UNIT 4.17 Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages
Published Online: 1 OCT 2003
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Nucleic Acid Chemistry
How to Cite
Guga, P. and Stec, W. J. 2003. Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages. Current Protocols in Nucleic Acid Chemistry. 14:4.17:4.17.1–4.17.28.
- Published Online: 1 OCT 2003
- Published Print: SEP 2003
A method for solid-phase synthesis of stereodefined PS-oligos via an oxathiaphospholane approach using pure P-diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5′-O-DMTr-N-protected deoxyribonucleosides with 2-chloro-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O-labeled mercaptoalcohol, the corresponding 18O-labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS-oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5′-O-DMTr-N-protected-deoxyribonucleoside-3′-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s into their P-diastereomers, and for manual solid-phase synthesis of PS-oligos. Oxidation of 5′-O-DMTr-N-protected-deoxyribonucleoside-3′-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s with selenium dioxide yields their 2-oxo-analogs, which are suitable either for elongation of stereodefined PS-oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O-labeled PO-oligos of predetermined P-chirality.
- nucleic acid;
- oligonucleotide probe;
- stereocontrolled synthesis;