Roberto Pagliarin

Inhibition of the catecholase activity of biomimetic dinuclear copper complexes by kojic acid

Abstract The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)-γ-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts.

Ternary Gd(III)L-HSA adducts: Evidence for the replacement of inner-sphere water molecules by coordinating groups of the protein. Implications for the design of contrast agents for MRI

Abstract Two novel gadolinium(III) chelates based on the structure of the heptadentate macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) ligand have been synthesized and their relaxometric and luminescent properties thoroughly investigated. They contain two water molecules in the inner coordination sphere in fast exchange with the bulk solvent and bear either a p-bromobenzyl or a p-phosphonatomethylbenzanilido substituent for promoting further interaction with macromolecular substrates. Upon interaction with human serum albumin the expected relaxation enhancement is not observed owing to displacement of the two inner-sphere water molecules of the complexes by a donor atom (likely from a carboxylate group) on the protein and possibly the phosphate anion of the buffered solution, respectively. We modeled the observed behavior by measuring the decrease of the relaxation rate of the water protons upon addition of malonate anion to aqueous solutions of the complexes. Conversely, no change in the hydratation state of the Gd(III) center for both complexes has been observed when the substrate for the formation of the macromolecular adduct is represented by poly-β-cyclodextrin.

NMR relaxometric studies of Gd(III) complexes with heptadentate macrocyclic ligands

The water 1H and 17O NMR relaxation properties of solutions containing Gd(III) chelates of the heptadentate DO3A, PCTA[12] and PCTP[12] ligands were thoroughly investigated and the results obtained are compared with those previously reported for other Gd(III) complexes with octadentate ligands {H3DO3A=1,4,7,10‐tetraazacyclododecane 1,4,7‐triacetic acid; H3PCTA[12]=3,6,9,15‐tetraazabicyclo[9.3.1]pentadeca‐1(15),11,13‐triene‐3,6,9‐triacetic acid; H6PCTP[12]=3,6,9,15‐tetraazabicyclo[9.3.1]pentadeca‐1(15),11,13‐triene‐3,6,9‐tris(methanephosphonic) acid}. The observed behaviour is consistent with a hydration number q=2 in the case of GdDO3A and GdPCTA[12] and q=1 in the case of PCTP[12]. The high relaxivity of the latter complex is accounted for in terms of the occurrence of an additional contribution arising from water molecules tightly bound to the phosphonate moieties on the surface of the paramagnetic chelate. Furthermore, it was found that the decreased relaxation rates observed at basic pH in the case of GdDO3A and GdPCTA[12] can probably be ascribed to a partial decrease in their hydration. The measurement of 17O NMR transverse relaxation rates, in the temperature range 273–342 K, allowed the assessment of the water exchange rate between the coordination site and the bulk solvent. A particularly short exchange lifetime was measured for the octacoordinate GdPCTP[12], which suggests the occurrence of an associative exchange mechanism. Further insights into the understanding of the structural properties of the three complexes were gained by measuring the magnetic field dependence (NMRD profiles) of the proton relaxivity on a Koenig–Brown field cycling relaxometer.

Non-covalent conjugates between cationic polyamino acids and GdIII chelates: a route for seeking accumulation of MRI-contrast agents at tumor targeting sites.

Three novel Gd chelates containing on their external surface pendant phosphonate and carboxylate groups, which promote the interaction with the positively charged groups of polyornithine and polyarginine, have been synthesized. Their solution structures have been assessed on the basis of 1H- and 31P-NMR spectra of the Eu and Yb analogues. A thorough investigation of the relaxometric (1H and 17O) properties of the Gd chelates has been carried out and the observed relaxivities have been accounted for the sum of three contributions arising from water molecules in the first, second, and outer coordination layers, respectively. It has been found that the occurrence of a tight second coordination coating renders the dissociation of the water molecule directly coordinated to the Gd ion more difficult. The binding interactions between the negatively charged Gd chelates and the positively charged groups of polyornithine (ca. 140 residues) and polyarginine (ca. 204 residues) have been evaluated by means of the proton relaxation enhancement (PRE) method. Although the binding interaction decreases markedly in the presence of competitive anions in the solution medium, the affinity is strong enough that in blood serum it is possible to meet the conditions where most of the chelate is bound to the polyamino acid substrate. On this basis one may envisage a novel route for a MRI location of tumors as it is known that positively charged polyamino acids selectively bind to tumors having a greater negative charge than non-tumor cells.

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

Abstract A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM–1 s–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature 17O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA.

A straightforward entry into enantiomerically enriched β-amino-α-hydroxyphosphonic acid derivatives

Abstract The asymmetric aminohydroxylation (AA) reaction of β-substituted vinylphosphonates under Sharpless protocol followed by hydrolysis afforded β-amino-α-hydroxyphosphonic acids in moderate to good ee.

New Insights for Pursuing High Relaxivity MRI Agents from Modelling the Binding Interaction of GdIII Chelates to HSA

It was recognized early on that the relaxivity of a Gd complex at 0.5–1 T can be strongly enhanced if its molecular correlation time is lengthened by linking it to a slowly moving macromolecule. 2] In this context, a huge amount of attention has been devoted in the past decade to the study of systems able to form noncovalent adducts with serum albumin, which also has the advantage of yielding systems that remain confined in the blood vessels. Theory foresees the attainment of relaxivities >100 mm 1 s 1 for macromolecular monoaquo Gd chelates characterized by a molecular reorientation time of 10–30 ns. 6] However, in spite of a number of investigated systems, relaxivities of such magnitude for Gd chelates bound to HSA have never been found. One major limiting factor has been recognized to be the occurrence of an insufficiently fast exchange rate of the coordinated water (tM). [7, 8]

