Synthesis Of Oligodeoxyribonucleotides Research Articles

ChemInform Abstract: Solid‐Phase Synthesis of Oligodeoxyribonucleotides Without Base Protection Utilizing O‐Selective Reaction of Oxazaphospholidine Derivatives.

AbstractChlorooxazaphospholidine (II) exclusively reacts with the 3‐OH group of nucleosides (III) in the presence of unprotected nucleobase amino groups to afford the nucleoside O‐oxazaphospholidine monomers (IV) in moderate to good yields.

Improved synthesis of oligodeoxyribonucleotides.

An improved synthesis of oligodeoxyribonucleotides was achieved with NPE- and NPEOC-protected phosphoramidites leading to easier and time-saving purification and isolation of the gene fragments.

ChemInform Abstract: Development of New Protecting Groups for Guanine Residue in Oligodeoxyribonucleotide Synthesis.

Attempts were made to develop new protecting groups for 1,6-lactam function of 2-N-acyl guanine in oligodeoxyribonucleotide synthesis. Several acyl groups, aryl groups, and carbamoyl groups were tested. Dimethylcarbamoyl and phenylacetyl groups are shown to be a good combination for guanine residue. 6-O-Di-methylcarbamoyl-2-N-pheylacetyl-2'-deoxy guanosine have been successfully used in the synthesis of d[AAGCTT], which is Hind Ⅲ recognition sequence.

ChemInform Abstract: A New Approach to the Synthesis of Oligodeoxyribonucleotides with Alkylamino Groups Linked to Internucleotide Phosphate Groups.

Abstract An efficient method for the synthesis of oligodeoxyribonucleotides with alky1amino groups based on the H-phosphonate method and the Atherton- Todd reaction has been developed. During the synthesis internucleotide phosphorodiester groups did not react with the activated H-phosphonate.

ChemInform Abstract: Synthesis of Antisense Oligodeoxyribonucleotide Analogues by Use of Deoxyribonucleoside 3′-Bis(1,1,1,3,3,3-hexafluoro-2-propyl) Phosphites as New Key Intermediates.

The deoxyribonucleoside 3′-bis(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite units (3) are used in the chemical synthesis of oligodeoxyribonucleotides on solid supports. The new phosphite units (3) were prepared easily by reaction of nucleosides and tris-(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite (2). They are readily activated by N-methylimidazole under very mild conditions on a solid support. This operation involves a one step reaction, which is an advantage over both the phosphite and H-phosphonate approaches. The use of deoxyribonucleoside phosphite intermediates in the synthesis of antisense oligodeoxyribonucleotides is also described.

ChemInform Abstract: Synthesis, Base Pairing, and Structural Transitions of Oligodeoxyribonucleotides Containing 8-Aza-2′-deoxyguanosine.

The synthesis of oligonucleotides containing 8-aza-2′-deoxyguanosine (z8Gd; 1) or its N8-regioisomer z8Gd* (2) instead of 2′-deoxyguanosine (Gd) is described. For this purpose, the NH2 group of 1 and 2 was protected with a (dimethylamino)methylidene residue (5, 6), a 4,4′-dimethoxytrityl group was introduced at 5′-OH ( 7, 8), and the phosphonates 3a and 4 as well as the phosphoramidite 3b were prepared. These building blocks were used in solid-phase oligonucleotide synthesis. The oligonucleotides were characterized by enzymatic hydrolysis and melting curves (Tm values). The thermodynamic data of the oligomers 12–15 indicate that duplexes were stabilized when 1 was replacing Gd. The aggregation of d(T-G-G-G-G-T) (18) was studied by RP 18 HPLC, gel electrophoresis and CD spectroscopy and compared with that of oligonucleotides containing an increasing number of z8Gd residues instead of Gd. Similarly to [d(C-G)]3 (12a), the hexamer d(C-z8G-C-z8G-C-G) (14) underwent salt-dependent B-Z transition.

