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.     

Oligonucleotides Containing N7-Cyanoborane-2′-deoxyguanosine

Abstract

For synthesis of N⁷-cyanoborane-containing oligonucleotides, the 5′-DMT protecting group is not a suitable precursor because the boronated nucleoside is incompatible with DMT cations released during deprotection of the oligonucleotide. As an alternative to DMT, we have investigated use of the 5′-Fmoc protecting group. We found that the cyanoborane group is stable during synthesis and deprotection conditions used with Fmoc derivatives.     

A Short,Novel, and Cheaper Procedure for Oligonucleotide Synthesis Using Automated Solid Phase Synthesizer

Abstract

Dimethylthiuram disulfide (DTD) has been developed as an efficient thiolation reagent during automated synthesis of oligonucleotides using phosphoramidite chemistry. Simultaneous thiolation and capping was accomplished by mixing DTD with capping solution B, which saved 20% of solvent consumption and compressed the four-step synthesis cycle to three. Large-scale (1 mmol) synthesis of phosphorothioate oligonucleotides has been demonstrated with excellent yield and purity.     

A Reinvestigation of Sulfenyl Groups as Amino Protecting Groups for the Synthesis of Oligonucleotides on Solid Support by Phosphoramidite Chemistry†

Abstract

Although sulfenyl groups as protectors of heterocyclic amines of nucleosides appeared satisfactory during the synthesis of DNA and RNA via the phosphotriester approach, their usefulness in automated synthesis of oligonucleotides using phosphoramidite chemistry has not been investigated. Herein, we examined the stability and efficiency of 2-nitrophenylsulfenyl- and tritylsulfenyl-nucleosides upon the conditions applied in oligonucleotide synthesis by the phosphoramidite approach.

     

SYNTHESIS OF 5-(2,2′-BIPYRIDINYL AND 2,2′-BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: PRECURSORS TO METALLO-DNA CONJUGATES

ABSTRACT

The synthesis of 2,2′-bipyridinyl-2′-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2′-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2′-deoxyuridine nucleoside (Bipy²⁺-dU) with an ethylenyl linker. The Bipy²⁺-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

New Synthesis of 5-Carboxy-2′-deoxyuridine and Its Incorporation into Synthetic Oligonucleotides

Abstract

5-Carboxy-2′-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2′-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2′-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

Stability of Phosphorothioate Oligonucleotides in Aqueous Ammonia in Presence of Stainless Steel

Abstract

Antisense oligonucleotides as modulators of gene expression represent an exciting new drug technology. Oligodeoxyribonucleotide phosphorothioates are now among the most intensively investigated nuclease-resistant antisense analogs, as evidenced by a number of ongoing clinical trials against several disease targets. Structurally, these differ from natural oligonucleotides by the replacement of one of two nonbridging oxygen atoms by a sulfur atom at each internucleotide linkage. Among factors in the successful development of these complex molecules to support broad clinical trials have been advances made in automation, analysis and purification. The large scale synthesis of oligonucleotide phosphorothioates is presently carried out by initial formation of the internucleotide phosphite linkage using solid-phase phosphoramidite chemistry followed by sulfurization. Efficient synthesis of 20-mer oligophosphorothioates has been achieved on 0.15 mole scale with only 1.75-fold excess of amidite synthons. However, as the scale of synthesis increases to meet future market demands, issues related to fast and efficient synthesis, automation, scalability and product purification are also being investigated. One issue has been the protocol for final product deprotection. Since deprotection involves large quantities of saturated aqueous ammonium hydroxide, one might consider use of stainless steel reactors to withstand resulting vapor pressure at 55°C. A recent report,¹ however, discusses the instability of dimer phosphorothioates in aqueous ammonia in the presence of metal ions. As this is potentially an important issue for phosphorothioate oligonucleotide synthesis, we describe herein our findings regarding deprotection of a 20-mer oligodeoxyribonucleotide phosphorothioate with aqueous ammonia during process development studies.     

Boranophosphate Oligonucleotides: New Synthetic Approaches

Abstract

Recently our laboratory reported a new backbone-modified class of oligonucleotides, with a borane (B₃3?) group replacing one of the non-bridging oxygen atoms. Here we present two new approaches to synthesize the boranophosphate oligonucleotides. All-stereoregular boranophosphate oligonucleotides can be prepared by enzymatic template extension reactions using nucleoside a-boranotriphosphates, which are good substrates for a number of polymerases. Larger scale synthesis of boranophosphate oligonucleotides can be carried out by effective chemical synthesis using the H-phosphonate approach, instead of previously used phosphoramidite methodology. The main advantage of H-phosphonate methodology is the ability to carry out one boronation reaction, after oligonucleotide chain elongation has been completed, using mild conditions without base damage and producing the desired boranophosphate oligonucleotides in high yield.     

Synthesis and DNA Binding Properties of a New Benzo[f]Imidazo[1,5b]-Isoquinoline-Butan-1,2-Diol Derivative

A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA, DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was used for the synthesis of a zipping duplex.     

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 Incorporation of 2′-O-Methyl-Pseudouridine into Oligonucleotides

Abstract

A short multigram synthesis of 2′-O-methylpseudouridine and its phosphoramidite derivative is described which avoids the use of protecting groups on the nitrogens. A binding study of oligonucleotides containing this modification suggest an increased binding affinity to RNA when compared to oligonucleotides incorporating 2′-O-methyluridine.     

