Automated synthesis of oligonucleotides

Scheme 1. Automated Synthesis of Oligonucleotides. Synthetic Cycle for the Phosphoroamidite Method.

CPG = Controlled Pore Glass (Solid Support) 1 and 2 (B], 62, 63, 64) Commercially available Scheme 1. Automated Synthesis of Oligonucleotides. Synthetic Cycle for the Phosphoroa-midite Method. 

The most important strategy for the linear synthesis of oligonucleotides has been developed by M. Caruthers. Nowadays, the phosphite triester method is widely used in the automated synthesis of oligonucleotides on silica. The principle is shown in Scheme 25.1. 

FIGURE 25.19 The steps involved in automated synthesis of oligonucleotides using the phosphoramidite coupling method. 

An important application of solid-phase automated synthesis is the production of oligonucleotides via phosphoramidite solid-phase chemistry. Modem instmments work under cost-effective and high-throughput synthesis with 96-well, 384-well, and even 1536-well microplates. " The use of automated synthesis of oligonucleotides was reviewed in the nineties. " " ... 

The popularity of the deoxyribonucleoside phosphoramidites 8a-d (Fig. 3) in the automated synthesis of oligonucleotides has nonetheless been hampered by the thiolate treatment required for the removal... 

Fig 4 Alternate plumbing diagram for the automated synthesis of oligonucleotide phosphodiesters and phosphorothioates. This scheme also incorporates an output for a capping reagent... 

Andrus, A., Vu, H, Ramstad, P., and Pallas, M. (1991) Large scale automated synthesis of oligonucleotides. Nucl. Acids Symp. Ser. 24,41,42. 

Asseline, U and Thuong, N. T (1990) New solid-phase for automated synthesis of oligonucleotides containing an amino-alkyl linker at their 3 -end Tetrahedron Lett. 31,81-84. 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction. The techniques for chemical synthesis of oligonucleotides are highly developed. Very efficient automated methodologies based on solid phase synthesis are used extensively in fields that depend on the availability of defined DNA sequences.52... 

For the automated solid-phase synthesis of oligonucleotides, however, silica was found to be the support of choice [192-197]. Silica with large pore size (25-300 nm), so-called controlled pore glass (CPG), is generally used for this purpose. The main advantages of CPG, as compared with silica gel, are its more regular particle size and shape, and greater mechanical stability. 

Automated synthesis of peptide and oligonucleotide libraries was initiated about 10 years ago [4], Within the last three years, there has been much attention focused on the generation of combinatorial libraries of small molecules. As with biopolymers, the use of solid resin support was central to the advance of this field. In solid-phase synthesis, one of the reactants is covalently bound to the solid support and an excess of the other reactants may be used in each step to drive reactions to completion. Purification of the intermediates and final product is easily achieved through extensive washing of the resin after each chemical step. For the purpose of high throughput synthesis, cleavage of the final... 

While the assembly of oligopeptides and oligonucleotides is routine and has become highly automated, the automated synthesis of oligosaccharides is still in its infancy despite some notable advances [82,83,84]. The situation becomes even more complex when the protecting group requirements for both the oligosaccharide assembly, and peptide assembly combine in the same molecule to stretch this aspect of chemical synthesis to its limits. Conse-... 

Chemical synthesis of single-stranded DNA probes of defined sequence can be accomplished by the series of reactions shown in Figure 9-18. With automated Instruments now available, researchers can program the synthesis of oligonucleotides of specific sequence up to about 100 nucleotides long. Alternatively, these probes can be prepared by the polymerase chain reaction (PCR), a widely used technique for amplifying specific DNA sequences that is described later. 

The automated synthesis of platinated oligonucleotides has been achieved using /f-phosphonate oligonucleotide assembly. The platinated thymidine derivative (182) was synthesised from the corresponding /f-phosphonate derivative of thymidine by deprotonation with potassium hydroxide and then reaction with trans-[(NH3)2PtCl2]. 

Usman N, Pon RT, Ogilvie KK. Preparation of ribonucleoside 3 -0-phos-phoramidites and their application to the automated solid-phase synthesis of oligonucleotides. Tetrahedron Lett 26 4567-4570, 1985. 

Navarro, A. E. Spinelli, N. Moustrou, G. Ghaix, G. Mandrand, B. Brisset, H. Automated synthesis of new ferrocenyl-modified oligonucleotides study of their properties in solution. Nucl. Acid. Res. 2004, 32, 5310-5319. 

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