Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS (original) (raw)
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Nucleosides, Nucleotides and Nucleic Acids, 2005
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.
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.
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.
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.
2007
Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m 7 Gp 3 G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3 0 -OH on both m 7 Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m 7 Gp 3 G cap by ''anti-reverse'' cap analogs (ARCAs) in which the 3 0 -OH of m 7 Guo moiety was substituted by 3 0 -deoxy or 3 0 -O-methyl groups, leading to m 7 3 0 dGp 3 G or m 2 7,3 0 -O Gp 3 G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2 0 of m 7 Guo. We have also developed a series of ARCAs containing tetra-and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1-to 2.6-fold more efficiently than their counterparts capped with m 7 Gp 3 G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the aand b-, or band g-phosphate moieties, leading to m 2 7,3 0 -O Gpp CH2 pG and m 2 7,3 0 -O Gp CH2 ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m 2 7,3 0 -O Gpp CH2 pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.
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 cap analogs stabilize mRNA and increase translational efficiency in mammalian cells
RNA, 2007
Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the a-, b-, or g-phosphate moieties, m 2 7,29-O Gppp S G, m 2 7,29-O Gpp S pG, and m 2 7,29-O Gp S ppG, respectively. The new compounds were also modified at the 29-O position of the m 7 Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the aand b-phosphates. Oligonucleotides capped with m 2 7,29-O Gpp S pG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m 2 7,29-O Gpp S pG (D1) and both isomers of m 2 7,29-O Gppp S G were partially resistant. Luciferase mRNA capped with m 2 7,29-O Gpp S pG (D2) had a t 1/2 of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m 7 Gp 3 G-capped mRNA. Luciferase mRNAs capped with m 2 7,29-O Gpp S pG (D1) and m 2 7,29-O Gpp S pG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m 7 Gp 3 G. The greater yield of protein due to combining higher translational efficiency with longer t 1/2 of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.