Mauro Fasano

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

Abstract The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (KA) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of KA. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM–1 s–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate.

Crystal structure and solution dynamics of the lutetium(III) chelate of DOTA

Abstract The X-ray structure of Na[Lu(DOTA)(H 2 O)]·4H 2 O consists of (−1) complex anions balanced by sodium cations. The ninecoordinate Lu(III) is linked to the four nitrogen and to four oxygen atoms of the macrocyclic ligand DOTA in a square antiprismatic arrangement with a coordinated water molecule in capping position. In aqueous solution the complex is present under two isomeric forms in the relative ratio of 10:1, as evaluated from the low temperature limiting 13C NMR spectrum. The variable temperature behavior is consistent with the presence of two dynamic processes. One exchanges enantiomeric pairs for both isomers and involves concerted conformational changes of the ethylenic groups of the macrocyclic ring and rotation of the acetate arms. The other corresponds to the interconversion between the two isomers. Complete line-shape analysis of the variable-temperature 13C NMR spectra of Lu-DOTA allowed a quantitative evaluation of the two processes. The enantiomerization is slower than the isomerization: the corresponding calculated free energies of activation are 65.9 ± 1.2, 62.7 ± 2.0 (exchange major ⇌ minor) and 58.7 ± 2.I kJ mol −1 (exchange minor ⇌ major), respectively.

EPR investigations of the iron domain in neuromelanin

The interactions between iron and neuromelanin (NM) have been studied by means of EPR spectroscopy. The variable temperature EPR spectral features of a specimen of NM extracted from normal human midbrains clearly indicate that iron is present as polynuclear oxy-hydroxy ferric aggregates as well as isolated Fe(III) centres. Ferric oxy-hydroxy phases are typical of the iron storage proteins ferritin and hemosiderin, but the comparison of the variable temperature EPR spectra of ferritin and NM highlights significant differences between the two iron(III)oxy-hydroxy domains. Moreover, further investigations on melanin models synthesised in the presence of either ferritin or a ferric salt as iron sources suggest that the same pathway of formation and inclusion of the polynuclear iron oxide is operating in NM and in the model systems, whatever is the source of iron.

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinsons disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.

Non-ionic Ln(III) chelates as MRI contrast agents: Synthesis, characterisation and 1H NMR relaxometric investigations of bis(benzylamide)diethylenetriaminepentaacetic acid Lu(III) and Gd(III) complexes

Abstract The syntheses of the ligand bis(benzylamide)diethylenetriaminepentaacetic acid (BBA-DTPA) and its Gd(III) and Lu(III) complexes are reported. The solid state structure of Lu(III) BBA-DTPA was determined in a single crystal X-ray diffraction study. The lutetium ion adopts a mine-coordinate geometry, which is best described as a distorted tricapped prism. Eight coordination sites are occupied by the ligand (three nitrogen atoms, two carboxamide oxygens and three carboxylate oxygens) and the ninth site is occupied by the oxygen atom of a water molecule. The VT 13 C NMR spectra of Lu(III) BBA-DTPA are consistent with the presence of three isomers in solution. Water proton relaxation rates have shown that Gd(III) BBA-DTPA displays an analogous behaviour to that observed for other Gd(III) complexes of related bisamide derivatives of DTPA. The solubility of Gd(III) BBA-DTPA has been markedly improved by the presence of an excess of hydroxypropyl-β-cyclodextrin. The thermodynamic stability of the inclusion complex has been evaluated by measuring the proton relaxation enhancement upon addition of β-cyclodextrin.

Isolation and 13C-NMR characterization of an insoluble proteinaceous fraction from substantia nigra of patients with Parkinson's disease

Neuromelanin is a dark brown pigment suspected of being involved in the pathogenesis of Parkinsons disease. This pigment can be isolated from normal human substantia nigra by a procedure that includes an extensive proteolytic treatment. In this study we used such a procedure to extract the neuromelanin pigment from a pool of substantia nigra from patients affected by Parkinsons disease. 13C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize the solid residue obtained from the extraction procedure. We found that the pigment extracted from the substantia nigra of parkinsonian patients was mainly composed of highly cross‐linked, protease‐resistant, lipo‐proteic material, whereas the neuromelanin macromolecule appears to be only a minor component of this extract. A synthetic model of melanoprotein has been prepared by enzymatic oxidation of dopamine in the presence of albumin. Once it has undergone the same proteolytic treatment, this model system yields a 13C‐NMR spectrum which is similar to that observed for the parkinsonian midbrain extract. These results are consistent with the view that oxidative stress has a relevant role in the pathogenesis of Parkinsons disease.

