[en] Phosphorothioate (PS) modification is one of the most widely used oligonucleotide chemical alterations in the oligonucleotide backbone. It has proven to be crucial in the field of therapeutic oligonucleotides regarding the optimization of their physicochemical and biological properties. In this study, a capillary electrophoresis (CE) method with an acidic background electrolyte (BGE) containing a combination of β- and γ-cyclodextrins derivatives as chiral selectors is proposed for the diastereomeric separation of 5-mer oligonucleotides containing 0, 1, 2, or 3 phosphorothioate linkages (5´-TCGTG-3´). The effects of the BGE pH, organic modifier addition, and type of cyclodextrin (CD) on chemo- and stereoselectivity and resolution were studied. A mixture of 25 mM (2-hydroxy-3-N,N,N-trimethylamino)propyl-γ-CD and 10 mM carboxymethyl-β-cyclodextrin in a pH 3 buffer was found to be the most appropriate system for the qualitative evaluation of the short oligonucleotides investigated. These phosphorothioate oligonucleotides were separated with high efficiency in less than 11 min with no capillary treatment. The suggested approach can be the basis for purity testing of this new generation of therapeutics.
Research center :
CIRM - Centre Interdisciplinaire de Recherche sur le Médicament - ULiège [BE]
Disciplines :
Pharmacy, pharmacology & toxicology
Author, co-author :
Ghassemi K, Maryam; Laboratory for the Analysis of Medicines, Center for Interdisciplinary Research on Medicines (CIRM), University of Liege, Quartier hopital, Avenue Hippocrate 15, 4000 Liege, Belgium. Electronic address: marianne.fillet@uliege.be
Demelenne, Alice ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Crommen, Jacques ; Université de Liège - ULiège > Département de pharmacie
Servais, Anne-Catherine ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Fillet, Marianne ; Université de Liège - ULiège > Département de pharmacie > Analyse des médicaments
Language :
English
Title :
Improvement of chemo- and stereoselectivity for phosphorothioate oligonucleotides in capillary electrophoresis by addition of cyclodextrins.
Catani, M., De Luca, C., Medeiros Garcia Alcântara, J., Manfredini, N., Perrone, D., Marchesi, E., Weldon, R., Müller-Späth, T., Cavazzini, A., Morbidelli, M., Sponchioni, M., Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification. Biotechnol. J., 15(8), 2020, e1900226, 10.1002/biot.201900226 Aug.
Crooke, S.T., Liang, X.H., Baker, B.F., Crooke, R.M., Antisense technology: A review. J. Biol. Chem., 296, 2021, 100416, 10.1016/j.jbc.2021.100416 Jan-Jun.
J Jahns, H., Taneja, N., Willoughby, J.L.S., Akabane-Nakata, M., Brown, C.R., Nguyen, T., Bisbe, A., Matsuda, S., Hettinger, M., Manoharan, R.M., Rajeev, K.G., Maier, M.A., Zlatev, I., Charisse, K., Egli, M., Manoharan, M., Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo. Nucleic Acids Res. 50:3 (2022), 1221–1240, 10.1093/nar/gkab544 Feb.
Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J.S., Broder, S., Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus. Proc. Natl. Acad. Sci. U S A 84:21 (1987), 7706–7710, 10.1073/pnas.84.21.7706 Nov.
Islam, M.A., Fujisaka, A., Kawakami, J., Yamaguchi, T., Obika, S., Different reactivity of Sp and Rp isomers of phosphorothioate-modified oligonucleotides in a duplex structure. Bioorg. Med. Chem. Lett., 30(14), 2020, 127166, 10.1016/j.bmcl.2020.127166 Jul 15Doi.
Meena Lemaitre, M.M., Stereocontrolled Oligonucleotides for Nucleic Acid Therapeutics: A Perspective. Nucleic. Acid Ther. 31:1 (2021), 1–6, 10.1089/nat.2020.0906 Feb.
Iwamoto, N., Butler, D.C.D., Svrzikapa, N., Mohapatra, S., Zlatev, I., Sah, D.W.Y., Meena Standley, S.M., Lu, G., Apponi, L.H., Frank-Kamenetsky, M., Zhang, J.J., Vargeese, C., Verdine, G.L., Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat. Biotechnol. 35:9 (2017), 845–851, 10.1038/nbt.3948 Sep.
Sakamuri, S., Eltepu, L., Liu, d., Lam, S., Meade, B.R., Liu, B., Dello Iacono, G., Kabakibi, A., Luukkonen, L., Leedom, T., Foster, M., Bradshaw, C.W., Impact of Phosphorothioate Chirality on Double-Stranded siRNAs: A Systematic Evaluation of Stereopure siRNA Designs. ChemBioChem 21:9 (2020), 1304–1308, 10.1002/cbic.201900630 May.
Alkadi, H., Jbeily, R., Role of Chirality in Drugs: An Overview. Infect. Disord. Drug Targets 18:2 (2018), 88–95, 10.2174/1871526517666170329123845 Doi.
Ribeiro, A.R., Maia, A.S., Cass, Q.B., Tiritan, M.E., Enantioseparation of chiral pharmaceuticals in biomedical and environmental analyses by liquid chromatography: an overview. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 968 (2014), 8–21, 10.1016/j.jchromb.2014.02.049 Oct 1Doi.
