Stepwise synthesis of oligonucleotides

Russian Chemical Bulletin -     

Solid-phase enzymatic synthesis of oligonucleotides

[formula: see text] The controlled and selective synthesis of oligonucleotides on the solid phase is possible under mild aqueous conditions using the enzyme T4 RNA ligase, the resins being tentagel or kieselguhr/polydimethylacrylamide.     

A novel solid support for the synthesis of 3-aminoalkylated oligonucleotides

A novel improved controlled pore glass (CPG) support based on the 2-(hydroxymethyl)-6-nitrobenzoyl (HMNB) protecting group was developed for the synthesis of 3^′-aminoalkylated oligonucleotides. The release of oligonucleotides with free 3^′-amino groups from the support is complete within 2 h at 55 °C in concentrated ammonia.     

Automated chromatography of protected oligonucleotides with high separation efficiency

Summary During the total chemical synthesis of a structural gene for the human peptide hormone angiotensin II [1] some difficult problems in ion-exchange chromatography of protected oligodeoxyribonucleotides were solved using an adsorption and a chromatographic column in combination with an automatic gradient generator and gradienttermination controller. This arrangement allows a well-timed gradient termination and therefore the separation of substances with only a small difference in charge. On the other hand the automatic run of the chromatography of protected oligonucleotides reduces the time factor in oligonucleotide chemistry. This technique is applicable for ion-exchange chromatography in general.     

Automated oligosaccharide synthesis

Peptides and oligonucleotides are prepared by automated synthesizers that can be operated by non-specialists. Carbohydrates have been hard to assemble, but the increasing awareness of the biological importance of this class of complex repeating biopolymers has prompted efforts to accelerate their synthesis. This tutorial review defines the state of the art of automated solid phase oligosaccharide synthesis and identifies areas in need of further innovation. Application of the automated synthesis method to prepare a malaria vaccine candidate is briefly highlighted.     

An efficient and versatile solid-phase synthesis of 5'- and 3'-conjugated oligonucleotides

[reaction: see text] An easy and efficient solid-phase strategy to obtain 5'- and 3'-oligonucleotide conjugates in highly pure form has been developed. Ad hoc derivatized solid supports, to which the first nucleoside unit can be attached through a phosphate linkage, have been exploited both in a pre- and post-DNA assembly conjugation approach. A number of 5'- or 3'- oligonucleotide conjugates, incorporating a variety of labels covalently linked through a phosphodiester or a phosphoramidate bond, have been synthesized and characterized.     

A convenient synthesis of the cytidyl 3′-terminal monomer for solid-phase synthesis of RNG oligonucleotides

Replacing the phosphodiester backbone of RNA with positively charged guanidinium linkages has been shown to enable RNA oligomers to overcome electrostatic repulsion and bind double-stranded DNA in a triplex with high affinity. Ribonucleotide monomers with the ability to form guanidinium linkages have been synthesized for the generation of ribooligonucleotides with guanidinium linkages (RNGs) through solid-phase synthesis. We report herein an efficient method for the synthesis of N⁴-benzoyl-2′-O-(tert-butyldimethylsilyl)-5′-N-(4-monomethoxytritylamino)-3′-O-succinyl-5′-deoxycytidine, a new monomer required for the solid-phase synthesis of cytidyl RNG oligonucleotides.     

含手性磷酸酯基的寡核苷酸类似物的立体控制性合成

本文总结了近十几年来立体控制性合成含手性磷酸酯基的寡核苷酸类似物的进展, 重点介绍了寡核苷酸硫代磷酸酯和甲基膦酸酯的立体控制性合成方法 .     

Solid-phase ‘phosphite’ synthesis of oligonucleotides

A simple modification of the Letsinger's phosphite approach is shown to have a very good potential for fast, efficient and economical synthesis of oligonucleotides on silica gel support.     

Optical tweezers directed one-bead one-sequence synthesis of oligonucleotides

An optical tweezers directed parallel DNA oligonucleotide synthesis methodology is described in which controlled pore glass (CPG) beads act as solid substrates in a two-stream microfluidic reactor. The reactor contains two parallel sets of physical confinement features that retain beads in the reagent stream for synthetic reaction but allow the beads to be optically trapped and transferred between the reagent and the inert streams for sequence programming. As a demonstration, we synthesized oligonucleotides of target sequence 25-nt, one deletion and one substitution using dimethoxytrityl (DMT) nucleoside phosphoramidite chemistry. In detecting single-nucleotide mismatches, fluorescence in situ hybridization of the bead-conjugated probes showed high specificity and signal-to-noise ratios. These preliminary results suggest further possibilities of creating a novel type of versatile, sensitive and multifunctional reconfigurable one-bead one-compound (OBOC) bead array.     

