Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides

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 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 R p followed by S p, 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 R p and S p diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation. Electronic supplementary material The online version of this article (10.1007/s00216-019-01813-2) contains supplementary material, which is available to authorized users.

Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents

“…At 30 mM, all injection volumes give Langmuirian elution profiles, but at 10 mM, there is a significant fronting, likely due to the insufficient amount of ionpairing reagent in the eluent. Similar observations have been made for peptides [23]. As a conclusion, it is necessary to use Fig.…”

Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase

“…Oligomer hydrophobicity and number of stereoisomers increase with increasing number of phosphorothioated sites [10,12], and so does the strength of the stationary phase-oligomer interaction. Hence, previous works employed ion-pairing reagents to separate S-oligos [9][10][11] and could not avoid the use of these reagents to discriminate between analytes differing by a single phosphorothioated site. In contrast, we could successfully analyze multiple S-oligos without using any ion-pairing reagents by TRC on an IB5D5 hydrogel column, as S-oligos moderately interacted with the stationary phase.…”

“…organic solvent-containing mobile phases and/or gradient methods [5]. In particular, several S-oligo analysis methods use ion-pairing reagents such as protonated triethylamine [9][10][11]. Generally, S-oligo molecules are more hydrophobic than their non-modified versions [10].…”

Phosphorothioate Substitutions in RNA Structure Studied by Molecular Dynamics Simulations, QM/MM Calculations and NMR Experiments

“…12 Separate synthesis of the two enantiomers or purification of their mixtures are non-trivial issues and subject of ongoing experimental research. [13][14][15] On the other hand, the PT enantiomer can be unambiguously selected in theoretical studies. Despite their significant biological effects on RNA metabolism, the structural and dynamical impacts of PT modifications are poorly understood.…”