Synthesis Of Oligodeoxyribonucleotides Research Articles

Solution-phase synthesis of oligodeoxyribonucleotides using the H-phosphonate method with N-unprotected 5'-phosphite monomers.

Recent advances in nucleic acid therapeutics increase the requirements for developing efficient methods for the chemical synthesis of oligodeoxyribonucleotides (ODNs). In this study, we report a new approach for the solution-phase synthesis of ODNs using the H-phosphonate method with N-unprotected 5′-phosphite monomers. The 5′-phosphite monomers are synthesized in a single step from unprotected 2′-deoxyribonucleosides using 5′-O-selective phosphitylation and can be applied to the synthetic cycle of the H-phosphonate method. We synthesized four kinds of 5′-phosphite monomers and then optimized the conditions for the condensation between the 3′-hydroxy groups of the 5′-phosphite monomers and the H-phosphonate monoesters. As a result of various investigations, solution-phase synthesis of trithymidine diphosphate (TTT) and tetramers containing four kinds of nucleobases was achieved according to the procedure consisting of repeated condensation, deprotection, and purification using simple extraction or precipitation.

Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of O6-methylguanine and their recognition by MGMT and Atl1

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.

Synthesis of damaged DNA containing the oxidative lesion 3′-oxothymidine

Oxidative events that take place during regular oxygen metabolism can lead to the formation of organic or inorganic radicals. The interaction of these radicals with macromolecules in the organism and with DNA in particular is suspected to lead to apoptosis, DNA lesions and cell damage. Independent generation of DNA lesions resulting from oxidative damage is used to promote the study of their effects on biological systems. An efficient synthesis of oligodeoxyribonucleotides (ODNs) containing the oxidative damage lesion 3′-oxothymidine has been accomplished via incorporation of C3′-hydroxymethyl thymidine as its corresponding 5′-phosphoramidite. Through oxidative cleavage using sodium periodate in aqueous solution, the lesion of interest is easily generated. Due to its inherent instability it cannot be directly isolated, but must be generated in situ. 3′-Oxothymidine is a demonstrated damage product formed upon generation of the C3′-thymidinyl radical in ODN.

Preparation of a disulfide-linked precipitative soluble support for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks.

The preparation of a disulfide-tethered precipitative soluble support and its use for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks is described. To obtain the building blocks, N-acyl protected 2´-deoxy-5´-O-(4,4´-dimethoxytrityl)ribonucleosides were phosphorylated with bis(benzotriazol-1-yl) 2-chlorophenyl phosphate. The “outdated” phosphotriester strategy, based on coupling of PV building blocks in conjunction with quantitative precipitation of the oligodeoxyribonucleotide with MeOH is applied. Subsequent release of the resulting phosphate and base-protected oligodeoxyribonucleotide trimer 3’-pTpdCBzpdGibu-5’ as its 3’-(2-chlorophenyl phosphate) was achieved by reductive cleavage of the disulfide bond.

Solid-Phase Synthesis, Hybridizing Ability, Uptake, and Nuclease Resistant Profiles of Position-Selective Cationic and Hydrophobic Phosphotriester Oligonucleotides.

Analogues of oligonucleotides and mononucleotides with hydrophobic and/or cationic phophotriester functionalities often generate an improvement in target affinity and cellular uptake. Here we report the synthesis of phosphotriester oligodeoxyribonucleotides (ODNs) that are stable to the conditions used for their preparation. The method has been demonstrated by introducing phosphoramidite synthons where N-benzyloxycarbonyl (Z) protected amino alcohols replace the cyanoethyl group. After synthesis these ODNs were found to be stable to the condition required to remove base labile protecting groups and the ODNs from the solid support. Moreover the use of 1-(4,4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde) in place of Z protection on the amino alcohol has allowed us to introduce cationic aminoethyl phosphotriester modifications into ODNs. Melting temperatures of duplexes containing cationic or hydrophobic Z modified ODNs indicate that the backbone-phosphotriester modifications minimally affect duplex stability. Nuclease stability assays demonstrate that these phosphotriesters are resistant toward 5'- and 3'-exonucleases. Fluorescently labeled 23-mer ODNs modified with four cationic or hydrophobic Z phosphotriester linkages show efficient cellular uptake during passive transfection in HeLa and Jurkat cells.

Synthesis and characterization of oligodeoxyribonucleotides modified with 2'-thio-2'-deoxy-2'-S-(pyren-1-yl)methyluridine.

