Structure of an 11-mer DNA Duplex Containing the Carbocyclic Nucleotide Analog: 2′-Deoxyaristeromycin

Abstract

2′-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the action of DNA glycosylases. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of an 11-mer DNA containing a single dAr?T base pair at its center. Analysis of the spectra revealed the existence of a right-handed duplex in solution, stabilized by Watson-Crick hydrogen bonding and base-stacking interactions. The carbocyclic sugar adopted a C1′-exo conformation and sugars of the 3′-flanking base pair had puckers in the O4′—endo range. The dAr?T base pair was mildly propeller twisted, and the dAr analogue showed a positive roll with the 3′-flanking base. Our findings indicate that the observed resistance of dAr-containing oligodeoxynucleotides to the catalytic action of DNA glycosylases relates to its electronic properties rather than structure, and validate the use of dAr and related carbocyclic nucleoside analogues for biological and structure/function relationship studies.     

Synthesis and DNA Duplex Stabilities of Oligonucleotides Containing C-5-(3-Methoxypropynyl)-2′-deoxyuridine Residues

Abstract

5′-Dimethoxytrityl-5-(3-methoxypropynyl)-2′-deoxyuridine phosphoroamidite was synthesized with the use of commercial 3- methoxypropyne. Oligonucleotides (ODNs) containing 5-(3- ethoxypropynyl)- 2′-deoxyuridine in different positions were prepared. The stabilities of the duplexes formed by these ODNs with the complementary templates are increased in comparison with the unmodified counterparts. On average modified residue incorporated, the Tm is raised by 1°C.     

Synthesis of Oligonucleotides Containing 2′-Deoxyguanosine Adducts of Nitropyrenes

Two different approaches to synthesize oligonucleotides containing the 2 ′-deoxyguanosine adducts formed by nitropyrenes are described. A direct reaction of an unmodified oligonucleotide with an activated nitropyrene derivative is a convenient biomimetic approach for generating the major adducts in DNA. A total synthetic approach, by contrast, involves several synthetic steps, including Buchwald-Hartwig Pd-catalyzed coupling, but can be used for incorporating both the major and minor adducts in DNA in high yield.     

Spin Labeled Nucleoside Analogues: 4′-Hydroxymorpholin-2′-Ylpurines and Pyrimidines

Abstract

Upon borane-pyridine reduction, a series of nucleoside dialdehyde dioximes 2 underwent cyclization to the corresponding 4′-hydroxymorpholin-2′-ylpurines or pyrimidines 3 from which the peracetyl derivatives 4 were prepared. At room temperature, compounds 3 and 4 exist as a mixture of invertomers in which the 4′S (equatorial 4′-OH or 4′-OAc) predominates. A 14 kcal/mol, nitrogen inversion barrier was estimated from variable temperature experiments. N.O.E. and ³J_CH measurements established the anti conformation of the base-“sugar” bond. Compounds 3 spontaneously oxidized to the corresponding aminoxyl free radicals, EPR spectra of which showed that they existed in a chair conformation.     

Synthesis and Biological Activity of a Bis-Substituted 3′-Deoxyadenosine Analog of 2–5A

Abstract

The 5′-monophosphate, p5′(3′dA)2′p5′A2′5′(3′dA), was synthesized and found to bind to the 2–5A-dependent endonuclease of mouse L cells only two-three times less effectively than the parent p5′A2′p5′A2′p5′A. When evaluated for its ability to activate the 2–5A-dependent endonuclease, ppp5′(3′dA)2′p5′A2′p5′(3′dA) was found to be fifty times more effective than ppp5′A2′p5′(3′dA)2′p5′A and ten times less effective than 2–5A as an endonuclease activator     

Convenient Synthesis of Oligodeoxynucleotides Containing 2′-Deoxy-6-thioinosine

Abstract

A facile synthesis of oligodeoxynucleotides (ODN) containing 2′-deoxy-6-thioinosine (dI^6S) based on the convertible nucleoside O6-phenyl-2′-deoxyinosine is presented. After standard solid-phase DNA synthesis and removal of the cyanoethyl protecting groups with DBU treatment with aqueous sodium hydrogen sulfide introduces the sulfur functionality, deprotects the other nucleobases and cleaves the ODN from the solid support in a one-pot reaction. In addition, the extinction coefficient of 2′-deoxy-6-thioinosine is determined by enzymatic fragmentation of the resulting ODN in the presence of adenosine deaminase.     

Hoogsteen-Duplex DNA: Synthesis and Base Pairing of Oligodeoxynucleotides Containing 1-Deaza-2′-deoxyadenosine

Abstract

Solid-phase synthesis of oligonucleotides containing 1-deazaadenine was carried out employing phosphonate and phosphoramidite chemistry. Hoogsteen base pairing was established for the duplex d(c¹A₂₀)·d(T₂₀).     

Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.     

Synthesis and Photochemical Conversion of Oligonucleotides Containing 2-Chloro-2′-Deoxyadenosine

Abstract

The rate and velocity of the photoconversion of 2-chloro-2′-deoxyadenosine into 2′-deoxyisoguanosine within oligonucleotides was found to be sequence-specific and depends on the nearest neighbor.     

