Studies on Synthesis and Structure of O-β-D-Ribofuranosyl(1″→2′)-ribonucleosides and Oligonucleotides (original) (raw)
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Structure and Function of Nucleosides and Nucleotides
Angewandte Chemie International Edition in English, 1973
The nucleosides participating in biological processes consist of a sugar and a heterocyclic nucleobase; the nucleotides, which occur as monomers and as building units of polymeric nucleic acids, contain an additional phosphoester group. The complexity of the molecules leads to a complex stereochemistry with which the present progress report is concerned. Particular attention will be devoted to conformational considerations at the sugar groups, the syn-anti conformation, the position of the C(5')-O(5') bond relative to the sugar group, and the conformation of the phosphoester bonds. The article touches upon base pairing and base stacking, as well as forces stabilizing the syn conformation, and also deals with the reaction mechanism of the enzyme pancreatic ribonuclease as established from the stereochemistry of nucleotides and the mechanisms of action of the antileukemia drug 6-azauridine and the antibiotic actinomycin D. Views on the effects of the unusual structures of the "rare" nucleosides 4-thiouridine, isopentenyladenosine, and dihydrouridine on the structure of transfer ribonucleic acid are also presented.
The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme
RNA, 1998
Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an a-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 39-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 29-fluoro A, but not with 29-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C29-endo conformation. In other cases where there is interference with 29-deoxy A, the presence or absence of 29-fluoro A interference helps to establish whether the 29-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.
Tetrahedron, 1995
The syntheses of 3'-deoxy-3'-C-hydroxymethyl-aldopentopyranosyl nucleosides usmg an intramolecular radical C-C bond formation reactton is described. This method gives good results for the synthesis of thymme and adenine nucleosides, but not for cytosme and guanine nucleosides Dependent on the configuration (f%D-tXythrO or a-L-three), the conformanon of the adenine nucleosides is clearly different (axial base moiety for cc-n-erythro and equatorial adenine base for cr-L-three nucleosides) which could be explained by the gauche effect.
Synthesis of oligodeoxynucleotides containing 6-N-([13C]methyl)adenine and 2-N-([13C]methyl)guanine
J Chem Soc Perkin Trans 1, 1997
Oligonucleotides containing 6-N-([ 13 C]methyl)adenine and 2-N-([ 13 C]methyl)guanine have been prepared for NMR studies using the deprotection step to introduce the [ 13 C]methylamine group. For this purpose, the use of 2'-deoxy-6-O-(pentafluorophenyl)inosine 1 and 2'deoxy-2-fluoro-6-O-[2-(4-nitrophenyl)-ethyl]inosine 2 as precursors of the N-methylated nucleosides is described. Preliminary NMR characterization of the 13 C-labelled oligonucleotides shows that the 13 C chemical shift of the methyl group in N-methylguanine is sensitive to duplex formation, making it a useful local probe.
Cleavage of 3‘,5‘-Pyrophosphate-Linked Dinucleotides by Ribonuclease A and Angiogenin
Biochemistry, 2001
Recently, 3′,5′-pyrophosphate-linked 2′-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphatelinked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3′-pyrophosphate (P′f5′) adenosine (CppA) and uridine 3′-pyrophosphate (P′f5′) adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5′-AMP and cytidine or uridine cyclic 2′,3′-phosphate. The k cat /K m values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K m values (10-16 µM) are lower than those reported for any earlier small substrates (e.g., 500-700 µM for CpA and UpA). The k cat /K m value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K m is more modest. Ribonucleotide 3′,5′-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2′,3′-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t 1/2 > 15 days for uncatalyzed degradation of UppA at pH 6 and 25°C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by ∼10 5 -and 10 3.3 -fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5′-O leaving group, is not required for UppA cleavage because the pK a of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2′-deoxyuridine 3′-pyrophosphate (P′f5′) adenosine (dUppA), determined at 1.7 Å resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through π-π stacking with the six-membered ring of adenine.
Chemistry & Biodiversity, 2005
tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is wellknown based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-b-d-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5''-O-phoshorylated 2'-O-(b-dribofuranosyl)-b-d-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA Met will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.