RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis.Natural nucleotides exist as tautomers in solution, and tautomerization of the nucleobases in DNA has been recognized as a likely mutagenic mechanism ever since the double-helical structure of the DNA molecule was first deduced by Watson and Crick (33,36). The keto (for G and U) and amino (for A and C) tautomers of the natural nucleotides are the predominant species with tautomeric constants (K T ) on the order of 10 5 . However, tautomeric conversion to the rare enol or imino forms of the nucleobases can lead to altered hydrogen bonding specificity and thus mutagenesis through noncanonical basepairing interactions. This has become known as the "rare tautomer" hypothesis of mutation (19,29).The tautomerization of bases to yield ambiguous base-pairing properties has also been exploited in the design of novel nucleoside drugs, including 5-hydroxy-2Ј-deoxycytidine (22) and 5-aza-5,6-dihydro-2Ј-deoxycytidine (KP-1212) (18, 25) (Fig. 1A). However, attempts to design clinically useful antiviral compounds around this premise have met with only limited success (13,16,18,25).Nucleobases exhibiting multiple conformations due to rotation or tautomerization have received attention as potential antiviral agents acting through lethal mutagenesis (11,22). Since the discovery that ribavirin (Fig. 1B) can act as a lethal mutagen (7), likely through rotation of the exocyclic carboxamide moiety, the concept of lethal mutagenesis as an antiviral strategy has received considerable attention (5,11,12).Recently, we have demonstrated that the ribonucleoside analogue rP (Fig. 1C) can act as a potent mutagen of poliovirus (PV) in vitro (13). Tautomerization of the nucleobas...

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

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...

Small molecules that mimic natural nucleosides and nucleotides comprise a major class of antiviral agents. A new approach to the design of these compounds focuses on the generation of lethal mutagens: [1,2] compounds that further accelerate the high rate of viral mutagenesis [3,4] to confer antiviral effects. By incorporating artificial nucleobases with degenerate base-pairing abilities into viral genomes, lethal mutagens increase viral genomic mutagenesis to intolerable levels during replication, a process termed "error catastrophe", which results in the loss of viral viability. [5, 6] The antiviral drug ribavirin (1) is one such lethal mutagen effective against the RNA viruses A C H T U N G T R E N N U N G poliovirus (PV) [7] and hepatitis C virus. [8] Ribavirin is converted intracellularly to the 5'-triphosphate (RTP), which is a substrate for viral RNA-dependent RNA polymerases (RdRP). By mimicking the natural purines, RTP is misincorporated opposite pyrimidines in the enzyme-bound viral RNA template. The incorporated nucleobase of ribavirin promotes genomic mutatagenesis by templating C and U during subsequent rounds of viral replication; this facilitates error catastrophe and loss of viral viability. [7,[9][10][11] As part of our efforts to identify more efficacious antiviral lethal mutagens, we report the synthesis, X-ray structure, and antiviral evaluation of the 5-nitroindole-containing ribonucleoside 3, and incorporation of the related ribonucleotide 5 into RNA by a viral RdRP. These compounds represent RNA analogues of the previously reported "universal" deoxyribonucleoside 2, a compound shown to base pair with all four natural DNA pseudobases. [12,13] By eliminating strong hydrogen-bond donors/acceptors, and possessing a large aromatic p system, 5-nitroindole 2 stabilizes DNA duplexes by aromatic p-stacking interactions with adjacent DNA bases. [14,15] The utility of 2 has been demonstrated in applications that range from incorporation into DNA hairpins, [16,17] primers for PCR and DNA sequencing, [13,18,19] detection of single nucleotide polymorphisms, [20,21] and (pseudobase) incorporation into peptide nucleic acids.[22]We hypothesized that ribonucleoside analogues with universal base-pairing properties might possess enhanced antiviral activity relative to ribavirin (a purine mimic) by accelerating lethal viral mutagenesis. We demonstrate here that 5-nitroindole ribonucleotide 5 is universally incorporated opposite each native RNA base by a viral (poliovirus) RdRP (3D pol ). Although triphosphate 5 becomes incorporated into RNA by poliovirus 3D pol more slowly than ribavirin triphosphate (RTP), 5 represents a much more potent inhibitor of this viral enzyme, and nucleoside 3 exhibits antiviral activity in cell culture.5-Nitroindole ribonucleoside 3 and phosphorylated analogues 5-7 were synthesized as shown in Scheme 1. Commercially available 9 was chlorinated with TiCl 4