Biomedical subjects

Tiziana Beringhelli

Publications and source records attributed to Tiziana Beringhelli.

11 recordsLinked to original sources

PubMed2006

Computational and experimental approaches for assessing the interactions between the model calycin beta-lactoglobulin and two antibacterial fluoroquinolones.

Norfloxacin and levofloxacin, two fluoroquinolones of different bulk, rigidity and hydrophobicity taken as model ligands, were docked to one apo and two holo crystallographic structures of bovine beta-lactoglobulin (BLG) using different computational approaches. BLG is a member of the lipocalin superfamily. Lipocalins show a typical b-barrel structure encompassing an internal cavity where small hydrophobic molecules are usually bound. Our studies allowed the identification of two putative binding sites in addition to the calyx. The rigid docking approximation resulted in strong repulsive forces when the ligands were docked into the calyx of the apo form. On the contrary, hindrance was not experienced in flexible docking protocols whether on the apo or on the holo BLG forms, due to allowance for side chain rearrangement. K(i) between 10(-7) and 10(-6) M were estimated for norfloxacin at pH 7.4, smaller than 10(-5) M for levofloxacin. Spectroscopic and electrophoretic techniques experimentally validated the occurrence of an interaction between norfloxacin and BLG. Changes in chemical shift and dynamic parameters were observed between the (19)F NMR spectra of the complex and of the ligand. A K(i) (ca 10(-7) M) comparable with the docking results was estimated through a NMR relaxation titration. Stabilization against unfolding was demonstrated by denaturant gradient gel electrophoresis on the complex versus apo BLG. NMR experimental evidence points to a very loose interaction for ofloxacin, the racemic mixture containing levofloxacin. Furthermore, we were able to calculate in silico K(i)'s comparable to the published experimental values for the complexes of palmitic and retinoic acid with BLG.

Animals↗

PubMed2006

Synthesis and reactivity of N-heterocycle-B(C6F5)3 complexes. 4. Competition between pyridine- and pyrrole-type substrates toward B(C6F5)3: structure and dynamics of 7-B(C6F5)3-7-azaindole and [7-Azaindolium]+[HOB(C6F5)3]-.

Reaction between 7-azaindole and B(C6F5)3 quantitatively yields 7-(C6F5)3B-7-azaindole (4), in which B(C6F5)3 coordinates to the pyridine nitrogen of 7-azaindole, leaving the pyrrole ring unreacted even in the presence of a second equivalent of B(C6F5)3. Reaction of 7-azaindole with H2O-B(C6F5)3 initially produces [7-azaindolium]+[HOB(C6F5)3]- (5) which slowly converts to 4 releasing a H2O molecule. Pyridine removes the borane from the known complexes (C6F5)3B-pyrrole (1) and (C6F5)3B-indole (2), with formation of free pyrrole or indole, giving the more stable adduct (C6F5)3B-pyridine (3). The competition between pyridine and 7-azaindole for the coordination with B(C6F5)3 again yields 3. The molecular structures of compounds 4 and 5 have been determined both in the solid state and in solution and compared to the structures of other (C6F5)3B-N-heterocycle complexes. Two dynamic processes have been found in compound 4. Their activation parameters (DeltaH = 66 (3) kJ/mol, DeltaS = -18 (10) J/mol K and DeltaH = 76 (5) kJ/mol, DeltaS = -5 (18) J/mol K) are comparable with those of other (C6F5)3B-based adducts. The nature of the intramolecular interactions that result in such energetic barriers is discussed.

Journal Article↗

PubMed2006

NMR investigation of the dihydrogen-bonding and proton-transfer equilibria between the hydrido carbonyl anion [HRe2(CO)9]- and fluorinated alcohols.