An Improved Method for Large Scale Synthesis of Oligonucleotides Applying the NPE/NPEOC-Strategy

Abstract

Several oligonucleotides were synthesized in scales up to 60 μmol in a standard 10 μmol cartridge on a standard DNA synthesizer. The advantage of a special phosphoramidite approach using only β-eliminating protecting groups over the commonly practised automated oligonucleotide synthesis using ammonia-labile blocking groups could be demonstrated by ¹H-NMR-spectroscopy.     

Characterization of oligodeoxyribonucleotide synthesis on glass plates

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5′-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using ³²P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5′-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.     

A Biotin Phosphoramidite Reagent for the Automated Synthesis of 5′-Biotinylated Oligonucleotides

Abstract

A bis(DMTr)biotin phosphoramidite containing serine and 6-aminohexanol moieties was prepared by a multiple-step reaction, and used successfully in the solid phase synthesis of 5′-biotinylated oligonucleotides.     

Synthesis of an Oligonucleotide Analogue of Ethenoadenosine

Abstract

A phosphoramidite building block derived from 11-carboxy-1,N ⁶-ethenoadenosine has been prepared to be used in a solid supported oligonucleotide synthesis.     

The Synthesis of Branched Oligonucleotides as Signal Amplification Multimers for Use in Nucleic Acid Assays

Abstract

Branched oligonucleotides have been synthesized using phosphoramidite derivatives with two protected hydroxyl functions. These molecules are employed for a label amplification strategy used in DNA probe diagnostics.     

Picomole Syntheses of High Quality Oligonucleotide Primers in Combination with the Preparation of Oligonucleotide Arrays

Abstract

The establishment of a new synthesis procedure for the preparation of oligonucleotide arrays is described. A modified phosphoramidite chemistry allowed the in situ synthesis of oligomer arrays on specially derivatized polypropylene membranes which can be used both for hybridisation experiments and for the isolation of the individual oligonucleotides.     

Improved synthesis of 5-hydroxymethyl-2'-deoxycytidine phosphoramidite using a 2'-deoxyuridine to 2'-deoxycytidine conversion without temporary protecting groups

5-Hydroxymethylcytosine has recently been characterized as the ‘sixth base’ in human DNA. To enable research on this DNA modification, we report an improved method for the synthesis of 5-hydroxymethyl-2′-deoxycytidine (^5-HOMedC) phosphoramidite for site-specific incorporation into oligonucleotides. To minimize manipulations we employed a temporary protecting group-free 2′-deoxyuridine to 2′-deoxycytidine conversion procedure that utilizes phase transfer catalysis. The desired ^5-HOMedC phosphoramidite is obtained in six steps and 24% overall yield from 2′-deoxyuridine.     

Synthesis of Deoxyribonucleoside Phosphorodithioate Dimers by a Triester Method

Abstract

Modified oligodeoxynucleotides have recently received much attention due to their therapeutic applications. Among the more promising are phosphorodithioates where both nonbridging oxygen atoms in the phosphate diesters are replced by sulfur. Deoxynucleoside phosphorodithioate dimers have been prepared in several ways, using H-phosphonate, phosphordiamidite, phosphoramidite, and thiophosphoramidite methods. Reports have also appeared on the synthesis of oligonucleotides with alternating phosphodiester and dithiophosphodiester linkages, as well as one on ribonucleoside dimers. Of the above methods, the thiophosphoramidite method has been applied successfully for the preparation of mixed base oligonucleotides containing contiguous phosphorodithioate linkages. However, this method gives products which contain varying amounts of phosphorothioate linkages (2 ? 10%) due to factors associated with the involvement of trivalent thiophosphorus compounds. In addition, the thiophosphoramidite synthons are difficult to purify on silica gel column, and have a tendency to dismutate in presence of acidic catalysts such as tetrazole. The thiophosphite intermediate which is formed is also unstable to tetrazole. Similarly in the thio- and dithio-H-phosphonate method, the primary coupling products are unstable to catalysts, pivaloyl chloride and iodine. Recently, Dahl et al reported^1–2 synthesis of dimers and oligomers upto octamer which also leads to formation of small amounts of phosphorothioate linkages. In additon, about 1.2% per phosphorodithioate linkage of the oligomer is cleaved during     

Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10–23 to enhance target affinity: Implications for DNAzyme design

Deoxyribozymes (DNAzymes) are important catalysts for potential therapeutic RNA destruction and no DNAzyme has received as much notoriety in terms of therapeutic use as the Mg²⁺-dependent RNA-cleaving DNAzyme 10–23 (Dz10–23). As such, we have investigated the synthetic modification of Dz10–23 with a guanidinium group, a functionality that reduces the anionic nature and can potentially enhance the membrane permeability of oligonucleotides. To accomplish this, we synthesized a heretofore unknown phosphoramidite, 5-(N,N′-biscyanoethoxycarbonyl)-guanidinoallyl-2′-deoxyuridine and then incorporated it into oligonucleotides via solid phase synthesis to study duplex stability and its effect on Dz10–23. This particular modification was chosen as it had been used in the selection of Mg²⁺-free self-cleaving DNAzymes; as such this will enable the eventual comparison of modified DNAzymes that do or do not depend on Mg²⁺ for catalysis. Consistent with antecedent studies that have incorporated guanidinium groups into DNA oligonucleotides, this guanidinium-modified deoxyuridine enhanced the thermal stability of resulting duplexes. Surprisingly however, Dz10–23, when synthesized with modified residues in the substrate binding regions, was found to be somewhat less active than its non-modified counterpart. This work suggests that this particular system exhibits uniform binding with respect to ground state and transition state and provides insight into the challenge of re-engineering a Mg²⁺-dependent DNAzyme with enhanced catalytic activity.