PMID:18430890

Citation	Kowalska, J, Lewdorowicz, M, Zuberek, J, Grudzien-Nogalska, E, Bojarska, E, Stepinski, J, Rhoads, RE, Darzynkiewicz, E, Davis, RE and Jemielity, J (2008) Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS. RNA 14:1119-31
Abstract	Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K(AS)) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7)Gpp(S)pG) was characterized by a K(AS) that was more than fourfold higher than that of its unmodified counterpart (m(7)GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7)Gppp(S)G and m(2) (7,2'-O )Gppp(S)G) were established as S(P) and R(P) , respectively, using enzymatic digestion and correlation with the S(P) and R(P) diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.
Links	PubMed PMC2390807 Online version:10.1261/rna.990208
Keywords	Animals; Caenorhabditis elegans Proteins/chemistry; Endoribonucleases/chemistry; Eukaryotic Initiation Factor-4E/chemistry; Guanosine/analogs & derivatives; Guanosine/chemistry; Humans; Hydrolysis; Molecular Structure; Phosphates/chemistry; Phosphorothioate Oligonucleotides/chemical synthesis; Phosphorothioate Oligonucleotides/chemistry; Phosphorothioate Oligonucleotides/pharmacology; Protein Biosynthesis/drug effects; Pyrophosphatases/chemistry; RNA Cap Analogs/chemical synthesis; RNA Cap Analogs/chemistry; RNA Cap Analogs/pharmacology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CAEEL:D8FRQ0	involved_in	GO:0006402: mRNA catabolic process	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
CAEEL:D8FRQ0	enables	GO:0050072: m7G(5')pppN diphosphatase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
CAEEL:DCPS	involved_in	GO:0006402: mRNA catabolic process	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
CAEEL:DCPS	enables	GO:0050072: m7G(5')pppN diphosphatase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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