Jean-Claude Rossi

Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1.

We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4). We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.

Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha.

F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2α, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2α, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2αis the only identified metabolite of 8-epi-PGF2α in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2α, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5,6-dihydro-8-epi-PGF2α as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2α. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another β-oxidation product, 2,3,4,5-tetranor-8-epi-PGF2α, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major β-oxidation products of 8-epi-PGF2α are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2α formation and degradationin vivo.

Tandem mass spectrometric quantification of 8-iso-prostaglandin F2α and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2α in human urine

Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC–tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2α, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2α. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2α and 2,3-dinor-5,6-dihydro-8-iso-PGF2α, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2α. 2,3-Dinor-5,6-dihydro-8-iso-PGF2α was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC–tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2α measured by GC–MS and GC–tandem MS (r=0.84).

The Handy Use of Brown’s P2‐Ni Catalyst for a Skipped Diyne Deuteration: Application to the Synthesis of a [D4]‐Labeled F4t‐Neuroprostane

Browns P2-Ni does the job: An efficient synthesis of tetradeuterated neuroprostane (see structure) has been accomplished by using an eneˆdiyne stereoselective mots cles : 15-D2-IsoP; 15-E2t-IsoP; total synthesis; selective enzymatic chemical differentiation of a non-meso-1,5-diols

Total Synthesis of the Four Enantiomerically Pure Diasteroisomers of the Phytoprostanes E1Type II and of the 15-E2t-Isoprostanes

Syntheses of the four enantiomerically pure diastereoisomers of the phytoprostanes E1 type II and 15-E2t-isoprostanes (1-4) are described. The key steps included the preparation of the Freïmanis (+/-)-hydroxycyclopentenone 5, enzymatic resolution of this racemic hydroxycyclopentenone, Wittig and Horner-Wadsworth-Emmons (HWE) coupling reactions and finally enantioselective reductions.

Isoprostanes and phytoprostanes: Bioactive lipids

Polyunsaturated fatty acids (PUFA) are important constituents in all eukaryotic organisms, contributing to the structural integrity of biological membranes and serving as precursors for enzymatically-generated local hormones. In addition to these functions, PUFA can generate by a free radical-initiated mechanism, key products which participate in a variety of pathophysiological processes. In particular, free radical-catalyzed peroxidation of PUFA leads to in vivo formation of isoprostanes (IsoP), neuroprostanes (NeuroP), and phytoprostanes (PhytoP) which display a wide range of biological actions. IsoP are now the most reliable indicators of oxidative stress in humans. In this review, we will discuss some advances in our knowledge regarding two cyclic PUFA derivatives, IsoP and PhytoP, and how their biological roles may be clarified through new approaches based on analytical and synthetic organic chemistry.

The 5‐series F2‐isoprostanes possess no vasomotor effects in the rat thoracic aorta, the human internal mammary artery and the human saphenous vein

Among the F2‐isoprostanes, the 15‐ and the 5‐series are currently used as markers of lipid peroxidation in vascular diseases. 15‐F2t‐IsoP (also named iPF2α‐III) exerts a vasoconstriction in most vessels, whereas no data is available concerning 5‐F2t‐IsoP (also named iPF2α‐VI), which is more abundant in plasma. The aim of this study was to determine whether 5‐F2t‐IsoP possess any vascular effects on various vessels including the isolated rat thoracic aorta, the human internal mammary artery and the saphenous vein. In organ baths, 5‐F2t‐IsoP and its 5‐epimer did not affect the basal tone of any vessel, unlike 15‐F2t‐IsoP. These compounds possessed no antagonist effects on 15‐F2t‐IsoP‐induced contractions, No dilator effect was observed in comparison with sodium nitroprusside and acetylcholine on the rat aorta. In conclusion, we show that unlike 15‐F2t‐IsoP, 5‐F2t‐IsoP and its 5‐epimer possess no vasomotor effects and as such are unlikely to be involved in the pathogenesis of vascular diseases. Further studies are required to test whether these mediators may have effects on systems not being measured in the current study.

Convenient method for the preparation of trityl ethers from secondary alcohols.

Abstract The preparation of trityl ethers from secondary alcohols (10 mmol) with triphenylmethyl chloride (1.2 eq.) is carried out at room temperature by using DBU (1.4 eq.) as base in CH2Cl2. The high yielding procedure is very simple and its applicability is general.

Total synthesis of 4(RS)-F4t-isoprostane methyl ester

Abstract The first total synthesis of 4( RS )-F 4t -isoprostane methyl ester 1 is described using diacetone- d -glucose as starting material. This new isoprostane (neuroprostane) would be very useful in neurological studies as a potent lipid peroxidation index to obtain an integrated assessment of oxidative stress in the human brain.

Leukotrienes stimulate acid secretion from isolated gastric parietal cells

Leukotrienes LTC4 and LTD4 display contractile effect on the stomach. The stimulation of acid secretion by LTC4, LTD4 and LTE4 was evidenced on a crude isolated cell preparation from rabbit gastric mucosa using the (14C)aminopyrine accumulation method. LTs were in the same order of potency. No potentiation with histamine, carbachol or IBMX was observed suggesting a specific mechanism for LTs on parietal cell.