COMPLEXATION OF CALIX[4]ARENE HYDROXYMETHYL- PHOSPHONIC ACID WITH TRYPTOPHAN AND N - ACETYL -TRYPTOPHAN AMIDE

The Host-Guest complexation of calixarene hydroxymethylphosphonic acid with tryptophan and N-acetyltryptophan amide has been investigated by the RP HPLC method in H 2 O/MeCN (99/1) solution (column support Hypersil CN, UV-detector , λ = 254 nm). Adsorption of calixarene hydroxymethylphosphonic acid on the Hypersil CN surface has been studied. It has been found that hydroxymethylphosphonic acid is characterized by reversible sorption on the Hypersil CN surface. The binding constants (K A = 23000 M -1 and 39000 M -1 for tryptophan and N-acetyltryptophan amide, respectively) of the supramolecular complexes have been calculated from the ratio between the capacity factors k’ of the Guest and the calixarene hydroxymethylphosphonic acid Host concentration in the mobile phase. The Gibbs free energies of the tryptophan and N-acetyltryptophan amide complexes are - 24.84 and - 26.15 kJ/mol, respectively. The molecular modelling of calixarene hydroxymethylphosphonic acid and its complexes with tryptophan and N-acetyltryptophan amide (Hyper Chem, version 8, force field PM3) has indicated that the complexes are stabilized by hydrogen bonds, electrostatic, π-π , and solvatophobic interactions. The geometric parameters of the energy minimized calixarene macrocycle and its complexes with tryptophan and N-acetyltryptophan amide have been calculated. According to the calculations it has been shown that the Host-Guest complexation does not change the flattened-cone conformation of calixarene. Finally, the inverse correlation has been found between the K A values of the complexes and the Log P v а lues of the guest molecules.


The Host-Guest complexation of calixarene hydroxymethylphosphonic acid with tryptophan and N-acetyltryptophan amide has been investigated by the RP HPLC method in H 2 O/MeCN (99/1) solution (column support Hypersil
CN, UV-detector, λ = 254 nm). Adsorption of calixarene hydroxymethylphosphonic acid on the Hypersil CN surface has been studied. It has been found that hydroxymethylphosphonic acid is characterized by reversible sorption on the Hypersil CN surface. The binding constants (K A = 23000 M -1 and 39000 M -1 for tryptophan and N-acetyltryptophan amide, respectively) of the supramolecular complexes have been calculated from the ratio between the capacity factors k' of the Guest and the calixarene hydroxymethylphosphonic acid Host concentration in the mobile phase. The Gibbs free energies of the tryptophan and N-acetyltryptophan amide complexes are -24.84 and -26. 15 kJ/mol, respectively. The molecular modelling of calixarene hydroxymethylphosphonic acid and its complexes with tryptophan and N-acetyltryptophan amide (Hyper Chem, version 8, force field PM3) has indicated that the complexes are stabilized by hydrogen bonds, electrostatic, π-π, and solvatophobic interactions. The geometric parameters of the energy minimized calixarene macrocycle and its complexes with tryptophan and N-acetyltryptophan amide have been calculated. According to the calculations it has been shown that the Host-Guest complexation does not change the flattened-cone conformation of calixarene. Finally, the inverse correlation has been found between the K A values of the complexes and the Log P vаlues of the guest molecules. L-Tryptophan is an essential amino acid that is low abundant in proteins (1.4% only). As a consequence, Trp residues frequently play a key role in studying the protein structure and functions. For instance, soluble Trp residues in proteins have been shown to be critical for the specific recognition of nucleic acid sequences [1,2,3,4]. Moreover, it is worth mentioning that Trp has the peculiar property to exhibit a significant intrinsic fluorescence that is environment sensitive, and therefore, can be used to investigate the properties and interactions of proteins with ligands [5].

