One-Pot Synthesis of Nucleoside 5′-Triphosphates from Nucleoside 5′-H-Phosphonates

Abstract

Nucleoside 5′-triphosphates (NTPs) play key roles in biology and medicine. However, these compounds are notoriously difficult to synthesize. We describe a one-pot method to prepare NTPs from nucleoside 5′-H-phosphonate monoesters via pyridinium phosphoramidates, and we used this approach to synthesize ATP, UTP, GTP, CTP, ribavirin-TP, and 6-methylpurine ribonucleoside-TP (6MePTP). Poliovirus RNA-dependent RNA polymerase efficiently employed 6MePTP as a substrate, suggesting that the cognate nucleoside, a poorly understood antiviral agent, may damage viral RNA.Nucleoside 5′-triphosphates (NTPs) are critical mediators of myriad biological processes including DNA replication, transcription, and translation. Correspondingly, synthetic mimics of NTPs have been widely used as molecular probes and biological assay components and represent active metabolites of certain drugs such as the antiviral agent ribavirin. Despite their importance in biology and medicine, the diversity of commercially available NTPs is limited because these compounds are often difficult to prepare and isolate in pure form. 1 Traditional approaches for the synthesis of NTPs include the "one-pot, three-step" method and the method of Ludwig and Eckstein. 2,3 Although these strategies work well for some substrates, others are plagued by low yields and difficulties in purification. 4 More recently, Ahmadibeni reported 5 a solid-phase route to NTPs, and Borch reported 6 a method for the preparation of activated phosphoramidates that can be converted to NTPs by reaction with tris(tetra-n-butylammonium) hydrogen pyrophosphate. The final coupling step employed by Borch was found to proceed in remarkably high yield, but the required four-step synthesis of We hypothesized that nucleoside 5′-H-phosphonates might provide novel and more readily synthesized precursors to NTPs. This hypothesis was based on reported syntheses of phosphates, phosphoramidates, and other phosphate derivatives from these precursors. [7][8][9][10] After conversion to silyl phosphites with TMSCl, H-phosphonate monoesters can be oxidized by elemental iodine and other reagents to generate electrophilic intermediates. 11,12 These intermediates are known to react with a variety of nucleophiles to afford addition products, but surprisingly, this strategy has not been previously investigated for the synthesis of NTPs.We report here a one-pot approach for the synthesis of NTPs from nucleoside 5′-Hphosphonate monoesters, relatively stable compounds that can be easily prepared from 2′,3′-O-isopropylidene-protected nucleosides. 13 We demonstrate that fully deprotected ribonucleoside 5′-H-phosphonate monoesters can be converted in situ to pyridinium phosphoramidate intermediates. Upon addition of nucleophilic tris(tetra-n-butylammonium) hydrogen pyrophosphate, NTPs can be isolated by use of a two-step purification protocol.As shown in Scheme 1, starting with the known 2′,3′-O-isopropylidene-protected nucleosides 1-6, 13-15 phosphitylation 16 with salicyl phosphorochlorodite or PCl 3 provi...

Solid‐Phase Synthesis of Oligonucleotide 5′‐(α‐P‐Thio)triphosphates and 5′‐(α‐P‐Thio)(β,γ‐methylene)triphosphates

“…Taking into account the high susceptibility of H-phosphonothioate monoester to oxidation by iodine, the method developed by Sun et al [12] represented an alternative to achieve the oxidative phosphorylation. The conditions used for the synthesis of NTPs via a pyridinium phosphoramidate intermediate were adapted to our synthesis route.…”

Synthesis of 4‐Cyanoindole Nucleosides, 4‐Cyanoindole‐2ʹ‐Deoxyribonucleoside‐5ʹ‐Triphosphate (4CIN‐TP), and Enzymatic Incorporation of 4CIN‐TP into DNA

“…After considering extensive reviews on nucleoside triphosphate synthesis (Burgess & Cook, ; Roy, Depaix, Perigaud, & Peyrottes, ), we decided to forego the use of the popular “one pot, three step” chemistry (Ludwig, ; Yoshikawa, Kato, & Takenishi, ) and instead utilized a more recent methodology that employs a phosphonate precursor before elaborating to the full triphosphate (Sun et al., ), which we found to be easier to purify and would also allow for simple adaption to synthesize mono‐ and diphosphate derivatives if desired.…”

How To Form a Phosphate Anhydride Linkage in Nucleotide Derivatives

“…The intermediate salicylphosphite 6 (Scheme ) is very sensitive to traces of water in the reaction mixture, and the main product of the triphosphate synthesis according to Scheme might be nucleoside H ‐phosphonate. This disadvantage was overcome by Peterson and co‐workers, who specially prepared nucleoside 5′‐ H ‐phosphonates by the treatment of partially protected nucleosides with 4 or PCl 3 and subsequent hydrolysis . After deprotection, nucleoside 5′‐ H ‐phosphonates 14 were activated by forming silyl esters and oxidized by I 2 in the presence of pyridine (Scheme ).…”