ChemInform Abstract: Repair and Replication of Oxidized DNA Bases Using Modified Oligodeoxyribonucleotides

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Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N6 -carboxymethyl-2′-deoxyadenosine or N4 -carboxymethyl-2′-deoxycytidine

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N6-carboxymethyl-2′-deoxyadenosine (N6-CMdA) and N4-carboxymethyl-2′-deoxycytidine (N4-CMdC), liquid chromatography–ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N6-CMdA or N4-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N6-CMdA and dC with N4-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N4-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N6-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N6-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N4-CMdC. The formation and the mutagenic potential of N6-CMdA and N4-CMdC suggest that these lesions may bear important implications in the etiology of NOC-induced tumor development.

Solid-Phase Synthesis of Oligodeoxyribonucleotides without Base Protection Utilizing O-Selective Reaction of Oxazaphospholidine Derivatives

A study on the development of a novel method to synthesize oligodeoxyribonucleotides without base protection is described. We found that nucleoside 3′-O-oxazaphospholidine derivatives exclusively react with the hydroxy group of nucleosides in the presence of unprotected nucleobase amino groups. Since the O-chemoselectivity of the oxazaphospholidine derivatives is likely due to their ring structure, which allows the regeneration of the oxazaphospholidine derivatives from the corresponding base phosphitylation adducts via an intramolecular recyclization, the method is expected to be compatible with any kinds of acidic activators.

Sulfonium derivatives of thioxanthenone, a new class of photodetritylating agents for microarray oligonucleotide synthesis

The usability of a new class of photo acids, namely, sulfonium hexaphosphates based on thioxanthenone, for the removal of the dimethoxytrityl protective group in the process of oligonucleotide synthesis has been studied in order to search for new detritylating agents for microarray oligodeoxyribonucleotide synthesis. 2,4-Diethyl-9-oxo-10-(4-heptyloxyphenyl)-9H-thioxanthenium hexafluorophosphate has been successfully used for the solid-phase synthesis of (dT)(10).

Automated Oligonucleotide Solid-Phase Synthesis on Nanosized Silica Particles Using Nano-on-Micro Assembled Particle Supports

This article describes an original strategy to enable solid-phase oligodeoxyribonucleotide (ODN) synthesis on nanosized silica particles. It consists of the reversible immobilization of silica nanoparticles (NPs) on micrometric silica beads. The resulting assemblies, called nano-on-micro (NOM) systems, are well adapted to ODN synthesis in an automated instrument. First, NPs are derivatized with OH functions. For NOM assembly preparation, these functions react with the silanols of the microbeads under specific experimental conditions. Furthermore, OH groups allow ODN synthesis on the nanoparticles via phosphoramidite chemistry. The stability of the NOM assemblies during ODN solid-phase synthesis is confirmed by scanning and transmission electron microscopy (SEM and TEM, respectively), together with dynamic light scattering analyses. Then, the release of ODN-functionalized nanoparticles is performed under mild conditions (1% NH(4)OH in water, 1 h, 60 degrees C). Our technique provides silica nanoparticles well functionalized with oligonucleotides, as demonstrated by hybridization experiments conducted with the cDNA target.

Synthesis and Properties of Oligodeoxyribonucleotides Bearing a Polyamino Group at the 2′ Position via 2′-O-Carbamoylmethyl and 2′-S-Carbamoylmethyl groups

An oligodeoxyribonucleotide containing 2 ′-O-methoxycarbonylmethyluridine was synthesized and converted into several 2 ′-modified oligodeoxyribonucleotides by a postsynthetic modification method. Using this method, oligodeoxyribonucleotides bearing a polyamine at the 2 ′ position were easily prepared, which could form duplexes with either complementary DNA or RNA.

Oligonucleotide−RGD Peptide Conjugates for Surface Modification of Titanium Implants and Improvement of Osteoblast Adhesion

