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Preparation of C4′-modified nucleoside analogs

Nature Protocols (2026)Cite this article

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Abstract

C4′-modified nucleoside analogs continue to attract global attention for treating infectious diseases and as components in oligonucleotide therapeutics. Current preparations mostly employ lengthy semi-synthetic approaches that do not allow for the efficient exploration of the chemical space associated with this valuable nucleoside subclass. Here we describe the pilot-scale (250 mg) and process-scale (85 g) preparation of 4′-methyl-ribothymidine (4′,5-dimethyluridine) using a de novo strategy. Both l- and d-nucleoside analogs are accessible, and 10 different C4′ modifications and 20 different nucleobases can be used interchangeably to create new analogs. This protocol involves the use of an enantioselective proline-catalyzed aldol between 2,2-dimethoxyacetaldehyde and a dioxanone. A 1,2-addition into the aldol product installs the C4′ modification. Subsequent cyclization via intramolecular trans-acetalization delivers the modified ribose core of the nucleoside analog. Peracetylation, followed by Vorbrüggen glycosylation, completes the route. The pilot- and process-scale protocols can be completed in ~5 and ~7 d, respectively, to deliver C4′-modified nucleoside analogs in good yields and excellent enantiopurity.

Key points

  • C4′-modified nucleoside analogs can be prepared via this five-step process. Pilot-scale (250 mg) and process-scale (85 g) procedures are described.

  • Although not limited to these, 10 different C4′ modifications and 20 different nucleobases have been shown to be compatible with this procedure.

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Fig. 1: Examples of C4′-modified NAs in drug discovery.
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Fig. 2: Overview of de novo five-step synthesis of C4′-modified NAs.
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Fig. 3: A proposed route to C4′-2-deoxy-NAs from intermediate 13.
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Fig. 4: Process- and pilot-scale synthesis of C4′-modified NAs.
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Fig. 5: Summary of yields and anomeric ratios for different scales of glycosylation (Stage 5).
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Fig. 6: Overview of C4′ modifications and nucleobases that are compatible with our process.
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Data availability

The relevant data for this protocol can be found in the text and Supplementary Information of this paper and/or its supporting primary research paper. A preprint article containing the raw data for the additional C4′ modifications and nucleobases has been deposited at ChemRxiv https://doi.org/10.26434/chemrxiv.10001711/v1.

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Acknowledgements

We thank funding from the Alberta Ministry of Technology and Innovation through SPP–ARC (Striving for Pandemic Preparedness–The Alberta Research Consortium). M.W.M. is thankful for the support from the Manley and Marian Johnston Professorship in Chemistry. G.K. and L.K.M. acknowledge the Canada Excellence Research Chairs Program (CERC in Glycomics). We thank M. Ferguson for his work on structural confirmation with X-ray crystallography. We also thank E. Fu (Hall Lab) for his work on %ee determination.

Author information

Author notes

  1. These authors contributed equally: Gautam Kumar, Jason W. Wang.

Authors and Affiliations

  1. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Thirupathi Nuligonda, Gautam Kumar, Jason W. Wang, Lara K. Mahal & Michael W. Meanwell

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  1. Thirupathi Nuligonda
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  2. Gautam Kumar
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Contributions

T.N. optimized the process-scale protocol, wrote the pilot- and process-scale procedures and the Supplementary Information. T.N. and G.K. expanded the substrate scope of the protocol. J.W.W. helped optimize the process-scale protocol. L.K.M. edited the manuscript and the pilot- and process-scale procedures. M.W.M. wrote the manuscript and supervised the project.

Corresponding author

Correspondence to Michael W. Meanwell.

Ethics declarations

Competing interests

M.W.M. and T.N. declare the following competing interests: the University of Alberta holds a patent (WO 2025/199647 A1) describing the synthesis of C4ʹ-modified NAs via the process presented in this manuscript. The other authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Stanislaw Wnuk, who co-reviewed with Mukesh Mudgal; Noriko Saito-Tarashima; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference

Nuligonda, T. et al. Nat. Commun. 15, 7080 (2024): https://doi.org/10.1038/s41467-024-51520-5

Extended data

Extended Data Fig. 1

A) Aldol reaction: synthesis of aldol product. B) Purification of process scale aldol reaction. The aldol reaction needs to be maintained at between 0 - 4oC, which can be done in a refrigerator as shown in panel A. Purification on process scale requires the use of column with an internal diameter of 10 cm.

Extended Data Fig. 2

A) Cannulation setup for Grignard reaction. B) Setup for the filtration through celite for quenched Grignard reaction. On process scale, the Grignard should be slowly cannulated into the reaction mixture (panel A). The quenched reaction mixture is filtered through a pad of celite to remove insoluble salts (panel B).

Extended Data Fig. 3

A) pilot scale setup of glycosylation reactions; B) Process setup of glycosylation reaction. On pilot scale, it is possible to set up multiple glycosylation reactions in the same heating bath (panel A). A 1 L round-bottom flask is required for process scale glycosylation (panel B).

Extended Data Fig. 4

Concentration of crude 4′-methylribothymidine (19) (process scale); B) Blow drying pilot scale sample of 19. Rotary evaporators are used to remove solvents from crude mixtures and purified samples (panel A). In order to remove the acetamide by-product from the glycosylation, blow drying the sample with a steady stream of air for several hours is required (panel B).

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Nuligonda, T., Kumar, G., Wang, J.W. et al. Preparation of C4′-modified nucleoside analogs. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01353-x

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