Natural purine derivatives e.g. 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are used in medicine as anticancer drugs. To obtain the new thiopurines of potential biological activity, a series of 2,6-disubstituted 7-methylpurines which possess one or two S, S-Me, S-Et groups was synthesized.

The pK values (site N(1), N(3) or N(9) of dissociation) of some of the compounds [1, 2] suggest that they are dissociated significantly at physiological and higher pH values. They exist in solution, as a mixture of the neutral and anionic forms. At pH 7.4 6-MP and 6-TG are ionized in 33.4 and 13.7 %, whereas at pH 9.3 they are ionized 97.5 and 92.6 %, respectively.

The pH-dependence of the binding of purine series (ligand, L) to bovine serum albumin (BSA) was examined by fluorescence. The ligand and BSA were dissolved in TRIS-HCl buffer (pH 6.1, 7.4 and 8.0) and borate buffer (pH 9.3). Fluorescence emission spectra taken with a Kontron SFM spectrofluorimeter at excitation wavelength 295 nm, were recorded from 300 to 450 nm. The increasing concentrations of ligand (6 µM – 400 μM) were added to BSA (10 μM) and fluorescence of tryptophan residues (Trp-134 and Trp-212) was measured. The relationship between a quantity of unbound ligand and pH was analyzed. Quenching of BSA fluorescence by L was analyzed with the Stern-Volmer equation (K_Q). Binding data (K_a, n) were evaluated basing on the Scatchard model for a single class of binding site. In the case of two or more classes of non-interacting binding sites (when the Scatchard plot was curved) calculations were initiated by the use of reciprocal Klotz plots [3] and refined by nonlinear regression based on the Levenberg-Marquardt algorithm using the OriginPro 7.5 program [4].

References

[1] The Merck Index, Publisher by Merck Research Laboratories Division of Merck CO., Inc, 1996.

[2] Sochacka J, Kowalska A (2006) J. Planar Chromatogr 15: 320.

[3] Kragh-Hansen U, Hellec F, Foresta B, Maire M, Moller J (2001) Biophys J 80: 2898.

[4] Paal K, Mueller J, Hegedus L (2001) Eur. J. Biochem 268: 2187.

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