КОМПЛЕКСОУТВОРЕННЯ КАЛІКС[4]АРЕНГІДРОКСИМЕТИЛ-ФОСФОНОВОЇ КИСЛОТИ З ТРИПТОФА-НОМ ТА N-AЦЕТИЛ-ТРИПТОФАНАМІДОМ
To characterize the role of the given Trp residue in the protein properties and functions, the common strategy is to site-selectively mutate this residue into another one. To disturb the protein structure minimally the aromatic Phe or Tyr residues are frequently selected as a substitute. Nevertheless, due to the key role of Trp residues in protein folding, these mutations can result in improperly folded proteins, and it does not allow characterizing the specific role of the Trp residues mutated.
To characterize the role of soluble Trp residues in proteins it would be interesting to use complexing agents that can selectively bind these Trp residues as an alternative strategy, and therefore, promote the interaction of the target proteins with their ligands.
In this respect, calix [4]arene hydroxymethylphosphonic acids [13,14,15] which have been shown to form selectively Host-Guest supramolecular complexes with amino acids [16,17], appear to be good candidates to bind soluble Trp residues. To test this possibility the Host-Guest complexation of calixarene hydroxymethylphosphonic acid (CPA) with Trp and N-аcеtyltryptophan amide (NATA) used as models of Trp residues in proteins has been investigated by RP HPLC and molecular modelling (Scheme).

Experimental Part
CPA was synthesized by the reaction of formylcalixarene with Na salt of ethylphosphite followed by dealkylation of the ester formed by the consecutive action of trimethylbromosilane and methanol in accordance with [13]. Because of its poor solubility in water CPA was analysed as a monosodium salt obtained by addition of one equivalent of sodium methylate to CPA solution in methanol. Trp and NATA were obtained from Sigma-Aldrich (St. Louis, MO, USA), acetonitrile was obtained from Acros Organics (Janssen Pharmaceuticalaan 3A 2440 Geel Belgium).

HPLC analysis
Chromatographic analysis was performed in isocratic conditions using a Hitachi liquid chromatogra-Scheme ISSN 2308-8303 phy system (Hitachi, Ltd, Tokyo, Japan) equipped with a high-pressure pump, a Rheodyne Model Sample 7120 injector (20 μl) and an UV-detector. The column (250×4.6 mm i.d.) was packed with Hypersil CN (Merck, Germany, Darmstadt). The samples of CPA for RP HPLC analysis were prepared by dissolution in the mobile phase (H 2 O/MeCN, 99/1 v/v). The choice of the solvent was dictated by solubility of CPA, Trp and NATA under the same conditions. The flow rate of the mobile phase was 0.8 ml/min. The final CPA concentrations were in the range of 0.10-0.70×10 -4 M. The ultraviolet detector was operated at 254 nm. The Trp and NATA samples for HPLC analysis were prepared in the same solvent (C = 0.05×10 -4 M). The amount of the sample injected was 20 μL. Each of the samples was analyzed five times. The mobile phase that contained the CPA as an additive was equilibrated for 3 h before analysis. Under these conditions the column was saturated with the CPA additive. All chromatograms were obtained at 32 o C.

Моlecular modelling
The initial molecular modelling of CPA and its complexes with Trp or NATA was carried out by the molecular mechanics ММ+ method (the force field PM3). The structures obtained were optimized by the semi-empirical method (the HyperChem software package, version 8) [http://www.hyper.com/ Download/AllDownloads/tabid/470/Default.aspx].