The interaction of fluorinated alcohols with the anionic hydrido complex [HRe2(CO)9]- (1) has been investigated by NMR spectroscopy. According to the acidic strength of the alcohols, the interaction may result not only in the formation of dihydrogen-bonded ROH...[HRe2(CO)9]- adducts 2, but also in proton transfer to give the neutral species [H2Re2(CO)9] (3). With the weaker acid trifluoroethanol (TFE) evidence for the occurrence of the dihydrogen-bonding equilibrium was obtained by 2D 1H NOESY. The dependence of the hydride chemical shift on TFE concentration at different temperatures provided values for the constants of this equilibrium, from which the thermodynamic parameters were evaluated as deltaH(degrees) = -2.6(2) kcal mol(-1), deltaS(degrees) = -9.3(2) cal mol(-1) K(-1). This corresponds to a rather low basicity factor (E(j) = 0.64). Variable-temperature T1 measurements allowed the proton-hydride distance in adduct 2 a to be estimated (1.80 angstroms). In the presence of hexafluoroisopropyl alcohol (HFIP) simultaneous occurrence of both dihydrogen-bonding and proton-transfer equilibria was observed, and the equilibria shifted versus the protonated product 3 with increasing HFIP concentration and decreasing temperature. Reversible proton transfer between the alcohol and the hydrido complex occurs on the NMR timescale, as revealed by a 2D 1H EXSY experiment at 240 K. For the more acidic perfluoro-tert-butyl alcohol (PFTB) the protonation equilibrium was further shifted to the right. Thermal instability of 3 prevented the acquisition of accurate thermodynamic data for these equilibria. The occurrence of the proton-transfer processes (in spite of the unfavorable pK(a) values) can be explained by the formation of homoconjugated RO...HOR- pairs which stabilize the alcoholate anions.

Anions↗

PubMed2005

Oxygen-bridged borate anions from tris(pentafluorophenyl)borane: synthesis, NMR characterization, and reactivity.

The previously known anion [(C6F5)3B(mu-OH)B(C6F5)3]- (2) has been prepared by a two-step procedure, involving deprotonation of (C6F5)3BOH2 to give [B(C6F5)3OH]- (1), followed by addition of B(C6F5)3. The solution structure and the dynamics of 2 have been investigated by 1H and 19F NMR spectroscopy. The reaction of [NHEt3]2 with NEt3 resulted in the formation of [NHEt3]+ [(C6F5)3BOH]-, [NHEt3]+ [(C6F5)3BH]-, and (C6F5)3B- (CH2CH=N+ Et2). This indicates that in the presence of a nucleophile anion 2 can dissociate to B(C6F5)3 and 1. The reaction of [HDMAN]2 with 1,8-bis(dimethylamino)naphthalene (DMAN) confirmed this trend. In the presence of water, 2 transformed into the adduct [(C6F5)3BO(H)H...O(H)B(C6F5)3]- (3), containing the borate 1 hydrogen-bonded to a water molecule coordinated to B(C6F5)3. The same compound is formed by treating (C6F5)3BOH2 with 0.5 equiv of a base. A competition study established that for 1 the Lewis acid-base interaction with B(C6F5)3 is about 5 times preferred over H-bonding to (C6F5)3BOH2. The X-ray single-crystal analysis of [2-methyl-3H-indolium]3 provided the first experimental observation of an asymmetric H-bond in the [H3O2]- moiety, the measured O-H and H...O bond distances being significantly different [1.14(2) vs 1.26(2) A]. The reaction of NEt3 with an equimolar mixture of B(C6F5)3 and bis(pentafluorophenyl)borinic acid, (C6F5)2BOH, afforded the novel borinatoborate salt [NHEt3]+ [(C6F5)3BOB(C6F5)2]- ([NHEt3]4). X-ray diffraction showed that the B-O bond distances are significantly shorter than in [(C6F5)3B(mu-OH)B(C6F5)3]-. Variable-temperature 19F NMR revealed high mobility of the five aryl rings, at variance with the more crowded anion 2. 2D NMR correlation experiments showed that in CD2Cl2 the two anions [(C6F5)3BOH]- and [(C6F5)3BH]- form tight ion pairs with [NHEt3]+, in which the NH proton establishes a conventional (BO...HN) or an unconventional (BH...HN), respectively, hydrogen bond with the anion. The diborate anions 2-4, on the contrary, gave loose ion pairs with the ammonium cation, due both to the delocalized anionic charge and to the more sterically encumbered position of the oxygen atoms that should act as H-bond acceptors.

Journal Article↗

PubMed2005

Solution conformation and dynamics of the ion pairs originating from the reaction of B(C6F5)3 with bisindenyl dimethyl zirconium complexes.

The two ion pairs [(4,7-Me(2)indenyl)(2)ZrMe](+)[MeB(C(6)F(5))(3)](-) (1 b) and [(indenyl)(2)ZrMe](+) [MeB(C(6)F(5))(3)](-) (2 b) have been generated in situ by reaction of stoichiometric B(C(6)F(5))(3) with the corresponding dimethyl zirconocenes. It has been shown that molecular mechanics computations, guided by experimental (1)H/(1)H NOE correlations, can provide information on the conformers present in solution. The dynamics of the ion pairs has also been investigated, showing the occurrence of both the processes previously characterized for this class of compounds, namely the B(C(6)F(5))(3) migration between the two methyl groups and dissociation-recombination of the whole [MeB(C(6)F(5))(3)](-) anion, the latter process being much faster than the first one (about three order of magnitude). Moreover, it has been shown that in certain conditions intermolecular processes can occur, which mimic the above-mentioned dissociative exchanges. In particular, the presence of species containing loosely bound [MeB(C(6)F(5))(3)](-) anion fastens the exchange of this anion, while the presence of free B(C(6)F(5))(3) accelerates its exchange between the two methyl sites.

