Synthesis and Metabolic Fate of 4-Methylthiouridine in Bacterial tRNA.

Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s4 U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms4 U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms4 U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal in vivo. Our findings reveal the complex nature of bacterial RNA damage repair.

Prebiotic phosphorylation of 2-thiouridine provides either nucleotides or DNA building blocks via photoreduction.

Breakthroughs in the study of the origin of life have demonstrated how some of the building blocks essential to biology could have been formed under various primordial scenarios, and could therefore have contributed to the chemical evolution of life. Missing building blocks are then sometimes inferred to be products of primitive biosynthesis, which can stretch the limits of plausibility. Here, we demonstrate the synthesis of 2'-deoxy-2-thiouridine, and subsequently 2'-deoxyadenosine and 2-deoxyribose, under prebiotic conditions. 2'-Deoxy-2-thiouridine is produced by photoreduction of 2,2'-anhydro-2-thiouridine, which is in turn formed by phosphorylation of 2-thiouridine-an intermediate of prebiotic RNA synthesis. 2'-Deoxy-2-thiouridine is an effective deoxyribosylating agent and may have functioned as such in either abiotic or proto-enzyme-catalysed pathways to DNA, as demonstrated by its conversion to 2'-deoxyadenosine by reaction with adenine, and 2-deoxyribose by hydrolysis. An alternative prebiotic phosphorylation of 2-thiouridine leads to the formation of its 5'-phosphate, showing that hypotheses in which 2-thiouridine was a key component of early RNA sequences are within the bounds of synthetic credibility.

Synthesis of 2'-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides.

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions.

Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C-I and C-Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion.

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA(Leu(UUR)) and tRNA(Lys).

5-Taurinomethyluridine (τm(5)U) and 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) are located at the wobble position of human mitochondrial (hmt) tRNA(Leu(UUR)) and tRNA(Lys), respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys) are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm(5)U and τm(5)s(2)U into 17-mers related to the sequence of the anticodon arms hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys), respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF3) are compatible with the blockage of the canonical monomeric units. The synthesis of τm(5)s(2)U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.

5-Fluoro-4-thiouridine phosphoramidite: new synthon for introducing photoaffinity label into oligodeoxynucleotides.

The synthesis of phosphoramidite of 5-fluoro-4-thio-2'-O-methyluridine is described. An appropriate set of protecting groups was optimized including the 4-thio function introduced via 4-triazolyl as the 4-(2-cyanoethyl)thio derivative, and the t-butyldimethyl silyl for 2' and 3' hydroxyl protection, enabling efficient synthesis of the phosphoramidite. These protecting groups prevented unwanted side reactions during oligonucleotide synthesis. The utility of the proposed synthetic route was proven by the preparation of several oligonucleotides via automated synthesis. Photochemical experiments confirmed the utility of the synthon.

5-Nitro-2-pyridyl-1-thioglucosides: application in synthesis of analogues of glycosyltransferases natural substrates.

5-Nitro-2-pyridyl-1-thioglucosides were used in synthesis of complex uridine derivatives (13-16) in two different sequences of reactions. In one route, the first step was glycosylation of selectively protected 5-nitro-2-pyridyl-1-thioglucoside 1 with two different glycosyl donors (5 or 6), next, the nitro group in aglycone of obtained disaccharides 7 or 8 was reduced and then obtained products 9 or 10 were condensed with uridine derivatives 3 or 4 using DMT-MM as condensing agent under microwave irradiation. In the second route, condensation and glycosylation reactions were applied in reverse order. As it turned up, a sequence of reactions affected the yield of final glycoconjugates 13-16 and depended on the type of uridine derivatives used.

Chemical synthesis of LNA-2-thiouridine and its influence on stability and selectivity of oligonucleotide binding to RNA.

