 1:2 atenolol–CD inclusion complexes in methanol:water 1:9 v:v.

Fig. 13. The temperature dependence of the fluorescence intensity of the atenolol–*γ*-CD system in methanol:water 1:9 v:v (A) and the activation energies of the respective processes

indicated the presence of 1:2 complexes (inset of Fig. 14).

300 350 400 450

 25o C 70o C 25o

C (on cooling)

λ **(nm)**

Differing from the spectra in methanol:water, using dimethylformamide as solvent we observed an enhanced decrease of the B1 band intensity, together with a significant increase of B2 (Fig. 14). Despite the significant intensity change of B1, the readings on this band could not be used for the stoichiometry determination. On band B2, the fits, according to eq. 6,


0.0

 ln(F0 /F)300 nm ln(F/F0 ) 359nm

Ea

=1.40 kcal/mol (R=0.988)

Ea

=1.23 kcal/mol (R=0.990)

0.1

**ln(F0/F)** & **ln(F/F0**

**)**

0.2

0.3

(B)

0.43±0.10 0.51±0.09 0.81±0.11

3.67±0.77 4.91±0.90 5.54±0.75

0.94±0.19 1.02±0.19 1,88±0,30

B2, 345 nm 358 nm 380 nm

B2, 345 nm 359 nm 380 nm

B2, 345 nm 359 nm 380 nm

*λex* = 282 nm.

(B). *λex* = 250 nm.

0

200

400

**F (a.u.)**

600 (A)

To obtain more data on the atenolol complexation process, several other methods were used, including circular dichroism and absorption spectroscopies, together with molecular modeling. Therefore, the stoichiometry of the inclusion complex of atenolol with *β*-CD has been determined *via* circular dichroism spectroscopy. While in absence of CD no signal is recorded for atenolol, upon CD addition a negative dichroic signal appears at 275 nm (Fig. 15A). The corresponding calculated transition moment is located in the plane of the aromatic ring, perpendicular to the long molecular axis (*vide infra*). The appearance of an induced circular dichroism signal of atenolol confirms its inclusion into the asymmetric CD cavity and indicates that the aromatic ring is perturbed by CD incorporation. Moreover, its negative sign indicates the perpendicular orientation of the transition moment with respect to the symmetry axis of *β*-CD.

The data for the system atenolol–*β*-CD were fitted with eq. 3, revealing the formation of complexes of 1:1 stoichiometry (Fig. 15B). One can observe the greater scattering of the experimental points, characteristic to the measurements by circular dichroism. The data were also analyzed using the Scott model (eq. 2, inset of Fig. 15B), which yielded a K11 value in good accordance with the results of the nonlinear model, although the fit was of somewhat lower quality. The value of the association constant also correlates to that obtained by us (Fig. 16) and by Borodi *et al.* (Borodi et al., 2008) (110±22 M-1, Scott's model) from UV-vis absorption data.

Fig. 14. The fluorescence spectrum of atenolol (2×10-5 M in dimethylformamide) in the absence (1) and presence (2–30) of increasing amounts of *β*-CD (up to 8×10-4 M). *λex* = 275 nm. The DMF spectrum has been subtracted. Inset: Plot of the fluorescence intensity of B2 *vs*. the *β*-CD concentration.

The Determination of the Stoichiometry

respect to the β-CD symmetry axis;

on the amino group.

of atenolol is plotted in grey.

a single experiment is not sufficient.

obtained by fitting the data to different models.

**4. Conclusions** 

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 67

This is rationalized in terms of the equatorial orientation of the corresponding transition moment of atenolol calculated at the TDDFT level (represented in grey in Fig. 17A) with

b. the increased and temperature irreversible emission correlated to the amino group

This behaviour can be explained on the basis of the inclusion of the isopropylamino moiety of atenolol in one of the two CD cavities of the 1:2 complex (Fig. 17B). The molecule is "fixed" by the cavity in a conformation where the excited state is predominantly localized

Fig. 17. Proposed structures for the (A) 1:1 and (B) 1:2 atenolol–*β*-CD inclusion complexes, *via* molecular modeling. The dotted lines represent hydrogen bonds. The transition moment

The estimation of the stoichiometry of a CD inclusion complex represents the first step in the characterization of the complex, leading to the determination of reliable association constants and offering a hint of the supramolecular structure. Unfortunately, the unequivocal estimation of the stoichiometry is not always straightforward and in some cases

In the four discussed examples we have described some cases we have encountered, in order of increasing complexity. In the first case we have had no problems in determining the stoichiometry, the 1:1 inclusion complex formed by the phenoxathiin derivative being clearly defined, with all the other models leading to absurd results. For the two following guests, DEAC and indapamide, the uncertainties arose in the determination of the stoichiometry were solved in two ways, starting with the careful examination of the results

a. the negative signal of the dichroic band of atenolol in the 1:1 complex.

observed upon the formation of the 1:2 complex.

