Solid oligonucleotides

To estimate the concentration of an oligonucleotide solution, the absorption is measured at 260 nm. Correct weights of solid oligonucleotides are difficult to obtain due to non-stoichio-metric inclusion of water molecules into the solid. UV spectra of nucleic acids exhibit a pronounced hypochromicity. In double-stranded nucleic acids, base stacking with a distance of about 3.5 A lowers UV-absorption by up to 20% due ii-systems interaction when compared with single-stranded oligomers (Fig. 25). 

The protected nucleoside-3-phosphoramidite monomer units such as 671 are used in the solid-phase oligonucleotide synthesis. In the 60mer synthesis, 104 allylic protective groups are removed in almost 100% overall yield by the single Pd-catalyze reaction with formic acid and BuNH2[432], N,(9-protection of uridine derivatives was carried out under pha.se-transfer conditions[433]. 

Synthetic organic polymers, which are used as polymeric supports for chromatography, as catalysts, as solid-phase supports for peptide and oligonucleotide synthesis, and for diagnosis, are based mainly on polystyrene, polystyrene-divinylbenzene, polyacrylamide, polymethacrylates, and polyvinyl alcohols. A conventional suspension of polymerization is usually used to produce these organic polymeric supports, especially in large-scale industrial production. 

Cleavage of oligonucleotide from solid support and removal of A benzoyl and A isobutyryl blocking groups. 

One widely used method involving protected compounds is solid-phase synthesis (polymer-supponed reagents). This method has the advantage of requiring only a simple workup by filtration such as in automated syntheses, especially of polypeptides, oligonucleotides, and oligosaccharides. 

The disadvantage of this method is that the dichloridites and monochloridites are sensitive to water and thus could not be used readily in automated oligonucleotide synthesis. This problem was overcome by Beaucage and Caruthers, who developed the phosphoramidite approach. In this method, derivatives of the form R 0P(NR2)2 react with one equivalent of an alcohol (catalyzed by species such as l//-tetrazole) to form diesters, R OP(OR")NR2, which usually are stable, easily handled solids. These phosphoroamidites are easily converted to phosphite triesters by reaction with a second alcohol (catalyzed by l//-tetrazole). Here, again, oxidation of the phosphite triester with aqueous iodine affords the phosphate triester. Over the years, numerous protective groups and amines have been examined for use in this approach. Much of the work has been reviewed. ... 

The antisense oligonucleotide LErafAON against the serine/threonine kinase c-Raf has been tested in phase I clinical trials. The antisense oligonucleotides ISIS-5132, which also inhibits c-Raf, and ISIS-3521, which inhibits PKC, went through different phase clinical trials with solid tumour patients. Unfortunately, no objective responses occurred with these PKI. GEM-231, an oligonucleotide targeting the RIa subunit of protein kinase A is currently undergoing phase I/II clinical trials alone or in combination with traditional therapy for the treatment of solid cancers [3]. 

Solid-phase chemistry is an efficient synthetic tool that, compared with solution-phase chemistry, simplifies the work-up of the reaction, allows the process to be driven to completion by using excess of reagents, and can be automatized [2a]. In recent years, many studies have been devoted to developing both surface-mediated and resin-supported synthesis. Today the solid-phase approach is not limited to peptides and oligonucleotides but is also used to synthesize molecules of lower molecular weight. 

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... 

Silica or porous glass is usually used as the solid phase in oligonucleotide synthesis. The support is functionalized through an amino group attached to the silica surface. There is a secondary linkage through a succinate ester to the terminal 3 -OH group. 

Over the past decade the techniques of combinatorial synthesis have received much attention. Solid phase synthesis of polypeptides and oligonucleotides are especially adaptable to combinatorial synthesis, but the method is not limited to these fields. The goal of combinatorial synthesis is to prepare a large number of related... 

Fig. 45. (a) Introduction of M(phen)31 complex into DNA oligomers, (b). Steady-state emission spectra of modified oligonucleotides in 0.01 m sodium phosphate buffer, pH 7.0, 0.1 NaCl. Top Ru(phen)3+-modified 20-mer duplex (solid line), a 1 1 mixture of non-comple-mentary Ru(phen)3+- and Os(phen)g+-modified 20-mers (- -) and a 20-mer duplex containing Ru(phen)g+ and Ps(phen)3+groups on different strands separated by one base pair (---). Bottom 20-mer duplex with 5 -terminal Rufphen) (solid line), and Ru(phen)jf/Os(phen)3 -containing analog (---). Reproduced with permission from Ref. (157). Copyright 1998, American Chemical Society. 

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