Synthetic models for biological trinuclear copper clusters. Trinuclear and binuclear complexes derived from an octadentate tetraamine-tetrabenzimidazole ligand

The new tetraamino-tetrabenzimidazole ligand N,N ′{bis[3-(1-methyl-2-benzimidazolyl]amino}piperazine (L) has been synthesised together with a series of trinuclear and binuclear complexes. Two terminal binding sites with tridentate linkages (A sites) and one central binding site with the bidentate piperazine residue (B site) are used by the ligand to bind divalent metal centres in the trinuclear complexes [Cu H 3 L] 6+ , [Cu H 2 Zn H L] 6+ , and [Cu H 2 Co H L] 6+ . In the binuclear complex [Cu H 2 L] 4+ each nitrogen donor of the piperazine rine acts as an axial ligand for the two coppers bound to A sites, but these piperazine donors can be easily displaced by protonation to form the [Cu H 2 LH 2 )] 6+ species. The structure of this protonated complex has been determined by X-ray analysis. The crystals of composition [Cu H 2 (LH 2 )(CH 3 CN) 4 ][ClO 4 ] 6 ·2H 2 O·3CH 3 CN belong to the monoclinic system. space group P 2 1 /n with cell parametrs a = 10.661 (3), b = 23.014(3), c = 17.217(4)A, β = 96.58(2)°, Z = 2. The citation is arranged on a crystallographic symmetry centre, which is located at the middle of the protonated piperazine ring. The protonation at the piperazine N atoms is supported by the total charge of the cation and by the analysis of the difference Fourier map. The copper ions are five-coordinated, with ligation of the two benzimidazole residues and the tertiary N donor in the basal plane of a distorted square pyramid. Two CH 3 CN molecules, one at the basal, the other at the apical position, complete the coordination polyhedron. The centrosymmetric spacer, between the tertiary N atoms, deviates from the higher C 2h symmetry, so that the two approximately planar N (CH 2 ) 3 groupings lie in two parallel planes. Complexes containing reduced copper ions, [Cu H 2 L 2+ and [Cu H 2 Cu II L] 4+ , have also been obtained, but these ions do not bind to the piperazine B site which can only be used to coordinate divalent metal ions. The complexes containing Cu(II) centres exhibit EPR signals indicative of mononuclear species with tetragonal symmetry. The different coordination environment of [Cu H 2 L] 4+ with respect to [Cu H 2 Zn H L] 6+ , [Cu H 2 Co H L] 6+ or [Cu H 2 (LH 2 ] 6+ is reflected by a difference in the magnetic parameters of the complexes. The EPR spectrum of [Cu H 3 L] 6+ is very similar to those of [Cu H 2 Zn H L] 6+ , typical for Cu(II)-A sites, but the integrated intensity accounts for only about 2.2 paramagnetic centres per molecule. It is likely that a dipolar interaction between one of the Cu(II)-A centres and the Cu(II)-B centre produces severe broadening of the corersponding signals and apparent reduction in the overall EPR intensity. Voltammetric data in acetonitrile solution exhibit quasi-reversible electron transfer for the Cu(II)/Cu(II) couples with estimated reduction potentials in the range 0.39–0.56 V versus NHE. The voltammogram of [Cu H 3 L] 6+ run at low concentration and sweep rate shows that the three-electron transfer in split into one-electron and two-electron steps. Binding experiments show that the complexes bind azide molecules in the terminal mode to the copper (II) centres with affinity constants decreasing in the series: [Cu H 3 L] 6+ > [Cu H 2 L] 4+ . The complexes derived from L are catalytically active in the air oxidation of di-tert-butylcatechol (DTBC). The oxidations are biphasic, with a fast initial stoichiometric phae corresponding to reduction of a pair of copper(II) centres and oxidation of DTBC to quinone followed by the catalytic reaction. The catalytic reaction follows substrate saturation behaviour, with kinetic constants decreasing in the order: [Cu H 3 L 6+ > [Cu H 2 Co H L] 6+ > [Cu H 2 Zn H L] 6+ ≈ [Cu H 2 L] 4+ . Anaerobic experiments show that the two-electron oxidation of DTBC involves reduction of one Cu(II)-A site and the Cu(II)-B site for [Cu H 3 L] 6+ , both the Cu(II)-A sites of Cu H 2 Zn H L] 6+ and [Cu H 2 K] 4+ , but one Cu(II)-A and Co(III) in the case of the mixed [Cu H 2 Co 11 L] 6+ complex, since the Co(II) ion is rapidly oxidised to Co(III) in the conditions in which the catalytic reaction is carried out.

Contrast Agents for Magnetic Resonance Imaging: A Novel Route to Enhanced Relaxivities Based on the Interaction of a GdIII Chelate with Poly-β-cyclodextrins

The formation of a macromolecular adduct between a suitably functionalized GdIII complex and an exogeneous substance such as a poly-β-cyclodextrin (see picture) forms the basis of the novel route to contrast agents for magnetic resonance imaging endowed with high relaxivity discussed herein.

Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: activation of adenosine monophosphate-activated protein kinase and induction of apoptosis

A series of novel 1,4-diaryl-2-azetidinones were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was observed with the best compounds (±)-trans-20, (±)-trans-21, and enantiomers (+)-trans-20 and (+)-trans-21, which exhibited IC(50) values of 3-13 nM against duodenal adenocarcinoma cells. They induced inhibition of tubulin polymerization and subsequent G2/M arrest. This effect was accompanied by activation of AMP-activated protein kinase (AMPK), activation of caspase-3, and induction of apoptosis. Additionally, the most potent compounds displayed antiproliferative activity against different colon cancer cell lines, opening the route to a new class of potential therapeutic agents against colon cancer.