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 Terminal Nucleoside Phosphates and Thiophosphates via Phosphoramidite Chemistry

Abstract

Synthetic oligonucleotides for use in biological processes often require a 5′-terminal phosphate function. To study enzymatic reactions this group can be replaced by a thiophosphate function. Most chemical methods for the synthesis of these termini base on phosphotriester chemistry.     

The Application of H-Phosphonate Chemistry in the HELP Synthesis of Oligonucleotides

Abstract

The use of the H-phosphonate chemistry for its possible utilization in the liquid phase synthesis of oligonucleotides has been investigated.     

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.     

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.     

Nanotechnology and Potential of Microorganisms

ABSTRACT

There is a growing need to develop clean, nontoxic and environmentally friendly (“green chemistry”) procedures for synthesis and assembly of nanoparticles. The use of biological organisms in this area is rapidly gaining importance due to its growing success and ease of formation of nanoparticles. Presently, the potential of bio-organisms ranges from simple prokaryotic bacterial cells to eukaryotic fungus and even live plants. In this article we have reviewed some of these biological systems, which have revolutionized the art of nano-material synthesis.     

Phosphoramidite Chemistry for the Synthesis of cycloSal-Pro-Nucleotides

Abstract

An alternative synthesis of 3-methyl-cycloSal-nucleotides 3–6 using phosphor-amidite chemistry is described. This protocol clearly shows advantages for the cycloSal-introduction into cytosine containing nucleoside analogues.     

Rapid Synthesis of Oligodeoxyribonucleotides VIII.1 An Improved Procedure for Solid-Phase Oligodeoxyribonucleotide Synthesis Using a Controlled Pore Glass Support and Phosphotriester Chemistry

Abstract

An improved method for solid phase oligodeoxyribonucleotide synthesis is described that uses phosphotriester chemistry, controlled pore glass as support, a new and more stable linkage agent and a better protecting group combination.     

Automated Synthesis of Cyclic Oligodeoxyribonucleotides via Phosphoramidite Method

Abstract

The functionalized polyethylene glycol/polystyrene copolymer support 4 was shown to be suitable for a completely automated synthesis of small- to medium-sized cyclic oligodeoxy-ribonucleotides. Syntheses of the linear precursors were achieved by the phosphoramidite method, whereas the cyclization reactions were based on the phosphotriester chemistry.     

DNA synthesis in the elongating nondividing cells of the lentil epicotyl and its promotion by gibberellin

Two-day-old lentil seedlings, (Lens culinaris Med.) were incubated for a 48-hour period with and without gibberellin (GA) in the presence and absence of 5-fluorodeoxyuridine (FUDR). The number of cells per epicotyl did not increase during this period. Growth of the epicotyl was thus due to cell elongation alone.

The elongating cells of this tissue synthesized DNA. GA promoted and FUDR inhibited cell elongation, DNA synthesis, and RNA synthesis in the tissue.

FUDR promoted uptake of thymidine and thymidine incorporation into cellular DNA, presumably by inhibiting synthesis of endogenous thymidine. Presence of GA promoted thymidine incorporation into cellular DNA and uridine incorporation into cellular RNA. In either case, there was no effect on the uptake of the precursor into the tissue.

Fractionation of thymidine-labeled nucleic acids on a MAK column showed that thymidine was exclusively incorporated into the DNA fraction. Presence of GA promoted thymidine incorporation into this fraction and also increased the amount of ribosomal RNA.

The data provide direct evidence for the conclusion that DNA synthesis is necessary for elongation of certain plant cells.

     

White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses

Functionalized compounds, which are difficult to produce by classical chemical synthesis, are of special interest as biotechnologically available targets. They represent useful building blocks for subsequent organic syntheses, wherein they can undergo stereoselective or regioselective reactions. “White Biotechnology” (as defined by the European Chemical Industry [http://www.europabio.org/white_biotech.htm], as part of a sustainable “Green Chemistry,”) supports new applications of chemicals produced via biotechnology. Environmental aspects of this interdisciplinary combination include:

• Use of renewable feedstock

• Optimization of biotechnological processes by means of:

  • New “high performance” microorganisms

  • On-line measurement of substrates and products in bioreactors

  • Alternative product isolation, resulting in higher yields, and lower energy demand

In this overview we describe biotechnologically produced pyruvic, 2-oxopentaric and 2-oxohexaric acids as promising new building blocks for synthetic chemistry. In the first part, the microbial formation of 2-oxocarboxylic acids (2-OCAs) in general, and optimization of the fermentation steps required to form pyruvic acid, 2-oxoglutaric acid, and 2-oxo-d-gluconic acid are described, highlighting the fundamental advantages in comparison to chemical syntheses. In the second part, a set of chemical formula schemes demonstrate that 2-OCAs are applicable as building blocks in the chemical synthesis of, e.g., hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. Finally, some perspectives are discussed.

     

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.     

Improvements to the Chemical Synthesis of Biologically Active RNA Using 2′-O-Fpmp Chemistry

Abstract

The synthetic cycle protocol for the solid phase synthesis of RNA using 5′-O-(DMTr)-2′-O-(Fpmp)-ribonucleoside phosphoramidites is optimised. A simple and reliable two step deprotection procedure is developed to isolate biologically active RNA. It is demonstrated that fully deprotected RNA is completely stable under the deprotection conditions and that it does not undergo internucleotide cleavage and/or migration. Ribozymes and substrate RNAs synthesized using this chemistry were found to be catalytically active.     

