Abstract
Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry; however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered by their rapid in vivo metabolism. Although a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. This operationally simple approach provides access to pure stereoisomers of nucleoside α-thiodiphosphates and α-thiotriphosphates, as well as symmetrical or unsymmetrical dinucleoside thiodiphosphates and thiotriphosphates (including RNA cap reagents). We demonstrate that ligand–receptor interactions can be dramatically influenced by P-stereochemistry, showing that such thioisosteric replacements can have profound effects on the potency and stability of lead candidates.
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Data availability
All data generated or analysed during this study (including experimental procedures, optimization details and spectral data for all new compounds) is available within the paper and Supplementary Information.
X-ray crystallographic data for compound (−)-11 have been deposited with the Cambridge Crystallographic Data Centre (CCDC deposition no. 2172688). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. Source data are provided with this paper.
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Acknowledgements
We are grateful to D.-H. Huang and L. Pasternack (Scripps Research) for NMR spectroscopic assistance, B. Sanchez, Q.N. Wong and J. Chen for HRMS assistance, M. Gembicky for X-ray crystallographic analysis, A. Bauer, M. Meanwell, M. Bielecki, A.F. Garrido Castro, C. He, Y. Kawamata and S. Gnaim for insightful discussions, and T.E.-H. Ewing for analytical assistance. Financial support for this work was provided by Bristol-Myers Squibb. M.O. was supported by the Polish National Agency for Academic Exchange (Bekker programme no. PPN/BEK/2020/1/00111/U/00001). H.-J.Z. was supported by Shanghai Institute of Organic Chemistry Fellowship. M.N. thanks the Council for Higher Education, Fulbright Israel. K.A.J thanks the NIH, NIDDK, for funding (ZIADK031116). C.E.M. and co-workers were funded by the Deutsche Forschungsgemeinschaft (SFB 1328). J.J. and co-workers were supported by the Polish National Science Centre (grant no. 2019/33/B/ST4/01843). Bristol Myers Squibb assisted with the conceptualization of this work. The other funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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H.-J.Z., M.O., M.N., M.A.S., M.D.E. and P.S.B. conceptualized the study. H.-J.Z., M.O., M.N. and B.Z. developed the reagents. H.-J.Z., M.O. and M.N. conducted optimization and scope experiments. H.-J.Z., M.O., M.N. and Z.L. analysed the data. H.B., J.N., S.M., H.A.-H., B.B. and C.E.M. designed and performed biological experiments on the P2Y13 and P2X receptors and ecto-enzymes. S.A.L., V.S. and K.A.J. designed and performed biological evaluation on the P2Y6 and P2Y14 receptors. V.S. and K.A.J. performed molecular modelling. O.P., J.K. and J.J. designed and performed CleanCap thioisosteres biophysical studies. H.-J.Z., M.O., M.N., C.E.M., K.A.J., J.K., J.J. and P.S.B. wrote the manuscript. P.S.B., M.A.S. and M.D.E. acquired funding. P.S.B. administered and supervised this work.
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A provisional US patent application on this work has been filed by Bristol-Myers Squibb (application no. 63/376,249), with H.-J.Z., M.O., M.N., B.Z., M.A.S., M.D.E. and P.S.B. listed as inventors. The patent covers the synthesis of new reagents and their applications to the synthesis of nucleotide thioisosteres. P.S.B. is a paid consultant for Bristol Myers Squibb. B.Z., Z.L., M.A.S. and M.D.E. are employees of Bristol Myers Squibb.
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Materials and methods, Supplementary Figs. 1–47, Tables 1–18 and spectral data for all new compounds.
Supplementary Data
Crystallographic data for compound (−)-11; CCDC reference 2172688.
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Unprocessed numerical data for graphs 3c, 3d, 3g, 3h, 3i, 3j.
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Zhang, HJ., Ociepa, M., Nassir, M. et al. Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates. Nat. Chem. 16, 249–258 (2024). https://doi.org/10.1038/s41557-023-01347-2
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DOI: https://doi.org/10.1038/s41557-023-01347-2
