phosphoramidite and hydroxide-ion

This unit describes the chemical synthesis of phosphoramidite building blocks that carry a protecting group at the 3' position. These inversely oriented synthons expose a 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) group as the photolabile protecting group of choice. Among other applications, the building blocks can be employed for light-controlled in situ synthesis of DNA microarrays, producing arrayed oligonucleotides that are attached to the support via their 5' ends, leaving their 3' termini available to act as substrates for polymerases.

Topics: Hydroxides; Models, Biological; Oligodeoxyribonucleotides; Organophosphorus Compounds; Photolysis

To develop oligonucleotides containing new 2'-O-modified ribonucleosides as nucleic acid drugs, we synthesized three types of ribonucleoside derivatives modified at the 2'-hydroxyl group with 2-(methoxycarbonyl)ethyl (MOCE), 2-(N-methylcarbamoyl)ethyl (MCE), and 2-(N,N-dimethylcarbamoyl)ethyl (DMCE) groups, as key intermediates, via the oxa-Michael reaction of the appropriately protected ribonucleoside (U, C, A, and G) derivatives. Among them, the 2'-O-MCE ribonucleosides were found to be the most stable under basic conditions. To study the effects of the 2'-O-modification on the nuclease resistance of oligonucleotides incorporating the 2'-O-modified ribonucleosides and their hybridization affinities for the complementary RNA and DNA strands, 2'-O-MCE-ribonucleoside phosphoramidite derivatives were successfully synthesized and subjected to the synthesis of 2'-O-MCE-oligonucleotides and 2'-O-methyl-oligonucleotides incorporating 2'-O-MCE-ribonucleosides. The 2'-O-MCE-oligonucleotides and chimeric oligomers with 2'-O-MCE and 2'-O-methyl groups thus obtained demonstrated complementary RNA strands and much higher nuclease resistances than the corresponding 2'-O-methylated species. Finally, we incorporated the 2'-O-MCE-ribonucleosides into antisense 2'-O-methyl-oligoribonucleotides to examine their exon-skipping activities in splicing reactions related to pre-mRNA of mouse dystrophin. The exon-skipping assay of these 2'-O-methyl-oligonucleotide incorporating 2'-O-MCE-uridines showed better efficacies than the corresponding 2'-O-methylated oligoribonucleotide phosphorothioate derivatives.

Topics: Acrylates; Animals; Base Sequence; Dystrophin; Exons; Hydrogen-Ion Concentration; Hydroxides; Mice; Nucleic Acid Hybridization; Oligoribonucleotides; Organophosphorus Compounds; Purine Nucleotides; Ribonucleases; RNA Splicing; Substrate Specificity; Uridine

Topics: Animals; Base Sequence; Hydroxides; Mice; NIH 3T3 Cells; Oligoribonucleotides; Organophosphorus Compounds; Ribonucleosides; RNA

The 2-(4-tolylsulfonyl)ethoxymethyl (TEM) as a new 2'-OH protecting group is reported for solid-supported RNA synthesis using phosphoramidite chemistry. The usefulness of the 2'-O-TEM group is exemplified by the synthesis of 12 different oligo-RNAs of various sizes (14-38 nucleotides long). The stepwise coupling yield varied from 97-99% with an optimized coupling time of 120 s. The synthesis of all four pure phosphoramidite building blocks is also described. Two new reliable parameters, delta(C2')-delta(C3') and delta(H2')-delta(H3'), have been suggested for the characterization of isomeric 2'-O-TEM and 3'-O-TEM as well as other isomeric mono 2'/3'-protected ribonucleoside derivatives. The most striking feature of this strategy is that the crude RNA prepared using our 2'-O-TEM strategy is sufficiently pure (>90%) for molecular biology research without any additional purification step, thereby making oligo-RNAs easily available at a relatively low cost, saving both time and lab resources.

Topics: Base Sequence; Chemistry, Organic; Chromatography, High Pressure Liquid; DNA Adducts; Electrons; Hydroxides; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Sequence Data; Oligonucleotides; Organophosphorus Compounds; Ribonucleosides; RNA; Sulfones; Time Factors

1-(2,3-Dideoxy-3-amino-alpha-D-arabino-hexofuranosyl)thymine is considered as a conformationally restricted acyclic nucleoside using the furanose ring to link the diol backbone to the nucleobase. The appropriately substituted phosphoramidites were synthesised via 1-(5,6-di-O-acetyl-2,3-dideoxy-3-phthalimido-alpha-D-arabino-hexofuranosyl)thymine and used in oligodeoxynucleotide (ODN) synthesis. However, the binding affinity of the mixed ODNs towards complementary DNA and RNA was decreased compared to the wild-type oligos. The decrease was smaller when the monomer was inserted near the end of the sequence. The insertions into an alpha T sequence or in a beta T sequence gave nearly the same dropping in melting temperature per modification which indicates that the new nucleotide modifications behave both as alpha and beta nucleotides.

Topics: Arabinonucleosides; Binding Sites; DNA, Complementary; Hydroxides; Molecular Structure; Oligodeoxyribonucleotides; Organophosphorus Compounds; RNA; Temperature; Thymidine