Synthesis of 2‘-C-Difluoromethylribonucleosides and Their Enzymatic Incorporation into Oligonucleotides

Abstract

[Chemical reaction: See text] Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of these analogues, 2'-C-beta-difluoromethylribonucleosides. We constructed the glycosylating agent 4 in three steps from 1,3,5-tri-O-benzoyl-alpha-D-ribofuranose 1. The key steps included nucleophilic addition of difluoromethyl phenyl sulfone to 2-ketoribose 2 followed by mild and efficient reductive desulfonation. Ribofuranose 4 glycosylated bis(trimethylsilyl)uracil directly, giving difluoromethyluridine 7 efficiently after deprotection. Conversion of 4 to the corresponding ribofuranosyl bromide allowed efficient access to C, A, and G analogues. A related approach starting from methyl D-ribofuranose offered synthetic entry into the diastereomeric manifold, 2'-C-alpha-difluoromethyl-arabino-alpha-pyrimidine. To incorporate 2'-C-beta-difluoromethyluridine into an oligodeoxynucleotide we converted 7 to the bisphosphate and carried out successive ligation reactions using T4 RNA ligase and T4 DNA ligase. Analogous to natural RNA linkages, the resulting oligonucleotide undergoes hydroxide-catalyzed backbone scission at the difluoromethyluridine residue via internal transphosphorylation.

An improved strategy for the synthesis of [18F]-labeled arabinofuranosyl nucleosides

“…It has been shown that NH 3 can efficiently remove the benzoyl group of protected uridine when the incubation was performed at 4°C for 24–48 h [24–25]; however, in our case, incubation with NH 3 at 50°C for 5 min resulted in incomplete deprotection and unidentified byproducts. We also observed that presence of SnCl 4 reduced the overall yield of the desired final product (β-anomer) than expected from the coupling step.…”

α-(Difluoromethyl)styrene: Improved approach to grams scale synthesis

“…The difluoromethyl moiety has been widely used to modify physical, chemical and biological properties of organic molecules. Although, several methods for the synthesis of difluorometylated derivatives are available [7][8][9][10][11][12][13], only few protocols of FMST synthesis on a large scale have been reported to date [14][15][16]. According to the literature synthetic strategies the difluoromethyl α-substituted styrene was prepared by two-step procedure, where a key step involved a nucleophilic addition of Page 6 of 17 A c c e p t e d M a n u s c r i p t either Gringard's reagent (phenylmagnesium bromide [14]) or organolithium (methyllithium [15]) to corresponding gem-difluorinated ketones.…”

Recent advances in the synthesis of fluorinated nucleosides

“…303 Their method commenced with the construction of the glycosylating agent 792 in several steps from 1,3,5-tri-O-benzoyl-a-D-ribo-furanose 789, and the key steps included the nucleophilic addition of PhSO 2 CF 2 H to the ketone 790 followed by mild and efficient reductive desulfonation (Scheme 121). Glycosylation of the compound 792 with bis (trimethylsilyl)uracil and subsequent debenzoylation gave the difluoromethyluridine 793 (B¼U).…”