Synthesis of Fluorophosphate Nucleotide Analogues and Their Characterization as Tools for ¹⁹F NMR Studies

Abstract

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).

Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

“…The O-to-S substitution results in a higher preference for soft divalent metal cations, whereas the O-to-BH 3 substitution generally decreases affinity to metal cations and reduces H-bond acceptor properties (Stivers and Nagarajan 2006;Li et al 2007;Guga and Koziolkiewicz 2011). The O-to-F substitution at the terminal phosphate results in a smaller net negative charge and impairs H-bonding (Baranowski et al 2015). The bridging O-to-CH 2 substitution provides resistance to hydrolysis since the P-C bonds are remarkably stable, however, the inability to form H-bonds can deteriorate affinity to some proteins (Klein et al 2002;Grudzien et al 2006).…”

Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

“…Such an approach provides a relatively simple way to obtain a wide scope of chemically modified oligophosphate mono-and diesters (Fig. 7), including phosphorothioates [79][80][81], seleno- [82], borano- [83], and fluorophosphates [84], as well as phosphoramidates [85], C-phosphonates [86], and bisphosphonates [87]. P-imidazolides were shown to react readily with other inorganic nucleophiles such as fluorides [84] and sulfates [88] to yield fluorophosphates and phosphosulfates, respectively.…”

“…The P-imidazolide approach has been used for the synthesis of fluorophosphate analogs of 7-methylguanosine mononucleotides: m 7 GMPF, m 7 GDPF, and m 7 GTPF (Fig. 2a) [84]. The compounds were tested as 19 F NMR probes for cap-related processes.…”

“…The compounds were tested as 19 F NMR probes for cap-related processes. m 7 GTPF was used as a binding probe for eIF4E in a 19 F NMR assay based on changes in the transverse relaxation rate upon binding by the protein [84]. All three compounds: m 7 GMPF, m 7 GDPF, and m 7 GTPF, were found useful for monitoring activity of DcpS as they were cleaved by the enzyme to m 7 GMP and a corresponding fluorine-containing leaving group: a fluoride anion, fluoromonophosphate, and fluorodiphosphate, respectively.…”

“…Each of the products could be detected due to different d F chemical shifts compared to the corresponding substrate (Fig. 10a) [84]. As a more sensitive alternative to 19 F NMR experiments, a fluorescent inhibitor screening assay for DcpS based on P-F bond cleavage in m 7 GMPF and the use of a fluoride-sensitive chemosensor for product quantitation has been developed (Fig.…”