Methoxymethyl and (p-nitrobenzyloxy)methyl groups in the synthesis of oligoribonucleotides by the phosphotriester method

An efficient synthetic method for monomer ribonucleotide synthons containing 2'-O-methoxymethyl and 2'O-(p-nitrobenzyloxy)methyl groups used for oligonucleotide phosphotriester method with O-nucleophilic intramolecular catalysis at the stage of formation of internucleotide bond is developed. It is shown that synthons containing protecting 2'-O-(p-nitrobenzyloxy)methyl group may be used for automatic synthesis of phosphotriester oligoribonucleotides with high yields and synthons containing methoxymethyl group--to get 2'-O-modified oligonucleotides.     

Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages.

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.     

Facile and Rapid Deprotection Conditions for the Cleavage of Synthetic Oligonucleotides from 1,4-Anhydroerythritol-Based Universal Polymer Support

In our previous report [Kumar, P.; Dhawan, G.; Chandra, R.; Gupta, K.C. Polyamine-assisted rapid and clean cleavage of oligonucleotides from cis-diol bearing universal support. Nucl. Acids Res. 2002, 30, e130 (1-8)], we demonstrated polyamine-mediated deprotection of oligonucleotides from cis-diol group bearing universal polymer support (I). However, vulnerability of the conventional dC^bz to modifications under these conditions compelled us to employ dC^ac during synthesis of oligonucleotide using conventional synthons. Here, a new set of simple and rapid deprotection conditions has been developed for the complete cleavage of oligonucleotides from the 1,4-anhydroerythritol-based universal polymer support employing conventional dC^bz synthon. Using manganese-imidazole complex in aqueous ammonium hydroxide (～30%), fully deprotected oligonucleotide sequences were obtained in 40 min, which were analyzed on reverse phase-HPLC and compared with the standard oligomers in terms of their retention time. Finally, their biological compatibility was established by analyzing PCR amplified products of npsA gene of N. meningitidis.     

A convenient method for the synthesis of phosphoramidite non-nucleotide inserts for preparation of functionalized oligonucleotides

A simple approach to the synthesis of amidophosphite synthons of achiral non-nucleotide inserts using 4-(2-(4,4??-dimethoxytrityloxy)ethyl)morpholine-2,3-dione as a backbone of key precursor was suggested. Non-nucleotide synthons were synthesized using this approach that were suitable for synthesis of acridine-containing oligonucleotide derivatives, as well as oligonucleotides with branched carbohydrate-phosphate backbone.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligonucleotide phosphotriester method using O-nucleophilic intramolecular catalysis at the stage of the internucleotide bond formation. The former synthons may be used for the automatic synthesis of 2′-modified oligonucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high yields.     

Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2'-fluoronucleoside and arabinofuranosylcytosine]

A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.     

Oxidative DNA cleavage by free and oligonucleotide-conjugated O-bromobenzoic acid in the presence of copper(II) ions

o-Bromobenzoic acid was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base modification and backbone cleavage. The oligonucleotide conjugate bearing 5-(4'-aminopropyl-sulfomoyl)-2-bromobenzoic acid as a reactive group was synthesized and DNA cleavage activity of this oligonucleotide conjugate was tested on a model deoxyoligonucleotide.     

Carboranyl oligonucleotides: 4. synthesis and physicochemical studies of oligonucleotides containing 2'-O-(o-carboran-1-yl)methyl group

Boronated oligonucleotides are potential candidates for antisense oligonucleotide technology (AOT), boron neutron capture therapy (BNCT), and as tools in molecular biology. A method was developed for the solid phase synthesis of oligonucleotides containing 2'-O-(o-carboran-1-yl-methyl) (2'-CBM) group. Synthesis was performed using a standard beta-cyanoethyl cycle and automated DNA synthesizer. Manual steps were performed for the insertion of a modified monomer bearing the 2'-CBM group. Several tetradecanucleotides complementary to DNA-HCMV, and bearing 2'-CBM modification near the 3'-end or 5'-end or in the middle of the oligonucleotide chain were synthesized. The resulting oligomers were characterized by polyacrylamide gel electrophoresis (PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and ultraviolet spectroscopy (UV), circular dichroism (CD), and melting temperature (Tm) measurements. Tm of duplexes formed between 2'-CBM-modified tetradecanucleotides and complementary DNA and RNA template were compared with those formed by the unmodified oligonucleotide and complementary sequence. The stability of 2'-CBM oligonucleotides in the presence of phosphodiesterase I from Crotalus atrox venom and in human serum was studied. Oligonucleotides bearing the 2'-CBM group are characterized by increased resistance to enzymatic digestion, increased lipophilicity, and the ability to form stable duplexes with complementary templates.     

Oligodeoxyribonucleotide Phosphorothioates: Substantial Reduction of (N-1)-mer Content Through the Use of Trimeric Phosphoramidite Synthons

Abstract

Use of fully protected trimeric phosphoramidite synthons in the synthesis of oligonucleotide phosphorothioate shows a substantial reduction (>85%) in (n-1)-mer content as compared to oligomers synthesized through coupling of standard phosphoramidite monomers. A 20-mer oligodeoxyribonucleotide phosphorothioate which is in phase I clinical trials was chosen as an example for the studies.     

