Nucleoside H-Phosphonates. V. The Mechanism of Hydrogenphosphonate Diester Formation Using Acyl Chlorides as Coupling Agents in Oligonucleotide Synthesis by the Hydrogenphosphonate Approach

Abstract

The H-phosphono-acyl mixed anhydrides of type III were found to be the main intermediates during H-phosphonate diester formation using acyl chlorides as coupling agents in the reaction of hydrogenphosphonate monoesters with hydroxylic components.     

The Prooligonucleotide Approach: Synthesis of Mixed SATE-Phosphotriester Phosphodiester Oligonucleotides

Abstract

Synthesis of mixed SATE phosphotriester-phosphodiester prooligos was done following two ways. Phosphodiesters were generated by selective β-elimination of cyanoethyl using DBU or were introduced with H-phosphonate chemistry.     

Chemical Synthesis of Oligonucleotides Containing Damaged Bases for Biological Studies

Since nucleic acids are organic molecules, even DNA, which carries genetic information, is subjected to various chemical reactions in cells. Alterations of the chemical structure of DNA, which are referred to as DNA damage or DNA lesions, induce mutations in the DNA sequences, which lead to carcinogenesis and cell death, unless they are restored by the repair systems in each organism. Formerly, DNA from bacteria and bacteriophages and DNA fragments treated with UV or γ radiation, alkylating or crosslinking agents, and other carcinogens were used as damaged DNA for biochemical studies. With these materials, however, it is difficult to understand the detailed mechanisms of mutagenesis and DNA repair. Recent progress in the chemical synthesis of oligonucleotides has enabled us to incorporate a specific lesion at a defined position within any sequence context. This method is especially important for studies on mutagenesis and translesion synthesis, which require highly pure templates, and for the structural biology of repair enzymes, which necessitates large amounts of substrate DNA as well as modified substrate analogs. In this review, the various phosphoramidite building blocks for the synthesis of lesion-containing oligodeoxyribonucleotides are described, and some examples of their applications to molecular and structural biology are presented.     

Synthesis and Enzymatic Studies of Modified Oligonucleotides with Abiological Monomer Units

Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block¹ and the modified PNA-spacer (acyclic serinol nucleoside)² can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.     

Synthesis of Thymine-Modified Oligonucleotides

Abstract

Oligonucleotide-resins containing N-nitrothymidine residues yield N3-thymine modified oligonucleotides by reaction with a variety of amines followed by the standard ammonia treatment.     

Three-phase - Synthesis of Oligonucleotides

Abstract

Stable reagents were made from nucleoside-phospho-rochloridites and polymeric sec. amines. Treatment of these with tetrazole/CH₃CN and transfer of the resulting solution to immobilized oligonucleotide gave 95% chain elongation.     

Solid-Phase Synthesis of Modified Oligonucleotides

Synthetic oligonucleotides are ubiquitously found in most laboratories since solid-phase synthesis protocols have become highly optimized. These protocols make it possible to synthesize a large variety of modified oligonucleotides. As one example, we will review some of the developments regarding oligonucleotide synthesis from our own group. In particular, we will describe the synthesis of oligonucleotides carrying non-natural bases, of oligonucleotide–peptide conjugates, and of modified oligonucleotides used in the assembly of nanomaterials. This work is dedicated to the memory of Bruce Merrifield.     

Synthesis and Biophysical Studies of Modified Oligonucleotides Containing Acyclic Amino Alcohol Nucleoside Analogs

Abstract

Novel serine derivative of thymine was prepared and incorporated into oligonucleotides. These modified oligonucleotides were studied for their binding affinity with complementary DNA/RNA.     

Autoligation of Oligonucleotides via Nucleophilic Substitution Reaction

Abstract

A Fast and efficient template - driven autoligation reaction between oligonucleotides derivatized with bromoacetyl and thiol groups at their opposing termini is described. The product of reaction is capable of forming a stable duplex with a complementary DNA strand.     

