title: Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS.
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.
authors.affiliation: Division of Biophysics, University of Warsaw, 02-089 Warsaw, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kowalska
authors.forename: Magdalena
authors.initials: M
authors.lastname: Lewdorowicz
authors.forename: Joanna
authors.initials: J
authors.lastname: Zuberek
authors.forename: Ewa
authors.initials: E
authors.lastname: Grudzien-Nogalska
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Bojarska
authors.forename: Janusz
authors.initials: J
authors.lastname: Stepinski
authors.forename: Robert E
authors.initials: RE
authors.lastname: Rhoads
authors.forename: Edward
authors.initials: E
authors.lastname: Darzynkiewicz
authors.forename: Richard E
authors.initials: RE
authors.lastname: Davis
authors.forename: Jacek
authors.initials: J
authors.lastname: Jemielity
mesh_terms: D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry*; D004722:Endoribonucleases / Q000737:chemistry*; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry*; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D015394:Molecular Structure; D010710:Phosphates / Q000737:chemistry*; D054735:Phosphorothioate Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011755:Pyrophosphatases / Q000737:chemistry*; D012314:RNA Cap Analogs / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D029742:Caenorhabditis elegans Proteins; D039561:Eukaryotic Initiation Factor-4E; D010710:Phosphates; D054735:Phosphorothioate Oligonucleotides; D012314:RNA Cap Analogs; D006151:Guanosine; C016578:7-methylguanosine; C083120:phosphorodithioic acid; D004722:Endoribonucleases; C485332:DcpS protein, human; D011755:Pyrophosphatases; C509343:dcs-1 protein, C elegans