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Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
AstraZeneca, IMED Biotech Unit, Pharmaceut Sci, Adv Drug Delivery, S-43183 Gothenburg, Sweden.
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2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 15, p. 3383-3394Article in journal (Refereed) Published
Abstract [en]

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C-18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.

Place, publisher, year, edition, pages
SPRINGER HEIDELBERG , 2019. Vol. 411, no 15, p. 3383-3394
Keywords [en]
IP-RPLC, Ion-pair, Oligonucleotide, Antisense, Phosphorothioate, Diastereomer
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-387935DOI: 10.1007/s00216-019-01813-2ISI: 000469757300016PubMedID: 31020370OAI: oai:DiVA.org:uu-387935DiVA, id: diva2:1331657
Funder
Swedish Research Council, 2015-04627ÅForsk (Ångpanneföreningen's Foundation for Research and Development), 17/500Available from: 2019-06-27 Created: 2019-06-27 Last updated: 2019-06-27Bibliographically approved

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