An astute synthesis of locked nucleic acid monomers
© Sharma et al.; licensee Springer. 2015
Received: 2 September 2014
Accepted: 13 February 2015
Published: 4 March 2015
Novel attributes of Locked Nucleic Acid (LNA) makes it preferable over most of the other classes of modified nucleic acid analogues and therefore, it has been extensively explored in different synthetic oligonucleotide based therapeutics. In addition to five oligonucleotides of this class undergoing clinical trials, a healthy pipeline in pre-clinical studies validates the tenacity of LNA. Due to the increasing demand, an efficient biocatalytic methodology has recently been devised for the convergent synthesis of LNA monomers via selective enzymatic monoacetylation of diastereotopic hydroxymethyl functions of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose. This commentary article provides an insight into the different synthetic strategies followed for the synthesis of LNA monomers and their triumphs in clinical biotechnology.
KeywordsLocked nucleic acid Nucleic acid therapeutics Bio-catalysis Novozyme®-435 Modified oligonucleotides Linear synthesis Convergent synthesis Miravirsen
Seminal papers on LNA were independently instigated by Wengel [9,10] and Imanishi  groups. It is well known that the B-form DNA duplex possesses C2′-endo (S-type) and the A-form RNA duplex has C3′-endo (N-type) sugar puckering [12,13]. LNA is considered to be RNA mimic as the ancillary methylene bridge locks the sugar moiety into N-type sugar ring conformation (Figure 1). This conformational restriction results in preorganization of the backbone of LNA ONs, which leads to energetically favorable duplex formation via increased base stacking interactions according to standard Watson-Crick base pairing rules . Generally, the melting temperature (T m ) of duplexes is raised by 2-8°C per LNA nucleotide incorporation when compared to the corresponding unmodified duplexes, depending on the sequence context and number of modifications [14-16]. This makes LNA the prime nucleotide modification candidate for the applications where high hybridization affinity is desirable.
Current clinical trials of oligonucleotides modified with LNA 
LNA modified oligonucleotide
Hepatitis C virus (HCV)
Hypoxia-inducible factor-1 alpha (HIF-1α)
Following similar strategy, Koshkin et al.  synthesized LNA-A monomer taking adenosine as the starting material. Despite having some advantages, such as cheap and readily available RNA nucleosides as starting material and short synthetic route to LNA monomers, the linear approach suffers from poor yields. The two key reactions in the synthetic pathway, i.e. the introduction of the additional hydroxymethyl group at the C-4′-position of the protected RNA nucleoside 4 and the regioselective tosylation of the introduced 4′-C-hydroxymethyl group, generally proceeds with very low yields (Scheme 1).
Unprecedented success of Locked Nucleic acid (LNA) in oligonucleotide based therapeutics demands a cost efficient, convenient and environment friendly synthetic route for LNA monomers. Therefore, Novozyme®-435 mediated selective protection of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose has been highlighted which lead to relatively efficient and environment friendly synthesis of LNA monomers in comparison to the earlier reports.
Locked nucleic acid
Hepatitis C virus
We are grateful to the University of Delhi for providing financial support under DU-DST Purse Grant and under scheme to strengthen research and development. VKS and PR thank CSIR, and VKM thanks DBT, New Delhi for the award of JRF/SRF Fellowships.