Hybridization to RNA is important for many applications, including antisense therapeutics, RNA interference, and microarray screening. Similar thermodynamic stabilities of A-U and G-U base pairs result in difficulties in selective binding to RNA. Moreover, A-U pairs are weaker than G-C pairs so that binding is sometimes weak when many A-U pairs are present. It is known, however, that replacement of uridine with 2-thiouridine significantly improves binding and selectivity. To test for additional improvement of binding and of the specificity for binding A over G, LNA-2-thiouridine was synthesized for the first time and incorporated into many LNA-2'-O-methyl-RNA/RNA duplexes. UV melting was used to measure the thermodynamic effect of replacing 2'-O-methyluridine with 2'-O-methyl-2-thiouridine or LNA-2-thiouridine. The 2-thiouridine usually enhances binding and selectivity. Selectivity is optimized when a single 2-thiouridine is placed at an internal position in a duplex.

A direct synthetic approach to uracil anhydrothionucleoside derivatives.

Direct conversion of peracylated N(1)-(beta-D-glucopyranosyl)-2-thiouracil derivatives into the corresponding anhydrothionucleosides has been studied under various conditions including: gas-phase pyrolysis, heating without a solvent, and by heating in a solvent of high boiling point (DPE) in the presence of a base (DABCO) and reaction in a microwave reactor. Heating at 210-220 degrees C was found to give the best yield of a single isomer. The structures of the new anhydrothionucleosides were confirmed by NMR techniques.

Preparation of short interfering RNA containing the modified nucleosides 2-thiouridine, pseudouridine, or dihydrouridine.

Modified uridine derivatives such as 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D) are naturally existing nucleoside units identified in tRNA molecules. Recently, we have shown that such base-modified units introduced into functionally important sites of siRNA modulate thermodynamic stability of the duplex and its gene silencing activity. In this unit, we describe chemical synthesis of 3'-phosphoramidite derivatives of s(2)U and D units (the 3'-phosphoramidite derivative of Psi is commercially available), and their use for the synthesis of RNA oligonucleotides according to the routine phosphoramidite protocol. The only exception concerns the oxidation step with I(2)/pyridine/water which, if applied towards oligonucleotides containing s(2)U units, would lead to the loss of sulfur. Therefore, to avoid this side reaction, tert-butyl hydroperoxide is used as an oxidizing reagent. After the oligonucleotide chain assembly is completed, the resulting oligomer is deprotected under mild basic conditions (MeNH(2)/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side-reaction during the routine synthesis of dihydrouridine-containing RNA. Oligonucleotides modified with s(2)U, D, or Psi units are useful models for structure-function studies. Here, the procedure for preparation of siRNA duplexes is described.

Chemical synthesis and properties of 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine.

Unique taurine-containing uridine derivatives, 5-taurinomethyluridine (tau m5U) and 5-taurinomethyl-2-thiouridine (tau m5s2U), which were discovered in mammalian mitochondrial tRNAs, exist at the first position of the anticodon. In this paper, we report the first efficient synthesis of tau m5U and tau m5s2U and describe their physicochemical properties. These modified ribonucleosides were synthesized by the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents. UV and 1H NMR spectrometric studies revealed the structural properties of the taurine-containing base moieties and the sugar conformations of these modified ribonucleosides.

Activity of siRNAs with 2-thio-2'-O-methyluridine modification in mammalian cells.

In a search to identify chemical modifications to improve the properties of siRNA, we have investigated the effect of the 2 '-O-methyl-2-thiouridine modification on the biological activity of siRNA. Our results indicate that judicious placement of 2 '-O-methyl-2-thiouridine residues could lead to modified siRNA with activity in mammalian cells.

Chemical synthesis of RNA including 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine.

