SYNTHESIS AND PROPERTIES OF mRNA CAP ANALOGS CONTAINING PHOSPHOROTHIOATE MOIETY IN 5′,5′-TRIPHOSPHATE CHAIN (original) (raw)
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Bioorganic & Medicinal Chemistry, 2012
We describe synthesis and properties of eight dinucleotide mRNA 5 0 cap analogs containing imidodiphosphate moiety within 5 0 ,5 0 -tri-or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5 0 -imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying capdependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5 0 capped mRNAs in vitro to be rather moderate.
Nucleos Nucleot Nucleic Acids, 2003
A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.
Nucleosides, Nucleotides and Nucleic Acids, 2003
A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3 -tri-, P 1 , P 4 -tetra-and P 1 , P 5 -pentaphosphates, linked by a 5 0 -to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by -OCH 3 or -H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.
Bioorganic & Medicinal Chemistry Letters, 2013
Synthetic mRNA cap analogs are valuable tools in the preparation of modified mRNA transcripts with improved translational activity and increased cellular stability, and have recently attracted more attention because of their great potential in therapeutic applications. We have synthesized and tested isopropylidene dinucleotide cap analogs bearing a phosphorothioate group at the b position of the 5 0 ,5 0 -triphosphate bridge (two diastereomers of 2 0 ,3 0 -iPr-m 7 Gpp S pG), as synthetically simpler alternatives to previously obtained phosphorothioate cap analogs. To evaluate the utility of the new compounds in biological systems we determined their affinity to translation initiation factor 4E (eIF4E), and tested their translational properties in rabbit reticulocyte lysates (RRL) and in human immature dendritic cells (hiDCs). In order to explain the properties of isopropylidene analogs we performed 1 H NMR conformational analysis and correlated the absolute configuration at the b-phosphorous atom with previously synthesized m 7 Gpp S pG.
Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of cap-dependent translation
Bioorganic & Medicinal Chemistry Letters, 2009
We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5 0 -(1-thiotriphosphate). These new analogs of mRNA 5 0 cap (referred to as m 7 GTPaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m 7 GTPaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate 8−foldand8-fold and 8−foldand15-fold more efficiently than m 7 GTP and m 7 GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.
Biochemistry, 1987
New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of-0 with-H,-CH3, or-NH2. Additional analogues were synthesized with &methyl-or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 'H and 31P N M R for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by-H and-CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by-NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the a-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation. T a n s l a t i o n of eukaryotic mRNA is dependent on several aspects of mRNA structure. These macromolecules are for the most part monocistronic, possess 5'-termini of the form m7G(5')ppp(5')N, and are polyadenylylated on the 3'-end. The 5' modification is known as a cap and is necessary for optimum translation. It is recognized by at least three translation initiation factors, namely, eIF-4A,' eIF-4B, and eIF-4F (Grifo et al., 1983; Edery et al., 1983, 1985). These are also collectively termed cap binding proteins (CBPs; Shatkin, 1985). These factors bind at or near the 5'-cap of mRNA early in the initiation phase of protein biosynthesis and facilitate mRNA attachment to the 40s ribosomal subunit. Cap analogues inhibit this step of initiation both in complete translation assay (
RNA, 2008
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 a, b, or g position of the 59,59-triphosphate chain. Three of them were also modified with methyl groups at the 29-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 b-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 g 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 a position (i.e., m 7 Gppp S G and m 2 7,29-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 59-O-(1-thiodiphosphate) (GDPaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.
Biochemistry, 1989
New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -0 with -H, -CH3, or -NH2. Additional analogues were synthesized with &methyl-or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 'H and 31P N M R for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the a-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.
Bioorganic & Medicinal Chemistry, 2006
Four novel 5 0 mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl 2 . Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3 0 to 5 0 cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.
New Journal of Chemistry, 2010
We describe the chemical synthesis and preliminary biophysical and biochemical characterization of a series of mRNA 5' end (cap) analogs designed as reagents for obtaining mRNA molecules with augmented translation efficiency and stability in vivo and as useful tools to study mRNA metabolism. The analogs share three structural features: (i) 5',5'-bridge elongated to tetraphosphate to increase their affinity to translation initiation factor eIF4E (ii) a single phosphorothioate modification at either the α, β, γ or δ-position of the tetraphosphate to decrease their susceptibility to enzymatic degradation and/or to modulate their interaction with specific proteins and (iii) a 2'-O-methyl group in the ribose of 7-methylguanosine, characteristic to Anti-Reverse Cap Analogs (ARCAs), which are incorporated into mRNA during in vitro transcription exclusively in the correct orientation. The dinucleotides bearing modified tetraphosphate bridge were synthesized by ZnCl 2 mediated coupling between two mononucleotide subunits with isolated yields of 30-65%. The preliminary biochemical results show that mRNAs capped with new analogs are 2.5-4.5 more efficiently translated in a cell free system than m 7 GpppG-capped mRNAs, which makes them promising candidates for RNA-based therapeutic applications such as gene therapy and anti-cancer vaccines. § Electronic Supplementary Information (ESI) available: , HPLC profiles, 1 H and/or 31 P NMR spectra of compounds 1-6.