Challenges and Solutions in Oligonucleotide Analysis, Part V: Troubles with Phosphorothioate Oligonucleotides

In this article, we focus on analysis of phosphorothioate oligonucleotides (ONs). Thiolation of the phosphate backbone of an ON forms diastereomers that complicate their analysis. We describe recent advances in the separation and characterization of phosphorothioate oligonucleotides by liquid chromatography and ion mobility mass spectrometry.

The recent expansion of pharmaceutical portfolios to include more therapeutic oligonucleotides (ONs) has led to a dramatic increase in research of chromatographic methods for analysis and purification of these molecules. This has been accompanied by a new set of method development and troubleshooting challenges, particularly for those whose background has been focused on small molecule separations, which have not previously been addressed in any detail in the “LC Troubleshooting” column. Thus, I am thrilled to have Martin Gilar join me for a series of LC Troubleshooting installments focused on challenges encountered in liquid chromatography (LC) analyses of ONs. Gilar is one of the world’s experts on this topic, and this series of articles should be a rich resource for LC practitioners working in this area.

—Dwight Stoll

In Part I of this series, we outlined liquid chromatography (LC) methods for oligonucleotide (ON) analysis;¹ in Part II, we elaborated the principles of ion-pairing reversed-phase LC (IPRP-LC);² in Part III, we focused on LC-mass spectrometry (MS);³ and in Part IV we highlighted some of the less-known challenges of oligonucleotide analysis, such as nonspecific adsorption, peak splitting, and others.⁴ In Part V, we explain the reasons why analysis of phosphorothioate (PS) oligonucleotides is more complicated, and why the separation of PS diastereomers is becoming more important for oligonucleotide drug development.

Stereochemistry of PS Diastereomers and Its Impact on Oligonucleotide Properties

Figure 1 illustrates the structures of the two diastereomers of a single phosphorothioate linkage in the backbone of an ON molecule. Oligonucleotide synthesis uses mononucleotide building blocks consisting of pentoses with chiral carbons (carbons 1, 3, and 4 in DNA or 1, 2, 3, and 4 in RNA molecules; carbon numbers are shown in Figure 1). Replacement of one of the oxygens in the phosphate group linking two pentoses with a sulfur converts the achiral phosphodiester group to a phosphorothioate group and creates a new chiral center. The addition of this chiral phosphate group to a stereochemically pure ON molecule yields S_p and R_p stereoisomers (Figure 1), which are not enantiomers (because they are not mirror images), but diastereomers. The number of diastereomers (Z) in the ON increases with the number of PS linkages (n) according to formula Z = 2ⁿ. In other words, the isomeric complexity of the ON increases exponentially as the number of PS groups increases. One, two, three, or 24 PS linkages will produce two, four, eight, or 16.8 million diastereomers!

PS Oligonucleotides as Therapeutic Compounds

The first generation of antisense oligonucleotide (ASO) therapeutic compounds relied on full phosphorothioation of the ON backbone to improve the ASO resistance against exo- and endonucleases in vivo.^5,6 The properties and utility of PS oligonucleotides for therapeutic applications were studied and reviewed by Eckstein and others.^7–13 Both S_p and R_p forms (Figure 1) are more resistant to enzymatic hydrolysis, but the S_p isomer is exceptionally resistant to exonucleases, and the R_p isomer against endonucleases.¹⁰ Stereochemistry of PS linkages also affects hybridization strength with complementary ONs: R_p forms hybridize with greater stability resulting in higher melting temperatures than S_p forms.^13,14

While the first and second generations of ASO compounds were heavily PS modified, recent therapeutic oligonucleotides use 2’ fluorination or 2’ O-methylation (among others) to maintain in vivo stability and use PS modifications less extensively.^15–17 Typically, one or two PS modifications protect 5’ and/or 3’ termini of small interfering RNA (siRNA) or guide RNA (gRNA) therapeutic molecules.¹⁸ Stereopure PS oligonucleotides were also proposed for therapeutic purposes, containing either only Sp or Rp PS linkages.^14,19

It has been reported that PS oligonucleotide synthesis does not yield a 50:50 ratio of Sp:Rp diastereomers (as is the case for achiral synthesis of enantiomers);^11,12 different nucleotide coupling activators used for synthesis produce different yields of Rp:Sp linkages. The ability to characterize diastereomeric composition of PS ONs has been requested by regulatory agencies, prompting studies aimed at assessing the equivalency of therapeutic PS ONs.^13,20

Effects of PS ON Diastereomer Composition on LC Analysis

It is known that achiral columns exhibit selectivity for diastereomers of PS ONs—that is, stationary phases such as C18 (that don’t explicitly have chiral characteristics), can chromatographically resolve diastereomers.^18,21 While this is obviously beneficial in cases where separation of the diastereomers is desirable, it also means that LC analyses of PS ON samples consisting of thousands-to-millions of diastereomers exhibit wider peaks compared to phosphodiester ONs of the same sequence (that is, no PS linkages)^22–24 or to stereochemically pure PS ONs.¹⁴ The partial separation of PS diastereomers (which leads to peak broadening) makes it more challenging to resolve ON metabolites or truncation products carried over from the synthesis of the parent PS ON.²⁵

