Jean-Luc Décout

Is the NAD(P)H:Flavin Oxidoreductase from Escherichia coli a Member of the Ferredoxin-NADP+ Reductase Family? EVIDENCE FOR THE CATALYTIC ROLE OF SERINE 49 RESIDUE

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH. The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group. It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies. A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring. Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine. kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme. Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified. This suggests that serine 49 is involved in the binding of the isoalloxazine ring. Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation. This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction. In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor. Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.

Hydrolysis of 2′-deoxypurine nucleosides. The effect of substitution at the C-8 position

Abstract The hydrolytic stability of 2′-deoxypurine nucleosides is decreased by introduction of electron-withdrawing substituents at the C-8 position in the series of compounds 2–8 , 10–14 . The sulfone group causes a 2.9 × 10 4 rate acceleration for glycosidic bond cleavage in compound 14 .

RECENT DEVELOPMENTS IN THE SYNTHESIS, CHEMICAL MODIFICATIONS AND BIOLOGICAL APPLICATIONS OF SULFUR MODIFIED NUCLEOSIDES, NUCLEOTIDES AND OLIGONUCLEOTIDES

INTRODUCTION ....... . .. ...... .. ... .. ... ... .. .. . .. ..... .. .. ... .. .. . .. .. .. .... . .. .. .. ...................... ._.... .......... .... .... .. .. .. .. .. 29 1. MODIFICATIONS IN THE HETEROCYCLIC BASE ............................................................. 29 1. Thiobases Containing Nucleosides, Nwleotides ......................................................................... 29 2. Thiobases Containing Oligonucleotides, Photochemical Probes and Tools .................... .. .. ..... 3 1 3. Synthesis and Modi@&ns of Nucleosides and Oligonwleotkh?s Using Sulfur Chem ...................................................................................................................... 35 a) Sulfur in Glycosidation Methods, Synthesis of Thiobases Containing Nucleosides, Control of Stereochemistry ........................................................................................................ 35 b) Modijications of Thionucleosides and Nucleotides. .... ............. ........ .... .. .. .. .... .. .. .. .. .. .... .. .. ..... .. . 31 c) Modijications of Oligonucleotides ............................................................................................ 39

Decomposition of FK 409, a new vasodilator: Identification of nitric oxide as a metabolite

Abstract (±)-(3E)-4-Ethyl-2-hydroxyimino-5-nitro-3-hexenamide, FK 409, is a natural product selected for its strong vasorelaxant properties. In neutral aqueous solution, it decomposes spontaneously giving rise to a diketone as shown by HPLC and mass spectrometry. Also, during decomposition, nitric oxide intermediate formation was demonstrated by EPR 1 spectroscopy specific assays. An analog of FK 409, in which the oxime group was selectively hydrolyzed, has lost the ability to generate NO and shows reduced vasorelaxant potency. These results provide a molecular basis for the vasorelaxant effects of FK 409.

Hydrolysis of oligonucleotides containing 8-substituted purine nucleosides. A new route for preparing abasic oligodeoxynucleotides

Abstract 2′-Deoxyadenosine substituted at C-8 by a propylthio group was introduced into oligodeoxyribonucleotides by solid phase synthesis. Oxidation by potassium persulfate (oxone) occurred selectively on the sulfur containing nucleoside causing a weakening of the glycosidic bond. Subsequent hydrolytic treatment led to selective removal of the modified base and generation of an abasic site. This constitutes a novel and convenient route for the chemical synthesis of oligodeoxyribonucleotides containing an abasic site at a preselected position in the sequence.

Possible prebiotic catalysts formed from adenine and aldehyde

Abstract Careful examination of the present metabolism and in vitro selection of various catalytic RNAs strongly support the “RNA World” hypothesis of the origin of life. However, in this scenario, the difficult prebiotic synthesis of ribose and consequently of nucleotides remain a major problem. In order to overcome this problem and obtain nucleoside analogs, we are investigating reactions of the nucleic acid base, adenine 1 , with different aldehydes under presumably prebiotic conditions. In the reaction of adenine and pyruvaldehyde 2 in water, we report here the formation in high yield of two isomeric products. These compounds possessing alcohols functions as nucleosides result from condensation of two molecules of pyruvaldehyde on the 6-amino group of one adenine molecule. Their catalytic activities in the model hydrolysis of p-nitrophenylesters appeared interesting in the search of prebiotic catalysts.

New stereoselective reaction of methylglyoxal with 2-aminopyridine and adenine derivatives: Formation of imino acid-nucleic base derivatives in water under mild conditions

A remarkable stereoselective reaction of methylglyoxal with 2-aminopyridine, the nucleic base adenine and adenine nucleosides leads in good yield to heterocycles of a new family in water under mild conditions and should be of interest in the understanding of the biological effects of methylglyoxal which is toxic, mutagenic and involved in diabetic complications.

8-azidoadenosine and ribonucleotide reductase

Inhibitors of ribonucleotide reductase are potential antiproliferative agents, since they deplete cells from DNA precursors. Substrate nucleoside analogues, carrying azido groups at the base moiety, are shown to have strong cytostatic properties, as measured by the inhibition of the incorporation of thymidine into DNA. One compound, 8-azidoadenosine, inhibits CDP reduction in cytosolic extracts from cancer cells. The corresponding diphosphate behaves as a substrate for ribonucleotide reductase while the triphosphate is an allosteric effector.

Antibiotic drugs aminoglycosides cleave DNA at abasic sites: shedding new light on their toxicity?

Abasic sites are probably the most common lesions in DNA resulting from the hydrolytic cleavage of glycosidic bonds that can occur spontaneously and through DNA alkylation by anticancer agents, by radiotherapy, and during the repair processes of damaged nucleic bases. If not repaired, the abasic site can be mutagenic or lethal. Thus, compounds able to specifically bind and react at abasic sites have attracted much attention for therapeutic and diagnostic purposes. Here, we report on the efficient cleavage activity of characteristic antibiotic drugs of the major aminoglycosides (AG) family at abasic sites introduced either by depurination in a plasmidic DNA or site specifically in a synthetic oligonucleotide. Among the antibiotic AG drugs selected for this study, neomycin B is the most efficient (a 0.1 μM concentration induces 50% cleavage of an abasic site containing DNA). This cleavage activity could be related to aminoglycoside toxicity but also find medicinal applications through potentiation of cancer radiotherapy and chemotherapy with alkylating drugs. In the search for antibiotic and antiviral agents, we have previously described the synthesis of derivatives of the small aminoglycoside neamine, which corresponds to rings I and II of neomycin B constituted of four rings. The cleavage activity at abasic sites of four of these neamine derivatives is also reported in the present study. One of them appeared to be much more active than the parent compound neamine with cleavage efficiency close to that of neomycin.

Unusual addition of amines to C-2 of vinyl sulfone-modified-β-D-pent-2-enofuranosyl carbohydrates: synthesis of a new class of β-anomeric 2-amino-2,3-dideoxy-D-threo-pentofuranosides

When 3-C-sulfonyl-pent-2-enofuranosides and 3-C-sulfonyl-hex-2-enofuranosides were reacted with primary and secondary amines, only the beta-anomeric methoxy group of the pent-2-enofuranoside did not cause any hindrance to incoming nitrogen nucleophiles. This resulted in the unusual addition of amines, in which the diastereoselectivity of the reaction was overwhelmingly in favor of amino sugars of the D-arabino configuration. Selected products were desulfonylated to obtain a new class of beta-anomeric 2-amino-2,3-dideoxy-D-threo-pentofuranosides.