Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides

Koshkin, Alexei A.; Rajwanshi, Vivek K.; Wengel, Jesper

doi:10.1016/s0040-4039(98)00706-0

Cited by 131 publications

(101 citation statements)

References 9 publications

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“…Synthesis of 2,4-BNA Monomers and Their TFO Derivatives As shown in Chart 1, 2Ј,4Ј-BNA amidite units bearing imidazole and 2-aminoimidazole were synthesized by using 1 20) as the starting material. Coupling reaction of 1 with 2-nitroimidazoles gave 2b, whereas the reaction of unsubstituted imidazole gave the desired compound 2a along with an imidazolium salt 2a.…”

Section: Resultsmentioning

confidence: 99%

“…Under a N 2 atmosphere, 1-trimethylsilylimidazole (0.73 ml, 5.00 mmol) and trimethylsilyl trifluoromethanesulfonate (1.00 ml, 5.53 mmol) were added to a solution of 1 20) (1.00 g, 1.67 mmol) in anhydrous 1,2-dichloroethane (20 ml) at room temperature and the mixture was stirred at room temperature for 24 h. After addition of a saturated aqueous NaHCO 3 2-nitroimidazole (2b) Under a N 2 atmosphere, 2-nitroimidazole (277 mg, 2.45 mmol) and N,O-bis(trimethylsilyl)acetamide (0.71 ml, 2.87 mmol) were added to a solution of 1 (1.22 g, 2.04 mmol) in anhydrous 1,2-dichloroethane (20 ml) at room temperature and the mixture was refluxed for 1 h. After the mixture cooled, trimethylsilyl trifluoromethanesulfonate (0.15 ml, 0.83 mmol) was added to the mixture and the mixture was refluxed for 5 h. After addition of a saturated aqueous NaHCO 3 , the mixture was extracted with AcOEt. Usual work-up and purification by flash column chromatography [n-hexane/AcOEt (12/5)] afforded 2b (1.24 g, 93%) …”

Section: Methodsmentioning

confidence: 99%

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Synthesis and Triplex-Forming Ability of 2',4'-BNAs Bearing Imidazoles as a Nucleobase

Hari

Obika

Inohara

et al. 2005

CHEMICAL & PHARMACEUTICAL BULLETIN

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Triplex-forming oligonucleotide (TFO) is able to hybridize with a double-stranded DNA (dsDNA) target in a sequencespecific manner to form a triplex DNA, and it has attracted great interest because of its applications to gene therapy and diagnosis. [2][3][4] There are two fashions in triplex formation. One is a parallel motif triplex, where a TFO consisting of a homopyrimidine sequence hybridizes with a dsDNA target in parallel with a purine strand of the target duplex via Hoogsteen hydrogen bond. The other is an antiparallel motif triplex, in which the TFO and the purine strand of the duplex are in an antiparallel orientation. In both triplex motifs, the TFOs are able to hybridize only with a homopurine-homopyrimidine tract in the dsDNA target. This is a severe limitation of the practical use of TFOs. To overcome this problem, extensive research on nucleic acid analogues has so far been carried out. [5][6][7][8] We have focused on the parallel type triplex, and synthesized 2Ј-O,4Ј-C-methylene-bridged nucleic acid (2Ј, [9][10][11][12][13] /locked nucleic acid (LNA) 14) ) bearing unnatural nucleobases to recognize pyrimidine-purine interruption in the target dsDNA. [15][16][17][18][19] Recently, it was found that the 2Ј,4Ј-BNA bearing oxazole 15,16) : Fig. 1) and triplex-forming ability of the oligonucleotide derivatives are described. Results and DiscussionSynthesis of 2,4-BNA Monomers and Their TFO Derivatives As shown in Chart 1, 2Ј,4Ј-BNA amidite units bearing imidazole and 2-aminoimidazole were synthesized by using 1 20) as the starting material. Coupling reaction of 1 with 2-nitroimidazoles gave 2b, whereas the reaction of unsubstituted imidazole gave the desired compound 2a along with an imidazolium salt 2a.21) Next, 2a and 2b were reacted with K 2 CO 3 in MeOH to give 3a and 3b. Reduction of 3a and 3b smoothly proceeded to afford 2Ј,4Ј-BNA monomers 4a and 4b, respectively. Protection of an amino group in 4b gave the corresponding N,N-dimethylformamidine derivative 4b. The 5Ј-hydroxy groups of 4a and 4b were then protected with a dimethoxytrityl (DMTr) group to afford 5a and 5b, respectively. The preparation of phosphoramidites 6a and 6b was carried out by phosphitilation of 5a and 5b, respectively. Incorporation of the obtained amidites 6a and 6b into oligonucleotides was successfully achieved by using a standard phosphoramidite method on an automated DNA synthesizer. After purification by reverse-phase HPLC, composition of the oligonucleotides was confirmed by MALDI-TOF-MS. To expand the sequence of double-stranded DNA (dsDNA) targets in a triplex formation, 2,4-BNAs (2-O,4-C-methylene bridged nucleic acids) having imidazoles as a nucleobase were synthesized and incorporated into oligonucleotides. Triplex-forming ability of the modified oligonucleotides was evaluated by using melting temperature (T m ) measurements.Key words methylene bridged nucleic acid (BNA); locked nucleic acid (LNA); triplex; imidazole; oligonucleotide Reagents: a) 1-trimethylsilylimidazole, trimethylsilyl trifluoromethanesulfonate,...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Synthesis and Triplex-Forming Ability of 2',4'-BNAs Bearing Imidazoles as a Nucleobase

