Jean-Claude Beloeil

Conformation of Taxotere® and analogues determined by NMR spectroscopy and molecular modeling studies

Abstract Taxol 1 and Taxotere® 2 are antitumor compounds interacting with tubulin proteins. In order to find the best conformational fit to the receptor site, the structures of taxotere and twelve analogues showing various in vitro biological activity on tubulin, have been investigated by 1H NMR spectroscopy and molecular modeling studies. These structures were compared to that of Taxotere® 2 obtained by X-ray analysis. The results obtained from these studies suggest that the most active 2′R,3′S compounds possess a conformation in which the benzoate group at C-2 holds the side chain in a defined position due to hydrophobic interactions between this group and the N-amido or N-carbonyloxy group at C-3′. This situation together with the presence of hydrogen bonding between 2′OH-3′NH and 2′OH-1′CO gives rise to a specific orientation of the hydroxyl and phenyl groups at C-2′ and C-3′. On the other hand, the 2′S,3′R isomers which display low in vitro biological activity (ie: on tubulin), such as isotaxotere 8, possess a different conformation with no hydrophobic interactions between the side chain and the taxan skeleton.

2D COSY 1H NMR: a new tool for studying in situ brain metabolism in the living animal

2D COSY 1H NMR with surface coil has been used to resolve and assign cerebral metabolites which had previously been detected but could not be resolved or assigned in situ in the living animal by conventional ID 1H NMR. A wide range of cerebral metabolites, including alanine, N‐acetyl aspartate, aspartate, choline derivatives, creatine/phosphocreatine pool, GABA, glucose, glutamate/glutamine pool, inositol, lactate and taurine were simultaneously resolved and assigned in situ in the whole animal using the 2D COSY correlation graphs. Global irreversible ischemia caused the appearance and the disappearance of cross‐peaks in the 2D COSY 1H NMR map, corresponding to increases in alanine, GABA and lactate and glucose depletion.

CK flux or direct ATP transfer: Versatility of energy transfer pathways evidenced by NMR in the perfused heart

How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes (myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR. To get insight in the energy transfer pathway in the perfused rat heart, we developed a combined analysis of several protocols of magnetization transfer associated with biochemical data and quantitatively evaluated which scheme of energetic exchange best describes the NMR data. This allows to show the kinetic compartmentation of subcellular CKs and to quantify their fluxes. Interestingly, we could show that the energy transfer pathway shifts from the phosphocreatine shuttle in the oxygenated perfused heart to a direct ATP diffusion from mitochondria to cytosol under moderate inhibition of ATP synthesis. Furthermore using NMR measured fluxes and the known kinetic properties of the enzymes, it is possible to model the system, estimate local ADP concentrations and propose hypothesis for the versatility of energy transfer pathway. In the normoxic heart, a three fold ADP gradient was found between mitochondrial intermembrane space, cytosol and ADP in the vicinity of ATPases. The shift from PCr to ATP transport observed when ATP synthesis decreases might result from a balance in the activity of two populations of ANT, either coupled or uncoupled to CK. We believe this NMR approach could be a valuable tool to reinvestigate the control of respiration by ADP in the whole heart reconciling the biochemical knowledge of mitochondrial obtained in vitro or in skinned fibers with data on the whole heart as well as to identify the implication of bioenergetics in the pathological heart.

Evidence by NMR for mobility of the cytochrome domain within flavocytochrome b2

According to a model proposed by Gervais, M, Groudinsky, O., Risler, Y. and Labeyrie, F. ((1977) Biochem. Biophys. Res. Commun. 77, 1543-1551) flavocytochrome b2 is composed of a central flavodehydrogenase entity of 4 X 45 kDa to which are attached four cytochrome b2 globules of approx. 11 kDa that are released after proteolysis of the connective loops. A possible inherent mobility of the latter with functional significance was suspected. Proton NMR spectra at 400 MHz of the isolated and of the flavodehydrogenase-bound ferricytochrome b2 units have been compared. In the ranges downfield of +12 ppm and upfield from -4 ppm, where hyperfine-shifted heme proton resonances reside, the chemical shifts are identical for the two forms, but the linewidths are markedly broader for flavocytochrome b2. The linewidths of three heme resonances, a methyl at +19 ppm, two single protons at -6 and -8 ppm (most probably from one vinyl) and an unassigned line at -2.4 ppm, all increase by a factor of about 4. Since, in the present case, linewidths are controlled mainly by proton/proton dipolar relaxations which are caused by molecular tumbling, a change in linewidths of about 15 would be expected if the cytochrome b2 globule had no free motion relative to the flavodehydrogenase domain. The present results thus support the previous hypothesis that such a relative mobility, of unknown correlation time and amplitude, actually exists.

