Andrée Marquet

Influence of the amino acids of substance P in the recognition of its receptor: affinities of synthesized SP analogues for the specific 125I-BHSP binding site on rat brain synaptosomes

Substance P analogues have been synthesized, by solid-phase methodology, in order to get a better knowledge of the structural requirements for the 125I-BHSP binding on rat brain synaptosomes. Assuming that the core of SP exists in an alpha-helicoidal structure three major points should be underlined: the SP receptor recognizes probably the side of the helix bearing the two side chains of Phe and Phe; the arginine guanidinium interacts with either a carboxylate or a phosphate function of the binding site; the C-terminal tripeptide undergoes a conformational change allowing the interactions of the C-terminal amide with a carboxylate and that of the sulfur atom with an electrophile of the binding site. The specificity of these peptides have been further estimated by comparing their binding potencies to those observed for the 125I-BHE specific binding on rat cortical synaptosomes and their bioactivities on guinea-pig ileum.

Synthesis of 5,5′-dihydroxyleucine and 4-fluoro 5,5′-dihydroxyleucine, the reduction products of 4-carboxyglutamic and 4-carboxy-4-fluoroglutamic acids

Abstract Schemes for the synthesis of 5-5′-dihydroxyleucine 3 and its 4-fluoro analog 7 involving the condensation of a suitable “aminoacid moiety” with 2,2-dimethyl-5-iodomethyl-1,3-dioxane 15D or its fluoro analog 27A were tested. The anion of the ethyl N-diphenylmethylene-glycinate 25 gave better yields of 3 than the classical anion of diethyl acetamidomalonate. This strategy could not be successfully applied to the synthesis of 7 , which could be prepared by reduction of a suitably protected 4-fluoro-4-carboxyglutamate with BMS.

Synthese des 4α et 4β-deuterio 3β, 17β-dihydroxy androsta-5-ene

Abstract The synthesis of 4β and 4α-deuterio 3β, 17β-dihydroxyandrost-5-ene, based on the reduction of 6β-bromo-17β-hydroxyandrost-4-ene-3 one benzoate and of its deuterated homologue by LiAlH4, is reported. This SN2 reaction requires a syn relationship of entering and departing groups. The conditions for the synthesis of androst-5-ene-3,17-dione sterospecifically labelled at the 4 position are discussed.

Acetals Oxidation Into Esters with Electrophilic Halogens

Abstract A preparative oxidation of dioxolanes groups into esters is described. Among the different electrophilic halogens and solvents which have been tested, ICl/H2O in methylene chloride and Br2/H2O in acetonitrile gave the best results.

Search for the biochemical basis of biotin dependent multiple carboxylase deficiencies: determination of biotin activation in cultured fibroblasts

Abstract The activation step of biotin into biotinyl-AMP, which precedes the fixation of the prosthetic group on the apocarboxylases, has been examined in cultured fibroblasts from patients with biotin-dependent multiple carboxylase deficiencies. Two kinds of patients were tested, those with a defective holocarboxylase synthetase activity and those with a low level of the vitamin in the plasma. In both cases, the activation step of biotin was found to be very close to normal values.

Synthesis and biochemical properties of Z- and E-4,5-dehydrodethiobiotin

Radical species are postulated intermediates in the formation of the carbon-sulfur bonds of biotin. It was of interest to examine the behaviour of unsaturated analogues which should give rise to allylic radicals. The two isomers of 4,5-dehydrodethiobiotin have been synthesized and labelled with 14C on their carboxylic acid group. When incubated with an in vitro system capable of transforming dethiobiotin into biotin, they covalently label biotin synthase.

Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21

The ability of a glucocorticoid (triamcinolone acetonide: TA) and three progesterone derivatives with photoreactive groups at different positions (promegestone: R5020; 18-oxo-18-vinylprogesterone: 18OVP; 21-diazoprogesterone: 21DP) to bind covalently to the human mineralocorticoid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimentation gradient analysis and exchange assays with aldosterone showed that [3H]TA, a partial mineralocorticoid agonist, and [3H]R5020, a pure antimineralocorticoid, were covalently bound to hMR after UV irradiation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradiated [3H]TA- and [3H]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band labelled by [3H]TA or [3H]R5020, having a molecular mass of 120 kDa. This band was not detected in the presence of an excess of the corresponding unlabelled steroid or when the cytosol was recovered from non-infected Sf9 cells. Electrophoresis of a truncated hMR (hMRDelta(1-351)) photolabelled with [3H]TA revealed a 80 kDa band, compatible with the molecular mass of the truncated hMR. Limited chymotrypsin proteolysis of the [3H]TA photolabelled hMR generated a 30 kDa fragment covalently associated with [3H]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S. Jalaguier, M. Lombès, A. Souque, M.E. Rafestin-Oblin, Biochem. J. 315 (1996) 421-427), the present experiments provide evidence that [3H]TA is covalently bound to the ligand binding domain of the hMR. Exchange assays with [3H]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are covalently bound to hMR with an approx. 30-35% labelling efficiency.

SYNTHESIS OF PROTEIN CONJUGATES OF 2-CARBOXY-L-ARABINITOL 5-PHOSPHATE AND 2-CARBOXY-L-RIBITOL 5-PHOSPHATE

Abstract The synthesis of 5-O-[5-(pentanoic acid)]-phosphono-L-erythro-pent-2-ulose 2 was successfully achieved by coupling benzyl 5-hydroxypentanoate 9b and 1-O-benzyl-L-erythro-pent-2-ulose ethane- 1,2-diyl dithioacetal 13 with the enediol pyrophosphate 18. Compound 2 was coupled to carrier proteins, porcine thyroglobuline and bovine serum albumin and the conjugates were treated with KCN to give conjugates of the hapten 1, designed to raise catalytic antibodies.

On the Mechanism of C-S Bonds Formation in the Biosynthesis of Biotin

The biosynthesis of biotin, 1, has already been intensively studied (1). All the intermediates in the biosynthetic pathway from pimelic acid to dethiobiotin, 2, are presently known and formed by classical biochemical reactions. But the mechanism of the conversion of dethiobiotin into biotin, a very unusual transformation, is still completely unknown.