Algorithms↗

PubMed2005

Hemoglobin and heme scavenging.

Release of hemoglobin into plasma is a physiological phenomenon associated with intravascular hemolysis. In plasma, stable haptoglobin-hemoglobin complexes are formed and these are subsequently delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Heme arising from the degradation of hemoglobin, myoglobin, and of enzymes with heme prosthetic groups could be delivered in plasma. Albumin, haptoglobin, hemopexin, and high and low density lipoproteins cooperate to trap the plasma heme, thereby ensuring its complete clearance. Then hemopexin releases the heme into hepatic parenchymal cells only after internalization of the hemopexin-heme complex by CD91 receptor-mediated endocytosis. Moreover, alpha1-microglobulin contributes to heme degradation by a still unknown mechanism, with the concomitant formation of heterogeneous yellow-brown kynurenine-derived chromophores which are very tightly bound to amino acid residues close to the rim of the lipocalin pocket. During hemoglobin synthesis, the erythroid alpha-chain hemoglobin-stabilizing protein specifically binds free alpha-hemoglobin subunits limiting the free protein toxicity. Although highly toxic because capable of catalyzing free radical formation, heme is also a major and readily available source of iron for pathogenic organisms. Gram-negative bacteria pick up the heme-bound iron through the secretion of a hemophore that takes up either free heme or heme bound to heme-proteins and transports it to a specific receptor, which, in turn, releases the heme and hence iron into the bacterium. Here, hemoglobin and heme trapping mechanisms are summarized.

Animals↗

PubMed2004

Reorganization in apo- and holo-beta-lactoglobulin upon protonation of Glu89: molecular dynamics and pKa calculations.

Molecular dynamics (MD) simulations starting from crystallographic data allowed us to directly account for the effects of the protonation state of Glu89 on the conformational stability of apo- and holo-beta-lactoglobulin (BLG). In apo-BLG simulations starting from the protonated crystal structure, we observe a long-lived H-bond interaction between the protonated Glu89 and Ser116. This interaction, sequestering the proton from the aqueous medium, explains a pK(half) value evaluated at pH 7.3 by continuum electrostatics/Monte Carlo computation on MD data, using linear response approximation. A very large root-mean-square deviation (RMSD; 5.11 A) is observed for the EF loop between protonated and unprotonated apo-BLG. This results from a quite different orientation of the EF loop that acts either as a closed or as an open lid above the protein calyx. Proton exchange by Glu89 in apo- but not in holo-BLG is associated with a reorganization energy of 4.7 kcal/mol. A 3-ns MD simulation starting from the crystal structure of protonated apo-BLG, but considering the Glu89 as unprotonated, shows the progressive opening of the lid giving rise to the Tanford transition. In both holo-BLG forms, the lid is most probably held in place by hydrophobic interactions of amino acid side-chains of the EF loop with the palmitate hydrocarbon tail.

Animals↗

PubMed2003

Synthesis and reactivity of (C6F5)3B-N-heterocycle complexes. 1. Generation of highly acidic sp3 carbons in pyrroles and indoles.

The reaction of pyrroles and indoles with B(C(6)F(5))(3) and BCl(3) produces 1:1 B-N complexes containing highly acidic sp(3) carbons, for example, N-[tris(pentafluorophenyl)borane]-5H-pyrrole (1) and N-[tris(pentafluorophenyl)borane]-3H-indole (2), that are formed by a new formal N-to-C hydrogen shift, the mechanism of which is discussed. With some derivatives, restricted rotation around the B-N bond and/or the B-C bonds was observed by NMR techniques, and some rotational barriers were calculated from experimental data. The acidity of the sp(3) carbons in these complexes is shown by their ability to protonate NEt(3), with formation of pyrrolyl- and indolyl-borate ammonium salts. The driving force for this reaction is given by the restoration of the aromaticity of the heterocycle.

Journal Article↗

PubMed2002

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B.

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).

Animals↗

PubMed2002

An intramolecular N-H...(mu-H)Re2 dihydrogen bond and a novel mu 3-eta 2 coordination mode of the pyrazolate anion on a triangular cluster face.

