METHODS FOR THE SYNTHESIS OF OLIGONUCLEOTIDES
Publication Date: 2012-Jul-27
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Oligonucleotides are typically synthesized chemically using automatic instruments. These oligonucleotides are synthesized from nucleotide monomers. In some situations, it is also possible to include unnatural nucleotide monomers (i.e., modified monomers) in the synthesis. The products of such syntheses may be oligonucleotides that include one or more modified bases. The inclusion of modified bases may provide desired properties.
Nucleic acids are building blocks of life; they store and relay genetic information. The storage of genetic information is carried out by deoxyribonucleic acids (DNA), and the information is determined by particular sequence combinations of nucleobases adenine (A), guanine (G), cytidine (C) and thymine (T). This genetic information carried by DNAs may be transcribed into messenger ribonucleic acids (mRNA), which are mediators for protein expression. The mRNA can be translated into protein molecules with the help of transfer RNA (tRNA, amino acid carriers) and ribosomes (made up of RNA and protein), where amino acids are stitched together to form proteins, which in turn function as the major machinery of life.
DNA is made up of 2'-deoxyribonucleotides and RNA is made up of ribonucleotides. In the grand scheme of genetic information storage and transmission, RNAs have long been regarded as passive members. Their roles were thought to be well understood. However, it has recently become apparent that RNA molecules can also play complex roles.
Both DNA and RNA oligonucleotides are joined together by 3'-5'-phosphodiester linkages:
Nucleotides are the basic constituents of both DNA and RNA molecules; they are the phosphate esters of nucleosides (Scheme 1). Nucleosides contain a nitrogenous heterocyclic base and a ribose (pentose). DNA contains 2-deoxyribose, while RNA contains ribose. In aqueous solution, ribose exists predominantly in the pyranose form. However, the furanose form is also common for biomolecules.
Scheme 1. Structures of the four deoxyribonucleosides (top) and ribonucleosides (bottom).
Chemical Synthesis of Oligonucleotides
The major challenge in oligonucleotide synthesis lies in how to stitch together nucleotides via the 5'-3'-internucleotide phosphate bonds. Michelson and Todd.35 were among the first to achieve the chemical synthesis of such molecules. They used protected nucleoside and nucleotide monomers for the successful synthesis of oligonucleotide s (Scheme 2).
Scheme 2. i) Product obtained from the reaction between ammonium monobenzyl phosphate and (PhO)2P(O)Cl, 2,6-lutidine, benzene; ii) NCS, MeCN, benzene; ii) 2,6-lutidine, MeCN; iv) H2SO4, EtOH, H2O; v) Ba(OH)2, H2O.
The Phosphodiester Approach
Khorana and co-workers later developed the phosphodiester app...