Immunostimulant Activity of the Coordination Compounds of Isoleucine - Tryptophan Dipeptides with Zinc Ions

Abstract

Secondary immunodeficiency states related to T-cell immunity disorders are treated by immunomodulants such as tactivin (T-activin) and thymogen [1]. Tactivin is a mixture of peptides isolated from thymus [2], while thymogen is based on a synthetic glutamyl – tryptophan dipeptide [3]. Hyperimmunity and immunodeficiency states are usually treated with a composition comprising a dipeptide mixture with the general formula X-Trp, where X is glutamic acid, glutamine, leucine, and isoleucine [4]. It is known that complexation with metal (zinc, iron, platinum) ions leads to an increase in the specific activity of medicinal preparations [5]. In this context, we have studied the effect of complex formation with zinc ions on the immunostimulant activity of an isoleucine – tryptophan dipeptide. The dipeptide was obtained by condensation of carbobenzoxy-L-isoleucine with L-tryptophan using dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a nucleophilic additive. The carboxy group of tryptophan was protected by forming a salt with triethylamine. Upon purification, the protected peptide was unblocked by catalytic hydrogenation in the presence of palladium (10% Pd on activated charcoal). Preliminary purification of the free dipeptide was performed by extraction with n-butanol, while the final purification was effected by HPLC. The free dipeptide yield after lyophilization amounted to 98%. Before the synthesis of coordination compounds, it was necessary to determine the pH range in which the target dipeptide occurs in the zwitterion form. For this purpose, we performed the pH titration of isoleucine, tryptophan, and the isoleucine – tryptophan dipeptide and constructed the corresponding distribution diagrams using a method described in [6]. According to these data (Fig. 1), isoleucine, tryptophan, and the isoleucine – tryptophan dipeptide occur in the zwitterion form in the pH range 4.0 – 9.0, 3.0 – 10.0, and 6.0 – 8.0, respectively. The experimental dissociation constants are as follows: isoleucine, K 1 = 7.41 10 – 4 (pK 1 = 3.13) and K 2 = 2.45 10 – 10 (pK 2 = 9.61); tryptophan, K 1 = 1.12 10 – 2 (pK 1 = 1.95) and K 2 = 3.63 10 – 11 (pK 2 = 10.44); isoleucine – tryptophan dipeptide, K 1 = 7.4 10 – 6 (pK 1 = 5.13) and K 2 = 1.45 10 – 11 (pK 2 = 10.84). According to the available published data [7], the dissociation constants of amino acids are pK 1 = 2.36 and pK 2 = 9.68 for isoleucine and pK 1 = 2.38 and pK 2 = 9.39 for tryptophan. The difference between our values and the published data can be explained by the fact that our pH titration procedure was performed in aqueous amino acid solutions without maintaining constant ionic strength. Solutions of the zinc-containing coordination compounds were obtained by interaction of dilute aqueous solutions of dipeptide and zinc acetate at at a reagent concentration of 3.133 10 – 4 mole/liter, pH 6.0, and a temperature of 110 – 120°C. The reaction was conduced in the dark without access to air in order to eliminate oxidation of the indole group of tryptophan. The formation of a target coordination compound was confirmed by pH titration measurement, the results of which are presented in Fig. 2. The presence of an equivalence point in the titration curve confirms the coordination of dipeptide to zinc ion. We also attempted to determine the composition of the coordination compound using the method of isomolar series [8]. As can be seen from the results of these experiments (Figs. 3 and 4), an increase in the optical density of the isomolar solution with decreasing dipeptide concentration takes place at a wavelength of 310 – 345 nm. The calculation conducted for various wavelengths showed that the coordination compounds are characterized by a zinc to dipeptide ratio of 1 : 2 and 1 : 6, which corresponds to the possible complex ion compositions of [ZnL 2 (H 2 O) 4 ] and [ZnL 6 ], respectively (here, L denotes the isoleucyl – tryptophan fragment). The immunostimulant activity of dipeptide and the coordination compounds was evaluated in vivo by determining the titer of specific antibodies in the blood serum of calves Pharmaceutical Chemistry Journal Vol. 35, No. 7, 2001