Rapid and efficient synthesis of nucleoside 5'-0-(1-thiotriphosphates), 5'-triphosphates and 2',3'-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one

Ludwig, János; Eckstein, Fritz

doi:10.1021/jo00264a024

Cited by 373 publications

(318 citation statements)

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“…Unfortunately, we have been able to assign the absolute configuration only for the analogs substituted at the a position. However, our observation of the upfield shift of the m Gp S ppG (D1) and m 2 7,29-O Gp S ppG (D1), together with the previously reported correlation between the retention time on the RP HPLC column and the absolute configurations of nucleoside 59-O-(1-thiotriphosphates) and 59-O-(1-thiodiphosphates) (i.e., S P is the faster) (Ludwig and Eckstein 1989;Li et al 2005), suggest that the D1 isomers are also of the S P configuration in the case of g-substituted S analogs. GpppG for non-ARCA S analogs and m 2 7,29-O GpppG for ARCAs) within 40-90 min.…”

Section: Discussionsupporting

confidence: 84%

Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Kowalska¹,

Lewdorowicz²,

Zuberek³

et al. 2008

RNA

122

141

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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.

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Section: Discussionsupporting

confidence: 84%

Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Kowalska¹,

Lewdorowicz²,

Zuberek³

et al. 2008

RNA

122

141

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“…Except where noted, reagents were obtained commercially and used without further purification+ Dimethylformamide (DMF) was distilled over CaH 2 under reduced pressure and stored over 4A molecular sieves+ Pyridine was distilled over P 2 O 5 and stored over 4A molecular sieves+ Tri-n-butylamine was distilled twice over NaOH under reduced pressure+ Triethylphosphate was distilled over CaH 2 under reduced pressure and stored over 4A molecular sieves+ Thiophosphoryl chloride (PSCl 3 ) was freshly distilled before every use in thiophosphorylation+ Anhydrous of bis(tri-n-butylammonium)-pyrophosphate in DMF was prepared as described elsewhere (Ludwig & Eckstein, 1989)+ 7-Deazaadenosine (Tubercidin) was purchased from Sigma+ 7-Deazaguanosine was synthesized according to Seela et al+ (1990) from commercially available 7-deazaguanine (Sigma) and 2,3-O-isopropylidene-D-ribono-1,4-lactone (ICN Biomedicals)+ Satisfactory TLC, UV, and 1 H NMR data were obtained for every intermediate compound+ UV and 1 H NMR spectra of the product nucleoside were identical to that of b-anomer of 7-deazaguanosine (Seela et al+, 1990)+ 29-Deoxy and ribonucleotide-59-O-(1-thiotriphosphates) (G, A, T, U, and C) and all radioactive nucleotides were purchased from Amersham+ 31 P NMR spectra (161+978 MHz) were recorded using a Bruker AM 400 spectrometer+ For proton decoupling, a WALTZ-16 CPD pulse-sequence was applied+ Sodium phosphate, pH 7+0, was added to the samples to 30 mM concentration as an internal reference+ UV spectra were recorded using a Shimadzu Biospec-1601 spectrophotometer+…”

Section: Generalmentioning

confidence: 99%

Identification by modification-interference of purine N-7 and ribose 2′-OH groups critical for catalysis by bacterial ribonuclease P

Kazantsev

Pace

1998

RNA

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The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 59 ends. A self-cleaving RNase P RNA-substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 29-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 29-hydroxyl that, when substituted with 7-deazaadenine or 29-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate-phosphate in the tertiary structure of the ribozyme-substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 29-deoxyinterference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.

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“…However, in vitro transcription cannot insert a modified nucleoside site specifically at an arbitrary position within RNA, and in such cases, solid-phase RNA synthesis is the best approach (Earnshaw and Gait 1998;Scaringe et al 1998;Scaringe 2000Scaringe , 2001. Although solid-phase synthesis of 5Ј-triphosphorylated RNA has been reported (Ludwig and Eckstein 1989;Gaur et al 1992), this approach is limited in utility because the 5Ј-triphosphate must be introduced while the oligonucleotide is still attached to the solid-phase support and before global RNA deprotection. Related m 7 Gpppcapped RNA may be prepared after solid-phase synthesis using guanyl transferase with 5Ј-diphosphate RNA (Brownlee et al 1995), but the 5Ј-diphosphate RNA must be prepared in a manner analogous to 5Ј-triphosphate RNA, with the associated limitations.…”

Section: Introductionmentioning

confidence: 99%

Practical and general synthesis of 5′-adenylated RNA (5′-AppRNA)

Silverman¹

2004

RNA

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A simple strategy is reported for 5-adenylation of nearly any RNA sequence of indefinite length. The 5-adenylated product (5-AppRNA) is an activated RNA that is structurally similar to 5-triphosphorylated RNA, which is usually prepared by in vitro transcription using T7 RNA polymerase. In the new 5-adenylation strategy, the RNA substrate is first 5-monophosphorylated either by T4 polynucleotide kinase, by in vitro transcription in the presence of excess GMP, or by appropriate derivatization during solid-phase synthesis. The RNA is then 5-adenylated using ATP and T4 RNA ligase, in an interrupted version of the natural adenylation-ligation mechanism by which T4 RNA ligase joins two RNA substrates. Here, the final ligation step of the mechanism is inhibited with complementary DNA blocking oligonucleotide(s) that permit adenylation to occur with good yield. The 5-AppRNA products of this approach should be valuable as activated RNAs for in vitro selection experiments as an alternative to 5-triphosphorylated RNAs, among other likely applications. The 5-terminal nucleotide of an RNA substrate to be adenylated using the new method is not restricted to guanosine, in contrast to 5-triphosphorylated RNA prepared by in vitro transcription. Therefore, using the new approach, essentially any RNA obtained from solid-phase synthesis or other means can be activated by 5-adenylation in a practical manner.

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Rapid and efficient synthesis of nucleoside 5'-0-(1-thiotriphosphates), 5'-triphosphates and 2',3'-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one

Cited by 373 publications

References 1 publication

Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Identification by modification-interference of purine N-7 and ribose 2′-OH groups critical for catalysis by bacterial ribonuclease P

Practical and general synthesis of 5′-adenylated RNA (5′-AppRNA)

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