ChemInform Abstract The synthesis of silyl-protected ribonucleoside 3'-O-phosphoramidites (I), (II) and the preparation of the controlled-pore glass supports needed for the solid-phase chemical synthesis of oligoribonucleotides are described. These reagents are evaluated in the synthesis of a series of oligomers consisting of the pentadecameric homopolymers of adenosine, cytidine, guanosine, uridine, and various sequences of mixed-base composition. The results of these studies are applied to the successful chemical synthesis of the 43-mer (III), corresponding to the 3'-terminus of a formylmethionine (fMet) + RNA of Escherichia coli, in which the modified bases are substituted by their unmodified parent nucleosides. The effectiveness of alkylsilyl ethers for the protection of the 2'-hydroxyl of the ribose ring, when used in conjunction with the phosphoramidite method for the formation of the phosphotriester linkages in RNA synthesis, is clearly established.
tert-Butyldimethylsilyl chloride reacts selectively with hydroxyl groups in nucleosides and preferentially with the 5'-hydroxyl of deoxynucleosides. The tert-butyldimethylsilyl group can be removed under conditions which do not affect other commonly used acid or base labile protecting groups, yet it is stable to phosphorylation conditions. This allows for the synthesis of a variety of protected deoxynucleosides in good yields and introduces a versatile new protecting group to the nucleoside and nucleotide field.Le chlor~lre de tert-butyldimethylsilyle rCagit ~Clectivement avec les groupes hydroxyles des nuclCosides et prCf6rentiellement avec les hydroxyl-5' des dCsoxynucleosides. Tout en etant stable dans les conditions de phosphorylation, le groupe tert-butyldin~ithylsilyle peut ttre enleve dans des conditions qui n'affectent pas d'autres groupes protecteurs utilises couramment et q~~i sont labiles en milieux acide ou basique. Ceci permet d'effectuer, avec de bons rendements. la synthese d'un grande nombre de dCoxynuclCosides proteges et d'introduire un nouveau groupement protecteur versatile au dornaine des nucleosides et des nuclCotides.[Traduit par le journal]Can. J. Chem. 51, 3799 (1973) During our investigations into the synthesis base labile protecting groups on the nucleoside. and indentification of nucleosides and nucleo-This has facilitated the preparation of a number tides ( I , 2) we have been seeking versatile of protected nucleosides. hydroxyl-protecting groups. We describe in this I report the use of the tert-butyldimethylsilyl
The synthesis of oligonucleotides derived from uridine and adenosine using the tert-butyldimethylsilyl protecting group and the trichloroethylphosphorodichloridite condensation procedure is described. These procedures involve rapid preparation of protected starting materials and equally rapid condensation of units to form nucleotides. Optimum yields using collidine as base in tetrahydrofuran as solvent are between 65 and 80%. The utility of the procedure is rendered complete by the capability to remove both the phosphate protecting groups and the alkylsilyl groups in a single 30 min step using tetrabutylammonium fluoride in tetrahydrofuran.
Procedures have been developed for the selective formation of (a) 2′,5′-silylated ribonucleosides and (b) 3′,5′-silylated ribonucleosides. These procedures also permit the selective silylation at either the 2′- or 3′-position of dimethoxytritylated ribonucleosides. The procedures involve nitrate or perchlorate ion catalysis for selective reaction at 2′-positions and a combination of silver ion and DABCO or 4-nitropyridine N-oxide for selective reaction at the 3′-position. During the course of this work a general and rapid procedure was developed for the preparation and isolation of the 5′-dimethoxytrityl derivatives of the four common ribonucleosides. Silver ion was found to have a marked effect on dimethoxytritylation reactions.
The RNA of viroids and virusoids in plants, and the RNA transcripts of some tandemly repeated DNA sequences in the newt, can undergo self-catalysed cleavage to generate RNA with 5'-OH and 2',3'-cyclic-phosphate termini. These catalytic RNAs, or ribozymes, form a stem-loop secondary structure called a 'hammerhead' in which the catalytic (ribozyme) and substrate sequences are brought close together. Catalytically active mimics of hammerhead ribozymes can be readily made using oligoribonucleotides. Consequently, hammerhead analogues in which certain ribonucleotides are replaced by different ones have been constructed both to identify consensus residues required for cleavage activity and to determine the details of the cleavage mechanism. But these ribonucleotide-replacements tend to alter the conformation of the hammerhead by changing hydrogen-bonding and stacking potential at the position of substitution. We have now constructed structurally less-disrupted hammerhead analogues in which deoxyribonucleotides, which lack 2'-OH groups, are substituted for ribonucleotides. These mixed RNA-DNA polymers were synthesized using a strategy for the chemical synthesis of RNA that is compatible with DNA synthesis. Analysis of the cleavage products of several of these hammerhead analogues confirms the involvement in the reaction of the 2'-OH adjacent to the cleavage site in the substrate, and demonstrates that some 2'-OH groups in the catalytic region strongly affect activity. The results also indicate that the three-dimensional structure producing nucleic acid-type catalysis is not restricted to RNA.
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