As shown in Fig. 2, an enantioselective aldol reaction between 2,2-dimethoxyacetaldehyde (8; $0.90/g) and 2,2-dimethyl-1,3-dioxan-5-one (9; $2.9/g) afforded the central chiral building block 6 (93–94 %ee)23,24. This step, which utilizes inexpensive and readily accessible starting materials, has been performed on up to 20 g without loss of yield or enantioselectivity allowing us to conveniently stockpile this intermediate. We proceeded to explore 1,2-additions of the ketone functionality in 6 for the introduction of the eventual C4ʹ-modification. With methylmagnesium bromide, this step proceeded smoothly to afford the syn-diol 10 in 69% yield and good diastereoselectivity (5:1), setting the stage for the intramolecular trans-acetalization reaction to construct the modified ribose core 13. Critically, this one-pot sequence relies on the selective deprotection of the dimethyl acetal over the acetonide to unveil the oxocarbenium (11) required for ribose ring formation. Initial attempts here with traditional conditions such as catalytic amounts of Brønsted acids (e.g., AcOH, HCl, TsOH, TFA) were unselective and led to either decomposition of the starting material 10 or a complex mixture of unidentifiable by-products. Other reported conditions such as InCl3/H2O25, I2/acetone26, and TBSOTf/collidine27,28 (entry 5) were also unsuccessful. Following an extensive investigation, we found that by carefully altering the temperature and stoichiometry of TMSOTf and 2,6-lutidine we could effect the desired transformation (entries 3 and 4). To this end, TMSOTf (2 equiv.) and lutidine (1 equiv.) at −10 °C followed by a water quench cleanly afforded the desired product 13 in 66% yield via a remarkable sequence of intramolecular trans-acetalizations. The use of excess TMSOTf relative to lutidine proved critical for executing this reaction. We posited the conversion of 10 to 13 proceeded by acetal deprotection and acetonide migration to afford intermediate 11 where 11 then cyclizes to ribopyranoside 12a via nucleophilic attack by the primary alcohol (5-OH) on the oxocarbenium. Under the Lewis acidic conditions of the reaction mixture, 12a is converted to oxocarbenium 12b which then undergoes a transannular cyclization to form 13. The plausibility of this transannular cyclization in hexose rings is supported by both literature reports29,30 and our own computational work. Using the ωB97X-D/Def2-SVP level of theory31, we computed a low energy barrier of +20 kJ/mol for the cyclization of 12b to 13 indicating this to be a highly feasible transformation at room temperature (see Supplementary Information page 86). Additionally, we carried out a few key experiments to support our mechanistic proposal. We hypothesized acetonide migration must occur prior to cyclization in order for the reaction to proceed. To verify this, we blocked the 2-OH in compound 10 as an OMe (see Supplementary Information page 84) to purposely prevent acetonide migration. Attempts to cyclize this material were unsuccessful as no cyclized products were observed, which strongly suggests that acetonide migration must occur prior to cyclization so that the primary 5-OH can be unveiled. An alternative cyclization (see Fig. 2, pathway B) occurring via the nucleophilic attack by the tertiary alcohol (4-OH) in intermediate 11 was also considered. Pathway B proceeds through the formation of 12c, a compound observed as a minor product in our reaction mixture. Upon isolation of 12c, we re-exposed it to the cyclization conditions, however, no product formation was observed. Thus, 12c is an undesired by-product in the reaction rather than a productive intermediate. As such, pathway B was ruled out based on this key result. To close out the route, TESOTf promoted ring opening/peracetylation of 13 and subsequent Vorbrüggen glycosylation using thymine, bis(trimethylsilyl)acetamide (BSA) and TMSOTf in a mixture of 1,2-DCE and MeCN gave 4ʹ-methylthymidine (15, β-anomer only) in only five total steps. The previous shortest chemical synthesis of this analogue was 13 steps, highlighting the improved efficiency of this process32. To demonstrate its scalability, we then used this route to produce 1.05 g of 15 in a single run without observing significant changes in yield.Fig. 2: Five-step synthesis of nucleoside analogue 15.A Process development. B Mechanistic investigations into the intramolecular trans-acetalization. aIsolated yields; bCarried out at −10 °C; cCarried out at 0 °C. BSA bis(trimethylsilyl)acetamide.Enabled by the development of a versatile intramolecular trans-acetalization reaction, we proceeded to evaluate other Grignard reagents and nucleobases for the generation of high-value 4ʹ-modified nucleoside analogues (Fig. 3). Remarkably, this process proved compatible with an array of topical 4ʹ-modifications including methyl (15, 18–23), ethyl (24, 