DNA or RNA Oligonucleotide 2′-Hydrazides for Chemoselective Click-Type Ligation with Carbonyl Compounds

An efficient method for the synthesis of DNA or RNA oligonucleotide 2′-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2′-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH₃CN. The resulting oligonucleotide 2′-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH₃CN reduction to afford novel 2′-conjugates.     

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.     

Trimethylsilyl Trifluoromethanesulfonate-promoted Reductive 2′-O-arylmethylation of Ribonucleoside Derivatives

Arylmethyl groups such as benzyl, p-methoxybenzyl, and 1-pyrenylmethyl groups were introduced to the 2′-O-position of nucleosides by reductive etherification. Combining corresponding aromatic aldehydes with 2′-O-trimethylsilylnucleoside derivatives in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) resulted in moderate to good yields of the 2′-O-arylmethyluridine derivatives, whereas the corresponding cytidine and adenosine derivatives were obtained in low yields. The reaction of ribonucleosides with aliphatic aldehydes did not proceed smoothly. Anomerization of the uridine derivatives by TMSOTf was observed in CH₂Cl₂, toluene, and CH₃CN, but was completely suppressed when the reactions were conducted in 1,4-dioxane.     

REACTION OF N3-BENZOYL-3′,5′-O-(DI-TERT-BUTYLSILANEDIYL)URIDINE WITH HINDERED ELECTROPHILES: INTERMOLECULAR N3 To 2′-O PROTECTING GROUP TRANSFER

ABSTRACT

The compound N³-benzoyl-3′,5′-O-(di-tert-butylsilanediyl)uridine 2 was alkylated with various alkyl iodides in CH₃CN in the presence of base. Normal 2′-O-alkylated products were obtained with methyl or benzyl iodide. if hindered alkyl iodides with β-branching such as 2-ethylbutyl iodide were used as electrophiles under the same conditions, N³-alkyl-2′-O-benzoyl uridine derivatives were produced. This unexpected transformation is usually dormant with reactive alkylating agents, but expressed with sterically hindered, less reactive electrophiles. This unwanted reaction gives isomeric products whose spectra differ in only subtle ways from target compounds.     

Chemical-Enzymatic Synthesis of 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of Natural Heterocyclic Bases and Their 5′-Monophosphates

Abstract

Treatment of O², 3′-anhydro-5′-O-trityl derivatives of thymidine (1) and 2′-deoxyuridine (2) with lithium azide in dimethylformamide at 150 °C resulted in the formation of the corresponding isomeric 3′-azido-2′, 3′-dideoxy-5′-O-trityl-β-D-ribofuranosyl N¹- (the major products) and N³-nucleosides (3/4 and 5/6). 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of thymidine [Thd(3′NH₂)], uridine [dUrd(3′NH₂)], and cytidine [dCyd(3′NH₂)] were synthesized from the corresponding 3′-azido derivatives. The Thd(3′NH₂) and dUrd(3′NH₂) were used as donors of carbohydrate moiety in the reaction of enzymatic transglycosylation of adenine and guanine to afford dAdo(3′NH₂) and dGuo(3′NH₂). The substrate activity of dN(3′NH₂) vs. nucleoside phosphotransferase of the whole cells of Erwinia herbicola was studied.     

Synthesis and Perkow Reaction of Uridine Derivatives

Abstract

Several uridine derivatives with 0-p-toluene sulfonyl groups in 2′- or 3′-position were prepared and their oxidation to the corresponding uloses studied. By Perkow reaction these α-tosyl ketones led to enol-phosphates which were subjected to hydrogenation and hydrolysis. This procedure represents a facile access to uridine derived deoxy uloses.     

Uridine diphosphate 2-deoxyglucose: Chemical synthesis,enzymic oxidation and epimerization

The paper describes chemical synthesis of uridine diphosphate 2-deoxyglucose (UDPdGlc) through reaction of uridine 5′-phosphomorpholidate with 2-deoxy-α-d-glucopyranosyl phosphate. The prepared analog of uridine diphosphate glucose (UDPGlc) served as a substrate for calf liver UDPGlc dehydrogenase (EC 1.1.1.22), the reaction product was identified as nucleotide deoxyhexuronic acid derivative. The apparent K_m for UDPdGlc was found to be 60 times that of UDPGlc, and the relative V value for the analog was 0.09. The peculiar lag-period in reaction kinetics has been observed for the analog, and is presumably connected with the slow rate of the initial stages of the reaction. UDPdGlc was found to be quite an efficient substrate for UDPGlc 4-epimerases (EC 5.1.3.2) from yeast, calf liver and mung bean seedlings.     

mt-tRNA Components: Synthesis of (2-Thio)Uridines Modified with Blocked Glycine/Taurine Moieties at C-5,1

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2′,3′-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)₃) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm⁵(s²)U) and 5-taurinomethyl(-2-thio)uridine (τm⁵(s²)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2′,3′-O-isopropylidene-2-thiouridine is also reported.     

