Modified nucleosides

In recent year s, clinical studies on the role of uiinai y luodified nucleosides as the biochemical mai kers of various types of cancer have been actively undertaken. Most of the urinai y modified nucleosides ai e piimai ily originated by methylation of either the base part, the sugar hydroxyl par t, or in some cases, both par ts of the course of biodegradation of tRNA molecules. Hence, their isolation and identification plays a major role in biochemical analysis. 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

MI3 G. Dirheimer, in Modified Nucleosides and Cancer" (G. Glass, ed.),... 

One-electron oxidation of the adenine moiety of DNA and 2 -deoxyadenos-ine (dAdo) (45) gives rise to related purine radical cations 46 that may undergo either hydration to generate 8-hydroxy-7,8-dihydroadenyl radicals (47) or deprotonation to give rise to the 6-aminyl radicals 50. The formation of 8-oxo-7,8-dihydro-2 -deoxyadenosine (8-oxodAdo) (48) and 4,6-diamino-5-formamidopyrimidine (FapyAde) (49) is likely explained in terms of oxidation and reduction of 8-hydroxy-7,8-dihydroadenyl precursor radicals 47, respectively [90]. Another modified nucleoside that was found to be generated upon type I mediated one-electron oxidation of 45 by photoexcited riboflavin and menadione is 2 -deoxyinosine (51) [29]. The latter nucleoside is likely to arise from deamination of 6-aminyl radicals (50). Overall, the yield of formation of 8-oxodAdo 48 and FapyAde 49 upon one-electron oxidation of DNA is about 10-fold-lower than that of 8-oxodGuo 44 and FapyGua 43, similar to OH radical mediated reactions [91]. 

Amann, N. and Wagenknecht, H. A. (2002). Preparation of pyrenyl-modified nucleosides via Suzuki-Miyaura cross-coupling reactions. Syn-lett 687-691. 

The unique properties of oligonucleotides create crosslinking options that are far different from any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically duplicated in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

Some form of chemical labeling process must be used regardless of whether the final oligo conjugate is created by enzymatic or strictly chemical means. If enzymatic modification is to be done, the initial label still must be incorporated into an individual nucleoside triphosphate, which then is polymerized into an existing oligonucleotide strand (Section 1, this chapter). Fortunately, many useful modified nucleoside triphosphates are now available from commercial sources, often eliminating the need for custom derivatization of individual nucleotides. 

Access to modified nucleosides. Base-modified nucleosides and nucleotides are very important for their biological properties. They can be found as antiviral agents (HBH, VZV, AIDS),93 95 in the study of DNA degradation,96,97 as fluorescent agents and as chemical probes of DNA structure.98 101 The access to nucleosides can be achieved by different methods ... 

Over the past years, synthetic base-modified nucleosides and nucleotides have displayed important impact in diverse fields. Their biological properties have found application as antiviral tools against hepatitis virus (HBV), herpes virus (VZV) and human immunodeficiency (HIV).94,95,136 Many of those compounds exhibit antiproliferative, antibiotic and antifungal activities and some have been used as probes for DNA damages96,97,137 as well as in the anti-sense approach and DNA-probe technology with fluorescence properties.99... 

The vast majority of research focused on selenium in biology (primarily in the fields of molecular biology, cell biology, and biochemistry) over the past 20 years has centered on identification and characterization of specific selenoproteins, or proteins that contain selenium in the form of selenocysteine. In addition, studies to determine the unique machinery necessary for incorporation of a nonstandard amino acid (L-selenocysteine) during translation also have been central to our understanding of how cells can utilize this metalloid. This process has been studied in bacterial models (primarily Escherichia colt) and more recently in mammals in vitro cell culture and animal models). In this work, we will review the biosynthesis of selenoproteins in bacterial systems, and only briefly review what is currently known about parallel pathways in mammals, since a comprehensive review in this area has been recently published. Moreover, we summarize the global picture of the nonspecific and specific use of selenium from a broader perspective, one that includes lesser known pathways for selenium utilization into modified nucleosides in tRNA and a labile selenium cofactor. We also review recent research on newly identified mammalian selenoproteins and discuss their role in mammalian cell biology. 

More than 60 per cent of all diseases in Europe, North America and Japan are caused by the action of viruses, amongst them bronchitis, hepatitis, influenza, infections by several strains of herpes as well as by human immunodeficiency viruses (HIV) [216]. Modified nucleosides that inhibit the replication of the... 

Glycosylated Compounds. The rationale for the use of modified nucleosides for the treatment of cancer and viral disease relies on the hope that cancer cells or virally infected cells will mistake the modified compounds for the natural substrates and incorporate them into a metabolic pathway. The altered stmc-ture of the false substrate will, it is hoped, then bring that process to a halt and result in cell death. 

Additionally, marine organisms have proven to be a rich source for a wide variety of modified nucleosides considered worthy for clinical application. For example, arabinoside-nucleosides, constituents of the Caribbean sponge C.ryptotethya crypta, have led researchers to synthesise analogues with improved antiviral and anticancer activity [4],... 

The structures of some modified nucleosides found in tRNA. The parent ribonucleosides are shown on the left in yellow screens. The other bases found in RNA result from post-transcriptional modification. 

SAR. Introduction of a (N)-methanocarba modification to NEC A in 56 increased the hA3AR binding affinity by sixfold. Thus, the (N) conformation is confirmed to be highly preferred over the (S) conformation for binding at the A3AR, for a range of structures of multiply modified nucleoside derivatives. 

Jacobson K, Gao Z, Chen A, Barak D, Kim S, Lee K, Link A, Rompaey P, Van Calenbergh S, Liang B (2001) Neoceptor concept based on molecular complementarity in GPCRs a mutant adenosine A3 receptor with selectively enhanced affinity for amine-modified nucleosides. J Med Chem 44 4125-4136... 

N. Hunt, G. McGall, S.P. Fodor, F.S. Collins, Enhanced High Density Oligonucleotide Array-based Sequence Analysis Using Modified Nucleoside Triphosphates , Nucl. Acids Res., 26(21), 4975 -4982 (1998). 

Heavy atom incorporation for phasing can also be accomplished through the use of modified nucleosides. Specifically, 5-bromouridine (Adams et al, 2004 Baugh etal, 2000 Kieft etal, 2002 Martickand Scott, 2006), 5-iodour-idine (Klein et al, 2009), STa-P-seleno-triphosphates (Brandt et al, 2006),... 

Moreover, the morpholino backbone s immunity to enzymatic attack avoids the potential for nonspecific interactions associated with degradation by-products. In contrast, modified nucleosides or nucleotides resulting from degradation of other antisense compounds might be toxic or might be incorporated into cellular genetic material and thereby lead to mutations or other undesired biologic effects. 

Fig. 5. Some modified nucleosides found in tRNA molecules.

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