Preparation of a 4′‐Thiouridine Building‐Block for Solid‐Phase Oligonucleotide Synthesis

Abstract

Starting from a commercially available thioether, we report a nine‐step synthesis of a 4′‐thiouridine phosphoramidite building‐block. We install the uracil nucleobase using Pummerer‐type glycosylation of a sulfoxide intermediate followed by a series of protecting group manipulations to deliver the desired phosphite. Notably, we introduce a 3′,5′‐O‐di‐tert‐butylsilylene protecting group within a 4′‐thiosugar framework, harnessing this to ensure regiospecific installation of the 2′‐O‐silyl protecting group. We envisage this methodology will be generally applicable to other 4′‐thionucleosides and duly support the exploration of their inclusion within related nucleic acid syntheses. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: (2R,3S,4R)‐2,3‐O‐Isopopropylidene‐5‐O‐tert‐butyldiphenylsilyl‐1‐(4‐sulfinyl)cyclopentane: SulfoxidationBasic Protocol 2: 2′,3′‐O‐Isopropylidene‐5′‐O‐tert‐butyldiphenylsilyl‐4′‐thiouridine: Pummerer glycosylationBasic Protocol 3: 4′‐Thiouridine: DeprotectionBasic Protocol 4: 2′‐O‐tert‐Butyldimethylsilyl‐3′,5′‐di‐tert‐butylsiloxy‐4′‐thiouridine: 2′,3′,5′‐O‐silylationBasic Protocol 5: 2′‐O‐tert‐Butyldimethylsilyl‐4′‐thiouridine: Selective 3′‐5′‐desilylationBasic Protocol 6: 2′‐O‐tert‐Butyldimethylsilyl‐5′‐O‐dimethoxytrityl‐4′‐thiouridine: 5′‐O‐dimethoxytritylationBasic Protocol 7: 2′‐O‐tert‐butyldimethylsilyl‐3′‐O‐[(2‐cyanoethoxy)(N,N‐diisopropylamino)phosphino]‐5′‐O‐dimethoxytrityl‐4′‐thiouridine: 3′‐O‐phosphitylation

Biocatalytic nucleobase diversification of 4’-thionucleosides and de novo RNA synthesis using 5-ethynyl-4’-thiouridine

“…Considering the importance of chemical modifications to the D-ribose core in developing nucleoside analogue therapeutics, we recently disclosed an efficient gram-scale synthetic entry to 4'-thioribopyrimidines, where the furanose ring oxygen is substituted with a sulfur atom. [13][14][15] This work complimented wider attention from several academic and industrial groups exploring the chemical synthesis of 4′-thionucleosides, since the first disclosure of 4′-thioadenosine in the early 1960s. [16][17][18][19] Bioisosteric replacement of furanose oxygen with larger sulfur can impart changes to the furanose ring conformation, improving the hydrolytic stability of the glycosidic linkage and has delivered several biologically active 4'-thionucleosides (Figure 1a).…”

Biocatalytic Nucleobase Diversification of 4′‐Thionucleosides and Application of Derived 5‐Ethynyl‐4′‐thiouridine for RNA Synthesis Detection

“…Considering the importance of chemical modifications to the d ‐ribose ring in developing nucleoside analogue therapeutics, we recently disclosed an efficient gram‐scale synthetic entry to 4′‐thioribopyrimidines, where the furanose ring oxygen is substituted with a sulfur atom [13–15] . This work complimented wider attention from several academic and industrial groups exploring the chemical synthesis of 4′‐thionucleosides, since the first disclosure of 4′‐thioadenosine in the early 1960s [16–19] .…”