A new concept for modular biosurface engineering of titanium implants based on the self-assembly of complementary oligonucleotides was biochemically investigated and optimized. This study describes the synthesis and characterization (RP-HPLC and Sakaguchi assay) of oligodeoxyribonucleotide (ODN) conjugates of the hexapeptide GRGDSP containing the RGD sequence as the recognition motif for cellular adhesion receptors (integrins). The peptide was chosen exemplarily as a model molecule, because it is a simple but potent bioactive molecule and relatively well investigated. The conjugation products must fulfill two main requirements: (I) the ability to hybridize and (II) the preservation of biological activity of the RGD peptide for the enhancement of osteoblast adhesion. In the following text, the term "hybridization" is generally used for Watson-Crick base pairing. The ability of the conjugates to hybridize to surface-immobilized complementary ODN was verified by competitive hybridization with radiolabeled ((32)P) complementary strands and by hybridization experiments using a quartz crystal microbalance (QCM). Surface hybridization was further characterized using different adsorption isotherms (e.g., Freundlich and Frumkin types), since the type of isotherm and the derived thermodynamic parameters may reveal characteristic differences between ODN and conjugates thereof. Biological activity of the conjugates was examined in vitro with osteoblasts. The cells were either cultured directly on the ODN-GRGDSP modified titanium implants or used for competition adhesion studies with dissolved ODN-GRGDSP conjugates. All results support the successful establishment of the new surface modification system. Hybridization of RGD peptide-modified nucleic acids to ODN-modified titanium implant materials is thus a promising method for osteoblast attachment in a modular and self-organizing system on implant surfaces.

Chemical synthesis of oligodeoxyribonucleotides containing N 3- and O4 -carboxymethylthymidine and their formation in DNA

Humans are exposed to N-nitroso compounds from both endogenous and exogenous sources. Many N-nitroso compounds can be metabolically activated to give diazoacetate, which can result in the carboxymethylation of DNA. The remarkable similarity in p53 mutations found in human gastrointestinal tumors and in shuttle vector studies, where the human p53 gene-containing vector was treated with diazoacetate and propagated in yeast cells, suggests that diazoacetate might be an important etiological agent for human gastrointestinal tumors. The O6-carboxymethyl-2′-deoxyguanosine was previously detected in isolated DNA upon exposure to diazoacetate and in blood samples of healthy human subjects. The corresponding modifications of thymidine and 2′-deoxyadenosine have not been assessed, though significant mutations at A:T base pairs were found in the p53 tumor suppressor gene in human gastrointestinal tumors and in shuttle vector studies. To understand the implications of the carboxymethylation chemistry of thymidine in the observed mutations at A:T base pairs, here we synthesized authentic N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT), incorporated them into DNA, and demonstrated, for the first time, that they were the major carboxymethylated derivatives of thymidine formed in calf thymus DNA upon exposure to diazoacetate. The demonstration of the formation of N3-CMdT and O4-CMdT in isolated DNA upon treatment with diazoacetate, together with the preparation of authentic oligodeoxyribonucleotide substrates housing these two lesions, laid the foundation for investigating the replication and repair of these lesions and for understanding their implications in the mutations observed in human gastrointestinal tumors.

Stereocontrolled synthesis of backbone-modified oligonucleotides via diastereopure H-phosphonate intermediates

Synthesis of stereoregulated backbone-modified oligodeoxyribonucleotides via the corresponding diastereopure H-phosphonate intermediates is described. The diastereopure H-phosphonate intermediates were synthesized by using the nucleoside 3'-oxazaphospholidine monomers, and were stereospecifically converted into a variety of P-stereoregulated backbone-modified oligonucleotides, such as phosphorothioates, boranophosphates, and phosphoramidates, on a solid-support.

Volume 1, Number 1

Volume 1, Number 1

Naphthyridine-tethered Oligodeoxyribonucleotides: Dye/DNA Conjugates for Homogeneous SNPs Assays

Abstract We here report the synthesis and fluorescence properties of naphthyridine-tethered oligodeoxyribonucleotides for discrimination of cytosine-related single nucleotide alterations in DNA. A/C alleles of cytochrome P450 variants (CYP2C19) were successfully discriminated simply by adding baeDANP-ODN under slightly basic conditions.

In vivo production of oligodeoxyribonucleotides of specific sequences: application to antisense DNA.

Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.

Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine.

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.

1,1-Dihydroperoxycyclododecane as a new, crystalline non-hygroscopic oxidizer for the chemical synthesis of oligodeoxyribonucleotides

A new oxidation method for DNA synthesis was developed by the use of 1,1-dihydroperoxycyclododecane in CH 2Cl 2–EtOAc under anhydrous conditions. This new oxidizer was successfully applied to the synthesis of oligodeoxyribonucleotides that involves an oxidation step for conversion of phosphite intermediates to phosphate derivatives.