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Synthesis 2002(6): 0802-0808
DOI: 10.1055/s-2002-25756
SPECIALTOPIC
© Georg Thieme Verlag Stuttgart · New York

Preparation of LNA Phosphoramidites

Daniel Sejer Pedersen, Christoph Rosenbohm, Troels Koch*
Cureon A/S, Fruebjergvej 3, 2100 Copenhagen, Denmark
Fax: +4570260097; e-Mail: tk@cureon.com;
Further Information

Publication History

Received 10 February 2002
Publication Date:
26 April 2002 (online)

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Abstract

A highly efficient method for the preparation of LNA (Locked Nucleic Acid) phosphoramidite monomers with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite and 4,5-dicyanoimidazole has been devised. The quality of the phosphoramidites prepared in this manner is equal to HPLC purified phosphoramidites and can easily be used for oligonucleotide synthesis without further purification. In addition the possibility of using 4,5-dicyanoimidazole in catalytic amounts has been investigated and showed optimum results when 0.7 equivalent was used, and that reducing the amount further leads to undesired phosphitylation of the nucleobase.
Furthermore it is demonstrated that LNA phosphoramidite monomers are exceedingly stable in acetonitrile solution thereby prolonging the effective lifetime of the reagent significantly.

Key words

phosphoramidites - phosphitylations - phosphitylation activators - nucleotide analogues - bicyclic compounds

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1

We have defined LNA as an oligonucleotide containing one or more 2′-O,4′-C-methylene-β-d-ribofuranosyl nucleotide monomer(s). LNA has recently also been named 2′,4′-BNA. [2]

24

1H-Tetrazole is an explosive chemical and has also been reported to be toxic. [23]

33

At no time during our catalytic studies with DCI did we detect any nucleobase phosphitylated products. We have attributed this to the expected higher reactivity of the nucleobase phosphoramidites resulting in their hydrolyses during the aqueous bicarbonate workup prior to HPLC analysis.

34

All phosphitylation reactions were completed in less than 2 h except for the G phosphoramidite 7b synthesis, which took up to 4 h for completion.