An Extended Phosphate Linkage: Synthesis,Hybridization and Modeling Studies of Modified Oligonucleotides

Abstract

Novel stretched oligonucleotides (A-D) containing a 3′-α-C-methylene phosphodiester bridge (5-atoms long) have been synthesized on an automated synthesizer utilizing phosphoramidite chemistry. The key building-block 1-[3′-O-β-cyanoethyldiisopropylaminophosphiryl-2,3-dideoxy-5-O-dimethoxytriphenylmethyl-3-C-(hydroxymethyl)-β-D-erythro-pentofuranosyl]thymine (21) was prepared in a stereoselective manner from thymidine. Hybridization studies indicated a drop (1.8–3.0°C/mod.) in affinity for the complementary RNA and DNA targets. Molecular modeling results indicated that the 5-atom modified backbone had a different geometry around the phosphodiester linkage compared to the natural phosphodiester linkage. The stretched backbone may not be useful for antisense or triplex constructs, however it may find applications in biochemical/enzyme studies.

     

Use of New Phosphonylating and Condensing Agents in The Synthesis of Oligonucleotides Via The H-Phosphonate Approach

Abstract

Bis (1, 1, 1, 3, 3, 3-hexafluoro-2-propyl) phosphonate was most promising as a phosphonylating agent for the preparation of nucleoside-3′-phosphonate units. 1, 3-Dimethyl-2-chloro-imida-zolinium chloride (UWC) as a coupling agent has successfully been used for the internucleotidic H-phosphonate bonds formation via the H-phosphonate approach on a solid support.     

xylo-Configured Oligonucleotides (XNA,Xylo Nucleic Acids): Synthesis and Hybridization Studies

Abstract

We report synthesis and high-affinity hybridization of fully modified home-thymine 2′-deoxy and 2′-deoxy-2′-fluoro xylo nucleic acids.     

Glycerol-Furcated Oligonucleotides: Synthesis and Aggregation

Abstract

The synthesis of the bi-furcated complementary oligonucleotides 2 and 3 is described and their complex formation is studied as a function of their molar ratio and ionic strength by means of temperature-dependent UV- and CD spectroscopy as well as by dynamic light scattering. Structural proposals for the different aggregates are given.     

Synthesis and Properties of New Fluorescein-Labeled Oligonucleotides

Abstract

2,4-Dinitroaniline is an efficient intramolecular fluorescence-quencher for fluorescein - labeled oligonucleotides and interacts with the heterocyclic bases on duplex formation. Consequently, intramolecular fluorescence quenching is disturbed in double labeled oligonucleotides of this type, and fluorescein shows strong fluorescence in a duplex form. There is a substential increase of the fluorescence-quantum yield when the marker and quencher is attached to a single guanosine residue. Two kinds of doubly labeled oligonucleotides have been synthesized, using the NPE/NPEOC strategy.     

Efforts Toward Synthesis of Oligonucleotides for Commercialization

Abstract

Recent progress on the development of oligonucleotide phosphorothioates to meet market demands is reported.     

A General Approach for the Hydroxy Group Functionalization of Synthetic Oligonucleotides

Abstract

Generally applicable non-nucleosidic solid supports and phosphoramidite building blocks that enable attachment of various tethers to the hydroxy groups of oligonucleotides were prepared. The key feature of the structure of these reagents is an ester bond of moderate reactivity which allows postsynthetic introduction of tethers by reaction with substituted alkylamines.     