Novel method of the synthesis and hybridization properties of an oligonucleotide containing non-ionic diisopropylsilyl internucleotide linkage

Efficient synthesis of a dithymidine dinucleotide analog bearing a diisopropylsilyl linkage instead of a phosphodiester linkage is described with respect to its incorporation into oligonucleotides. The diisopropylsilyl linkage was introduced into the oligonucleotide by preparation of the phosphoramidite derivative of a dithymidine dimer unit. The diisopropylsilyl-modified oligonucleotide exhibited hybridization behavior with both single strand and duplex DNA. The thermal stability of both the duplex and triplex showed a relative instability compared to the corresponding natural phosphodiester DNA, because of the steric hindrance of the isopropyl group on the silicon atom.     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: I. Chemistry and Dynamics of Nucleotide Arylation with Pentafluoropyridine; Obtaining of Synthons for Molecular Design of Nucleic Acid Analogues

Pentafluoropyridine reacts with thymidine, adenosine, and uridine hydroxy groups to give quantitative yields of the corresponding nucleoside di- and triaryl ethers. The nucleophilic substitution reactions proceed successively and in parallel, with the slowest step being the nucleophilic substitution of the nucleoside secondary hydroxyls. The resulting ethers contain tetrafluoropyridyl moieties, which could be smoothly modified by nucleophilic substitution of fluorine atoms. The ethers are useful intermediate synthons (both isolated and in situ) for molecular design of oligonucleotide analogues.     

Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides

We describe the preparation of oligonucleotides containing a 5'-linker bearing an aminooxy group. Use of the trityl protecting group for the aminooxy moiety allows purification of the modified oligonucleotide by reverse phase HPLC and cleavage in mild acidic conditions. Derivatization with an aldehydic reporter group is efficient and rapid.     

Oligonucleotides conjugated to short lysine chains

A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2'-amino-2'-desoxy-uridine nucleotide bearing a succinyl linker at the 2' position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.     

Immobilization of acrylamide-modified oligonucleotides by co-polymerization.

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.     

Dimeric Oligonucleotide Synthons Containing 2-Deoxy-3-O-Phosphonomethyl-α-D-erythro-pentofuranosyl Nucleosides

Abstract

The synthesis of 2′-deoxy-3′-O-phosphonomethyl-α-D-erythro-pentofuranosyl-thymine and -adenine (1, B is Thy or Ade) is described. Dimeric oligonucleotide synthons such as 2 containing isosteric phosphonate internucleoside linkage were prepared.     

C-Difluoromethylene-containing, C-trifluoromethyl and C-perfluoroalkyl carbohydrates. Synthesis by carbohydrate transformation or building block methods

The synthetic methods for preparing carbohydrates bearing a C-branched substituent of the type CF2-Y, with Y = F, Y = CnF(2n + 1) or Y = a carbon-attached or heteroatom-attached nonfluorinated residues, are reviewed. Both direct introduction of C-branched fluorinated substituents (direct trifluoromethylation, perfluoroalkylation or difluoromethylenation) and building block methods from fluorinated synthons are considered.     

Acyclic Nucleotide Analogues and Related Compounds

Abstract

Acyclic nucleotide analogues bearing amino- and N-substituted amino groups in the side chain were prepared by alkylation of the bases with corresponding oxiranes and subsequent introduction of phosphonomethyl ether function. Novel enantiomeric synthons for the preparation of HPMP-compounds were prepared from a common intermediate and applied to syntheses of novel compounds (e.g. 8-azaguanine derivatives).     

Synthesis of an Oligonucleotide Bearing a Phosphate Function at the 5′-Terminus Using a Novel Protecting Group in Terms of a Cellulose Acetate Derivative as a Polymer-Support

Abstract

Synthesis of the title oligonucleotide bearing a phosphate function at the 5′-terminus, i.e., pCpUpCpGpUpCpCpApCpCpA, by the use of the terminal cytidylic acid unit involving a novel protecting group of 2-[2-(monomethoxytrityloxy)ethylthio]ethyl group on its 5′-phosphoryl function in terms of a cellulose acetate polymer-support is described.     

A Facile One-pot Synthesis of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Urethane Protected 3-Amino Alkyl Isoxazole-5-Carboxylic Acids and their Utility for the Preparation of Isoxazole-Linked Peptidomimetics

Cu(I) catalyzed [3+2] cycloaddition of in situ generated N ^α-Fmoc/Boc/Z-protected amino alkyl nitrile oxide with propiolic acid has led to a new class of 3,5-disubstituted isoxazole-bearing amino acid derivatives. The click chemistry protocol described herein is efficient in furnishing the isoxazole embedded amino acids. These unnatural synthons were subjected to chain extension on N- as well as C-termini to obtain 3,5-disubstituted isoxazole bearing di and tripeptidomimetics.