(A) (B)

Fig. 15. (A) The circular dichroism spectra of atenolol (5×10-4 M in pH 7.4 phosphate buffer) in absence (1) and presence (2–7) of increasing concentrations of *β*-CD (up to 2×10- 2 M). (B) Determination of the stoichiometry and association constant of the atenolol–*β*-CD complex.

Fig. 16. (A) The absorption spectra of atenolol (5×10-4 M in pH 7.4 phosphate buffer) in absence (1) and presence (2–7) of increasing concentrations of *β*-CD (up to 2×10-2 M). (B) Determination of the stoichiometry and association constant of the atenolol–*β*-CD complex.

As stated above, molecular mechanics calculations were performed using the MM+ force field in order to gain theoretical support for two experimental results:

a. the negative signal of the dichroic band of atenolol in the 1:1 complex.

This is rationalized in terms of the equatorial orientation of the corresponding transition moment of atenolol calculated at the TDDFT level (represented in grey in Fig. 17A) with respect to the β-CD symmetry axis;

b. the increased and temperature irreversible emission correlated to the amino group observed upon the formation of the 1:2 complex.

This behaviour can be explained on the basis of the inclusion of the isopropylamino moiety of atenolol in one of the two CD cavities of the 1:2 complex (Fig. 17B). The molecule is "fixed" by the cavity in a conformation where the excited state is predominantly localized on the amino group.

Fig. 17. Proposed structures for the (A) 1:1 and (B) 1:2 atenolol–*β*-CD inclusion complexes, *via* molecular modeling. The dotted lines represent hydrogen bonds. The transition moment of atenolol is plotted in grey.

### **4. Conclusions**

66 Stoichiometry and Research – The Importance of Quantity in Biomedicine

Fig. 15. (A) The circular dichroism spectra of atenolol (5×10-4 M in pH 7.4 phosphate buffer) in absence (1) and presence (2–7) of increasing concentrations of *β*-CD (up to 2×10- 2 M). (B) Determination of the stoichiometry and association constant of the atenolol–*β*-CD

250 260 270 280 290 300

λ **(nm)**

(7)

(1)




θ **(mdeg)**


0.0

(B)

**1:1** K11=245±32 M-1

R2 =0.987 F-stat=1401

0.000 0.002 0.004 0.006 0.008

0.000 0.002 0.004 0.006

**1:1** K11=163±16 M-1

R2 =0.998 F-stat=5673

**[**β**-CD] (M)**



**[atenolol][**β**-CD]/**Δθ


**[**β**-CD] (M)**

0.000 0.003 0.006 0.009

**[**β**-CD] (M)**

**1:1** K11=237 M-1 R2 =0.969

Fig. 16. (A) The absorption spectra of atenolol (5×10-4 M in pH 7.4 phosphate buffer) in absence (1) and presence (2–7) of increasing concentrations of *β*-CD (up to 2×10-2 M). (B) Determination of the stoichiometry and association constant of the atenolol–*β*-CD

0.68

0.70

**A**

0.72

(B)

As stated above, molecular mechanics calculations were performed using the MM+ force

field in order to gain theoretical support for two experimental results:

250 260 270 280 290 300

λ **(nm)**

(7)

(1)

complex.


θ **(mdeg)**

(A)

complex.

0.0

0.2

0.4

**A**

0.6

0.8 (A)

The estimation of the stoichiometry of a CD inclusion complex represents the first step in the characterization of the complex, leading to the determination of reliable association constants and offering a hint of the supramolecular structure. Unfortunately, the unequivocal estimation of the stoichiometry is not always straightforward and in some cases a single experiment is not sufficient.

In the four discussed examples we have described some cases we have encountered, in order of increasing complexity. In the first case we have had no problems in determining the stoichiometry, the 1:1 inclusion complex formed by the phenoxathiin derivative being clearly defined, with all the other models leading to absurd results. For the two following guests, DEAC and indapamide, the uncertainties arose in the determination of the stoichiometry were solved in two ways, starting with the careful examination of the results obtained by fitting the data to different models.