Several strategies were proposed to suppress the separation of PS diastereomers, thereby reducing this “chemical” source of peak broadening. These include the addition of hexafluoroisopropanol (HFIP) to IPRP mobile phases,^24,25 the use of hydrophobic ion-pairing (IP) reagents,²³ and elevated column temperatures.²² The effect of ion-pairing reagent hydrophobicity on diastereomeric separation in IPRP-LC is illustrated in Figure 2.

Diisopropylethylamine acetate is more efficient in suppressing diastereomeric separation of PS ONs than triethylammonium acetate (TEAA) and thus provides narrower peaks in IPRP LC. Similar results were observed for dibutylamine or hexylamine ion-pairing systems.²³ An ion-pairing mobile phase system consisting of 5 mM tributylammonium acetate is also highly effective for suppression of diastereomeric resolution and often used for LC–MS analysis of PS ONs.^20,26

For ON drugs containing a limited number of PS modifications (1–4 PS linkages), it is feasible to resolve the modest number of diastereomers. It has been shown that 2–16 isomers can be resolved with reversed-phase liquid chromatography (RPLC),^12,25 anion-exchange LC,^18,27 or other methods.^17,28 Diastereomeric characterization of PS ONs is an active research subject of several research groups.^{17,21,26,29–33} Figure 2b illustrates that RPLC is an efficient method for the separation of diastereomers; however, the resolution success in RPLC (and with hydrophilic ion-pairing systems) strongly depends on the ON sequence motif.^11,12,34 PS ON sequences consisting of C and G nucleotides are better resolved than motifs consisting of A or U nucleotides; chemical modification (2’F, 2’OMe or nucleotides such as locked nucleic acid, LNA) often enhances the diastereomeric separation, but not always.³⁴ Despite the volume of current research, the LC separation of these diastereomers remains poorly understood, and difficult to predict.

Methods for Characterization of PS Diastereomeric Ratios by LC and Ion Mobility Spectrometry

Available reports suggest that successful separation of PS ONs is achievable up to 16 diastereomers, especially for 4–6 nt-long oligonucleotides.^18,33 However, chromatographic resolution is incomplete for longer oligonucleotides with more than four PS modifications, and not feasible for samples of fully thiolated ONs that contain thousands to millions of diastereomers. Roussis and colleagues developed chromatographic methods that enable relative estimation of diastereomeric distributions in PS oligonucleotides from the chromatographic peak retention shift. For example, under RPLC conditions, Sp species are more strongly retained than Rp diastereomers.^10–12,14 They showed that the retention shift pattern clearly differentiates between six model diastereoisomeric mixtures (of the same ON sequence) using several chromatographic methods and ³¹P nuclear magnetic resonance (NMR) spectroscopy.^20,35

In an alternative approach, PS oligonucleotides were fragmented via chemical³¹ or enzymatic cleavage,²⁹ or by MS/MS fragmentation, followed by ion mobility spectrometry (IMS) separation of the resulting 2-5 nt-long species.³⁶ It has been shown that the IMS method (separating charged molecules in the gas phase due to their different collisional cross-sections) provides useful diastereomeric resolution of 2-5 nt PS ON diastereomers. Separation was achieved with high-resolution ion mobility coupled with HRMS³⁰ or with cyclic IMS.³⁷ Good agreement between IMS and IPRP LC results were reported³⁶ for ONs up to 5 nt, containing two PS linkages (four diastereomers, Figure 3). The encouraging IMS capabilities for analysis of PS diastereomers will undoubtedly be further explored, optimizing the selection of charged ions, and conditions for best IMS resolution.

Summary

Thiolation of the backbone of therapeutic oligonucleotides is a common and useful modification that enhances their in vivo stability. Molecules with the specific Sp and Rp stereo configurations exhibit distinct biochemical activities, such as differences in resistance to nuclease enzymes, and different energies of hybridization with complementary ON sequences. However, this modification also presents challenges when analyzing these molecules using liquid chromatography. Thiolation leads to the formation of diastereomers due to newly formed chiral centers, which can in turn lead to a kind of chemical peak broadening due to partial separation of numerous diastereomers. In this installment of “LC Troubleshooting,” we’ve highlighted some of the difficulties encountered in the analysis of PS ONs by LC, and reviewed strategies that can be used when characterization of the diastereomeric composition of an ON sample is needed. Although there has been tremendous progress in this area recently, further research and development of LC and MS (IMS) methodologies are still needed to make the characterization of PS diastereomers more robust, explainable, and predictable.

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