Recently, novel base-modified uridine derivatives, 5-taurinomethyluridine (tau m(5)U) and 5-taurinomethyl-2-thiouridine (tau m(5)s(2)U) have been discovered from mammalian mitochondrial tRNAs, and these modified ribonucleosides were found to exist at the first position of the anticodon. We herein report efficient reactions for the synthesis of RNA oligomers including these base-modified ribonucleosides.

beta-Selective synthesis of 2'-deoxy-5,6-dihydro-4-thiouridine, a precursor of the unstable nucleoside product of ionising radiation damage 2'-deoxy-5,6-dihydrocytidine.

4-Thio oxathiaphosphepane nucleosides - undergo a rearrangement in pyridine that leads selectively to the beta anomer of the 2'-deoxy-5,6-dihydro-4-thiouridine derivative . This diastereoselective reaction proceeds through a multistep mechanism initiated by the addition of pyridine at the C1'position of - and concomitant opening of the oxathiaphosphepane. This was confirmed by the trapping of the corresponding intermediate in the closely related DMAP series. In contrast, LR thiation of in pyridine leads to a new class of modified nucleosides containing an oxathiaphospholane moiety. The quantitative conversion of into the corresponding 5,6-dihydrocytosine derivative with NH3-MeOH is also reported.

Efficient synthesis of 2',3'-dideoxy-2'-amino-3'-thiouridine.

[reaction: see text] Metal ion rescue experiments provide a powerful approach to establish the presence and role of divalent metal ions in the biological function of RNA. The utility of this approach depends on the availability of suitable nucleoside analogues. To expand the range of this experimental strategy, we describe the first synthesis of 2',3'-dideoxy-2'-amino-3'-thiouridine (12) in 19.5% overall yield starting from 2,2'-anhydrouridine (1).

4-Thio-5-bromo-2'-deoxyuridine: chemical synthesis and therapeutic potential of UVA-induced DNA damage.

4-Thio-5-bromo-2'-deoxyuridine (3a) is prepared from 5-bromo-2'-deoxyuridine (BrdU) and its key properties are explored. The thionucleoside (3a) can react readily with monobromobimane and produces high fluorescence. 3a has UV maximum absorption at 340 nm and can be incorporated into cellular DNA. The cells containing 3a become sensitive to UVA light, offering therapeutic potential for UVA-induced cell killing.

A new route to 2'-O-alkyl-2-thiouridine derivatives via 4-O-protection of the uracil base and hybridization properties of oligonucleotides incorporating these modified nucleoside derivatives.

Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without formation of a mismatched base pair with the guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine and various 2'-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified nucleoside derivatives were synthesized, and their hybridization and structural properties were studied in detail by the 1H NMR analysis of these modified nucleosides and Tm experiments of RNA duplexes with their complementary RNA strands.

Synthesis and properties of 2'-O-alkylated 2-thiouridine derivatives and oligonucleotides containing 2'-O-alkylated 2-thiouridine derivatives.

Oligonucleotides containing 2-thiouridine form stable RNA duplexes with complementary RNAs and show high selectivity in the base pair recognition. Moreover, alkylation of the 2'-hydroxyl group provides new insight into developments of new antisense RNAs capable of formation of more stable duplexes with target RNAs. Oligonucleotides having such properties are desirable as agents for the antisense strategy and SNPs analysis. In this study, we report a new method for the synthesis of 2-thiouridine and its 2'-O-alkylated derivatives. The hybridization and structural properties of these modified nucleosides were also studied in detail by the 1H NMR analysis and melting temperature (Tm) measurement of RNA duplexes.

Thiation of 2'-deoxy-5,6-dihydropyrimidine nucleosides with Lawesson's reagent: characterisation of oxathiaphosphepane intermediates.

Treatment of 2'-deoxy-3',5'-dithexyldimethylsilyl-5,6-dihydrouridine with Lawesson's reagent led to the expected C4-thiolated derivative together with a number of oxathiaphosphepane isomers which resulted from the heat reversible incorporation of an AnPS2 unit within the 2'-deoxyribose moiety explaining the subsequent anomerisation of the 5,6-dihydropyrimidine nucleosides.

MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli.

Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.