Retention of configuration in the action of human plasma 3'-exonuclease on oligo(deoxynucleoside phosphorothioate). A new method for assignment of absolute configuration at phosphorus in isotopomeric deoxyadenosine 5'-O-[(18)O]phosphorothioate |
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Authors: | Koziołkiewicz Maria Owczarek Alina Wójcik Marzena Domański Krzysztof Guga Piotr Stec Wojciech J |
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Institution: | Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 ?ód?, Poland. |
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Abstract: | A new method of analysis has allowed the exonucleolytic cleavage by human 3'-exonuclease to be determined. Hydrolysis by human plasma 3'-exonuclease proceeds with retention of configuration at phosphorus. The new method determines the sense of chirality at phosphorus in isotopomeric adenosine 5'-O-(18)O]phosphorothioates. This is based on stereospecific two-step conversion of the mono-thionucleotide into the corresponding deoxyadenosine 5'-O-alpha-(18)O]thiotriphosphate, followed by the use of terminal deoxyribonucleotidyl transferase and MALDI TOF mass spectrometry of the resulting elongated primer. Retention of configuration in the reaction of plasma 3'-exonuclease implies a two-step mechanism with two displacements on phosphorus. Inversion at each step leads to overall retention. |
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