Cerebral metabolic changes induced by MK-801: a 1D (phosphorus and proton) and 2D (proton) in vivo NMR spectroscopy study

The dynamic effects of the non-competitive NMDA receptor antagonist, MK-801 on brain metabolism were investigated over 105 minutes in unanesthetized rats by proton and phosphorus NMR spectroscopy. MK-801 (0.5 and 5 mg/kg, i.p) induced no changes in intracellular pH, and in phosphocreatine, ATP, and inorganic phosphate levels, indicating that the drug preserved energy and intracellular pH homeostasis. There were transient increases in lactate after both doses of MK-801, suggesting early activation of glycolysis, which was not immediately matched by enhanced oxidative metabolism or by enhanced blood flow. Thereafter, lactate control level was not restored after 0.5 mg/kg whereas it was restored after 5 mg/kg in spite of a sustained metabolic activation. The low dose of MK-801 also caused a continuous decrease in cerebral aspartate level (-38%) which is thought to match the enhanced energy demand, whereas the high dose caused shorter and smaller changes. The intracerebral glucose level rose after MK-801 injection, indicating that brain tissue had an adequate or even excessive supply of glucose. Glucose time course seemed to closely match the changes in blood flow elicited by MK-801. This is the first study giving the metabolic pattern of a pharmacological activation. We demonstrate an excess of glycolysis over oxidative metabolism in the early time similar to that following physiological and pathophysiological states such as photic stimulation and seizures. The difference between the effects of the two doses of MK-801 suggests that the adjustment of cerebral metabolism to MK-801 activation is faster and greater with the high dose than with the low dose.

In vitro study of newborn rat brain maturation: implication for sudden infant death syndrome ☆

We have used slice preparation from newborn rats to study the development of the nucleus tractus solitarius neuronal network and brain intracellular phosphorus metabolites. As shown previously on adults, the newborn preparation retains local excitatory and inhibitory synaptic connections and enables study of intrinsic electrical properties in the nucleus tractus solitarius. Electrophysiological investigation of inhibitory synaptic transmission demonstrated a maturational step at days 4-6 after birth. Nuclear magnetic resonance spectroscopy of brain slices revealed a metabolic maturation between postnatal days 11 and 17. Results emphasize the differential maturation steps during the postnatal development of rat central nervous system. Possibly, Sudden Infant Death Syndrome may result from the abnormal timing in the occurrence of these steps.

Model studies in the taxane diterpene series - Part. I

Abstract Photochemical [2 + 2] cycloaddition of enol acetate 10a to cyclohexene led to a 3 : 1 mixture of the tetracyclic photoadducts A and B which, through reverse aldol reactions, under mild alkaline conditions produced two compounds : a tetracyclic ketone 13 and a tricyclic diketone 14 . Hydrolytic cleavage in methanolic hydrochloric acid of 15 and 16 resulting from irradiation of the enol ether 10b to cyclohexene gave rise to the rearranged tricyclic diketone 14 only. The trans stereochemistry 3α, 8β of the fused-ring system in 14 established by X-ray measurements, is different from the stereochemistry of the naturally occuring taxane derivatives.

Cerebral intracellular pH regulation during hypercapnia in unanesthetized rats: a31P nuclear magnetic resonance spectroscopy study

The energy metabolism and the brain intracellular pH regulation under arterial CO2 tensions of 25-90 mm Hg were investigated in unanesthetized spontaneously breathing rats by in vivo phosphorus nuclear magnetic resonance spectroscopy (31P NMR). The 31P brain spectra, recorded with a high resolution spectrometer (AM 400 Brucker), allowed repeated non-invasive measurements of cerebral pH (pHi), phosphocreatine (PCr), inorganic phosphate (Pi) and adenosine triphosphate (ATP) levels in 15 rats breathing a gas mixture containing 21% O2, N2, and a varied percentage of CO2. The pHi decreased significantly when the paCO2 was increased by hypercapnia. The percentage of pH regulation, estimated from the linear regression analysis of pHi versus the logarithm of the paCO2 was 78%. This result indicates that spontaneously breathing unanesthetized animals have better pHi regulation under hypercapnia investigated than that estimated for higher levels of hypercapnia in previous studies on unanesthetized animals, suggesting that there is a threshold for this highly efficient regulation. Furthermore, there were no significant correlations between the PCr, ATP and Pi levels and the paCO2 levels during hypercapnia. This indicates that physiological variations of the CO2 tension in the blood, and consequently in the brain parenchyma, have little effect on cerebral energy metabolism in unanesthetized spontaneously breathing animals.

Metabolic acidosis induced by N-methyl-d-aspartate in brain slices of the neonatal rat: 31P- and 1H-magnetic resonance spectroscopy

1H- and 31P-magnetic resonance spectroscopy was used to monitor intracellular lactate, phosphorus metabolites and pH in superfused brain slices from 2- to 9-day-old rats. N-Methyl-D-aspartate (NMDA) (100 microM, 0.5-3 min) was applied in the extracellular magnesium-free perfusion medium. NMDA induced intracellular metabolic acidosis, i.e., an increase of freely mobile lactate levels and an 0.3 pH unit acidification. This was abolished when the extracellular glucose supply was reduced. Experiments also indicate that acidosis is not responsible for the cell damage resulting from activation of NMDA receptors in hypoglycemic conditions.

Manicol: a sesquiterpenoid hydroxytropolone from Dulacia guianensis; A revised structure (X-ray analysis)

Abstract Manicol, isolated from Dulacia guianensis and for which structure 1 was previously proposed, was shown to be the sesquiterpenoid hydroxytropolone 5 . This revised structure was established by X-ray analysis of manicol and its diacetate 4 . Methylation of manicol afforded three dimethyl ethers which were differentiated mainly by 13C nmr spectroscopy including the heteronuclear spin population inversion method. The major methylated product 6 was shown to undergo a LAH rearrangement leading to the benzylic alcohol 9a which was subsequently converted to the methyl ether 10 .