UNIT 4.17 Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages

  1. Piotr Guga,
  2. Wojciech J. Stec

Published Online: 1 OCT 2003

DOI: 10.1002/0471142700.nc0417s14

Current Protocols in Nucleic Acid Chemistry

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.

Author Information

  1. Polish Academy of Sciences, Lodz, Poland

Publication History

  1. Published Online: 1 OCT 2003
  2. 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;
  • analog;
  • oligodeoxyribonucleotide;
  • oligonucleotide probe;
  • P-chirality;
  • stereocontrolled synthesis;
  • phosphorothioate;
  • oxathiaphospholane