Pyrene-functionalized oligonucleotides are intensively explored for applications in materials science and diagnostics. Here, we describe a short synthetic route to 2'-S-(pyren-1-yl)methyl-2'-thiouridine monomer S, its incorporation into oligodeoxyribonucleotides (ONs), and biophysical characterization thereof. Pseudorotational analysis reveals that the furanose ring of this monomer has a slight preference for South-type conformations. ONs modified with monomer S display high cDNA affinity but decreased binding specificity. Hybridization is associated with bathochromic shifts of pyrene absorption bands and quenching of pyrene fluorescence consistent with an intercalative binding mode of the pyrene moiety. Monomer S was also evaluated as a building block for mixed-sequence recognition of double-stranded DNA via the Invader strategy. However, probes with +1 interstrand arrangements of monomer S were found to be less efficient than Invader probes based on 2'-O-(pyren-1-yl)methyluridine or 2'-N-(pyren-1-yl)methyl-2'-N-methyl-2'-aminouridine.

Facile Enzymatic Synthesis of Base J-Containing Oligodeoxyribonucleotides and an Analysis of the Impact of Base J on DNA Replication in Cells

We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.

ChemInform Abstract: Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′‐Amino‐α‐L‐LNA Adenine Monomers: High‐Affinity Targeting of Single‐Stranded DNA.

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-l-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-l-LNA adenine monomers W–Z. The synthesis of the target phosphoramidites 1–4 is initiated from pentafuranose 5, which upon Vorbruggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-l-LNA adenine intermediate 9, which after a series of nontrivia...

Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-l-LNA Adenine Monomers: High-Affinity Targeting of Single-Stranded DNA

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2'-amino-α-L-LNA adenine monomers W-Z. The synthesis of the target phosphoramidites 1-4 is initiated from pentafuranose 5, which upon Vorbrüggen glycosylation, O2'-deacylation, O2'-activation and C2'-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2'-amino-α-L-LNA adenine intermediate 9, which after a series of nontrivial protecting-group manipulations affords key intermediate 15. Subsequent chemoselective N2'-functionalization and O3'-phosphitylation give targets 1-4 in ~1-3% overall yield over 11 steps from 5. ONs modified with pyrene-functionalized 2'-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2'-pyrene-functionalized 2'-amino-α-L-LNAs of considerable interest for DNA-targeting applications.

Synthesis of Short Oligodeoxyribonucleotides by Phosphotriester Chemistry on a Precipitative Tetrapodal Support

The use of “outdated” phosphotriester chemistry, which avoids the need for oxidation after each coupling step, competes favorably with the widely applied phosphoramidite chemistry as an in-house method for the preparation of short oligonucleotides on hundred-milligram scales on a tetrapodal soluble support that may be quantitatively precipitated with methanol.

ChemInform Abstract: Frontiers and Approaches to Chemical Synthesis of Oligodeoxyribonucleotides

AbstractReview: 72 refs.

Frontiers and Approaches to Chemical Synthesis of Oligodeoxyribonucleotides

The advantages and disadvantages of existing approaches to the synthesis of oligodeoxyribonucleotides (ODN) are discussed focusing on large-scale methods. The liquid phase and solid supported synthesis and the synthesis on soluble polymers are discussed. Different problems concerning the methods and implementation of the ODN synthesis are outlined depending on goals of using target oligomers.

Preparation of Azido Containing Oligonucleotides Through Diazo Transfer Reaction

The use of diazo transfer reagent, imidazole-1-sulfonyl azide hydrochloride (ISAHC), in the presence of Cu(2+) cation enables clean and efficient conversion of aminated oligodeoxyribonucleotides (ODNs) into their azido counterparts under mild conditions. ODNs bearing amino tether either at the 3', 5', or any internal position could be modified in this way, thus demonstrating the versatility of this reaction. The method also benefits from the commercial availability or easy access by routine automated DNA synthesis of amino-containing oligodeoxyribonucleotide starting material. Easy access to such azido-modified ODNs is of great interest for conjugation in particular through copper catalyzed 1,3-dipolar cycloaddition (CuAAC reaction).