The Determination of the Stoichiometry

summarized in the scheme below.

Experimental data must be analysed: • In the spectral region free from band overlap • On the band showing the largest change in intensity

**5. Acknowledgement** 

**6. References** 

of Cyclodextrin Inclusion Complexes by Spectral Methods: Possibilities and Limitations 69

As a general conclusion, to establish the real stoichiometry of G:H systems, the use of multiple methods is recommended together with considering several required conditions,

> **SPECTRAL METHODS** SPECIFIC REQUIREMENTS

**Absorption Fluorescence Circular dichroism** 

• Spectra must be recorded with a large number of accumulations • Identify the position of the dichroic signals, their

Correlation with the theoretical results: • The sign of the band indicates the orientation of the guest transition

• Comparison of the experimental and simulated spectra

sign and intensity

moment

• A very careful study of the photophysical properties of the free guest is a must (emission wavelength, quantum yield, presence of several species, influence of solvent, temperature, *etc*.) • Perform experiments using several excitation wavelengths if the presence of several species is assumed

This work was supported by the grant CEEX-VIASAN-7/2008 entitled "Polymorphic forms and the encapsulation of bioactive substances into cyclodextrins for improving drug quality (CALIMED)" and by the strategic grant POSDRU/89/1.5/S/58852, Project "Postdoctoral programme for training scientific researchers" cofinanced by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007–2013.

• Examine carefully the statistical parameters of the fits • Compare when possible with theoretical simulations

GENERAL REQUIREMENTS

• Use several fitting models

Abi-Mosleh, L., Infante, R.E., Radhakrishnan, A., Goldstein, J.L. & Brown, M.S. (2009).

Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells*. Proceedings of the National Academy of Sciences*, Vol. 106, No. 46, (November 2009), pp. 19316-19321, ISSN 1091-6490

For DEAC, the slightly better statistical parameters, R2 and Fisher statistic coefficient, for the 1:1+1:2 stoichiometry, as compared to 1:1, was a first indication of the real stoichiometry. The presence of a mixture of complexes became clear by examining the residuals of the two corresponding fits. Thus, we have rejected the hypothesis of the 1:2 stoichiometry, for which the residuals were grouped in positive and negative values for different regions of the plot, and accepted the simultaneous presence of a mixture of two complexes, 1:1+1:2, for which the residuals were lower and randomly distributed, attesting the inherent experimental errors and not an inadequate model.

When the values of the statistical parameters are not sufficient to determine the adequate model, a theoretical simulation of the property *vs.* concentration curve for the assumed stoichiometry is necessary. In the case of the indapamide–CD systems, we succeeded to establish the presence of a 1:2 mixture of complexes by coupling an improvement of the experimental conditions, *i.e.* extending the concentration of the host up to the limit of solubility, with theoretical curves built assuming different stoichiometries.

In the last case, the atenolol–CD inclusion complexes represented more difficult systems and some uncertainties still remained. The problems are due to the high complexity of the fluorescence spectrum of the free drug, very sensitive to the experimental conditions such as the solvent, excitation wavelength, temperature, *etc*. We have observed that the emission band that is enhanced in intensity in the presence of CDs, and therefore assigned to the inclusion complex formation, is also present for some experimental conditions and to different extents in the spectrum of the free guest. Therefore, it is difficult to state that the fluorescence intensity measured in the presence of CDs is uniquely due to the inclusion complex and not to another species pre-existent in the system. The presence of several conformational equilibriums and/or of inter-/intra-molecular interactions in the free atenolol solution can be influenced by the CD addition. The second problem encountered was the very slight variation in the main emission band intensity, which prevented its use for quantitative estimations. With all these reservations, we considered that the stoichiometry of the inclusion complex, as revealed by the fluorescence measurements, is 1:2. In the circular dichroism spectra, working in other concentration domain and measuring a ground state property, we evidenced the presence of a 1:1 complex but the association constants are different as compared to those obtained by fluorescence. Getting different values for the association constants, by the use of several experimental methods, is a widely discussed topic in literature (Valeur et al., 2007), various explanations being given, the different range of concentrations used being the most frequent (Radi & Eissa, 2011). Another explanation invoked especially for the cases in which association parameters measured using fluorescence, on one side, and absorption and circular dichroism spectroscopies, on the other, are compared consists in the different features of the involved guest state, the excited state for the first method and the ground state for the other two. Last but not least, the isothermal calorimetric titration is a method also suitable for completing the information on the inclusion process, the method allowing for the determination, in a single experiment, of the stoichiometry, association constant and enthalpy change during the process. The experimental data can be fitted to several models, including one or more independent classes of binding sites or sequential binding [Xing et al., 2009].