Xie, S.M., Yuan, L.M., Recent progress of chiral stationary phases for separation of enantiomers in gas chromatography. J. Sep. Sci. 40:1 (2017), 124–137, 10.1002/jssc.201600808 JanDoi.
Wang, R.Q., Ong, T.T., Ng, S.C., Synthesis of cationic beta-cyclodextrin derivatives and their applications as chiral stationary phases for high-performance liquid chromatography and supercritical fluid chromatography. J. Chromatogr. A 1203:2 (2008), 185–192, 10.1016/j.chroma.2008.07.046 Sep 5Doi.
Bernardo-Bermejo, S., Sánchez-López, E., Castro-Puyana, M., Marina, M.L., Chiral capillary electrophoresis. TrAC – Trends Anal. Chem., 124, 2020, 115807, 10.1016/j.trac.2020.115807.
El Deeb, S., Silva, C.F., Junior, C.S.N., Hanafi, R.S., Borges, K.B., Chiral Capillary Electrokinetic Chromatography: Principle and Applications, Detection and Identification, Design of Experiment, and Exploration of Chiral Recognition Using Molecular Modeling. Molecules, 26(10), 2021, 2841, 10.3390/molecules26102841 May 11.
S Salido-Fortuna, S., Castro-Puyana, M., Marina, M.L., Chiral Micellar Electrokinetic Chromatography. J. Chromatogr. A, 2020, 461383, 10.1016/j.chroma.2020.461383 Aug 30;1626.
Scriba, G.K.E., Jáč, P., Cyclodextrins as Chiral Selectors in Capillary Electrophoresis Enantioseparations. Methods Mol. Biol., 2019, 339–356, 10.1007/978-1-4939-9438-0_18 1985.
Zhu, Q., Scriba, G.K.E., Advances in the Use of Cyclodextrins as Chiral Selectors in Capillary Electrokinetic Chromatography: Fundamentals and Applications. Chromatographia 79 (2016), 1403–1435, 10.1007/s10337-016-3167-0.
Peluso, P., Chankvetadze, B., Native and substituted cyclodextrins as chiral selectors for capillary electrophoresis enantioseparations: Structures, features, application, and molecular modeling. Electrophoresis 42:17-18 (2021), 1676–1708, 10.1002/elps.202100053 Sep.
Rezanka, P., Navrátilová, K., Rezanka, M., Král, V., Sýkora, D., Application of cyclodextrins in chiral capillary electrophoresis. Electrophoresis 35:19 (2014), 2701–2721, 10.1002/elps.201400145 Oct.
Chankvetadze, B., Lindner, W., Scriba, G.K., Enantiomer separations in capillary electrophoresis in the case of equal binding constants of the enantiomers with a chiral selector: commentary on the feasibility of the concept. Anal. Chem. 76:14 (2004), 4256–4260, 10.1021/ac0355202 Jul 15.
Blanco, M., Valverde, I., Choice of chiral selector for enantioseparation by capillary electrophoresis. TrAC – Trends Analyt. Chem. 22:7 (2003), 428–439, 10.1016/S0165-9936(03)00705-2.
Stavrou, I.J., Agathokleous, E.A., Kapnissi-Christodoulou, C.P., Chiral selectors in CE: Recent development and applications (mid-2014 to mid-2016). Electrophoresis 38:6 (2017), 786–819, 10.1002/elps.201600322 Mar.
Gilar, M., Belenky, A., Cohen, A.S., Polymer solutions as a pseudostationary phase for capillary electrochromatographic separation of DNA diastereomers. Electrophoresis 21:14 (2000), 2999–3009, 10.1002/1522-2683(20000801)21:14<2999::AID-ELPS2999>3.0.CO;2-1 Aug.
Demelenne, A., Gou, M.J., Nys, G., Parulski, C., Crommen, J., Servais, A.C., Fillet, M., Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides. J. Chromatogr. A, 2020, 460716, 10.1016/j.chroma.2019.460716 Mar 15;1614.
Frey, P.A., Sammons, R.D., Bond order and charge localization in nucleoside phosphorothioates. Science 228:4699 (1985), 541–545, 10.1126/science.2984773 May 3.
Thaplyal, P., Bevilacqua, P.C., Experimental approaches for measuring pKa's in RNA and DNA. Meth. Enzymol. 549 (2014), 189–219, 10.1016/B978-0-12-801122-5.00009-X.
Lee, Y.J., Price, W.E., Sheil, M., Effect of organic solvents on the separation of benzoic acids by capillary electrophoresis. Analyst 120 (1995), 2689–2694, 10.1039/AN9952002689.
Fillet, M., Hubert, P., Crommen, J., Enantiomeric separations of drugs using mixtures of charged and neutral cyclodextrins. J. Chromatogr. A 875:1–2 (2000), 123–134, 10.1016/s0021-9673(00)00084-4 Apr 14.
Blanco, M., Coello, J., Iturriaga, H., Maspoch, S., Pérez-Maseda, C., Separation of profen enantiomers by capillary electrophoresis using cyclodextrins as chiral selectors. J. Chromatogr. A 793:1 (1998), 165–175, 10.1016/s0021-9673(97)00893-5 Jan 9.