- Jordheim LP, Durantel D, Zoulim F, Dumontet C. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat Rev Drug Discov. 2013;12:447–64.View ArticleGoogle Scholar
- Sofia MJ, Chang W, Furman PA, Mosley RT, Ross BS. Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase. J Med Chem. 2012;55:2481–531.View ArticleGoogle Scholar
- De Clercq E. A 40-year journey in search of selective antiviral chemotherapy. Annu Rev Pharmacol Toxicol. 2011;51:1–24.View ArticleGoogle Scholar
- Watts JK. Locked nucleic acid: tighter is different. Chem Commun. 2013;49:5618–20.View ArticleGoogle Scholar
- Wengel J. Synthesis of 3′-C- and 4′-C-branched oligodeoxynucleotides and the development of Locked Nucleic Acid (LNA). Acc Chem Res. 1999;32:301–10.View ArticleGoogle Scholar
- Olsen AG, Nielsen C, Wengel J. Synthesis and evaluation of anti-HIV activity of 3-azido-4-(hydroxymethyl)tetrahydrofuran derivatives containing 2-(thymin-1-yl)methyl, 2-(cytosin-1-yl)methyl or 2-(adenin-9-yl)methyl substituents- a new series of AZT analogues. J Chem Soc Perkin Trans. 2001;1:900–04.View ArticleGoogle Scholar
- Sharma VK, Rungta P, Prasad AK. Nucleic acid therapeutics: basic concepts and recent developments. RSC Adv. 2014;4:16618–31.View ArticleGoogle Scholar
- Lundin KE, Højland T, Hansen BR, Persson R, Bramsen JB, Kjems J, et al. Biological activity and biotechnological aspects of locked nucleic acids. Adv Genet. 2013;82:47–107.View ArticleGoogle Scholar
- Singh SK, Nielsen P, Koshkin AA, Wengel J: LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem Commun 1998, 455-56
- Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, et al. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron. 1998;54:3607–30.View ArticleGoogle Scholar
- Obika S, Nanbu D, Hari Y, Morio K, In Y, Ishida T, et al. Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3′-endo sugar puckering. Tetrahedron Lett. 1997;38:8735–38.View ArticleGoogle Scholar
- Mitsuoka Y, Kodama T, Ohnishi R, Hari Y, Imanishi T, Obika S. A bridged nucleic acid, 2’,4’-BNA COC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2’,4’-BNA COC monomers and RNA-selective nucleic-acid recognition. Nucleic Acids Res. 2009;37:1225–38.View ArticleGoogle Scholar
- Sanger W. Principles of Nucleic Acid Structures. New York: Springer-Verlag; 1984.View ArticleGoogle Scholar
- Kaur H, Babu BR, Maiti S. Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem Rev. 2007;107:4672–97.View ArticleGoogle Scholar
- Veedu RN, Wengel J. Locked nucleic acids: promising nucleic acid analogs for therapeutic applications. Chem Biodivers. 2010;7:536–42.View ArticleGoogle Scholar
- Braasch DA, Liu Y, Corey DR. Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Res. 2002;30:5160–67.View ArticleGoogle Scholar
- Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol. 2012;199:407–12.View ArticleGoogle Scholar
- For details of clinical trials: https://clinicaltrials.gov/.
- Koshkin AA, Rajwanshi VK, Wengel J. Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides. Tetrahedron Lett. 1998;39:4381–4.View ArticleGoogle Scholar
- Koshkin AA, Fensholdt J, Pfundheller HM, Lomholt C. A simplified and efficient route to 2’-O,4’-C-methylene-linked bicyclic ribonucleosides (locked nucleic acid). J Org Chem. 2001;66:8504–12.View ArticleGoogle Scholar
- Kumar TS, Kumar P, Sharma PK, Hrdlicka PJ. Optimized synthesis of LNA uracil nucleosides. Tetrahedron Lett. 2008;49:7168–70.View ArticleGoogle Scholar
- Christensen SM, Hansen HF, Koch T. Molar-scale synthesis of 1,2:5,6-di-O-isopropylidene-α-D-allofuranose: DMSO oxidation of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose and subsequent sodium borohydride reduction. Org Process Res Dev. 2004;8:777–80.View ArticleGoogle Scholar
- Youssefyeh RD, Verheyden JPH, Moffatt JG. 4’-Substituted nucleosides. 4. Synthesis of some 4’-hydroxymethyl nucleosides. J Org Chem. 1979;44:1301–09.View ArticleGoogle Scholar
- Sharma VK, Kumar M, Olsen CE, Prasad AK. Chemoenzymatic convergent synthesis of 2-O,4-C-methyleneribonucleosides. J Org Chem. 2014;79:6336–41.View ArticleGoogle Scholar
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.