Abstract
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 C18 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.
Highlights
Antisense oligonucleotides (ASOs) are a growing and important class of therapeutic DNA or RNA intended for alteringElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Impurities are generated by the synthesis and degradation pathways of oligonucleotides [3, 4]
The aim of this study is to systematically investigate how the choice of tertiary ion-pairing alkylamine, ion-pair concentration, and modifier gradient slope influence the diastereomer selectivity of short PS-modified oligonucleotides
An investigation of how diastereomer selectivity varies with the carbon chain length of tertiary alkylamines is presented, followed by an indepth investigation of the effect of tributylammonium acetate (TBuAA) on oligonucleotide retention and selectivity
Summary
Impurities are generated by the synthesis and degradation pathways of oligonucleotides [3, 4]. These impurities include shortmers and longmers, as well as depurination and deamination products [5,6,7]. For separation and quantification of impurities, ion-pair reversed phase chromatography is currently the dominant separation mode but ion exchange chromatography, capillary electrophoresis, and hydrophilic interaction chromatography are used [3, 8, 9]. When using ion-pair chromatography, the most common ion-pairing reagents are tertiary alkylamines such as triethylammonium acetate (TEtAA) and tributylammonium acetate (TBuAA) [3, 10]. Triethylamine with addition of hexafluoro-2-propanol (HFIP) has been introduced as a way to maintain chromatographic performance while improving the mass spectrometry sensitivity [10]
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