the ReSeRVeD-PhASe hPLC StuDy of the ComPLexAtion of 5 , 17-BiS-( n-toLyLimino-methyL )-25 , 27-DiPRoPoxyCALix [ 4 ] ARene with ARomAtiC CARBoxyLiC ACiDS

The Host-Guest complexation of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with a number of aromatic carboxylic acids has been studied by reversed-phase high-performance liquid chromatography. The mobile phase was acetonitrile-water (80/20, v/v) with addition of 0.1% formic acid. The column was LiChrosorb RP 18, the UV detector operated at λ = 254 nm and at 26°C. The main chromatographic characteristics (retention time tR and capacity factor k’) of the aromatic carboxylic acids have been determined. The lipophilicity values of log P of carboxylic acids, as well as the binding constants KA (387-941 М-1) and Gibbs free energies ∆G (-14.74 – -16.94 kJ/mol) of the calixarene complexes with aromatic carboxylic acids have been calculated. The molecular modelling (Hyper Chem, version 8.0) of the calixarene complexes has revealed the presence of hydrogen bonds between carboxylic groups of the acids and nitrogen atoms of imino groups at the upper rim or oxygen atoms of hydroxyl groups at the lower rim of the calixarene macrocycle. The influence of log P lipophilicity of acids on KA values of the calixarene complexes has been assessed. The linear dependence of the binding constants on the acid lipophilicity indicates a significant role of solvophobic interactions on the complexation process. The relationship between supramolecular (KA) and physicochemical (molecular weight, log P, pKa) characteristics of acids has been found. The binding constants KA of the complexes increase with increase of their molecular weight and log P values.

The Host-Guest complexation of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix [4]arene with a number of aromatic carboxylic acids has been studied by reversed-phase high-performance liquid chromatography. The mobile phase was acetonitrile-water (80/20, v/v) with addition of 0.1% formic acid. The column was LiChrosorb RP 18, the UV detector operated at λ = 254 nm and at 26°C. The main chromatographic characteristics (retention time t R and capacity factor k') of the aromatic carboxylic acids have been determined. The lipophilicity values of log P of carboxylic acids, as well as the binding constants K A (387-941 М -1 ) and Gibbs free energies ∆G (-14.74 --16.94 kJ/mol) of the calixarene complexes with aromatic carboxylic acids have been calculated. The molecular modelling (Hyper Chem, version 8.0) of the calixarene complexes has revealed the presence of hydrogen bonds between carboxylic groups of the acids and nitrogen atoms of imino groups at the upper rim or oxygen atoms of hydroxyl groups at the lower rim of the calixarene macrocycle. The influence of log P lipophilicity of acids on K A values of the calixarene complexes has been assessed. The linear dependence of the binding constants on the acid lipophilicity indicates a significant role of solvophobic interactions on the complexation process. The relationship between supramolecular (K A ) and physicochemical (molecular weight, log P, pKa) characteristics of acids has been found. The binding constants K A of the complexes increase with increase of their molecular weight and log P values.  An important problem in chemistry and biology is molecular recognition, separation, membrane transport and analytical sensing of biorelevant molecules by artificial receptors [1][2][3][4][5][6][7]. Calixarenes -"macrocyclic vases", which are easily available through the cyclocondensation of para-substituted phenols with formaldehyde, -are widely used as molecular platforms for constructing specific receptors capable of highly selective recognition between fairly similar substrates [8][9][10]. Apparently, the outstanding receptor properties of functionalized calixarenes toward the biorelevant molecules make them highly promising materials for sensor technologies [11], as well as Host molecules for drug delivery systems in pharmaceutical science [5,6,8,[12][13][14][15][16][17].
The information on the supramolecular Host-Guest interaction of calix [4]arenes with the aromatic carboxylic acids will be useful in the design of artificial receptors for such biorelevant compounds.