The quantitative addition of pyrazole (Hpz) to the 44 valence-electron, triangular cluster anion [Re3(mu 3-H)-(mu-H)3(CO)9]- gives the novel unsaturated anion [Re3(mu-H)4(CO)9(Hpz)]- (1, 46 valence electrons), which contains a pyrazole molecule that is terminally coordinated on a cluster vertex. Solidstate X-ray and IR analyses reveal a rather weak hydrogen-bonding interaction between the NH proton and one of the hydrides bridging the opposite triangular cluster edge (delta H degree = -3.1 kcal mol-1 from the Iogansen equation). Both IR and NMR data indicate that such a proton-hydride interaction is maintained in the major conformer present in CD2Cl2, but also provide evidence of the presence of minor conformers of 1 in which the NH proton is involved in an intermolecular hydrogen bond with the solvent. The mu-H...HN bond length evaluated in solution through the T1 minimum value (2.07 A) and that determined in the solid state by X-ray diffraction (2.05 A) are in good agreement. NMR experiments show that, in acetone, intermolecular N-H...solvent interactions replace the intramolecular dihydrogen bond. At room temperature in CH2Cl2, the pyrazole ligand in 1 is labile and 1 slowly "disproportionates" to [Re3(mu 3-H)-(mu-H)3(CO)9]- and [Re3(mu-H)3(CO)9-(mu-eta 2-pz)(Hpz)]-, with H2 evolution. Slow H2 evolution also leads to the formation of the anion [Re3(mu-H)3-(CO)9(pz)]- (5), in which the pyrazolate anion adopts a novel mu 3-eta 2-coordination mode, as revealed by a single-crystal X-ray analysis. The analysis of the bond lengths indicates that the pyrazolate anion in 5 acts as a six-electron donor, with loss of the aromaticity. The formation of 5 from 1 is much faster in solvents with a high dielectric constant, such as acetone or DMF. Anion 5 was also obtained from the reaction of pyrazole with [Re3(mu-H)3(CO)9(mu 3-CH3)]- through the intermediate formation of two isomeric addition derivatives and following CH4 evolution.

Journal Article↗

PubMed2002

NMR and DFT analysis of [Re(2)H(2)(CO)(9)]: evidence of an eta(2)-H(2) intermediate in a new type of fast mutual exchange between terminal and bridging hydrides.

Protonation of the anion [Re(2)H(CO)(9)](-) (1) with a strong acid at 193 K affords the neutral complex [Re(2)H(2)(CO)(9)] (2), that in THF above 253 K irreversibly loses H(2) to give [Re(2)(CO)(9)(THF)], previously obtained by room-temperature protonation of 1. Treatment of 2 with NEt(4)OH restores the starting anion 1. Variable temperature (1)H and (13)C NMR spectra as well as T(1) measurements agree with the formulation of 2 as a classical [Re(2)H(mu-H)(CO)(9)] complex, in which two dynamic processes takes place. The "windshield-wiper motion" observed in several related complexes equalizes the two carbonyls trans to the hydrides (E(a) = 44(1) kJ mol(-)(1)), while another much faster process equalizes bridging and terminal hydrides already at 172 K. The variable temperature behavior of the (1)H transverse relaxation times revealed also proton exchange between 2, water, and the parent anion 1 (due to the acidity of 2), but such a process is too slow to account for the fast hydrides exchange in 2. The nature of the latter process has been investigated both experimentally and theoretically. Kinetic data, obtained by the analysis of the variable temperature (1)H spectra (E(a) = 24.5(5) kJ mol(-1)), revealed a small normal kinetic isotope effect (ca. 1.5). The (2)H chemical shift of the fully deuterated isotopomer 2-d(2) was found isochronous with 2, thus ruling out the presence of a significant concentration of a nonclassical [Re(2)(eta(2)-H(2))(CO)(9)] tautomer, in fast exchange with the classical dihydride. Density functional theory (DFT) calculations, carried out at the B3LYP level, confirmed the formulation of [Re(2)H(2)(CO)(9)] as a classical complex. However, when DFT was used to obtain a detailed description of the dynamic behavior of 2 in solution, a new type of hydride fast exchange emerged, involving the nonclassical tautomer as a relatively high energy (12.7 kJ mol(-1)) intermediate. Isotopic perturbation of the equilibrium by partial deuteration of 2 indicated the preference of deuterium for the bridging sites, with Delta H degrees = -475(4) J mol(-1) and Delta S degrees = -0.80(2) J K(-1) mol(-1). The same preference was observed in the anion [Re(2)H(mu-H)Cl(CO)(8)](-).

Journal Article↗