Template-free segmental synthesis of oligonucleotides containing nonnative linkages

Protected oligonucleotides containing 3'-alkyl carboxylic acids or 3'-alkylamines were obtained from photolabile solid-phase synthesis supports (1 and 4). Protected oligonucleotides containing 5'-alkylamines and 3'-hydroxyl groups were obtained using a photolabile solid-phase synthesis support (2) and a commercially available phosphoramidite reagent (3). Depending upon the source of alkylamine-containing oligonucleotide, the segments were coupled under mild conditions to form products containing either 5'-3' or 3'-3' linkages in good yield and high purity. Oligonucleotides as long as 40 nucleotides were prepared, and coupling yields of protected biopolymer segments were independent of length over the range examined. This method is particularly well suited for the convergent synthesis of oligonucleotides containing nonnative linkages and should be useful for the rapid assembly of modified biopolymers that are useful in biochemical studies.     

Reactive intermediates in the H-phosphonate synthesis of oligonucleotides

The formation of H-phosphonate diesters is an important step in the synthesis of oligonucleotides. Using diphenylchlorophosphate as the activator for the coupling step is often accompanied by side reactions as a result of self 'capping' and other reactions of the reactive intermediate. In the absence of base, the activation of ethyl H-phosphonate with diphenylchlorophosphate probably occurs through the intermediate formation of bis diethyl pyro-di-H-phosphonate rather than the expected diphenyl ethyl pyro-H-phosphonate. Pyridine acts as a nucleophilic catalyst converting diphenylchlorophosphate to its pyridinium adduct. Several side and unwanted reactions are quantified so that conditions to minimise these can be identified.     

Automated flow-through synthesis of heterocyclic thioethers

The fully automated, sequential flow-through synthesis of a 44-member array of thioethers 10[1-4,1-11] employing a resin "capture and release" reactor column is described. The array incorporates four different heterocyclic scaffolds, and the synthesis was performed using a custom-built robotic synthesizer that is able to (i) load and regenerate the reactor column and (ii) array each product into a single vial using UV threshold detection. All the compounds were obtained in high yield (>75%) and excellent purity (>95%) without the need for further purification.     

Nonenzymatic template-directed synthesis on hairpin oligonucleotides. 3. Incorporation of adenosine and uridine residues

We have used [32P]-labeled hairpin oligonucleotides to study template-directed synthesis on templates containing one or more A or T residues within a run of C residues. When nucleoside-5'-phosphoro(2-methyl)imidazolides are used as substrates, isolated A and T residues function efficiently in facilitating the incorporation of U and A, respectively. The reactions are regiospecific, producing mainly 3'-5'-phosphodiester bonds. Pairs of consecutive non-C residues are copied much less efficiently. Limited synthesis of CA and AC sequences on templates containing TG and GT sequences was observed along with some synthesis of the AA sequences on templates containing TT sequences. The other dimer sequences investigated, AA, AG, GA, TA, and AT, could not be copied. If A is absent from the reaction mixture, misincorporation of G residues is a significant reaction on templates containing an isolated T residue or two consecutive T residues. However, if both A and G are present, A is incorporated to a much greater extent than G. We believe that wobble-pairing between T and G is responsible for misincorporation when only G is present.     

Chemical synthesis of oligonucleotides. 3: Synthesis and characterization of N,O-protected ribophosphoesters for applications in RNA synthesis

The chemical synthesis of RNA, in contrast to that of DNA, poses problems due to fi) additional requirement of 2′-hydroxyl protection of the ribose moieties, and (ii) high lability of inter-ribophosphate bonds. Herein we report the synthesis and characterisation of N, O-protected ribophosphoesters 1 which are key monomeric derivatives in phosphotriester methodology for RNA synthesis. Both the isomeric 2′ and 3′-O-phosphates have been obtained and characterised. The utility of the t-butyl dimethylsilyl group for 2′-hydroxyl protection in the phosphotriester method is demonstrated by the synthesis of r(AUAU),r (UAUA) andr (CACA). Part 2, Rajendrakumaret al (1987) NCL communication number 4682     

Solid-phase synthesis of oligonucleotides representing fragments of trp promoters

The self-complementary oligonucleotides AACTAGTT and AGTTAACT, which are homologs of functionally important sections of trp promoters of three related bacteria, have been synthesized by the solid-phase method. A new block variant of synthesis on a polymer has been worked out and has been used for obtaining the octanucleotides AACTAGTTp and AGTTAACTp.M. V. Lomonosov Moscow State University. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 362–369, May–June, 1984.     

A base-stable dithiomethyl linker for solid-phase synthesis of oligonucleotides

A novel linkage, useful for the synthesis of oligonucleotides is described. The linking function is compatible with all conditions used for oligonucleotide synthesis, orthogonal to all other protecting groups, but regenerates 3′-OH rapidly upon mild reduction under aqueous conditions. This method is employed in the removal of depurinated fragments during the synthesis of oligonucleotides.