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The gallium(III) complex of a new tripodal 3-hydroxy-4-pyridinone (3,4-HP) chelator has been studied in terms of its physico-chemical and in vivo properties aimed at potential application as probe for nuclear imaging. In particular, based on spectrophotometric titrations, the hexa-coordinated (1:1) gallium complex appeared as the major species in a wide physiological acid-neutral pH range and its high stability (pGa = 27.5) should avoid drug-induced toxicity resulting from Ga(III) accumulation in tissues due to processes of transmetallation with endogenenous ligands or demetallation. A multinuclear ( 1 H and 71 Ga) NMR study gave some insights into the structure and dynamics of the gallium(III) chelate in solution, which are consistent with the tris-(3,4-HP) coordination and an eventual pseudo-octahedral geometry. Biodistribution and scintigraphic studies of the 67 Ga(III) labelled chelate, performed in Wistar rats, confirmed the in vivo stability of the radiolabelled complex, its non interaction with blood proteins and its quick renal clearance. These results indicate good perspectives for potential application of extrafunctionalized analogues in radiodiagnostic techniques.

Radiogalium chelates are important for diagnostic imaging in nuclear medicine (PET and ƴ-scintigraphy). Micelles are adequate colloidal vehicles for the delivery of therapeutic and diagnostic agents to organs and tissues. In this paper we describe the synthesis and in vitro and in vivo studies of a series of micelles-forming Ga(III) chelates targeted for the liver. The amphiphilic ligands are based on NOTA (NOTA = 1,4,7-triazacyclonoane-N,N’N’’-triacetic acid) and bear a α-alkyl chain in one of the pendant acetate arms (the size of the chain changes from four to fourteen carbon atoms). A multinuclear NMR study (1H, 13C, 27Al and 71Ga) gave some insights into the structure and dynamics of the metal chelates in solution, consistent with their rigidity and octahedral or pseudo-octahedral geometry. The critical micellar concentration of the chelates was determined using a fluorescence method and 27Al NMR spectroscopy (Al(III) was used as a surrogate of Ga(III)), both showing similar results and suggesting that the chelates of NOTAC6 form pre-micellar aggregates. The logP (octanol-water) determination showed enhancement of the lipophilic character of the Ga(III) chelates with the increase of the number of carbons in the α-alkyl chain. Biodistribution and Ƴ-scintigraphic studies of the 67Ga(III) labeled chelates were performed on Wistar rats, showing higher liver uptake for [67Ga](NOTAC8) in comparison to [67Ga](NOTAC6), consistent with a longer α-alkyl chain and a higher lipophilicity. After 24 hours both chelates were completely cleared off from the tissues and organs with no deposition in the bones and liver/spleen. [67Ga](NOTAC8) showed high kinetic stability in blood serum., Fundação para a Ciência e a Tecnologia (FCT)

The behaviour of three vanadium(V) systems, namely the pyridinone (V(V)-dmpp), the salicylaldehyde (V(V)-salDPA) and the pyrimidinone (V(V)-MHCPE) complexes, is studied in aqueous solutions, under aerobic and physiological conditions using (51)V NMR, EPR and UV-Visible (UV-Vis) spectroscopies. The speciations for the V(V)-dmpp and V(V)-salDPA have been previously reported. In this work, the system V(V)-MHCPE is studied by pH-potentiometry and (51)V NMR. The results indicate that, at pH ca. 7, the main species present are (V(V)O(2))L(2) and (V(V)O(2))LH(-1) (L=MHCPE(-)) and hydrolysis products, similar to those observed in aqueous solutions of V(V)-dmpp. The latter species is protonated as the pH decreases, originating (V(V)O(2))L and (V(V)O(2))LH. All the V(V)-species studied are stable in aqueous media with different compositions and at physiological pH, including the cell culture medium. The compounds were screened for their potential cytotoxic activity in two different cell lines. The toxic effects were found to be incubation time and concentration dependent and specific for each compound and type of cells. The HeLa tumor cells seem to be more sensitive to drug effects than the 3T3-L1 fibroblasts. According to the IC(50) values and the results on reversibility to drug effects, the V(V)-species resulting from the V(V)-MHCPE system show higher toxicity in the tumor cells than in non-tumor cells, which may indicate potential antitumor activity.