As a general conclusion, to establish the real stoichiometry of G:H systems, the use of multiple methods is recommended together with considering several required conditions, summarized in the scheme below.

### **SPECTRAL METHODS**

SPECIFIC REQUIREMENTS

68 Stoichiometry and Research – The Importance of Quantity in Biomedicine

For DEAC, the slightly better statistical parameters, R2 and Fisher statistic coefficient, for the 1:1+1:2 stoichiometry, as compared to 1:1, was a first indication of the real stoichiometry. The presence of a mixture of complexes became clear by examining the residuals of the two corresponding fits. Thus, we have rejected the hypothesis of the 1:2 stoichiometry, for which the residuals were grouped in positive and negative values for different regions of the plot, and accepted the simultaneous presence of a mixture of two complexes, 1:1+1:2, for which the residuals were lower and randomly distributed, attesting the inherent experimental

When the values of the statistical parameters are not sufficient to determine the adequate model, a theoretical simulation of the property *vs.* concentration curve for the assumed stoichiometry is necessary. In the case of the indapamide–CD systems, we succeeded to establish the presence of a 1:2 mixture of complexes by coupling an improvement of the experimental conditions, *i.e.* extending the concentration of the host up to the limit of

In the last case, the atenolol–CD inclusion complexes represented more difficult systems and some uncertainties still remained. The problems are due to the high complexity of the fluorescence spectrum of the free drug, very sensitive to the experimental conditions such as the solvent, excitation wavelength, temperature, *etc*. We have observed that the emission band that is enhanced in intensity in the presence of CDs, and therefore assigned to the inclusion complex formation, is also present for some experimental conditions and to different extents in the spectrum of the free guest. Therefore, it is difficult to state that the fluorescence intensity measured in the presence of CDs is uniquely due to the inclusion complex and not to another species pre-existent in the system. The presence of several conformational equilibriums and/or of inter-/intra-molecular interactions in the free atenolol solution can be influenced by the CD addition. The second problem encountered was the very slight variation in the main emission band intensity, which prevented its use for quantitative estimations. With all these reservations, we considered that the stoichiometry of the inclusion complex, as revealed by the fluorescence measurements, is 1:2. In the circular dichroism spectra, working in other concentration domain and measuring a ground state property, we evidenced the presence of a 1:1 complex but the association constants are different as compared to those obtained by fluorescence. Getting different values for the association constants, by the use of several experimental methods, is a widely discussed topic in literature (Valeur et al., 2007), various explanations being given, the different range of concentrations used being the most frequent (Radi & Eissa, 2011). Another explanation invoked especially for the cases in which association parameters measured using fluorescence, on one side, and absorption and circular dichroism spectroscopies, on the other, are compared consists in the different features of the involved guest state, the excited state for the first method and the ground state for the other two. Last but not least, the isothermal calorimetric titration is a method also suitable for completing the information on the inclusion process, the method allowing for the determination, in a single experiment, of the stoichiometry, association constant and enthalpy change during the process. The experimental data can be fitted to several models, including one or more independent classes of binding sites or

solubility, with theoretical curves built assuming different stoichiometries.

errors and not an inadequate model.

sequential binding [Xing et al., 2009].

Experimental data must be analysed: • In the spectral region free from band overlap • On the band showing the largest change in intensity

• A very careful study of the photophysical properties of the free guest is a must (emission wavelength, quantum yield, presence of several species, influence of solvent, temperature, *etc*.)

• Perform experiments using several excitation wavelengths if the presence of several species is assumed

### **Absorption Fluorescence Circular dichroism**

• Spectra must be recorded with a large number of accumulations • Identify the position of the dichroic signals, their sign and intensity

Correlation with the theoretical results:

• The sign of the band indicates the orientation of the guest transition moment

• Comparison of the experimental and simulated spectra

GENERAL REQUIREMENTS


### **5. Acknowledgement**

This work was supported by the grant CEEX-VIASAN-7/2008 entitled "Polymorphic forms and the encapsulation of bioactive substances into cyclodextrins for improving drug quality (CALIMED)" and by the strategic grant POSDRU/89/1.5/S/58852, Project "Postdoctoral programme for training scientific researchers" cofinanced by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007–2013.

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**Part 2** 

**Stoichiometry of Metal Complexes** 

