The study of calixarenes complexation with phenols by RP HPLC

The Host-Guest complexation of octakis(diphenoxyphosphoryloxy)tetramethylcalix[4]resorcinarene, 5,17-bis-(Ntolyliminomethyl)-25,27-dipropoxycalix[4]arene and 5,11,17,23-tetrakis(diisopropoxyphosphonyl)-25,26,27,28tetrapropoxycalix[4]arene with a series of 11 phenols (phenol, p-fluorophenol, p-chlorophenol, p-bromophenol, pyrogallol, p-cresol, p-aminophenol, p-nitrophenol, salicylic aldehyde, guaiacol and veratrole) has been studied by the high-performance liquid chromatography (RP HPLC) method. Chromatographic characteristics and log P of industrial phenols have been determined. Using the relationship of the phenol retention factor k’ vs the calixarene concentration in the mobile phase the stability constants of the supramolecular complexes KA (29-331 М-1) have been determined. The stability constants of the calixarene complexes show that the Host-Guest interaction strongly depends on the nature of the substituents in the Host and Guest molecules. Calixresorcinarene functionalized by diphenoxyphosphoryl groups and calixarene containing tolyliminomethyl groups formed more stable complexes with some phenols compared to calixarene functionalized by diisopropoxyphosphonyl groups. In accordance with the molecular modeling data the complexation does not change the C2v flattened-cone conformation of the calixarene skeleton. The Host-Guest complexes are stabilized by the intermolecular hydrogen bonds of phenolic OH groups with oxygen atoms of P = O groups at the upper rim, and OH groups at the lower rim of the macrocycle. Hydrophobic interactions also participate in the complexation.

The class of phenols is important as a component of many useful industrial materials, pharmaceuticals, herbicides, disinfectants, etc. Phenols are essential raw material and additives for laboratory processes, chemical industry, chemical engineering processes, wood and plastics processing [17].
Phenolic compounds are commonly synthesized industrially; however, they are also produced by plants and microorganisms [18].Organisms that synthesize phenolic compounds do so in response to ecological pressures, such as pathogen and insect attack, UV radiation and wounding [19].Phenols are found in the natural world, especially in the plant kingdom, such as fruits, vegetables, herbs, spices and unfiltered olive oil [20].
In animals and humans, after ingestion, natural phenols become part of the xenobiotic metabolism.These activated metabolites are conjugated with charged species, such as glutathione, sulfate, glycine or glucuronic acid.As they are present in food consumed in human diets and in plants used in traditional medicine of several cultures, their role in human health and disease is a subject of research.Some natural phenols can be used as biopesticides [21,22].
Phenols (aspirin and paracetamol, first of all) are widely used as inflammatory drugs [23].Crofelemer is applied for the treatment of diarrhea.Phenolic compound Combretastatin is active as an anticancer drug [24].
RP HPLC analysis RP HPLC analysis was performed using the liquid chromatographic system (Hitachi, Ltd., Tokyo, Japan).The column (250 × 4.6 mm i.d.) was packed with LiChrosorb RP-18 (Merck, Darmstadt, Germany).The UV detector was operated at 254 nm.The flow rate was 0.8 ml/min.Experiments were performed with isocratic conditions.All chromatograms were obtained at 26 °C.The mixture of acetonitrile-water (80 : 20, v/v) was used as a mobile phase.The mobile phases contained calixarene additives in the concentrations of 0.05-0.6mМ.The samples of analytes for injections were dissolved in the same mixture (C = 0.01 mM) of acetonitrile-water (80 : 20, v/v).The volume of the samples injected was 20 μL.For equilibration of the chromatographic column the mobile phase with the calixarene additive was fluxed for 3 h before analysis.Under these conditions the column was saturated with calixarene.
The stability constants K A of the calixarene complexes with phenols were determined by the RP HPLC method described in [28].The method is based on changing the phenols retention factor k' induced by the complexation with calixarenes in the mobile phase.

