Nicola Gaggelli

fac-{Ru(CO)3}2+ Selectively Targets the Histidine Residues of the β-Amyloid Peptide 1-28. Implications for New Alzheimer's Disease Treatments Based on Ruthenium Complexes

The reaction of the ruthenium(II) complex fac-[Ru(CO)(3)Cl(2)(N(1)-thz)] (I hereafter; thz = 1,3-thiazole) with human beta-amyloid peptide 1-28 (Abeta(28)) and the resulting {Ru(CO)(3)}(2+) peptide adduct was investigated by a variety of biophysical methods. (1)H NMR titrations highlighted a selective interaction of {Ru(CO)(3)}(2+) with Abeta(28) histidine residues; circular dichroism revealed the occurrence of a substantial conformational rearrangement of Abeta(28); electrospray ionization mass spectrometry (ESI-MS) suggested a prevalent 1:1 metal/peptide stoichiometry and disclosed the nature of peptide-bound metallic fragments. Notably, very similar ESI-MS results were obtained when I was reacted with Abeta(42). The implications of the above findings for a possible use of ruthenium compounds in Alzheimers disease are discussed.

Non-covalent inclusion of ferulic acid with α-cyclodextrin improves photo-stability and delivery: NMR and modeling studies

Ferulic acid (FA) is a highly effective antioxidant and photo-protective agent, already approved in Japan as a sunscreen, but it is poorly suited for cosmetic application because of its low physicochemical stability. We prepared the inclusion complex of FA with alpha-cyclodextrin by co-precipitation from an aqueous solution, and used (1)H NMR and molecular dynamics to investigate the most probable structure of the inclusion complex. In rotating frame nuclear Overhouser effect spectroscopy (ROESY) experiments FA penetrated the alpha-CD hydrophobic cavity with the alpha,beta-unsaturated part of the molecule and some of its aromatic skeleton. In proton chemical shift measurements of FA and alpha-cyclodextrins we determined the stoichiometry of the association complex (1:1) by Jobs method, and its stability constant (K(1:1) 1162+/-140 M(-1)) and described the molecular dynamics of the complex on the basis of theoretical studies. Encapsulation with alpha-cyclodextrin improves (i) the chemical stability of FA against UVB stress (10 MED [Minimal Erythemal Dose: 1 MED=25 mJ/cm(2) for skin phototype II: 30]), since no degradation products are formed after irradiation, and (ii) the bioavailability of FA on the skin, slowing its delivery (Strainer cell model).

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50μM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Capreomycin--a polypeptide antitubercular antibiotic with unusual binding properties toward copper(II).

Capreomycin is an important therapeutic agent having intriguing and diverse molecular features. Its polypeptidic structure rich in nitrogen donors makes the drug a promising chelating agent for a number of transition metal ions, especially for copper(II). The results of the model investigational studies suggest that capreomycin anchors Cu(2+) ion with an amino function of the α,β-diaminopropionic acid residue at pH around 5. At physiological pH copper(II) ion is coordinated by two deprotonated amide nitrogen atoms of the α,β-diaminopropionic acid, the serine residue as well as the amino function deriving from the β-lysine. Above that pH value we observe a rearrangement within the coordination sphere leading to movement of Cu(2+) to the center of the peptide ring with concurrent coordination of four nitrogen donors. Spin-lattice relaxation enhancements and potentiometric measurements clearly indicate that deprotonated amide nitrogen atom from the β-ureidodehydroalanine moiety is the fourth donor atom.

DETERMINATION OF INTRAMOLECULAR HYDROGEN BONDS IN AMIKACIN IN WATER SOLUTION BY NMR SPECTROSCOPY

Abstract An NMR investigation has been carried out on amikacin in water solution in the physiological pH range. Two-dimenstional heterocorrelated maps provide 1 H NMR chemical shifts from the unambiguous assignment of the 13 C NMR spectrum. Reorientational dynamics at the molecular level are interpreted in terms of a pseudoisotropic motion with a correlation time of 0.17 ns at 300 K. The pH and temperature dependences of 13 C NMR chemical shifts are interpreted to delineate protonation equilibria (all p K s are determined) and to assess the occurrence of two intermolecular hydrogen bonds, which are confirmed by molecular modelling.