The uptake of the oxidation products of two oxovanadium(IV) compounds, [N,N'-ethylenebis(pyridoxylaminato)]oxovanadium(IV), VIVO(Rpyr2en), and bis-[3-hydroxy-1,2-dimethyl-4-pyridinonato]oxovanadium(IV), VIVO(dmpp)2, by human erythrocytes was studied using 51V and 1H NMR and EPR spectroscopy. VIVO(Rpyr2en) in aerobic aqueous solution is oxidized to its VV counterpart and the neutral form slowly enters the cells by passive diffusion. In aerobic conditions, VIVO(dmpp)2 originates VV complexes of 1:1 and 1:2 stoichiometry. The neutral 1:1 species is taken up by erythrocytes through passive diffusion in a temperature-dependent process; its depletion from the extracellular medium promotes the dissociation of the negatively charged 1:2 species, and the protonation of the negatively charged 1:1 species. The identity of these complexes is not maintained inside the cells, and the intracellular EPR spectra suggest N2O2 or NO3 intracellular coordinating environments. The oxidative stress induced by the oxovanadium compounds in erythrocytes was not significant at 1 mM concentration, but was increased by both vanadate and oxidized VIVO(dmpp)2 at 5 mM. Only 1 mM oxidized VIVO(dmpp)2 significantly stimulated erythrocytes glucose intake (0.75 ± 0.13 against 0.37 ± 0.17 mM/h found for the control, pÂ

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the hydroxylation of l-Phe to l-Tyr. The non-heme iron in the enzyme (Fe(III) as isolated) is 6-coordinated to a 2-His-1-carboxylate motif and three water molecules (wat1, wat2 and wat3). Tyr325 is at the second coordination sphere, hydrogen-bonded to water (wat1). We prepared and expressed mutants with Leu, Ala, Ser and Phe at this position. Only Y325L and the conservative mutation Y325F resulted in stable enzymes, but the mutant Y325F has been found to be post-translationally hydroxylated and to revert back to wild-type PAH [S.D. Kinzie, M. Thevis, K. Ngo, J. Whitelegge, J.A. Loo, M.M. Abu-Omar, J. Am. Chem. Soc. 125 (2003) 4710-4711], being inadequate to investigate the early inferred functional role of Tyr325. On the other hand, compared to wild-type PAH, Y325L shows reduced specific activity, decreased coupling efficiency and decreased iron content. The mutant also reveals a very high affinity for l-Phe and BH4 and does not manifest positive cooperativity for the substrate. All together, our results support that the mutation Y325L causes the removal or increased delocalization of the iron-ligated wat1 and, in turn, a less tight binding of the metal. Tyr325 thus appears to have an important role ensuring stoichiometric binding of iron, correct geometry of the complexes with substrate and cofactor and, consequently, a right coupling efficiency of the PAH reaction. In addition, the residue appears to be important for the correct cooperative regulation by l-Phe. http://www.sciencedirect.com/science/article/B6TGG-4G24X8N-2/1/c2de94cf547830652188dae1c1f6ed94