Hari

Obika

Inohara

et al. 2005

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…The fluorescent signals attached to that probe are detected. The locked nucleic acids (LNAs) are nucleotide analogues that are conformationally locked in a C3′-endo/ N-type sugar conformation by O2′ to C4′ methylene linkage (Koshkin et al 1998;Ichikawa et al 1999;Wang et al 1999) that leads to reduced conformational flexibility (Braasch and Corey 2001). To increase duplex stability between LNAcontaining oligonucleotides and standard nucleic acids, using the LNA mediated TaqMan probe as an analog of the TaqMan probe is advantageous because it can increase thermal stability (about 3-8°C per modified base in the probe) (Letertre et al 2003;Kennedy et al 2005).…”

Section: Discussionmentioning

confidence: 99%

“…In the present study, two different methods to detect YMDD motif mutations were evaluated and compared with conventional direct sequencing: PCR-RFLP and real-time PCR by using locked nucleic acid (LNA)-mediated TaqMan probe, which is a nucleic acid analogue containing LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation (Koshkin et al 1998;Ichikawa et al 1999;Wang et al 1999). LNA oligonucleotides display unprecedented hybridization affinity toward complementary double-stranded DNA.…”

confidence: 99%

Rapid Detection of Lamivudine-Resistant Hepatitis B Virus Mutations by PCR-Based Methods

Chieochansin

Chutinimitkul

Payungporn

et al. 2006

Tohoku J. Exp. Med.

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Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide, and chronic HBV infection may progress to cirrhosis and hepatocellular carcinoma. Mutations at the active site of DNA polymerase of HBV, tyrosine-methionineaspartate-aspartate (YMDD) motif, render infected patients resistant to antiviral drug (Lamivudine) therapy. Hence, sensitive and specific methods aimed at detecting the mutants are essential. The purpose of this study was to develop methods for detecting the mutations at YMDD by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR using locked nucleic acid (LNA)-mediated TaqMan probes. The results obtained by these methods were compared with those examined by conventional direct sequencing on serum samples of 77 patients treated with lamivudine. Our results show that both PCR-RFLP and real-time PCR could detect wild type, YMDD, and its mutants, tyrosine-isoleucine-aspartate-aspartate and tyrosine-valineaspartate-aspartate. In addition, the mixtures of the wild-type virus and its mutants in the serum sample were detected. Importantly, real-time PCR is less time-consuming, and more sensitive for the detection of mixed populations than PCR-RFLP. The real-time PCR with LNA-mediated TaqMan probes is a sensitive, specific and rapid detection method for mutations at the YMDD motif, which will be essential for monitoring patients undergoing lamivudine antiviral therapy.HBV, YMDD, Real-time PCR, PCR-RFLP, LNA mediated TaqMan probes © 2006 Tohoku University Medical PressHepatitis B virus (HBV) is one of the major causes of liver disease worldwide, and chronic HBV infection can progress to cirrhosis and hepatocellular carcinoma (Jang et al. 2004). It is estimated that 350 million individuals are chronically infected with HBV and that 1 to 2% will die each year as a consequence of infection related complications (Allen et al. 1999). HBV is a member of the Hepadnaviridae, and has a relaxed-circular, partially double stranded DNA genome of approximately 3200 nucleotides. HBV replication occurs via reverse transcription using its own

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“…18) LNA oligonucleotides posses extraordinary mismatch sensitively to complementary nucleic acids in LNA/ DNA hybrids 19,20) and higher thermal stability than the DNA oligonucleotide. 21,22) Therefore, LNA oligonucleotides, which are homologous to the plant organelle SSU rRNA genes, can be annealed to the organelle genes with high temperature in which primers are inactive prior to the annealing step of primers. Moreover, LNA oligonucleotides block the annealing of primers by designing in the positions to compete with primers in the organelle genes, and are inoperative as primers upon phosphorylation of the 3′ end.…”

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confidence: 99%

Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

Ikenaga

Tabuchi

Oyama

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

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Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides

Cited by 131 publications

References 9 publications

Synthesis and Triplex-Forming Ability of 2',4'-BNAs Bearing Imidazoles as a Nucleobase

Synthesis and Triplex-Forming Ability of 2',4'-BNAs Bearing Imidazoles as a Nucleobase

Rapid Detection of Lamivudine-Resistant Hepatitis B Virus Mutations by PCR-Based Methods

Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

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