Synthesis of 5′-Thio-3′‑<i>O</i>‑ribonucleoside Phosphoramidites

The chemical synthesis of phosphoramidite derivatives of all four 5′-deoxy-5′-thioribonucleosides is described. These phosphoramidites contained trityl (A, G, C, and U), dimethoxytrityl (A and G), or <i>tert</i>-butyldisulfanyl (G) as the 5′-<i>S</i>-protecting group. The application of several of these phosphoramidites for solid-phase synthesis of oligoribonucleotides containing a 2′-<i>O</i>-photocaged 5′-<i>S</i>-phosphorothiolate linkage or 5′-thiol-labeled RNAs is also further investigated

Automated Solid-Phase Synthesis of RNA Oligonucleotides Containing a Nonbridging Phosphorodithioate Linkage via Phosphorothioamidites

This work describes a general method for the synthesis of oligoribonucleotides containing a site-specific nonbridging phosphorodithioate linkage via automated solid-phase synthesis using 5′-<i>O</i>-DMTr-2′-<i>O</i>-TBS-ribonucleoside 3′-<i>N</i>,<i>N</i>-dimethyl-<i>S</i>-(2,4-dichlorobenzyl) phosphorothioamidites (<b>2a</b>–<b>2d</b>). The 3′-phosphorothioamidites (<b>2a</b>–<b>2d</b>) can be conveniently prepared in good yields (86–99%) via a one-pot reaction from the corresponding 5′-<i>O</i>-DMTr-2′-<i>O</i>-TBS-ribonucleosides (<b>1a</b>–<b>1d</b>)

Highly Stereocontrolled Total Synthesis of β‑d‑Mannosyl Phosphomycoketide: A Natural Product from <i>Mycobacterium tuberculosis</i>

β-d-Mannosyl phosphomycoketide (<b>C</b>_<b>32</b><b>-MPM</b>), a naturally occurring glycolipid found in the cell walls of <i>Mycobacterium tuberculosis</i>, acts as a potent antigen to activate T-cells upon presentation by CD1c protein. The lipid portion of <b>C</b>_<b>32</b><b>-MPM</b> contains a <b>C</b>_<b>32</b><b>-mycoketide</b>, consisting of a saturated oligoisoprenoid chain with five chiral methyl branches. Here we develop several stereocontrolled approaches to assemble the oligoisoprenoid chain with high stereopurity (>96%) using Julia–Kocienski olefinations followed by diimide reduction. By careful choice of olefination sites, we could derive all chirality from a single commercial compound, methyl (2<i>S</i>)-3-hydroxy-2-methylpropionate (>99% ee). Our approach is the first highly stereocontrolled method to prepare <b>C</b>_<b>32</b><b>-MPM</b> molecule with >96% stereopurity from a single >99% ee starting material. We anticipate that our methods will facilitate the highly stereocontrolled synthesis of a variety of other natural products containing chiral oligoisoprenoid-like chains, including vitamins, phytol, insect pheromones, and archaeal lipids

Synthesis and Incorporation of the Phosphoramidite Derivative of 2′‑<i>O</i>‑Photocaged 3′‑<i>S</i>‑Thio­guanosine into Oligo­ribo­nucleo­tides: Substrate for Probing the Mechanism of RNA Catalysis

Oligo­ribo­nucleo­tides containing 3′-<i>S</i>-phosphoro­thiolate linkages possess properties that can reveal deep mechanistic insights into ribozyme-catalyzed reactions. “Photocaged” 3′-<i>S</i>- RNAs could provide a strategy to stall reactions at the chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the synthesis of 2′-<i>O-</i>(<i>o</i>-nitro­benzyl)-3′-thio­guano­sine phos­phorami­dite starting from <i>N</i>²-iso­butyryl­guano­sine in nine steps with 10.2% overall yield. Oligonucleotides containing the 2′-<i>O-</i>(<i>o</i>-nitro­benzyl)-3′-<i>S</i>-guano­sine nucleotide were then constructed, characterized, and used in a nuclear pre-mRNA splicing reaction

Transition State Features in the Hepatitis Delta Virus Ribozyme Reaction Revealed by Atomic Perturbations

Endonucleolytic ribozymes constitute a class of non-coding RNAs that catalyze single-strand RNA scission. With crystal structures available for all of the known ribozymes, a major challenge involves relating functional data to the physically observed RNA architecture. In the case of the hepatitis delta virus (HDV) ribozyme, there are three high-resolution crystal structures, the product state of the reaction and two precursor variants, with distinct mechanistic implications. Here, we develop new strategies to probe the structure and catalytic mechanism of a ribozyme. First, we use double-mutant cycles to distinguish differences in functional group proximity implicated by the crystal structures. Second, we use a corrected form of the Brønsted equation to assess the functional significance of general acid catalysis in the system. Our results delineate the functional relevance of atomic interactions inferred from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product in the degree of proton transfer to the leaving group