Synthesis of modified nucleosides for the incorporation into tRNAs

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).

Optimized methods for the enzymatic ligation of oligoribonucleotides and analogues

Frédéric David Meylan

The preparation of oligoribonucleotides is of great interest for structural and functional investigations in biomolecular chemistry and biology. Relevant RNAs are relatively long and contain often modifications. Two methods for their preparation are currently available: automated chemical synthesis and in vitro transcription. The chemical synthesis allows the preparation of relatively short (< 60mers) pure RNA sequences containing modified nucleotides. In contrast, in vitro transcription is appropriate for the synthesis of long RNA sequences but does not allow the incorporation of modifications. An elegant solution to this dilemma is offered by enzymatic ligations of RNA fragments with T4-DNA ligase, which is template-dependent and therefore highly selective. Here, we present a comprehensive study of parameters, which govern the effectiveness of this ligation. The influence of secondary structures present within templates were studied in detail. That allowed to design a new type of hybrid template, containing both DNA and 2'-OMeRNA nucleotides, which were particularly efficient (Chapter 3.2). Ligation reactions with structured substrates and different types of oligonucleotides were carried out, leading to a better comprehension of the enzyme mechanism. In this context, we found very strong indications for a different recognition of the donor and acceptor substrates by the enzyme (Chapter 3.2 and 3.3). Optimization of the enzymatic ligations with different substrates or templates has showed the importance of secondary structures for design of a successful reaction (Chapter 3.2 and 3.3). The lessons drawn from these preliminary experiments have been employed for the reliable preparative synthesis of natural oligoribonucleotides containing modifications (Chapter 3.4) and the preparation of long RNA sequences (Chapter 3.5).

EPFL2006

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

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