Synthesis and NMR Spectra of Some New Carbohydrate Modified Uridine Phosphoramidites

Abstract

The one step reaction of 2′- and 3′-keto derivatives of uridine with bromodifluoromethyl[tris(dimethylamino)]phosphonium bromide and zinc gives the corresponding 2′- and 3′-difluoromethylene nucleosides in good yield. Desilylation and phosphitylation of the resultant 2′- or 3′-hydroxyls provides the target 2′- and 3′-phosphoramidites 7 and 8 for use in oligonucleotide synthesis¹.     

Synthesis and properties of nucleoside derivatives acylated by chemically stable 2-(trimethylsilyl)benzoyl group

We report the synthesis and properties of nucleoside derivatives acylated by 2-(trimethylsilyl)benzoyl (TMSBz) that proved to be extremely stable under basic conditions when introduced into the 5′-hydroxyl group of thymidine, the 4-amino group of deoxycytidine and the 2′-hydroxyl group of uridine. In particular, 2′-O-TMSBz-uridine could be isolated and was more stable in pyridine, while it isomerized in CH₂Cl₂ in the presence of Et₃N to yield a mixture of the 2′-O- and 3′-O-acylated species.     

Nucleosides. 145. Synthesis of 2,5′-Anhydro-2-Thiouridine and its Conversion to 3′-O-Acetyl-2,2′-Anhydro-5′-Chloro-5′-Deoxy-2-Thiouridine. Studies Directed Toward the Synthesis of 2′-Deoxy-2′-Substituted arabino Nucleosides (7)

Abstract

In an attempt to introduce a substituent at C-2′ in the “up” arabino configuration directly by nucleophilic displacement reaction of a preformed pyrimidine ribonucleoside, we synthesized 2,5′-anhydro-5′-deoxy-2-thiouridine (6) in three steps from uridine. Compound 6 was converted into the 3′-O-acetyl derivative 7. Upon treatment of 7 with triflyl chloride in methylene chloride in the presence of triethylamine and p-dimethylaminopyridine, 2,2′-anhydro-1-(3-O-acetyl-5-chloro-2,5-dideoxy-β-D-arabinofuranosyl)-2-thiouracil (9) was obtained as the only isolable product. Obviously, the intermediate 3′-O-acetyl-2,5′-anhydro-2′-O-triflyl-2-thiouridine (8) was attacked by the chloride nucleophile at C-5′ first giving the 2′-O-triflyl-2-thiouridine intermediate from which 9 was formed by intramolecular nucleopilic reaction.     

Microwave-Assisted Synthesis of O′-Adamantylated Uracil-Derived Nucleosides

Abstract

Microwave-induced synthesis of O′-adamantyl derivatives of AZT, thymidine, 2′-deoxyuridine and uridine was investigated. Contrary to heterocyclus adamantylation of uracil and uridine in trifluoroacetic acid, the microwave-induced reaction provided sugar-substituted compounds.     

Bacterial Synthesis of Nucleotides

Inosine-5′, 2′(or 3′)-diphosphate was prepared by incubating 5′-IMP and p-nitrophenyl-phosphate with the bacteria characterized to phosphorylate at C_{3′ (&2′)}, or, on the contrary, by incubating 2′-IMP and a donor with the others capable of synthesizing 5′-nucleotide, via their phosphoryl transfer reactions.

Formation of the 5′, 2′(or 3′)-diphosphates of guanosine, cytidine, and uridine was also demonstrated to be carried out under the same relationship between nucleotide isomer as an acceptor and specificities of bacterial phosphotransferases, as observed in the phosphorylation of adenylic acid isomers, while 5′-dTMP was phosphorylated by both groups of bacteria.     

Some Aspects of Ribonucleoside Chemistry

Abstract

New routes to the preparations of 2′-deoxy-3′-C-methyl uridine (2c) and 1-(5′-0-trityl-3′-deoxy-β-D-glycero-pentofuran-2-ulosyl)uracil (4) from 5′-0-trityl-2′-0-tosyl uridine (1) and 5′-0-trityl-3′-0-tosyl uridine (3) respectively are described.     

Chromium(III) interactions with nucleotides. II

New derivatives were obtained from Cr(urea)₆Cl₃· 3H₂O in an ethyl acetate medium of chromium(III) with uracil, uridine, 5′UMP, 5′CMP, 5′GMP and 5′IMP. The new derivatives were characterized by elemental analysis, electronic and infrared spectroscopy and thermal analysis. These derivatives proved to be outer sphere complexes, in which the nucleotide, the nucleoside or the base interacts with the starting complex through intramolecular hydrogen bonding.Cr(XMP)(OH)·3H₂O (XMP: 5′UMP, 5′CMP, 5′GMP and 5′IMP) complexes were obtained by hydrolysis of the above derivatives of the nucleotides. In these reactions there is a total substitution of the urea molecules. The derivatives obtained by hydrolysis were characterized in solid state by electronic and infrared spectroscopy. These results provide more insight into the biological role of chromium.     

