Chemical approach for the study of the 'kissing complex' of Moloney murine leukaemia Virus

The replication of Moloney murine leukaemia virus relies on the formation of a stable homodimeric 'kissing complex' of a GACG tetraloop interacting through only two C center dot G base pairs flanked of 5'-adjacent Unpaired adenosines A9. Previous NMR investigations of a model stem loop I has not permitted to reveal the origin of this interaction. Therefore, with the aim of deeper comprehension of the phenomena, the model sequence 10 was prepared where position 9 has been substituted for a nucleoside offering a wider pi-stacking. In this context, the wyosine phosphoramidite building block 2 was prepared and incorporated by adapting the conditions of the automated synthesis and developing original templated enzymatic ligation. However, no 'kissing interaction' has been observed for this model sequence 10 due to steric hindrance as confirmed by computational simulation. Consequently. several other model sequences, 18, 23-26, containing modified nucleosides were prepared. Finally, the importance of the cross-loop H-bond between G8 and G11 nucleobases was revealed by preparing a 18mer RNA hairpin 27, where the guanosine G8 has been substituted for inosine. The latter, which does not possess a C-3 amino function compared to guanosine, is unable to form any 'kissing complex' demonstrating the importance of this secondary interaction in the formation of the complex.

Synthesis of 2'-O-[(triisopropylsilyl)oxy]methyl(=tom)-protected ribonucleoside phosphoramidites containing various nucleobase analogs

Sébastien Porcher

The first results of a study aiming at an efficient prepn. of a large variety of 2'-O-[(triisopropylsilyl)oxy]methyl(=tom)-protected ribonucleoside phosphoramidite building blocks contg. modified nucleobases are reported. All of the here presented nucleosides have already been incorporated into RNA sequences by several other groups, employing 2'-O-tbdms- or 2'-O-tom-protected phosphoramidite building blocks (tbdms=(tert-butyl)dimethyl-silyl). We now optimized existing reactions, developed some new and shorter synthetic strategies, and sometimes introduced other nucleobase-protecting groups. The 2'-O-tom, 5'-O-(dimethoxytrityl)-protected ribonucleosides N2-acetyl-iso-cytidine, O2-(diphenyl-carbamoyl)-N6-isobutyryl-isoguanosine, N6-isobutyryl-N2-(methoxy-acetyl)purine-2,6-diamine ribonucleoside (=N8-isobutyryl-2-[(methoxyacetyl)amino]adenosine), 5-methyluridine, and 5,6-dihydro-uridine were prepd. by first introducing the nucleobase protecting groups and the dimethoxytrityl group, resp., followed by the 2'-O-tom group. The other presented 2'-O-tom, 5'-O-(dimethoxytrityl)-protected ribonucleosides inosine, 1-methyl-inosine 18, N6-iso-pent-2-enyl-adenosine, N6-methyl-adenosine, N6,N6-dimethyl-adenosine, 1-methyl-guanosine, N2-methylguanosine, N2,N2-dimethyl-guanosine, N6-(chloroacetyl)-1-methyladenosine, N6-(1S,2R)-2-[(tert-butyl)dimethyl-silyl]oxy-1-[2-(4-nitrophenyl)ethoxy]carbonylpropylamino carbonyl-adenosine (derived from L-threonine) and N4-acetyl-5-methyl-cytidine were prepd. by nucleobase transformation reactions from std., already 2'-O-tom-protected. Finally, all these nucleosides were transformed into the corresponding phosphoramidites, which are fully compatible with the assembly and deprotection conditions for std. RNA synthesis based on 2'-O-tom-protected monomeric building blocks. [on SciFinder (R)]

2005

Synthesis of modified nucleosides for the incorporation into tRNAs

Sébastien Porcher

More than 100 modified nucleosides have been found in naturally occurring RNA sequences. These modifications are involved in the correct folding, in the maintenance of the reading frame during translation and in the proper interaction with enzymes and protein complexes. For an exhaustive study of their function it would be necessary to incorporate by chemical synthesis such modifications at selected positions of RNA sequences. Here we present optimized protocols for the preparation of a large variety of 2'-O-TOM protected ribonucleoside phosphoramidite building blocks containing the most frequently encountered modified nucleobases m2G, m22G, m1A, m5U, D, m5C, ψ, m1I, i6A, m62A, m6A, m1G, t6A, I and imG. In addition, the non-natural ribonucleoside phosphoramidites containing isoG, isoC and 4-desmethyl-5-methylwyosine have been prepared (Chapters I, II and III). For the introduction of base-labile modifications into RNA sequences, modified deprotection conditions in combination with the new N2-methoxyacetyl protected guanosine phosphoramidite have been developed (Chapter V). For the first time, the sensitive modified nucleoside wyosine was incorporated into a 18mer RNA sequence and into the anticodon loop of a truncated tRNA. For this purpose, enzymatic ligation strategies, based on T4 RNA and T4 DNA ligase, were evaluated and optimized (Chapters III and V). The decoding properties of modified anticodons have been reviewed and new models, based on the formation of secondary hydrogen bonds have been proposed (Chapter IV). For the efficient preparation of tRNA analogues esterified with unnatural amino acids, a synthesis of modified RNA sequences containing a 3'-terminal 2'-deoxy-2'-thioadenosine was developed. In analogy to the "native chemical ligation" of oligopeptides, its spontaneous and site-specific aminoacylation with weakly activated amino acid thioesters occurred efficiently in buffered aqueous solutions and under a wide range of conditions. This concept could be employed for a straightforward aminoacylation of analogously modified tRNAs (Chapter VI).

EPFL2006

Method for preparation of pentopyranosyl nucleosides

Title compds. consisting of ribo-, arabino-, xylo-, or lyxo-pyranoses in place of the usual ribo-furanose, were prepd. and used in solid-phase synthesis to construct (b-D-ribo)-(4'->2')-H-GGGCGGGC-H(I). Thus, 1,2,3,4-tetra-O-benzoyl-a/b-D-ribo-pyranose was substituted at the 1-position with a series of protected nucleoside bases, and the sugar manipulated through a series of steps to give either the 4'-O-dimethoxytrityl-3'-O-benzoyl-2'-O-(allyloxy)(diisopropyl-amino)phosphine compds. suitable for solid-phase synthesis of oligonucleotides, or 2'-O-CO(CH2)5CO2H compds. suitable for attachment to amine-resins to form the initial base of a growing chain. A key step in this synthesis was the simultaneous protection of 3'- and 4'-O by reaction with an aldehyde, ketone, acetal, or ketal protecting group to form cyclic acetals. These intermediates were then used in the construction of I. [on SciFinder (R)]

(Aventis Research and Technologies GmbH and Co. KG, Germany).1999

Synthesis of modified nucleosides for the incorporation into tRNAs

Sébastien Porcher

EPFL2006