Li+ is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li+ competes with Mg2+ for the Mg2+ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7Li T1 relaxation measurements and fluorescence spectroscopy with the Mg2+ fluorophore furaptra, we detected Li+/Mg2+ competition in three preparations: the purified G-protein transducin (Gt), stripped rod outer segment membranes (SROS), and SROS with purified Gt reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li+ in the presence of fixed amounts of Mg2+, the apparent Li+ binding constant decreased due to Li+/Mg2+ competition. Whereas for SROS the competition mechanism was monophasic, for Gt, the competition was biphasic, suggesting that in Gt, Li+/Mg2+ competition occurred with different affinities for Mg2+ in two types of Mg2+ binding sites. Moreover, as [Li+] increased, the fluorescence excitation spectra of both ROS-T and Gt were blue shifted, indicating an increase in free [Mg2+] compatible with Li+ displacement of Mg2+ from two low affinity Mg2+ binding sites of Gt. Gt release from ROS-T membrane was also inhibited by Li+ addition. In summary, we found evidence of Li+/Mg2+ competition in Gt-containing preparations. http://www.sciencedirect.com/science/article/B6TGG-4BJ2538-2/1/c5cc8bd5992b6629aba06705e0861119

Two DTPA derivatives, a mono-amide derivative containing an iodinated synthon, DTPA-IOPsp (L1) and the ligand DTPA(BOM)3 (BOM=benzyloxymethyl) (L2), radiolabelled with 153Sm3+ and 111In3+, were studied as potential hepatospecific gamma scintigraphic agents. In vivo studies with Wistar rats show that the main excretory pathway for all the chelates studied is the hepatobiliary system. The complexes of L2 show even greater hepatobiliary specificity than L1, perhaps as a consequence of longer blood circulation times due to their strong affinity towards HSA. The 153Sm3+ chelates are also more hepatospecific than the corresponding 111In3+ chelates. The La3+ and In3+ chelates of L1 and L2 show some structural and dynamic differences in aqueous solution, as studied by 1H NMR spectroscopy. While only two nona-coordinated isomers were observed for the La3+ complexes with both ligands, its number is much larger in the In3+ complexes, with both octa- and hepta-coordinated species (with unbound side arms), as well as structural isomers for each coordination number. http://www.sciencedirect.com/science/article/B6TGG-45HFJNC-1/1/26df0708dbb11a2e6f7218c1115fd80b

The solution chemistry and structure of the complex of the triazamacrocyclic ligand NOTP (1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate)) with Ga3+ in D2O have been investigated by 1H, 71Ga and 31P NMR spectroscopy. These NMR results show the presence of a 1:1 Ga(NOTP)3- complex, with a highly symmetrical, pseudo-octahedral geometry, possibly with a C3 axis. The 1H spectrum shows that the triazamacrocyclic chelate ring is very rigid, with all the ring protons non-equivalent. The complex is stable in aqueous solution in a wide pH range. Its high thermodynamic stability agrees well with previous results from biodistribution and [gamma] imaging studies in Wistar rats with 67Ga3+ chelates of triaza macrocyclic ligands, which showed that the neutral chelates 67Ga(NOTA) (where NOTA is 1,4,7-triazacyclononane-1,4,7-triacetate) and 67Ga(NOTPME) (where NOTPME is 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate monoethylester)) have similar in vivo behaviour, with high stability and rapid renal excretion, but the high negatively charged 67Ga(NOTP)3- has a considerably slower kidney uptake and elimination. http://www.sciencedirect.com/science/article/B6TGG-40X8D8M-26/1/b0839261f5ddd424e05a2da9204295a2

75As NMR (Nuclear Magnetic Resonance) was used as a probe of arsenate interactions in solution. The linewidth at half-height of the 75As NMR signal of arsenate was studied as a function of solution pH and temperature. Below pH 11.5, the 75As signal was too broad to be detected, but at higher pH, up to pH = 13.5, the signal became much narrower. This indicates that the arsenate species AsO4(3-) is quite symmetric, but the asymmetry of HAsO4(2-) is sufficient to cause extensive quadrupolar relaxation of the 75As nucleus. A full pH range 75As and proton NMR study of the interaction of arsenate with ethanol, ethylene glycol, glycerol, ribose, mannose, glucose, gluconic acid and acetate was undertaken in order to follow arsenate ester formation. The 75As line broadening effects and the proton ligand shifts observed indicate that complexation of arsenate by ribose, mannose, glucose, ethanol, ethylene glycol, and glycerol occurs at pH 12.7. However, no significant interaction is detected by NMR with gluconic acid or acetate. The effect of the nucleoside adenosine is quite small and those of phosphate and of the nucleotides AMP and ADP are negligible. The interaction of arsenate with potential cationic centers, such as the basic amino acids lysine and arginine and some macrocyclic triamines, was also studied. Such interaction depends on the pKa for protonation of the amine groups.