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).     

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

The emergence of antisense and antigene oligonucleotides as potential sequenceselective inhibitors of gene expression is evidenced by the growing number of ongoing clinicals trials against a variety of diseases. First generation antisense therapeutics utilize a uniformly modified oligodeoxyribonucleotide phosphorothioate where one non-bridging oxygen atom is formally replaced by sulfur, because natural DNA is unstable towards extra- and intracellular enzymes. Phosphoramidite chemistry has been widely used for the synthesis of phosphorothioate oligonucleotides because of its potential for automation, high coupling efficiency, ease of site-specific thioate linkage incorporation, and ready scalability. The large scale solid-supported synthesis of phosphorothioates is presently carried out by initial formation of the internucleotidic phosphite linkage followed by sulfurization of the phosphite triester to phosphorothioate using the Beaucage reagent. The resulting O,O-linked phosphorothioate diester linkage in the oligonucleotide is a chiral functional group. For a typical 20-mer there are 524,288 (2¹⁹) possible diastereoisomers. Separation and individual quantification of this number of diastereomers is currently not feasible. In addition, the best reported methods for stereocontrolled synthesis of phosphorothioate oligomers are not presently useful for drug synthesis; that is, since net 100% enantiomeric excess is not achieved in the coupling step, the oligomeric product still consists of the same mixture of Sp and Rp diastereomers, except that the levels of all but one isomer are reduced to low individual levels. As a result, even a small change in the and Sp phosphorothioate diesters, due to racemization during coupling, indicating that the overall synthetic process is stereo reproducible and under inherent process control.     

New Solid Supports Linking Nucleoside Scaffolds

Abstract

An easy and efficient strategy to obtain new nucleoside based solid supports in which the nucleoside moieties have been anchored to the solid support through the nucleobase is here proposed. A simple and efficient solid-phase synthesis of 5′ and 3′-derivatized uridine analogues has so been developed, following methodologies well established in organic chemistry.     

Synthesis and Properties of Modified Oligodeoxyribonucleotides Containing 9-(2-Amino-2-deoxy-β-D-arabinofuranosyl)adenine

Abstract

The synthesis of modified oligodeoxyribonucleotides# This publication is dedicated to Professor Tsujiaki Hata, who made a valuable contribution to the chemistry of nucleosides and nucleotides. containing 2′-amino-2′-deoxyarabinoadenosine residues (aAⁿ) was carried out by means of the standard phosphoramidite chemistry. A high reactivity of such compounds to electrophilic reagents was shown. The cross-link formation between 2′-amino group of aAⁿ and carboxyl function introduced into complementary strands occurs with 55% yield. The aAⁿ residues was shown to induce the increased resistance of modified oligomers towards the enzymatic cleavage and provide the insignificant destabilization of DNA duplexes.

     

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

Abstract

Sequence-specific modulation of gene expression for the treatment of diseases has come to reality. Multiple examples of oligodeoxyribonucleotide phosphorothioates, in which one nonbridging oxygen atom of the internucleotide phosphate group of DNA is replaced by a sulfur atom are currently in advanced clinical trials. Recent advances in phosphoramidite coupling chemistry and solid phase synthesis methodology, together with current state of the art large-scale synthesizers, allow complete assembly of a 20-mer deoxyribonucleotide phosphorothioate at 150 mmole scale in just 8 h. Very high average coupling efficiencies (>98.5%) have been achieved at these scales with only 1.75-fold molar amidite excess.     

Aboveground resource allocation in response to root herbivory as affected by the arbuscular mycorrhizal symbiosis

Aims

Arbuscular mycorrhizal (AM) fungi associate with the majority of terrestrial plants, influencing their growth, nutrient uptake and defence chemistry. Consequently, AM fungi can significantly impact plant-herbivore interactions, yet surprisingly few studies have investigated how AM fungi affect plant responses to root herbivores. This study aimed to investigate how AM fungi affect plant tolerance mechanisms to belowground herbivory.

Methods

We examined how AM fungi affect plant (Saccharum spp. hybrid) growth, nutrient dynamics and secondary chemistry (phenolics) in response to attack from a root-feeding insect (Dermolepida albohirtum).

Results

Root herbivory reduced root mass by almost 27%. In response, plants augmented investment in aboveground biomass by 25%, as well as increasing carbon concentrations. The AM fungi increased aboveground biomass, phosphorus and carbon. Meanwhile, root herbivory increased foliar phenolics by 31% in mycorrhizal plants, and increased arbuscular colonisation of roots by 75% overall. AM fungi also decreased herbivore performance, potentially via increasing root silicon concentrations.

Conclusions

Our results suggest that AM fungi may be able to augment plant tolerance to root herbivory via resource allocation aboveground and, at the same time, enhance plant root resistance by increasing root silicon. The ability of AM fungi to facilitate resource allocation aboveground in this way may be a more widespread strategy for plants to cope with belowground herbivory.

     

A Synthetic Approach to Carbocyclic Sinefungin

Abstract

An approach to an asymmetric synthesis of carbocyclic sinefungin (cSF) 2 is proposed. The sequence, which uses an original radical based chemistry for C-C bond formation, led to the immediate precursor 18 of the protected desired compound. While the overall yield is modest, it is noticeable that only a limited number of steps are needed to obtain the target compound.