To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).
Analogues of the mRNA 5′-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.
Several nucleotide-specific phosphohydrolases can cleave P-F bonds in substrate analogues containing a fluorophosphate moiety to release fluoride ions. In this work, by employing a fluoride-sensitive molecular sensor, we harnessed this cleavage reaction to develop a fluorescence assay to screen for phosphohydrolase inhibitors. The assay is rapid, sensitive, and based on simple and synthetically available reagents. The assay was adapted to the high-throughput screening (HTS) format and its utility was demonstrated by screening an 'in-house' library of small nucleotides against two enzymes: DcpS, a metal-independent mRNA decapping pyrophosphatase of the histidine triad (HIT) family; and PDE-I, a divalent cation-dependent nuclease. Our screening results agreed with the known specificities of DcpS and PDE-I, and led to the selection of several inhibitors featuring low-micromolar IC50 values. For DcpS, we also verified the results by using an alternative method with the natural substrate. Notably, the assay presented here is the first fluorescence-based HTS-adaptable assay for DcpS, an established therapeutic target for spinal muscular atrophy. The assay should be useful for phosphohydrolase specificity profiling and inhibitor discovery, particularly in the context of DcpS and other HIT-family enzymes, which play key roles in maintaining cellular functions and have been linked to disease development.
Protected guanosine and adenosine ribonucleosides and guanine nucleotides are readily functionalized with CF 3 substituents within the nucleobase. Protected guanosine is trifluoromethylated at the C8 position under radical-generating conditions in up to 95% yield and guanosine 5′-oligophosphates in up to 35% yield. In the case of adenosine, the selectivity of trifluoromethylation depends heavily on the functional group protection strategy and leads to a set of CF 3 -modified nucleosides with different substitution patterns (C8, C2, or both) in up to 37% yield. Further transformations based on phosphorimidazolide chemistry afford various CF 3 -substituted mono- and dinucleoside oligophosphates in good yields. The utility of the trifluoromethylated nucleotides as probes for 19 F NMR-based real-time enzymatic reaction monitoring is demonstrated with three different human nucleotide hydrolases (Fhit, DcpS, and cNIIIB). Substrate and product(s) resonances were sufficiently separated to enable effective tracking of each enzymatic activity of interest.
The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2′-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2′-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2′-O-MTase.
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