Locked Nucleic Acid (LNA) monomers and oligonucleotides were invented in 1997 (WO9914226) and have showed promising results as antisense drug candidates. However, there is no production method described in the literature for large scale LNA-phosphoramidite synthesis providing optimal yields of the LNA amidites necessary for efficient preparation of LNA based antisense drugs.
Oligonucleotide synthesis is typically performed using the phosphoramidite method, invented by Caruthers, U.S. Pat. No. 4,415,732, in 1980 and improved a couple of years later by Köster U.S. Pat. No. 4,725,677. LNA oligomers are being synthesised according to the phosphoramidite method, but so far the large scale supply of LNA monomers has been a problem due to slow reactions, side product formation during the reaction and reagents unstable at room temperature have been employed.
The mechanism of phosphoramidite activation and coupling in oligonucleotide syntheses has been studied in detail. Traditionally tetrazole has been used (Dahl et al. 1987, Nucleic Acid Research, 15, 1729-1743; Beaucage & Iver, 1992, Tetrahedron, 48, 2223-2311). The proposed mechanism of tetrazole is two step, first tetrazole protonates trivalent phosphorous followed by displacement of the N,N-diisopropylamine by the tetrazolide. This latter intermediate is very reactive with hydroxynucleophiles such as 5′-OH on nucleic acids. Therefore, tetrazole acts both as acid and as nucleophilic agent.
Vargeese et al. (Vargeese, C.; Carter, J.; Krivjansky, S.; Settle, A.; Kropp, E.; Peterson, K.; Pieken, W. Nucleic Acid Research, 1998, 26, 1046-1050; WO9816540;) have described an activator for the coupling of phosphoramidites to the 5′-hydroxyl group during oligonucleotide synthesis. The activator is 4,5-dicyanoimidazole (DCI) and its effectiveness is thought to be based on its nucleophilicity. It was shown that DCI significantly increased the yield in phosphoramidite oligomerisation.
Vargeese et al. has described a method of small scale DNA phosphoramidite thymine monomer synthesis using DCI, but the method they used provided a moderate yield (75%) after several re-precipitations of the amidite. Kittaka et al. (Kittaka A., Kuze T.; Amano M.; Tanaka H.; Miyasaka T.; Hirose K.; Yoshida T.; Sarai A.; Yasukawa T. and Ishii S. Nucleosides & Nucleotides 18, 2769-2783, 1999) have also used DCI for amidite synthesis but they reported an even lower yield of the product (65%). Kittaka et al. (Kittaka A.; Horii C.; Kuze T.; Asakura T.; Ito K.; Nakamura K. T.; Miyasaka T. and Inoue J., Synthetic letters, S1, 869-872, 1999) have also made a derivatised uridine phosphoramidite by 3′-O phosphitylation using DCI (0.7 eq), but without describing the reaction parameters further. The yield was reported to be 92%. Generally, phosphitylation of uracil and thymine provide the highest yields because these nucleobases usually do not interfere with the phosphitylation reagents.
Accordingly, there is nothing in the literature describing that DCI has been used as an activator for LNA phosphoramidite synthesis.
The previously reported oxy-β-D-ribo-LNA (FIG. 2) method of synthesis used 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite and diisopropylethylamine (Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tet. 1998, 54, 3607-3630). The reported yields were low (LNA-T 70%, LNA-U 58%, LNA-G 64%, LNA-A 73% and no yield for LNA-C was reported). The previously reported method for synthesising the oxy-α-L-ribo thymine phosphoramidite (FIG. 3) also utilized 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite and diisopropylethylamine and gave a yield of 60% (Hakansson, A. E.; Koshkin, A. A.; Sorensen M. D.; Wengel J. J.Org.Chem. 2000, 65, 5161-5166).