ribothymidine and 5-methylcytidine

Topics: Cytidine; Oligonucleotides; Organophosphorus Compounds; Uridine

5-Methylcytidine (m

Topics: Cytidine; Isomerism; Mass Spectrometry; Osmium; Oxidation-Reduction; RNA; RNA Processing, Post-Transcriptional; Uridine

Nucleobase methylations are ubiquitous posttranscriptional modifications of ribonucleic acids (RNA) that can substantially increase the structural diversity of RNA in a highly dynamic fashion with implications for gene expression and human disease. However, high throughput, deep sequencing does not generally provide information on posttranscriptional modifications (PTMs). A promising alternative approach for the characterization of PTMs, i.e. their identification, localization, and relative quantitation, is top-down mass spectrometry (MS). In this study, we have investigated how specific nucleobase methylations affect RNA ionization in electrospray ionization (ESI), and backbone cleavage in collisionally activated dissociation (CAD) and electron detachment dissociation (EDD). For this purpose, we have developed two new approaches for the characterization of RNA methylations in mixtures of either isomers of RNA or nonisomeric RNA forms. Fragment ions from dissociation experiments were analyzed to identify the modification type, to localize the modification sites, and to reveal the site-specific, relative extent of modification for each site.

Topics: Adenosine; Base Sequence; Cytidine; Ions; Methylation; Molecular Structure; RNA; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; Uridine

We describe an improved process to produce 2'-O-(2-methoxyethyl)-pyrimidines. Starting with commercially available O-2,2'-anhydro-5-methyluridine and tris-(2-methoxyethyl)borate, we modified the ring-opening reaction conditions and changed to a continuous extraction purification method to give 2'-O-(2-methaxyethyl)-5-methyluridine. The dimethoxytritylation 5'/3' ratios and yield were improved by the use of 2,6-lutidine as the base. Conditions to convert to the 5'-methylcytidine analog and its isolation by crystallization were optimized. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities.

Topics: Boron Compounds; Chemistry, Pharmaceutical; Chromatography; Cytidine; Esters; Hydrolysis; Models, Chemical; Pyridines; Pyrimidines; Uridine

Novel 5'-O-DMT- and MMT-protected 3'-C-methylene-modified thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluoro, hydrogen, and O-(2-methoxyethyl) substituents at the 2'-position have been synthesized by a new effective strategy from the corresponding key intermediates 3'-C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for the Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, and directly provided the desired 5'-O-DMT- and/or MMT-protected 3'-C-methylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation. The modified Arbuzov reaction of 3'-C-iodomethyl-5-methylcytidine 53, prepared from its 5-methyluridine derivative 42, with BTSP provided the 5-methylcytidine H-phosphonate 54, which was further transferred to the corresponding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7. 5'-O-MMT-protected 3'-C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3'-C-methylene-modified phosphonamidite monomers 8-10. Some of these new 3'-methylene-modified monomers 1-10 have been successfully utilized for the synthesis of 3'-methylene-modified oligonucleotides, which have shown superior antisense properties including nuclease resistance and binding affinity to the target RNA.

Topics: Anti-Infective Agents; Antineoplastic Agents; Cytidine; Oligonucleotides; Oligonucleotides, Antisense; Organophosphonates; Thymidine; Uridine

In DNA triple helices, methylation at C-5 of thymine or cytosine is reported to have similar stabilizing effects for both bases. Here we show, however, that methylation of the same positions in RNA triplexes has distinctly different effects than in DNA. We have previously described the use of circular triplex-forming RNA oligonucleotides to recognize RNA sequences. Here it is shown that addition of C-5 methyl groups to uracils in these compounds very significantly increases not only affinity but also sequence selectivity in binding a purine-rich RNA target, as measured by thermal denaturation with various target RNAs. Surprisingly, however, addition of C-5 methyl groups to cytosines actually decreases affinity in binding RNA, while the same substitution in DNA is thermally stabilizing. Possible sources of this divergent behavior are discussed. A synthesis of 5-methylcytidine ribonucleoside 2'-O-silyl-3'-O-phosphoramidite is also described.

Topics: Base Sequence; Cytidine; DNA, Circular; DNA, Complementary; Methylation; Methyltransferases; Nucleic Acid Conformation; RNA; RNA, Circular; RNA, Complementary; Substrate Specificity; Uridine