Jean-Marc Lhoste

Proton magnetic resonance characterization of the intermediate (S=1) spin state of ferrous porphyrins

The temperature dependence of the paramagnetic shifts in square planar ferrous complexes has been investigated for a number of porphyrin derivatives. Large anomalies in the Curie law have been observed in newly synthesized substituted tetraphenylporphins in which the environment of both faces of the tetrapyrrolic ring is strictly controlled. The pseudocontact and contact contributions to the measured hyperfine shifts as well as their temperature dependence have been interpreted in terms of a model with two electronic states. Following this model, the nature of the ground state of the ferrous ion depends critically upon the energy of the dz2 orbital relative to that of the degenerate dxz and dyz orbitals. For most of the porphyrin derivatives, the ground state is 3A2g, strongly perturbed by the closely lying 3Eg excited state. This strong mixing by spin–orbit coupling explains the large orbital contribution to the magnetic susceptibility of these complexes. A small axial perturbation can induce a reversal ...

Lithiation de furo- et pyrrolo[3,2-c]pyridines substituees sur leur position 4

Resume La lithiation des furo- et pyrrolo[3,2- c ]pyridines substituees sur leur sommet 4 effectuee par echange avec le t -butyl lithium, affecte selectivement le sommet 2 de leur heterocycle pentagonal.

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

The two metal-binding sites of human serotransferrin and lactotransferrin differences in histidine coordination as revealed by EPR of cupric complexes

We have demonstrated in a previous paper [l] , using the diethyl pyrocarbonate (DEP) as specific reagent, that in both human serotransferrin (STF) and lactotransferrin (LTF) histidine residues are directly involved in the iron-binding sites. It was also demonstrated that one of the binding sites contains 3 reactive histidine residues in STF and only one in LTF whereas the second site in both proteins was not affected by the DEP reaction. EPR spectra of ferric transferrin complex distinguish the 2 iron-binding sites [2-51 but they do not exhibit the hyperfine structure necessary for the identification of nitrogenous ligands. The nitrogen hyperfine structure is observed in the EPR spectra of copper-substituted transferrin [5,6] . Two different spectra were reported by Aasa et al. and Aisen et al. [4,5] for copper-STF in neutral solutions. They observed a signal corresponding to 3 or 4 nitrogen atoms bound to the cupric ions in a bicarbonate free system but to only 1 nitrogen atom in the presence of bicarbonate. However, the 2 binding sites could not be distinguished in these systems and the possibility of obtaining stable bicarbonate-free com-

NMR investigation of the electronic properties and magnetic states of hemes and hemoproteins : the S = 2 state in ferrous porphyrins*

The proton NMR data of a synthetic model of deoxy-myoglobin and hemoglobin show one that:(i) the ground state of the high spin iron (II) complex must be described by at least two electronic levels separated by ca.200 cm-1 and resulting from the removal of the degeneracy of the 5E level; (ii) this in-plane rhombic distorsion is directed towards the methine bridges of the porphyrin ring. It is proposed that the rhombicity results from specific interactions of the lambda orbitals of the axial base with the iron d lambda orbitals.

Synthese de s-triazolo[4,3-d] et [1,5-d] triazines-1,2,4

Abstract 2-substituted s-triazolo[1,5-d] and 3-substituted s-triazolo [4,3-d]-1,2-4-triazine 8-ones were obtained starting from 5-(3)-alkyl-3(5)-carboxyhydrazino-1,2,4 triazoles and ethyl orthoformate. However, the same reaction was not found to be an efficient one in the case of 5(3)-β-D-ribofuranosyl-3(5)-carboxyhydrazino-1,2,4 triazole.

ESR study of free and immobilized elastase

Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.

Conformational studies of some carbocyclic nucleoside analogues

Abstract Proton NMR spectra of some carbocyclic nucleoside analogs of tuberculin have been analyzed at 100 MHz in DMSO-d 6 . The spectral characteristics and nuclear Overhauser effects indicate a preferential syn conformation about the glycosidic bond when a hydroxyl group, oriented toward the base, is available for intramolecular hydrogen bond formation.

31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses

31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.

Synthesis and oxygenation studies of monomolecular hemoprotein models

Abstract The preparation and properties of molecules modeling the active site of natural oxygen carriers have received considerable attention in recent years for the elucidation of the factors that control the binding reactions of molecular oxygen and carbon monoxide in some hemoproteins. Such compounds have been synthesized following three requirements which seem necessary in the biological functions of natural systems: 1. pentacoordination of the iron(II) ion by a nitrogenous base on the proximal site; 2. steric protection of the heme in order to prevent it from irreversible oxidation into μ-oxo iron(III) dimers; 3. control of the dioxygen environment on the distal site. Several groups have investigated these three problems separately [1]. As part of our studies of the stereochemical influences on the formation and stability of oxygenated model compounds, we have prepared two series of porphyrin derivatives which correspond to the three structural conditions mentioned above. All the compounds were synthesized following the concept of both face hindered tetraphenylporphyrins (‘basket handle’ porphyrins) in which two opposite mesophenyl rings are bridged by a convenient chain [2,3]. The proximal base (pyridine or imidazole) was inserted into one of the handles. The size and polarity of the cage on the distal side can be modified to some extent by suitable chemical changes of the second handle. The newly ‘hanging base’ porphyrins were prepared either from 5,10,15,20-tetrakis(o-hydroxyphenyl)porphyrin (series A, compounds 1–4) or from 5,10,15,20-tetrakis(o-aminophenyl)porphyrin (series B, compounds 5–8). Thus the main structural difference between the two series lies in the presence of non polar ether groups in the former and of polar amide linkage in the latter [4]. Their iron(II) complexes have a magnetic high spin state (S = 2) characteristic of deoxymyoglobins and hemoglobins. Addition of O2 and CO to these complexes results in the formation of hexacoordinated diamagnetic species (S = O), as in oxy and carbooxyhemoproteins, without any paramagnetic perturbations by low lying thermally excited states. A systematic study of the O2 and CO rates and equilibrium binding constants has been carried out in toluene by laser flash photolysis using a modified exchange rate law [5]. This clearly establishes the important role of factors such as the distal steric hindrance, the proximal base constraint and the polarity of the dioxygen environment in the control of the O2 binding and the stability of oxygenated complexes. The association rate constants of O2 and CO are of the same order of magnitude in the two series, but a large variation in the dissociation rate constants is observed. The smaller dissociation rate constants are observed for the compounds having amide linkages as compared with their analogous ether bearings linkages. This results in an increase of the intrinsic stability of the oxygenated derivatives up to the values observed in the natural compounds. The proton magnetic resonance spectra of the oxygenated complexes indicate that the bound dioxygen molecule lies preferentially in a plane oriented toward the distal amide groups in the B series, in contrast with series A in which four nearly equivalent orientations are observed. This preferential orientation of the oxygen molecule results from an hydrogen bonding interaction with the amide groups as shown by the chemical shift of the corresponding protons. The role of this bond in the stability and the bent geometry of the oxygenated complex is of the same type as those proposed for hemoproteins involving distal histidine [6, 7].