How the α-hydroxymethylserine residue stabilizes oligopeptide complexes with nickel(II) and copper(II) ions

Potentiometric, spectroscopic and theoretical studies have shown that the α-hydroxymethylserine (HmS) residue is a very specific amino acid residue when inserted into a peptide sequence. The theoretical calculations as well as evaluated deprotonation microconstants indicated that in the HmS-HmS-His tripeptide the N-terminal ammonium group is more acidic than the imidazole nitrogen. The hydrogen bond formation between the N-terminal amino group and imidazole nitrogen stabilizes the cyclic conformation of the metal-free peptide. The unusual gain in the 4N complex stability in the copper(II) and nickel(II) complexes with HmS-HmS-His ligands seems to derive from the enhancement of the π-electron contribution to the metal–amide nitrogen bond.

Impact of Cu2 + ions on the structure of colistin and cell-free system nucleic acid degradation

Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV-vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health.

Metal Ion Effects on the cis/trans Isomerization Equilibrium of Proline in Short-Chain Peptides: A Solution NMR Study

The effect of copper(II) ions on the probabilities of existence of the four detectable conformers of the tetrapeptide Tyr‐Pro‐Phe‐Pro (β‐casomorphin 4) in [2H6]DMSO was investigated by 1H NMR spectroscopy. Integration of the Phe‐NH signals provided the relative populations in the free state as tt/tc/ct/cc=28:34:29:9 at 293 K (c=cis, t=trans). Copper(II) was shown to bind to all four isomers, yielding complexes with two different structures, depending on the conformation of Pro2. The interpretation of paramagnetic relaxation rates of Pro2‐Hα signals provided the corresponding isomeric probabilities in the metal‐bound state as 13:36:20:31. The observed stabilization of the conformation with the lowest probability of existence (cc) may be relevant for the biological role of copper and other metal ions.

Delineation of conformational and structural features of the amikacin–Cu(II) complex in water solution by 13C-NMR spectroscopy

The copper (II) complex of amikacin in water solution at pH 5.5 was investigated by 13C-NMR. The temperature dependence of spin-lattice relaxation rates was measured and fast exchange conditions were shown to apply. The motional correlation time of the complex was approximated by the pseudo-isotropic rotational correlation time of free amikacin in water solution (tau c = 0.17 ns at 300 K). Formation of a pseudo-tetrahedral 1:1 complex was demonstrated by relaxation rates analysis and also by UV-Vis spectrophotometry. Two amino nitrogens of amikacin, together with the amide nitrogen and the hydroxyl in the hydroxyl-aminopropyl carbonyl side chain, were assigned as the copper-binding sites and a model of the complex was built by using copper-carbon distances obtained by NMR analysis as input parameters.

Effect of CuII on the Complex between Kanamycin A and the Bacterial Ribosomal A Site

The solution structure of kanamycin A interacting with a ribosomal A‐site fragment was solved by transferred‐NOE techniques and found to agree with the structure of the complex observed in the crystal. Despite the fast exchange conditions found for the interaction, the bound form was identified by NOESY spectroscopy. At 600 MHz, NOE effects are only observed for the RNA‐associated antibiotic. Dissociation constants were measured by NMR spectroscopy for two sites of interaction (Kd1=150±40 μM; Kd2=360±50 μM). Furthermore, the effects of the CuII ion on the antibiotic, on the RNA fragment that mimics the bacterial ribosomal A site, and on the complex formed between these two entities were analyzed. The study led to the proposal of a model that localizes the copper ion within the kanamycin–RNA complex.