Role of Marine Natural Products in the Genesis of Antiviral Agents

Role of Marine Natural Products in the Genesis of Antiviral Agent

Efficient Synthesis of Exo-<i>N</i>-carbamoyl Nucleosides: Application to the Synthesis of Phosphoramidate Prodrugs

An efficient protection protocol for the 6-<i>exo</i>-amino group of purine nucleosides with various chloroformates was developed utilizing <i>N</i>-methylimidazole (NMI). The reaction of an <i>exo</i>-<i>N</i>⁶-group of adenosine analogue <b>1</b> with alkyl/and aryl chloroformates under optimized conditions provided the <i>N</i>⁶-carbamoyl adenosines (<b>2a</b>–<b>j</b>) in good to excellent yields. The reaction of <i>N</i>⁶-Cbz-protected nucleosides (<b>5a</b>–<b>c</b>) with phenyl phosphoryl chloride (<b>7</b>) using <i>t</i>-BuMgCl followed by catalytic hydrogenation afforded the corresponding phosphoramidate pronucleotides (<b>8a</b>–<b>c</b>) in excellent yield

Synthesis of 5′-Methylene-Phosphonate Furanonucleoside Prodrugs: Application to D‑2′-Deoxy-2′-α-fluoro-2′-β‑<i>C</i>‑methyl Nucleosides

A new and facile synthetic pathway to metabolically stable 5′-methylene-bis(pivaloyloxymethyl)(POM)phosphonate furanonucleoside prodrugs is reported. The key step involves a Horner–Wadsworth–Emmons reaction of a tetra(pivaloyloxymethyl) bisphosphonate salt with appropriately protected 5′-aldehydic nucleosides. This efficient approach was applied for the synthesis HCV related 2′-deoxy-2′-α-fluoro-2′-β-<i>C</i>-methyl nucleosides

Synthesis of Cyclopentanyl Carbocyclic 5‑Fluorocytosine ((−)-5-Fluorocarbodine) Using a Facially Selective Hydrogenation Approach

An efficient synthetic route to biologically relevant (−)-5-fluorocarbodine <b>6</b> was developed. Direct coupling of N⁶-protected 5-fluorouracil <b>15</b> with cyclopentenyl intermediate <b>13</b>, followed by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring-closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give, exclusively, the desired 4′-β stereoisomer

Synthesis of (2<i>S</i>)‑2-Chloro-2-fluororibolactone via Stereoselective Electrophilic Fluorination

A novel and efficient route for the preparation of (2<i>S</i>)-2-chloro-2-fluorolactone <b>29</b> is described. This approach takes advantage of a highly efficient diastereoselective electrophilic fluorination reaction (94% yield; >50:1 dr

LLV mutations and their abundance in the pre-suppression profile^a

a<p>Mutations are numbered beginning with the first nucleotide/amino acid of RT. The region encoding RT amino acids 65-210 was sequenced from LLV samples. Mutation abundance in the pre-suppression profile is expressed as the percentage of 454 sequence reads encoding the given mutation. Dashes (-) indicate that the specified mutation was not observed in sequence reads at a pre-determined 0.5% read threshold. LLV mutations that were not observed in the pre-suppression profile are underlined. LLV mutations associated with RT drug resistance are indicated by bold italics. ND, not determined: samples were either unavailable or not tested because viral loads were below the amplification sensitivity of the 454 sequencing assay. Nucleotide: (nt)</p>b<p>Number of LLV sequences containing a specific mutation (shown at left).</p>c<p>Sample was processed; however, sequence data was not generated at this position.</p>d<p>Sample analyzed by single-genome amplification (SGA) and not 454 sequencing. Five sequences were generated by SGA at week 10 PI for RM Mmu 37969.</p>e<p>The LLV predominant plasma clone (PPC) mutation in RM Mmu 37969 was characterized by the following ten linked RT nucleotide substitutions: C258T, C291T, A304C,T336C, G364A, A378G, A483G, A484T, T537C, and G612A. Nine of these mutations were linked on two individual sequences obtained at week 10 PI. G612A was only present in one of the week 10 PI sequences and is listed separately.</p

Sample viral load and number of LLV sequences/sample^a

a<p>The number of sequences generated in each plasma sample is shown to the left of the sample's viral load which is denoted in parentheses.</p>b<p>Viral load (VL) was determined by our standard viral load assay which has a sensitivity of 50 vRNA copies/mL.</p>c<p>Time points where some LLV sequences contained mutations which were not observed in the pre-suppression profile</p>d<p>Samples were obtained at necropsy and approximately 20 mL of plasma was analyzed to generate LLV sequences.</p>e<p>Viral load determined by an unmodified ultracentrifugation viral load assay (Deere <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone.0088258-Deere2" target="_blank">[48]</a> using triplicate 10 µL vRNA samples. Sensitivity was 2 vRNA copies/mL.</p

Longitudinal analysis of plasma viral loads.

<p>Plasma viral loads were determined by TaqMan RT-qPCR using our standard viral load assay. All RMs began HAART (4 NRTI, 1 NNRTI) after eight weeks of infection. Rhesus macaques Mmu 37969 and Mmu 38202 were necropsied during therapy at weeks 50 and 52 PI respectively. Viremia rebounded in RMs Mmu 38560, Mmu 38606, and Mmu 37774 upon cessation of therapy at week 50 PI. These RMs were necropsied on the following weeks PI: (Mmu 37774: week 65, Mmu 38560: week 67, and Mmu 38606: week 69). The dotted line indicates the lower limit of detection of the standard viral load assay (50 copies of viral RNA per mL of plasma).</p

Phylogenetic diversity of low-level viremia.

<p>For each RM, phylograms represent RT-SHIV LLV sequences that were obtained between weeks 18 and 50 PI. Sequences were derived by SGA using plasma vRNA samples that were collected after maximal viral load suppression (<50 vRNA copies/mL) was initially achieved. There were a total of 437 nucleotide positions in the alignment encoding reverse transcriptase amino acids 65 to 210. Neighbor-joining phylograms were constructed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone-0088258-g002" target="_blank">Figure 2</a> and were rooted on the consensus sequence of the RT-SHIV inoculum (open black circle). Each asterisk denotes that the indicated LLV sequence contained a single mutation which was not observed in the pre-suppression variant profile. The identity of these mutations has been annotated on the phylograms in black lettering. Drug resistance mutations to compounds which were not used in this study are annotated in red lettering to the right of LLV sequence in which they were observed. These mutations are associated with resistance to the NNRTI Nevirapine (S162N and Q174R) and the NNRTIs Etravirine and Rilpivirine (E138K). Finally, sequences pertaining to the putative predominant plasma clone sequence identified in RM Mmu 37969 have been annotated “PPC”. These sequences were observed at four separate time points (weeks 34, 45, 47, and 50 PI).</p

RT-SHIV mutations with putative selective pressures^a.

a<p>Capital letters indicate the following RMs: (A: Mmu 38202; B: Mmu 37969; C: Mmu 37774; D: Mmu 38560; E: Mmu 38606). In the category “#≥5%” bold RM designations separated in parentheses specify that the indicated mutation was observed in <5% of sequence reads.</p>b<p>Observed by our group in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone.0088258-VanRompay1" target="_blank">[46]</a>.</p>c<p>Observed in RT-SHIV_mne infected pigtail macaques (Shao et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088258#pone.0088258-Shao1" target="_blank">[47]</a>).</p