25), allyl (26, 27), trideuteromethyl (28–31), vinyl (32), and ethynyl (33, 34), that can be conveniently attached with a nucleobase of one’s choosing. We proceeded to generate 4ʹ-analogues of natural products adenosine (20, 26, 28), thymidine (15, 31, 32), and cytidine (22, 25, 29, 34). Non-canonical nucleobases that are in high demand in drug discovery such as 6-methoxy-adenine (18), 2-chloro-adenine (23), 6-chloro-adenine (27), iodouracil (19, 30), and 2-fluoro-adenine (21, 33) were also incorporated in excellent overall yields. In all cases, these syntheses represent roughly a two to threefold improvement in step count over the previous shortest syntheses for 1514,32, 1914, 2014,32, 21–2514,33,34,35, 3336, and 3436. For instance, during a drug discovery campaign pursuing treatments for RNA-dependent RNA viral infections, Isis Pharmaceuticals synthesized 22 in 14 steps via a semi-synthetic approach starting from diacetone-d-glucose33. In a very recent patent disclosing several DNA damage repair enzyme inhibitors, PrimeFour Therapeutics reported the only synthesis of 21 in 16 steps34. Even in comparison to routes that employed newly bioengineered enzymes, our sequence proved to be over twofold shorter (i.e., 15, 19, 20, and 23). For example, 23 was previously synthesized in 11 steps using a biocatalytic trans-glycosylation of 4ʹ-methyluridine with 2-chloroadenine14. The novel compounds (18, 26–32) synthesized with this expedited route map well onto structures previously disclosed in the recent patent literature and serve to highlight our process’ utility for exploring chemical space around this valuable chemotype. While a few analogues (i.e., 26 and 27) were obtained in lower overall yields, it is important to recognize that chemical synthesis in medicinal chemistry prioritizes routes for their efficiency and the structural diversity they can access. Furthermore, during pandemic emergencies the ability to rapidly generate and identify antivirals becomes even more important in fighting waves of infection and viral mutations.Fig. 3: Substrate scope of C4ʹ-modified nucleoside analogues.*Involves biocatalytic processes. BSA bis(trimethylsilyl)acetamide.C4ʹ-modified nucleoside analogues of some currently marketed antivirals have never before been synthesized presumably owing to the lengthy routes that would be required to make them. We sought to utilize our process in the synthesis of Ribavirin and Mizoribine analogues. Ribavirin is a broad-spectrum antiviral that is listed as an essential medicine by the World Health Organization (WHO)37 while Mizoribine is a natural product approved in Japan for use as an immunosuppressant during renal transplantation38. Uniquely, these nucleoside analogues contain unusual triazole and imidazole nucleobases respectively. We synthesized their C4ʹ analogues (see Fig. 4; 36–39) in just five steps with modest to good overall yields. In MTT cell viability assays, compounds 36–39 showed minimal cytotoxicity, an important criterion for antiviral drug development, and, in some instances, were even less cytotoxic than the parent compounds Ribavirin and Mizoribine (see Supplementary Information pages 88–89). Next, we turned our attention to the synthesis of C-linked nucleosides, which offer much improved metabolic stability compared to their N-linked counterparts. Using a modified protocol adapted from a recent report by Li et al.20, we attempted a nickel-catalyzed C-glycosylation of 14—this failed to provide the desired product. Surprisingly, using 40 instead of 14 as the glycosyl donor in the C-glycosylation afforded 41 in modest yield. 40 was generated through an alternate ring opening/activation of 13 using 5 equivalents of acetic anhydride in the presence of TESOTf. Finally, by performing the initial aldol step with d-proline instead of l-proline we made 4ʹ-ethyl analogue (43) of Levovirin (42), an l-nucleoside and investigational HCV antiviral39.Fig. 4: Synthesis of C4ʹ-modified ribavirin and mizoribine analogues, a C-linked analogue, and an l-nucleoside analogue.acac acetylacetone, Terpy terpyridine.In summary, we have developed a modular five-step de novo synthesis of C4ʹ-modified nucleoside analogues. This short sequence relies on an intramolecular trans-acetalization reaction to enable broad diversification via Grignard addition and Vorbrüggen glycosylation. Given the robustness of this protocol, several additional C4ʹ-modifications and nucleobases are anticipated to be compatible to support medicinal chemistry efforts. In all cases, this process is two to threefold shorter than the previous shortest syntheses reported for each analogue. This highly accessible and convenient protocol should facilitate the exploration of chemical space around this valuable chemotype and aid in the development of antiviral, anticancer, and oligonucleotide therapeutics.
