Jean-Louis Fourrey

THIONUCLEOBASES AS INTRINSIC PHOTOAFFINITY PROBES OF NUCLEIC ACID STRUCTURE AND NUCLEIC ACID-PROTEIN INTERACTIONS

In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).

An improved preparation of vinyl iodides

Abstract The oxidation of ketone hydrazones by iodine in the presence of a base to furnish vinyl iodides has been considerably improved. The three factors responsible are (1) absence of water, (2) the use of strong guanidine bases and (3) inverse addition.

Studies on the oxidation of hydrazones with iodine and with phenylselenenyl bromide in the presence of strong organic bases; an improved procedure for the synthesis of vinyl iodides and phenyl-vinyl selenides

Abstract The oxidation of hydrazones by iodine in the presence of strong organic bases (guanidines) has been studied. Improved yields of vinyl iodides can be obtained by inverse addition using dry solvents and with a final heating period where appropriate. The reaction has been extended to various dihydrazones with interesting results. In complementary studies hydrazones have been oxidized by phenylselenenyl bromide in the presence of strong organic bases to give the corresponding phenyl vinyl selenides in good yield.

A mild and effective iodination method using iodine in the presence of bis-(trifluoroacetoxy)iodobenzene

Abstract Herein is described a mild and effective iodination method using bis-(trifluoroacetoxy)iodobenzene-iodine as reagent to be applied to electron deficient heterocyclic systems (protected indoles, coumarin…) Moreover, sensitive protecting groups such as acetyl and tert-butyldimethylsilyl were found to be stable under the new iodination reaction conditions.

Design of artificial nucleobases for the recognition of the AT inversion by triple-helix forming oligonucleotides: A structure–stability relationship study and neighbour bases effect

We report herein on the synthesis, the incorporation into triplex forming oligonucleotides (TFO) and the recognition properties of a series of synthetic nucleosides designed for the specific recognition of an inverted A x T base pair in a pyrimidine triple helix motif. These analogues were designed on the basis of the results obtained with our previously reported compounds S and B(t), in order to define a structure-stability relationship. We report also on the chemical nature effect of the bases flanking S in the case of S-containing TFOs, in order to get further informations about the recognition process within the A x TxS triplet. This study establishes guidelines for the conception of more potent analogues for the recognition of both A x T and G x C inverted base pairs.

Chemical structures of the TU-C and TU-Cred products derived from E. coli tRNA

In 1969 we observed that irradiation at 335 nm of E. coli tRNA7 resulted in a specific and quantitative photoreaction of 4-thiouridine (4TU) [ 1 ] . The photoreaction takes place between the 4TU in 8 position and the cytidine in 13 position as shown by Yaniv et al. [2]. Moreover, sodium borohydride reduction of the TU-C photodimer quantitatively yields a fluorescent product TU-Cd, easily detected in the tRNA [3] . These reactions occur in a number of E. coli tRNA [4-51 and they have proved to be of value for the study of tRNA tertiary structure. They were also used to study the functioning of those molecules in the elongation [5-71 and initiation [8] processes of protein biosynthesis. Clearly a better understanding of these problems requires the determination of the chemical structure of the TU-C and TU-Cd products. We have previously described the isolation of TU-C from 335 nm irradiated E. coli tRNAya’ [4]. Since in the range 300-400 nm the absorption spectrum of TU-C remains very similar to that of 4TU it appeared possible that the cross-link formation does not greatly affect

Synthesis of chiral isoquinuclidines and determination of their absolute configuration

Abstract A route to 1,2-dihydropyridines N1-substituted with chiral auxiliaries has been developed starting from commercially available chiral amines. Cycloaddition between these dihydropyridines and methyl acrylate gave, in moderate d.e., isoquinuclidines of good enantiomeric purity whose absolute configuration has been established.

Improved procedure for the preparation of deoxynucleoside phosphoramidites: Arylphosphoramidites as new convenient intermediates for oligodeoxynucleotide synthesis

Abstract A simplified procedure for the preparation of deoxynucleoside methyl- and arylphosphoramidites is described. Both types of phosphoramidites can be conveniently activated by N-methylaniline trifluoracetate for their use in oligodeoxynucleotide synthesis.

An expeditious route to carbocyclic nucleosides : (-)-Aristeromycin and (-)-carbodine

Abstract The readily available bicyclic lactone (−)-1 was transformed into diacetate (−)-2 which served in an expeditious route to (−)-aristeromycin (3a) and (−)-carbodine (3b) in acceptable yields.

Expeditious enantioselective synthesis of carbocyclic nucleosides with antileishmanial activity

A short synthesis of the neplanocin A analogue 1, lacking the hydroxymethylene at C-4 of the parent naturally occurring carbocyclic nucleoside, is described. Moreover, the non toxic noraristeromycin 3, an intermediate in the synthetic scheme, was shown to exhibit a promising anti-leishmanial activity.