1H and 17O-NMR relaxometric investigations of paramagnetic contrast agents for MRI. Clues for higher relaxivities

Abstract The analysis of 1 H- and 17 O-NMR relaxometric data allows us to get a better insight into the understanding of the structural and dynamic factors responsible for the relaxivity of a given paramagnetic system. High relaxivities are obtained in the presence of a long molecular reorientational time and fast exchange of the water molecule(s) coordinated to the paramagnetic metal ion. Long molecular reorientational times are pursued either through the formation of covalent conjugates between a paramagnetic complex and a macromolecular substrate or through the formation of non-covalent adducts between suitably functionalized complexes and endogenous (e.g. serum albumin) or exogenous (e.g. poly-β-cyclodextrin) substrates. Within the class of covalent conjugates, it has been shown that the use of a DOTA-like chelate bearing the squaric acid as linking moiety leads to an improved relaxivity with respect to analogous systems obtained through reactions involving the bifunctional DTPA bisanhydride. As far as the exchange of the coordinated water in Gd(III) chelates is concerned, it depends on the energy difference between the ground ennea-coordinated state and the activated octa-coordinated state. In the presence of bulky substituents, the ground state is destabilized with a consequent increase of the exchange rate. An elongation of the exchange lifetime can occur upon the interaction with serum albumin. This behaviour may result in a decrease of the attainable relaxivity. Finally, it has been shown that Mn(II) chelates may represent a viable alternative to Gd(III) complexes. In fact, in spite of the lower effective magnetic moment, the non-covalent adducts between Mn(II) chelates and albumin display very high relaxivities. This result has been accounted for in terms of the very short exchange lifetime of the Mn(II) coordinated water molecule.

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.

Magnetic investigations of human mesencephalic neuromelanin

Pigmentation of neurons in substantia nigra is due to neuromelanin, a pigment that stores large amounts of iron. Human mesencephalic neuromelanin has been investigated by means of magnetic susceptibility measurements as a function of temperature. Magnetic measurements provide a physico-chemical characterization of the iron cluster buried in the organic melanin matrix and support the view that iron is not simply chelated, but rather is organized in a three-dimensional network. The paramagnetism of isolated iron ions is observed, in agreement with electron paramagnetic resonance spectroscopy. Furthermore, antiferromagnetic grains with a large size distribution function are present. These grains contain N spins coupled antiferromagnetically; however, N(1/2) spins are decoupled from the grain bulk and parallelly aligned. The latter subgrains are superparamagnetic with a blocking temperature ranging between 5 K and room temperature. This behavior has not been observed in synthetic melanin, where the paramagnetic contribution is strongly enhanced. Preliminary results on pigment isolated from patients affected by Parkinsons disease, a neurodegenerative pathology involving primarily pigmented neurons in substantia nigra pars compacta, show a lower total magnetization compared to control neuromelanin. The temperature behavior of zero field cooling and field cooling magnetizations is similar for both. The significant depletion of iron content in Parkinsons disease neuromelanin could indicate a progressive Fe migration from its storage environment to the cytosol.

Effect of bezafibrate and clofibrate on the heme-iron geometry of ferrous nitrosylated heme-human serum albumin: an EPR study.

The effect of bezafibrate (BZF) and clofibrate (CF), two therapeutic drugs displaying anticoagulant and antihyperlipoproteinemic activities, on the EPR-spectroscopic properties of ferrous nitrosylated heme-human serum albumin (HSA-heme-NO) has been investigated. In the absence of BZF and CF, HSA-heme-NO is a five-coordinate heme-iron system, characterised by an X-band EPR spectrum with a three-line splitting in the high magnetic field region. Addition of BZF and CF to HSA-heme-NO induced the transition towards a six-coordinate heme-iron species characterised by an X-band EPR spectrum with an axial geometry. These data indicate that HSA-heme-NO is a five-coordinate heme-iron system, BZF and CF acting as allosteric effectors, and show that the primary heme binding site and the CF cleft of HSA are conformationally-linked, regardless of their different location.