Results and Discussion
Cаlіxаrеne CPA, Trp and NATA in the given analysis conditions were registered on the chromatograms as sharp peaks (Fig. 1-3).   The binding constants of Host-Guest complexes of CPA with Trp or NATA were determined by the RP HPLC method as previously described [18,19]. The method is based on determination of the Guest retention time, t R , and the capacity factor, k', before and after CPA addition to the mobile phase. The binding constants K A of the CPA complexes with the Guest molecules were calculated by equation (1): whеre k 0 ' and k' -are the capacity factors of the Guest molecule determined in the absence and the presence of CPA in the mobile phase; [CA] is the concentration of CPA in the mobile phase. CPA (monosodium salt), Trp and NATA appear on the chromatograms as sharp symmetrical peaks (Fig. 1, 2) with the chromatographic characteristics given in Table 1.
The linear character of the adsorption isotherm of CPA (R 2 = 0.99) indicates its reversible sorption on the Hypersil CN surface. Addition of CPA to the mobile phase decreases the capacity factor values of Trp and NATA. The linear plots of their k' values νs the calixarene concentration (Tab. 2, Fig. 4) clearly show the formation of Host-Guest supramolecular complexes with 1:1 stoichiometry and allows calculating the К А values of the complexes by equation (1).
The binding constants K A and free Gibbs energies DG (DG = -RT lnK A ) for the CPA complexes are given in Tab It should be noted that the CPA-NATA complex is more stable than complexes of CPA with such aminoacids as Ala (21200 M -1 ), Phe (26600 M -1 ), Arg (27500 M -1 ), Asp (28800 M -1 ), His (31200 M -1 ), Lys (32500 M -1 ) [17]. Moreover, comparison of Trp and NATA indicates that changing the -OH group of Trp to the -NH 2 group and acylation of its alpha-amino group significantly increase the interaction with CPA.
To clarify the nature of the Host-Guest interaction, the molecular modelling study was carried out. The conformational search of the optimum geometry of CPA, Trp and NATA was performed by the method of molecular mechanics and the semi-empirical method.   Then the structures of the CPA complexes with the least energies were calculated (Fig. 5). Inclination (dihedral angles) of the calixarene benzene rings A, B, C, D in relation to the main macrocycle plane formed by CH 2 links for CPA and its complexes in the structures calculated is presented in Tab. 3.
The macrocyclic skeleton of CPA shows a flattened-cone conformation. The aromatic rings with phenolic OH groups are almost "coplanar" with the main macrocycle plane, but the propylated rings are "perpendicular" to the plane. The dihedral angle between the "coplanar" rings A and C is 110 о , while the angle between "perpendicular" rings B and D is 3 о . As seen from Table 3, the Host-Guest complexation does not almost change the flattened cone conformation of the calixarene skeleton.
For the structure of the Trp complex calculated an electrostatic contact of the negatively charged oxygen atom of the calixarene phosphonic group with the positively charged nitrogen atom of the amino acid is obvious (P-O ... H-N distance is 2.3 Ǻ). Additionally, the complex is stabilized by an intermolecular hydrogen bond between the indole NH group and the oxygen atom at the calixarene lower rim (NН ... O distance is 3.0 Ǻ).

ISSN 2308-8303
Similar to the Trp complex an intermolecular hydrogen bond (2.604 Ǻ) between the indole NH group and the oxygen atom at the calixarene lower rim is also observed. However, in contrast to the Trp complex, three intermolecular hydrogen bonds with the phosphonic group are formed in the NATA complex calculated: POH … O=CCH 3 (2.247 Ǻ), CH 3 C(O)NH … O=P (1.753 Ǻ) and C(O)NH … O=P (1.957 Ǻ). It is possible to predict that under experimental conditions hydrophobic and π-π interactions are also involved in stabilization of both NATA and Trp complexes. As is evidenced by the calculation data with the help of the molecular modelling, the structure of the CPA -NATA complex (the relative CPA -NATA complex energy DЕ = -14.9 kcal/mol) is more stable comparatively with the structure CPA -Trp complex (DЕ = -11.7 kcal/mol). These data are in a good agreement with the data obtained in the chromatographic calculations of the binding constants of the CPA -NATA complex (K A = = 39000 M -1 , DG = -6.25 kJ/mol) and the CPA -Trp complex (K A = 23000 M -1 and DG = -5.94 kJ/mol).

Conclusions
Summarizing all above-mentioned information it should be noted that experimental measurements of the complex stabilities show that CPA binds more effectively NATA than Trp or other aminoacids, such as Ala < Phe < Arg < Asp < His < Lys in the aqueous solution. It can be explained by formation of three intermolecular hydrogen bonds between the phosphonic group of the CPA-Host and the CNHC(O)CH 3 -C(O)NH 2 fragment of the NATA-Guest. The investigation of the molecular recognition and binding of calixarene phosphonic acids to Trp residues in proteins is in progress.