Synthesis of 2′-Modified Oligonucleotides Containing Aldehyde or Ethylenediamine Groups

Abstract

Oligonucleotides carrying 2′-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2′-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2′-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.     

Synthesis and Properties of Oligonucleotides Containing 5-Aza-2′-deoxycytidine

Abstract

The preparation of a protected derivative of 5-aza-2′-deoxycytidine carrying the 2-(p-nitrophenyl)ethyl group is described. The new derivative is useful for the preparation of oligonucleotides containing 5-aza-2′-deoxycytidine using a special methodology that avoids the use of ammonia.     

Synthesis and Properties of 2′-O-Methylribonucleotide Methylphosphonate Containing Chimeric Oligonucleotides

Abstract

2′-O-methylribonucleoside methylphosphonamidites are synthesized and incorporated into oligonucleotides to obtain chimeric antisense oligonucleotides. The resulting oligonucleotide binds to their target RNA/DNA sequences efficiently and stable in a medium containing bovine serum.     

Synthesis and Activity of Oligonucleotides Containing a Biologically Active Nucleoside at the 2′End

Abstract

The antiviral activity of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdUrd), acyclovir and other antiherpetic nucleosides depends on a selective phosphorylation by the herpesvirus-induced thymidine kinase in the infected cells. Viruses not encoding a specific thymidine kinase (TK) activity are resistant to the action of these nucleoside analogues. The nucleoside monophosphates are as such poorly taken up by the cells. In order to circumvent the necessity of intracellular phosphorylation, we synthesized four core oligonucleotides bearing a biological active nucleoside at the 2′end. It was hypothesized that these core oligonucleotides, like core 2–5A itself, would be taken up within the cell and that, following intracellular 2′-5′ phosphodiesterase cleavage, the 5′-monophosphate of the active product would be formed. In these circumstances, activity against TK- strains could be expected.     

Design and Facile Synthesis of New Dinucleotide Cap Analog Containing Both 2′ and 3′-OH Modification on M7Guanosine Moiety

The first example of the synthesis of new dinucleotide cap analog containing 2^′,3^′-diacetyl group on m⁷guanosine moiety is described. The desired modified cap analog, m^{7,2′,3′–diacetyl}G[5^′]ppp[5^′]G has been obtained by the coupling reaction of triethylamine salt of m^{7,2′,3′–diacetyl}GDP with ImGMP in presence of ZnCl₂ as a catalyst in 62% yield with high purity. The structure of new cap analog has been confirmed by ¹H and ³¹P NMR and mass data.     

Synthesis and Properties of an Initiation Codon Analog Consisting of 2′-O-Methyl Nucleotides

Abstract

An initiation codon analog consisting of 2′-O-methyl nucleotides (AmUmG) was synthesized and examined for its binding efficiency to E. coli ribosomes with fMetRNA^fMet and also its stability in binding assay systems for comparison with those of r(AUG) and d(ATG). AmUmG was found completely resistant to nucleases under the conditions used.     

Solid Phase Synthesis of 2′, 5′-Oligoadenylates Containing 3′-Fluorinated Ribose

Abstract

2′, 5′-phosphodiester bond-linked oligoadenylate trimers with 3′-fluoro-3′-deoxyadenosine residues incorporated at specific positions of the nucleotide sequence were synthesized by the solid phase phosphite triester (phosphoramidite) method. The syntheses were in the 2′ to 5′ direction and were performed manually using commercially available microcolumns. The oligonucleotides were 5′-end phosphorylated on the support before deprotection.     

The Synthesis of Oligonucleotides Containing Fluoro-2′-Deoxycytidine for Secondary Structure Determination of Tandem Tetraloop DNA Analogs

Abstract

A method to convert 5-FdU phosphoramidite to 4-triazol-5- FdU phosphoramidite was used to make deoxyoligonucleotides containing 5- FdC to follow a rare transition between a tandem hairpin and a single hairpin form, which results in moving a distant 5-FdC, 5-FdU bases into juxtaposition.     

Study of the Efficacy of a Pronucleotide of 2-Chloro-2′-Deoxyadenosine in Deoxycytidine Kinase-Deficient Lymphoma Cells

2-Chloro-2 ′-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 ′-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.     

5′-Substituted 2′-Deoxycytidines as Non-Substrate Inhibitors of Human Deoxycytidine Kinase

Abstract

Several 5′-substituted analogues of 2′-deoxycytidine (dC), including 5′-amino2′,5′-dideoxycytidine (5′-amino-ddC), 5′-azido-2′,5′-dideoxycytidine (5′-azido-ddC) and 5′-O-ethyl-2′-deoxycytidine (5′-O-ethyl-dC), are non-substrate inhibitors of human dC kinase. Whereas 5′-amino-ddC inhibits phosphorylation of deoxyadenosine (dA) with K_i ～ 1 μ inhibition of phosphorylation of dC is bimodal and more effective at low inhibitor concentrations. In particular 5′-O-ethyl-dC inhibits phosphorylation of dA 10-fold more effectively (K_i ～ 3 μ) than that of dC (K_i ～ 30 μ). For 5′-azido-ddC inhibition was shown directly to be competitive with respect to substrate.