Alkynyl Phosphonate DNA: A Versatile “Click”able Backbone for DNA-Based Biological Applications

Major hurdles associated with DNA-based biological applications include, among others, targeted cell delivery, undesirable nonspecific effects, toxicity associated with various analogues or the reagents used to deliver oligonucleotides to cells, and stability toward intracellular enzymes. Although a plethora of diverse analogues have been investigated, a versatile methodology that can systematically address these challenges has not been developed. In this contribution, we present a new, Clickable, and versatile chemistry that can be used to rapidly introduce diverse functionality for studying these various problems. As a demonstration of the approach, we synthesized the core analogue, which is useful for introducing additional functionality, the triazolylphosphonate, and present preliminary data on its biological properties. We have developed a new phosphoramidite synthon--the alkynyl phosphinoamidite, which is compatible with conventional solid-phase oligonucleotide synthesis. Postsynthesis, the alkynylphosphonate can be functionalized via "Click" chemistry to generate the 1,2,3-triazolyl or substituted 1,2,3-triazolyl phosphonate-2'-deoxyribonucleotide internucleotide linkage. This manuscript describes the automated, solid-phase synthesis of mixed backbone oligodeoxyribonucleotides (ODNs) having 1,2,3-triazolylphosphonate (TP) as well as phosphate or thiophosphate internucleotide linkages and also 2'-OMe ribonucleotides and locked nucleic acids (LNAs) at selected sites. Nuclease stability assays demonstrate that the TP linkage is highly resistant toward 5'- and 3'-exonucleases, whereas melting studies indicate a slight destabilization when a TP-modified ODN is hybridized to its complementary RNA. A fluorescently labeled 16-mer ODN modified with two TP linkages shows efficient cellular uptake during passive transfection. Of particular interest, the subcellular distribution of TP-modified ODNs is highly dependent on cell type; a significant nuclear uptake is observed in HeLa cells, whereas diffuse cytoplasmic fluorescence is found in the WM-239A cell line. Cytoplasmic distribution is also present in human neuroblastoma cells (SK-N-F1), but Jurkat cells show both diffuse and punctate cytoplasmic uptake. Our results demonstrate that triazolylphosphonate ODNs are versatile additions to the oligonucleotide chemist's toolbox relative to designing new biological research reagents.

Reduction of metal ions by boranephosphonate DNA

Oligodeoxyribonucleotides bearing boranephosphonate linkages (bpDNA) were shown to reduce a number of metal ions and form nanoparticles through a novel reaction pathway that leads to phosphate diesters or phosphate triesters in water or alcohols respectively. The synthetic utility of this reaction was further demonstrated through the synthesis of oligodeoxyribonucleotides containing phosphate triester linkages. This new reactivity also makes bpDNA promising for use in construction of DNA templated metallic nanostructures.

Synthesis of oligodeoxyribonucleotides containing a conformationally-locked anti analogue of O6-methyl-2′-deoxyguanosine and their recognition by MGMT and Atl1

We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.

Cover Picture: Highly Fluorescent 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐Deoxyuridine 5′‐Triphosphate as an Efficient Substrate for DNA Polymerases (ChemBioChem 15/2011)

The cover picture shows the synthesis by DNA polymerase (shown in green) of a fluorescent DNA probe containing a fluorescent nucleobase analogue, 5-(5,6-dimethoxybenzothiazol-2-yl)-2′-deoxyuridine (dbtU, shown as light blue spheres), that exhibits a very high quantum yield (0.701) and brightness (14 000) in aqueous 100 mm NaOH at 460 nm emission. Although the chemical synthesis of short oligodeoxyribonucleotides (ODNs) containing fluorescent nucleobase analogues at specific sites has been reported, the enzymatic incorporation of these fluorescent nucleobase analogues into DNA has not been substantially reported. On p. 2341 ff., A. Matsuda et al. describe the incorporation of dbtUTP (5′-triphosphate of dbtU) efficiently at the site opposite adenine in ODNs, and how dbtU was found to act not only as a triphosphate but also as a template in an identical manner to dT in its base-pairing pattern.

ChemInform Abstract: Further Optimization of Detritylation in Solid‐Phase Oligodeoxyribonucleotide Synthesis.

AbstractOptimized reaction conditions for the removal of dimethoxytrityl, pixyl, and dimethylpixyl protecting groups in compounds (I) are elaborated.

Further Optimization of Detritylation in Solid-Phase Oligodeoxyribonucleotide Synthesis

Various conditions for optimum detritylation (i.e., the removal of 5′-O-trityl protecting groups) during solid-phase synthesis of oligodeoxyribonucleotides were investigated. Di- and tri-chloroacetic acids of variable concentrations were used to study the removal of the 4,4′-dimethoxytrityl (DMTr) group. It was found that the DMTr group could be completely removed under much milder acidic conditions than what are currently used for automated solid-phase synthesis. The 2,7-dimethylpixyl (DMPx) is proposed as an alternative and more readily removable group for the protection of the 5′-OH functions both in solid- and solution-phase synthesis. The improved detritylation conditions are expected to minimize the waste and offer a protocol for incorporation of acid sensitive building-blocks in oligonucleotides.

Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

We report here the first synthesis of 5-phenyl–telluride–thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.