| Abstract |
The pyrimidinones mhcpe, 2-methyl-3H-5-hydroxy-6-carboxy-4-pyrimidinone ethyl ester (mhcpe, 1), 2,3-dimethyl-5-benzyloxy-6-carboxy-4-pyrimidinone ethyl ester (dbcpe, 2) and N-methyl-2,3-dimethyl-5-hydroxy-6-carboxyamido-4-pyrimidinone (N-MeHOPY, 3), are synthesized and their structures determined by single crystal X-ray diffraction. The acid-base properties of 1 are studied by potentiometric and spectrophotometric methods, the pK(a) values being 1.14 and 6.35. DFT calculations were carried out to determine the most stable structure for each of the H2L+, HL and L- forms (HL = mhcpe) and assign the groups involved in the protonation-deprotonation processes. The mhcpe(-) ligand forms stable complexes with (VO2+)-O-IV in the pH range 2 to 10, and potentiometry, EPR and UV-Vis techniques are used to identify and characterize the (VO)-O-IV-mhcpe species formed. The results are consistent with the formation of (VO)-O-IV, ((VO)-O-IV)L, ((VO)-O-IV)L-2, ((VO)-O-IV)(2)L2H-2, ((VO)-O-IV)L2H-1, ((VO)-O-IV)(2)L2H-3, ((VO)-O-IV)LH-2 species and (VO)-O-IV-hydrolysis products. Calculations indicate that the global binding ability of mhcpe towards (VO2+)-O-IV is similar to that of maltol (Hmaltol = 3-hydroxy-2-methyl-4H-pyran-4-one) and lower than that of 1,2-dimethyl-3-hydroxy-4-pyridinone (Hdhp). The interaction of (VO)-O-IV-complexes with human plasma proteins (transferrin and albumin) is studied by circular dichroism (CD), EPR and V-51 NMR spectroscopy. (VO)-O-IV-mhcpe-protein ternary complexes are formed in both cases. The binding of (VO2+)-O-IV to transferrin (hTF) in the presence of mhcpe involves mainly ((VO)-O-IV)(1)(hTF)(mhcpe)(1), ((VO)-O-IV)(2)(hTF)(mhcpe)(1) and ((VO)-O-IV)(2)(hTF)(mhcpe)(2) species, bound at the Fe-III binding sites, and the corresponding conditional formation constants are determined. Under the conditions expected to prevail in human blood serum, CD data indicate that the (VO)-O-IV-mhcpe complexes mainly bind to hTF; the formation of (VO)-O-IV-hTF-mhcpe complexes occurs in the presence of Fe-III as well, distinct EPR signals being clearly obtained for Fe-III-hTF and to (VO)-O-IV-hTF-mhcpe species. Thus this study indicates that transferrin plays the major role in the transport of (VO)-O-IV-mhcpe complexes under blood plasma conditions in the form of ternary V-IV-ligand-protein complexes. |
