A New RNA Synthetic Method with a 2‘-<i>O</i>-(2-Cyanoethoxymethyl) Protecting Group

Ohgi, Tadaaki; Masutomi, Yutaka; Ishiyama, Koichi; Kitagawa, Hajime; Shiba, Yoshinobu; Yano, Junichi

doi:10.1021/ol051151f

Cited by 75 publications

(69 citation statements)

References 20 publications

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“…To meet this need, we have developed an achiral 2′-hydroxyl protecting group, 2-cyanoethoxymethyl (CEM), that has low steric hindrance and that can be completely removed under mild conditions (25). CEM chemistry is also compatible with standard, unmodified DNA synthesizer equipment.…”

Section: Introductionmentioning

confidence: 99%

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

Shiba¹,

Masuda²,

Watanabe³

et al. 2007

Nucleic Acids Research

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A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2′-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-μmol scale corresponds to 1 mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100 nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.

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Section: Introductionmentioning

confidence: 99%

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

Shiba¹,

Masuda²,

Watanabe³

et al. 2007

Nucleic Acids Research

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“…[13][14][15] In this method, the CEM group is used as the 2′-hydroxyl protecting group. Because the CEM group is small, it offers minimal steric hindrance and so allows an increased yield at each coupling step.…”

Section: )mentioning

confidence: 99%

Improved Method for the Solid-Phase Synthesis of Oligoribonucleotide 5′-Triphosphates

Nagata

Takagaki

Wada

2012

CHEMICAL & PHARMACEUTICAL BULLETIN

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We have developed an improved solid-phase method for the synthesis of 5′-triphosphates (5′-TPs) of oligoribonucleotides. The method is based on the use of salicyl phosphorochloridite as the phosphitylating reagent and the improvement is characterized by the use of the highly reactive pyrophosphorylating reagent tris(tetra-n-butylammonium) hydrogen pyrophosphate instead of the conventional tri-n-butylammonium salt for the nucleophilic substitution reaction to form the cyclic ester intermediate. The improved method can be used to generate oligoribonucleotide 5′-TPs efficiently and reproducibly.Key words 5′-triphosphate; oligoribonucleotide; solid phase; RNA; triphosphorylation 5′-Triphosphates (5′-TPs) of nucleosides and nucleic acids play an important role in biochemical reactions and have many research and medical applications. Thus, nucleoside 5′-TPs have long been known to be substrates for enzymatic DNA and RNA synthesis. A more recent discovery is the fact that the immune response triggered by the binding of 5′-TPs of RNA oligomers to retinoic acid-induced protein I (RIG-I) works together in synergy with gene silencing mediated by small interfering RNAs to promote apoptosis in tumor cells. 1)As for applications, 5′-TPs of DNA and RNA oligomers are used as substrates for ligation in vitro, while modified nucleoside triphosphates are used as antiviral medicines and diagnostic reagents. The demand for 5′-TPs of DNA and RNA is increasing and will most likely be met by chemical synthetic rather than enzymatic methods because of advantages resulting from the ability to introduce chemically modified nucleosides at any point in the sequence as well as the practicality of scaling up the synthesis. Chemical methods for the synthesis of 5′-TPs of RNA in particular have the additional advantage that they allow the production of highly pure RNA.A number of chemical syntheses of 5′-TPs of DNA and RNA have been reported. 2-9) Among these, a liquid-phase synthesis of 5′-TPs by reaction of the oligonucleotide with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl phosphorochloridite) developed by Ludwig and Eckstein 10) is well known.11) However, there are few reports of the solid-phase synthesis of DNA and RNA 5′-TPs. 4,9,11) A solid-phase adaptation of the salicyl phosphorochloridite method of Ludwig and Eckstein 10) has been developed by Schmidt et al.,12) who used it to synthesize the 5′-TP of a 25-mer RNA.For triphosphorylation experiments in the present study, we synthesized a 25-mer RNA oligomer by the 2-cyanoethoxymethyl (CEM) method of RNA synthesis developed in our laboratory. [13][14][15] In this method, the CEM group is used as the 2′-hydroxyl protecting group. Because the CEM group is small, it offers minimal steric hindrance and so allows an increased yield at each coupling step. As a result, and also because of the ease of removal of the CEM group under mild conditions after synthesis, RNA can be synthesized more simply and easily by the CEM method than by methods involving conventional 2′-hydroxyl protect...

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“…We turned our attention to a publication describing the novel (2-cyanoethoxy)methyl (CEM) 2 -O-protecting group for RNA synthesis. [6] They found that this group is readily removable upon treatment with tetrabutylammonium fluoride (TBAF) in dry tetrahydrofuran (THF). We asked the question if the mentioned group could be used for protection of the 3 -O-position in deoxynucleotides, and further, if the deprotection under such nonbiological conditions is possible without affecting the duplex structure of the DNA-template.…”

Section: Introductionmentioning

confidence: 99%

The Fluoride Cleavable 2-(Cyanoethoxy)Methyl (CEM) Group as Reversible 3′-O-Terminator for DNA Sequencing-by-Synthesis—Synthesis, Incorporation, and Cleavage

Földesi¹,

Keller

Stura³

et al. 2007

Nucleosides, Nucleotides and Nucleic Acids

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A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast determination of DNA sequences. 2'-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM) group at the 3'-O-position are potential reversible terminators for the SBS technology. Herein we describe the synthesis, the incorporation by several polymerases, and the cleavage of this 3'-O-blocking group using 3'-O-CEM-thymidinyl-5'-O-triphosphate 7 as an example.

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A New RNA Synthetic Method with a 2‘-O-(2-Cyanoethoxymethyl) Protecting Group

Cited by 75 publications

References 20 publications

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

Improved Method for the Solid-Phase Synthesis of Oligoribonucleotide 5′-Triphosphates

The Fluoride Cleavable 2-(Cyanoethoxy)Methyl (CEM) Group as Reversible 3′-O-Terminator for DNA Sequencing-by-Synthesis—Synthesis, Incorporation, and Cleavage

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