Еxperimental Part
The RP HPLC study was performed on a Hitachi chromatograph (Hitachi, Ltd., Tokyo, Japan) consisting of a high-pressure pump connected to a Rheodyne sample 7120 injector with a 20 μL loop (Rheodyne, Scheme ISSN 2308-8303 Berkeley, USA) and an ultraviolet-visible detector. The column (250×4.6 mm i.d.) was packed with Li-Chrosorb RP 18 (Merck, Darmstadt, Germany). Acetonitrile was bought from the Acros Organics. Carboxylic acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). DPСА was synthesized by the method [25]. The acetonitrile-water (80/20, v/v) mixture was used as a blank mobile phase. The calixarene based mobile phases were prepared by dissolving DPСА in acetonitrile-water (80/20, v/v), 0.1% formic acid mixture to obtain the DPСА concentration of 0.05-0.6 mМ. The analytes for injections were dissolved in the mixture of acetonitrile-water (80/20, v/v) (C = 0.01 mM). The amount of the sample injected was 20 μL. All chromatograms were recorded at 26°C. The UV detector operated at 254 nm. The dead time t 0 was measured with NaNO 2 .

Determination of lipophilicity of log P of acids 1-6
Lipophilicity of log P of acids 1-6 (Tаble) was calculated by the HPLC method from equation log P = К x (log k').
The coefficient К being the relationship of log P value of benzoic acid 1 (1.87) [26] to its log k' was determined by RP HPLC in this work.
Моlecular modelling Molecular modelling of DPСА complexes with acids 1-6 were carried out using a Hyper Chem, version 8.0 programme [27]. The structures were optimized by the semi-empirical PM3 method.

Results and Discussion
Cаlіxаrеne DPСА and carboxylic acids 1-6 in the given conditions of analysis were registered on the chromatograms as sharp peaks. The chromatographic characteristics of carboxylic acids 1-6 (retention time t R , capacity factor k'), their binding constants К А and free Gibbs energies ΔG of their complexes with DPСА are presented in Tаble.
Binding constants of the inclusion Host-Guest complexes of DPСА with aromatic carboxylic acids 1-6 were determined by the RP HPLC method described in [29] and based on determination of retention factor k' of the Guest -carboxylic acids prior to and after the Host addition to the mobile phase. The DPСА addition to the mobile phase decreases retention factor k' of carboxylic acids 1-6. The linear character plots of 1/k'νs the DPСА concentration (Fig. 1) indicate formation of the Host-Guest supramolecular complexes with 1:1 stoichiometry.
In accordance with the data obtained (Table) the highest K A was observed for cinnamic acid (941 M -1 ), and the lowest K A was observed for the most bulky diphenylacetic acid (387 M -1 ). The binding constants K A strongly depended on the molecular weight (Fig. 2) and lipophilicity of log P (Fig. 3) of the acids.
There is the linear dependence of the binding constants K A on lipophilicity of log P of cinnamic, p-coumaric, gallic, caffeic, benzoic and diphenylacetic acid (Fig. 3). The increase of log P values of the acids leads to increase of K A values of their complexes with DPCA.
To clarify the nature of the supramolecular Host-Guest interactions the molecular modelling of DPAA complexes with cinnamic acid and diphenylacetic acid was carried out (Fig. 4).
Carboxylic acids deeply penetrate in the calixarene cavity ( Fig. 4)    The proportional dependence of the binding constants of the complexes on the long hydrogen bonds H … O is observed (Fig. 5). Additionally, the complexes can be stabilized by the van der Waals stacking and interactions between the Host and Guest molecules (Fig. 4). In the case of diphenylacetic and benzoic acids the other hydrogen bonding is observed. Carboxylic groups form intermolecular bonds with the basic nitrogen atoms of imino groups (Fig. 4C). Phenyl groups of these acids are included into the molecular cavity as a result of π-πstacking interactions. Plots of K A values vs H … O distances of the intermolecular hydrogen bonds between the Guest carboxylic groups and the Host hydroxyl groups at the lower rim of the macrocycle is presented in Fig. 5.

Conclusions
DPCA containing two imino groups at the upper rim of the macrocycle forms the Host-Guest inclusion complexes with biorelevant aromatic carboxylic acids. Their binding constants (387-941 М -1 ) in acetonitrile-water solution depend on the molecular weight and log P of the acids. The complexes are stabilized by intermolecular hydrogen bonds between the Guest carboxylic groups and the Host hydroxyl groups, van der Waals stacking and solvophobic interactions. Calixarene is a promising compound in the design of sensor devices or drug delivery systems for such biorelevant compounds.