Determination of the log P values of phenols
The values of log P of phenols 1-11 (Таb. 1) were calculated from equation 1: where the coefficient 2.11 is the ratio of the experimental value of log P of phenol 1.46 [29] to log k' of the phenol value of 0.69 determined by the RP HPLC method in this work.The reason of the experimental determination of lipophilicity was a mismatch of the literature data of the log P values of the phenols studied.For example, log P of p-nitrophenol 9 is 1.96 according to [30] and 1.68 according to [31].

Mоlecular modeling
Molecular modeling of calixarenes and their Host-Guest complexes were carried out using the Hyper Chem 8.0 program in the force field (PM3) [32].The structures were calculated by the semi-empirical method.The RMS (the standard deviation of the word root mean square) gradient was equal to 0.01 kcal/A mol.

Table 1
The values of log P of phenols 1-11, the stability constants K A (M -1 ) (RSD = 9-15 %) and free Gibbs energies ΔG (kDj/mol) of their Host-Guest complexes with CA- calixarene to the mobile phase decreases the retention factors k' of phenols 1-11 due to formation of the Host-Guest inclusion complexes.The linear character of plots of 1/k' ns the calixarene concentration (Fig. 2-4) indicates 1 : 1 stoichiometry of the Host-Guest supramolecular complexes formed.It allows using equation ( 2) correctly for calculation of the stability constants К А : whеre k 0 ' і k' -are capacity factors of the Guest molecule determined in the absence and the presence of the calixarene Host in the mobile phase.The stability constants К А of the calixarene complexes are within 29-331 M -1 (Tab.1).This is close to the stability constant of the β-cyclodextrin -pyrogallol complex determined by the UV-visible (K A = 78 M -1 ) and fluorescence (K A = 227 M -1 ) spectroscopy in the water solution [33].Free Gibbs energies DG of the cali-xarene complexes calculated by equation ( 3) are from -8.33 to -14.30 kJ/mol (Tab.1).

DG = -RT . ln K A
(3) To clarify the nature of the Host-Guest interaction the molecular modeling of the calixarene Hosts СА-1, СА-2 СА-3 and their complexes with p-aminophenol and phenol was carried out (Fig. 5).
In accordance with the molecular modeling data the calixarene macrocyclic skeleton of СА-1, СА-2 and СА-3 and their Host-Guest complexes exist in the flattened-cone conformation with C2v symmetry.Two aromatic rings in the distal position are ''coplanar'' oriented, but two other ones are "perpendicular" oriented to the main plane of the macrocycle.The dihedral angles characterized inclination of benzene rings A, B, C, D relatively to the main macrocycle plane formed by CH 2 links are presented in Tab. 2. As shown from Tab. 2, the complexation with phenols does not practically change dihedral angles in the calixarene macrocyclic skeleton.
In all complexes formation of the intermolecular hydrogen bonds between OH groups of phenols and oxygen atoms of calixarenes are observed (H···O distances are within 2.91-3.19Ǻ).In complex (a) ОH and NH 2 groups of p-аminоphenol form the hydrogen bonds with oxygen atoms of distal Р = О groups of СА-1.In complex (b) phenol forms the hydrogen bond with the oxygen atom of the ОН group at the lower rim of calixarene СА-2.In complex (c) phenоl forms the hydrogen bond with the Р = О group at the upper rim of the macrocycle СА-3.
Short contacts between calixarenes and phenol molecules (Fig. 5) indicate that the Host-Guest complexes can be additionally stabilized by various nonvalence Van der Waals, π-π, and hydrophobic interactions.Fig. 6 presents the linear dependences of the

Conclusions
Calix [4]arenes form stable supramolecular Host-Guest complexes with phenols in acetonitrile-water solutions.The stability constants of the complexes determined by the RP HPLC method show that the complex stability is strongly dependent on the nature of the substituents in the Host and Guest molecules.In accordance with the molecular modeling the complexation does not change the C2v flattened-cone conformation of the calixarene skeleton.The Host-Guest complexes are stabilized by the intermolecular hydrogen bonds of phenolic OH groups with oxygen atoms of P = O groups at the upper rim of the macrocycle and OH groups at the lower rim of the macrocycle.Hydrophobic interactions also participate in the Host-Guest complexation processes.