Efficient Synthesis of 2′-Bromo-2′-Deoxy-3′,5′-O-TPDS-pyrimidine Nucleosides by Boron Trifluoride Catalyzed Reaction of O2,2′-Anhydro-(1-β-D-Arabinofuran0Syl)pyrimidine Nucleosides with Lithium Bromide

Abstract

Efficient syntheses of 2′-bromo-2′-deoxy-3′,5′-O-TPDS-uridine (5a) and 1-(2-bromo-3,5-O-TPDS-β-D-ribofuranosyl)thymine (5b) from uridine and 1-(β-D-ribofuranosyl)thymine are described, respectively. The key step is a treatment of 3′,5′-O-TPDS-O²,2′-anhydro-1-(β-D-ardbinofuranosyl)uracil (4a) and -thymine (4b) with LiBr in the presence of BF₃-OEt₂ in 1,4-dioxane at 60°C to give 5a and 5b in 98%, and 96% yield, respectively.

     

Photochemistry of the O-Nitrobenzyl Protecting Group in RNA Synthesis

Abstract

UV irradiation of 2′-O (o-nitrobenzyl)adenylyl(3′-5′)uridine in the presence of O₂ yields the corresponding nitrobenzoyl derivative in addition to the expected A-U. A mechanism proposed for this oxidation involves the successive removal of the two benzylic protons with a hydroperoxide as the intermediate between the two steps.     

Effect of the sulfur atom on S2 and S4 positions of the uracil ring in different DNA:RNA hybrid microhelixes with three nucleotide base pairs

The effect of the sulphur atom on the uracil ring was analyzed in different DNA:RNA microhelixes with three nucleotide base-pairs, including uridine, 2-thiouridine, 4-thiouridine, 2,4-dithiouridine, cytidine, adenosine and guanosine. Distinct backbone and helical parameters were optimized at different density functional (DFT) levels. The Watson-Crick pair with 2-thiouridine appears weaker than with uridine, but its interaction with water molecules appears easier. Two types of microhelixes were found, depending on the H-bond of H2′ hydroxyl atom: A-type appears with the ribose ring in ³E-envelope C_3′-endo, and B-type in ²E-envelope C_2′-endo. B-type is less common but it is more stable and with higher dipole-moment. The sulphur atoms significantly increase the dipole-moment of the microhelix, as well as the rise and propeller twist parameters. Simulations with four Na atoms H-bonded to the phosphate groups, and further hydration with explicit water molecules were carried out. A re-definition of the numerical value calculation of several base-pair and base-stacking parameters is suggested.     

Studies on the ribothymidine content of specific rat and human tRNAs: a postulated role for 5-methyl cytosine in the regulation of ribothymidine biosynthesis.

Total mammalian tRNAs contain on the average less than one mole of ribothymidine per mole of tRNA. Mammalian tRNAs can be grouped into at least four classes, depending upon their ribothymidine content at position 23 from the 3′ terminus. Class A contains tRNA in which a nucleoside other than uridine replaces ribothymidine (tRNA_i^Met); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNA^Ser, tRNA^Trp, and tRNA^Lys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNA^Phe, tRNA₁^Gly, tRNA₂^Gly); Class D contains tRNA which totally lacks ribothymidine (tRNA^Val). Only those tRNAs in Class C are acceptable substrates for $\text{E.}\text{coli}$ uridine methylase, under the conditions used in these studies. These observations cannot be adequately explained solely on the basis of the presence or absence of a specific “universal” nucleoside other than U or rT at position 23 from the 3′ terminus. However, correlations can be made between the ribothymidine and 5-methylcytosine content of eucaryotic tRNA. We postulate that the presence of one or more 5-methylcytosines in and adjacent to loop III (minor loop) in individual tRNAs act to regulate the amount of ribothymidine formed by uridine methylase. Several experiments are proposed as tests for this hypothesis.     

Design and Synthesis of 2′-Deoxy-2′-[(1,2,3)Triazol-1-Yl]Uridines Using Click Chemistry Approach

A series of novel nucleosides bearing a 1,2,3-triazole moiety at the 2′-position of the sugar moiety has been synthesized starting from 2′-azidouridine and using the copper (I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction. The reactions proceeded in overall yield of 52–82% and gave almost exclusively the 1,4-disubstituted 1,2,3-triazoles. The 2′-azidouridine was synthesized from uridine in two steps, and reacted with a variety of differently substituted alkynes to give the desired 2′-triazole-substituted uridine derivatives.