The interaction of vanadate with 5'-mononucleotides, ADP, ATP, and various molecules containing some of their chemical moieties was studied in aqueous solution in the pH region of 5-9 using proton, 13C, 31P, and 51V nuclear magnetic resonance (NMR) spectroscopy. All the compounds studied formed noncyclic vanadate esters through interaction of monovanadate or divanadate with the hydroxyl groups of the ribose ring. Noncyclic anhydrides were also formed with the phosphate groups of ribose 5-phosphate, the mononucleotides, ADP, ATP, phosphate, pyrophosphate, and tripolyphosphate. In particular, ADP and ATP analogs resulted from AMP (AMPV and AMPV2) and from ADP (ADPV). Cyclic esters of trigonal bipyramidal geometry resulted from the interaction of vanadate with two ribose ring cis hydroxyl groups. AMP, CMP, and UMP formed two such complexes of 1:1 and 1:2 stoichiometries, similar to what has been observed for uridine and other nucleosides. However, 2'-deoxy-AMP does not yield this type of complexes. ADP and ATP also form similar cyclic ester complexes with vanadate, which does not chelate their pyrophosphate and tripolyphosphate moieties. Nevertheless, the separate pyrophosphate (PP) and tripolyphosphate (PPP) ligands form cyclic anhydrides of octahedral geometry with vanadate. However, their binding to vanadate is weaker than that of the ribose ring of nucleotides. Competition experiments between ethylene glycol and phosphate (P), pyrophosphate (PP), or tripolyphosphate (PPP) show that the relative strength of the interaction of these ligands with vanadate is PP greater than ethylene glycol greater than PPP greater than P.

Mo(VI) and W(VI) oxoions form strong complexes with 5' -mononucleotides in acidic aqueous solutions. Complexation occurs exclusively through the nucleotide phosphate ester groups, yielding complexes of stoichiometry [(ROPO3)2Mo5O15]4− and [(ROPO3)2W5O15]4−.

Proton, 13C and 51V nuclear magnetic resonance spectroscopy has been used to study the interaction of vanadate with several molecules containing more than one hydroxyl group, including various aldoses and nucleosides. The aldoses D-mannose and D-ribose mainly form tridentate complexes, of trigonal bipyramidal geometry, with vanadate at pH 7. These sugars use three consecutive hydroxyl groups, cis to each other, of their pyranose forms to bind vanadate in those cyclic triesters. Other aldoses, like D-glucose, which do not have this unique structural characteristic, do not form tridentate complexes, but can form weaker bidentate cyclic diesters using two consecutive pyranose cis hydroxyl groups. Of course, the pyranose forms of D-mannose and D-ribose, as well as the furanose form of D-ribose, also yield cyclic diesters of vanadate. All these aldoses form weak monodentate noncyclic monoesters of tetrahedral geometry using a single hydroxyl group. The nucleosides uridine, cytidine and adenosine form two complexes of trigonal bipyramidal geometry with vanadate. In these complexes, having 1:1 and 2:1 ligand-to-metal stoichiometries, the nucleosides form cyclic diesters with vanadate using their C2, and C3, hydroxyl groups.

ADP and ATP form in acidic aqueous solutions strong complexes with Mo(VI) oxocations in different stoichiometries. Complexation occurs predominantly, if not exclusively, through the phosphate groups of the nucleotides.