Helical Structure Formation Between Complementary Oligonucleotides. Minimum Chain Length Required for the Template-Directed Synthesis of Oligonucleotides

Helix formation between various combinations of 3–5 linked oligoribouridylates and oligoriboadenylates from dimer to dodecamer has been studied to gain information on the chain-length requirement for the template-directed condensation of oligoribonucleotides. We have measured the helix formation under high oligoribonucleotide concentration in the presence of magnesium ion at 0–50°C by UV or CD, as many model processes of oligoribonucleotides replication have been carried out under such conditions. Adenylic acid, (pA), diadenylic acid, (pA)₂, or triadenylic acid, (pA)₃, forms a helix with poly(U) or oligo(U) with a chain length of more than eight. On the other hand, neither uridylic acid, (pU), nor diuridylic acid, (pU)₂, can form a helix with oligo(A) or poly(A). Triuridylic acid, (pU)₃, or the longer oligo(U) forms a helix with oligo(A) with a chain length of over six. The results suggest that a trimer is the minimum unit as an incorporating nucleotide for conducting any set of nonenzymatic template-directed synthesis, AU and UA, as the nonenzymatic template-directed condensation of oligoribonucleotides correlates well with the results of helix formation of complementary oligoribonucleotides. We have further found the partial helix formation between 2–5 linked decauridylate, (pU)₁₀, and pA or 2–5 linked (pA)₂ at 0 °C, which indicates the possibility of the template activity of long 2–5 linked oligonucleotides for the nonenzymatic oligonucleotide synthesis.     

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels-Alder Bioconjugation

Abstract

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

Synthesis and Evaluation of Oligonucleotides of High Purity

Abstract

We have developed and evaluated methods for the production of highly pure oligonucleotides.

Presently the solid phase synthesis in an automated DNA synthesiser applying the phosphoramidite chemistry can be regarded as a standard. During the synthesis several undesirable by-products arise:

- incomplete coupling (1%) leads to 5′-truncated sequences. These sequences are acetylated at their 5′-hydroxyl group to prevent further elongation in subsequent coupling steps, but this “capping step” is incomplete, the capping-yield is 90%, leading to accumulation of sequences of the length n-1 with internal deletions.

- the glycosidic bond to N-protected purines, especially adenine, is susceptible to acid leading to depurination and subsequently to strand scission during alkaline deprotection of the oligonucleotide. This gives rise to 3′- and to 5′-truncated sequences. The 3′-truncated sequences will not be removed by standard Rp HPLC as they are tritylated.

- the reactions involved in synthesis and deprotection may cause base modifications (full length product with damaged bases).

- insufficient deprotection procedures may result in incomplete removal of protecting groups, especially from the bases (full length products with altered bases).

We have set up two different schemes (Fig. 1 and Fig. 2) for synthesis and purification, which should provide highly pure oligonucleotides with the potential of adapting to large scale production:

- accumulation of n-1 sequences (failure of capping) will be avoided by a double capping procedure using phosphite in the first capping step and an acetic anhydride capping reagent in the second capping step, as described in the literature¹.

- 3′-truncated sequences are removed by different methqds in the two schemes. In scheme I (Fig. 1) the 3′-truncated sequences can be washed off, as the 3′-full length product still is anchored to the solid support after deprotection. In scheme II (Fig. 2) the 3′truncated sequences are digested by snake venom phosphodiesterase. The 3′-full length product is protected against digestion by a 3′ - 3′-inverted end. An oligo with a correct 3′-end is, in both schemes, eventually obtained by cleaving with RNase between the ribo unit and the requested DNA-sequence.

- 5′-truncated sequences are removed by Rp HPLC using the DMTr group of the last coupling step (trityl-on synthesis) as a hydrophobic tag.

Very labile protecting groups will be used to avoid problems with deprotection.     

Synthesis,Biophysical Properties,and Stability Studies of Mixed Backbone Oligonucleotides Containing Novel Non-Ionic Linkages

Abstract

Mixed backbone oligonucleotides (MBOs) (containing ionic and non-ionic internucleotidic linkages) in which the non-ionic segments are either methylphosphotriester (PO-OMe) or primary phosphoramidate (PO-NH₂) linkages have been prepared using the recently described N-pent-4-enoyl (PNT) nucleoside phosphoramidates and H-phosphonates. Biophysical properties and stability studies suggest that these MBOs are novel antisense molecules.