- Open Access
An astute synthesis of locked nucleic acid monomers
Sustainable Chemical Processes volume 3, Article number: 2 (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.
Since the acclamation of nucleic acid therapeutics, modification in the sugar moiety of nucleosides has continuously reflected its supremacy for developing drug candidates for the treatment of cancer and viral infections [1-3]. After the pioneering development in dideoxy- and acyclic- nucleos(t)ides , currently the most promising modification in the ribofuranose moiety has appeared through the inclusion of an extra methylene bridge between 2′-O & 4′-C atom and synthesis of oligonucleotides (ONs) involving the modified nucleosides, termed as locked nucleic acid (LNA) (Figure 1) [4,5]. Although, no significant potency was observed against cancer or viral infections by LNA monomers or its analogues ; there is hardly any synthetic oligonucleotide (ON) based therapeutic strategy which has not been allured by their unique features [4,5,7,8].
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.
LNA-modified ONs have been extensively utilized in different approaches to target the corresponding nucleic acid counterparts. These primarily include, (a) antigene approach to block transcription of a particular gene; (b) antisense approach to induce RNA degradation; (c) siRNA mediated RNA degradation; and (d) blocking of microRNA [7,8]. Since LNA possess high hybridization affinity and target selectivity, it is unsurprising that increasing success in cell-line based experiments has paved their way to five LNA-based modified ONs under active clinical trials (Table 1). One of the most advanced LNA-based drugs Miravirsen which has entered Phase II clinical study is being developed by Santaris Pharma A/S. Miravirsen is an inhibitor of miR-122, a liver specific microRNA that is required by Hepatitis C virus (HCV) for replication. The liver-expressed miR-122 protects HCV from degradation. Miravirsen is designed to recognize and sequester miR-122, making it unavailable for HCV. As a result, the replication of the virus is effectively inhibited and the level of HCV is profoundly reduced (Figure 2) . If its phase III trial looks anything like its phase II, Miravirsen could be the first LNA-based drug to get FDA approval.
Two general strategies have been employed for the synthesis of LNA monomers; a linear strategy using commercially available RNA nucleosides as the starting material [11,19] and a convergent strategy where a common glycosyl donor is synthesized for coupling with different nucleobases [10,20,21]. Linear strategy was disclosed by Obika et al.  for the synthesis of LNA-U monomer 1a with uridine (2) as the starting material (Scheme 1).
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).
Alternatively, in the quest to establish a general method for the synthesis of all LNA monomers i.e. LNA-U, 1a; LNA-T, 1b; LNA-A, 1c and LNA-C, 1d; the convergent strategy was explored by Koshkin et al.  using 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (10) as a starting material, which can be synthesized easily from D-glucose [22,23]. Regioselective 5-O-benzylation of 10 followed by acetolysis afforded the furanose 12 in 55% yield, a key intermediate for coupling reactions with a variety of nucleobases. The Vorbrüggen coupling with silylated nucleobases afforded the nucleosides 13a-d, which on deacetylation led to the formation of benzylated nucleosides 14a-d. The tosylation of the primary hydroxyl group in benzylated nucleosides 14a-d followed by in situ base-induced intramolecular ring closure afforded the 2′-O,4′-C-linked locked nucleoside derivatives 15a-d. Debenzylation on dibenzylated nucleosides 15a-d efficiently yielded the LNA monomers 1a-d (Scheme 2).
Although, using the convergent strategy (Scheme 2), synthesis of LNA monomers with all natural nucleobases was standardized, regioselective benzylation of dihydroxy furanose derivative 10 remained unanswered . Hence, in order to avoid the regioselective transformation on the furanose diol 10, an alternate convergent synthesis was optimized by Koshkin et al.  (Scheme 3). Permesylation of furanose diol 10 afforded the dimesylated derivative 16, which on acetolysis followed by acetylation, afforded an anomeric mixture of 1,2-di-O-acetyl-3-O-benzyl-4-C-methanesulfonyloxymethyl-5-O-methanesulfonyl-D-ribofuranose (17). The glycosyl donor 17 was used as a common intermediate for coupling reactions with different nucleobases to afford the LNA monomers 1a-d [20,21] as shown in Scheme 3.
It seems easy to synthesize LNA monomers following the convergent strategy which utilizes the furanose diol 10 as the starting material. However, the use of 10 was found to be complicated due to the presence of two diastereotopic hydroxymethyl groups (Scheme 2 and Scheme 3). Therefore, we focused our attention towards lipase mediated diastereoselective protection of one of the hydroxymethyl groups in the crucial intermediate 10 with a base labile group such as acetyl, that can be hydrolyzed insitu concomitantly with 2′-O,4′-C-cyclization towards the end of the synthesis to get the LNA monomers . Screening of different lipases in organic solvents for the diasteroeselective acetylation of one of the two hydroxyl groups in dihydroxy compound 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (10) revealed that Candida antarctica lipase-B (Novozyme®-435) in diisopropyl ether (DIPE) in the presence of vinyl acetate as acetyl donor carries out the selective 5′-O-monoacetylation (Scheme 4).
In a successful biocatalytic transformation reaction, a solution of the compound 10 and vinyl acetate in DIPE was incubated with Novozyme®-435 (10% w/w of 10) at 45°C and 200 rpm in an incubator shaker. The progress of the reaction was monitored on analytical TLC. On completion, reaction was quenched by filtering off the enzyme and solvent was removed under reduced pressure. The crude product thus obtained was washed with hexane to afford monoacetylated compound 5-O-acetyl-3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (21) in quantitative yield. Using the optimized conditions, Novozyme®-435 was utilised for ten recycles of selective acetylation of compound 10 and was found to be equally regioselective for each cycle (Figure 3).
The synthesis of LNA monomers 1a-d was successfully achieved from enzyme-mediated monoacetylated compound 21. Tosylation of 21 afforded compound 22 which on subsequent acetolysis gave an anomeric mixture 23 in 95% overall yield. Aiming for the convergent synthesis of LNA monomers, the mixture 23 was used as common glycosyl donor for the Vorbrüggen’s coupling reaction with uracil, thymine, 6-N-benzoyladenine and cytosine to yield the corresponding 2′,5′-di-O-acetyl-3′-O-benzyl-4′-C-p-toluenesulfonyloxymethyl-ribonucleosides 24a-d in 71-89% yields. Subsequently, deacetylation and concomitant intramolecular cyclization under alkaline conditions afforded the 3′-O-benzyl-2′-O,4′-C-methylene-ribonucleosides 9a-d in 89-95% yields. Deprotection of the 3′-O-benzyl group in nucleosides 9a-d afforded the LNA monomers, i.e. 2′-O,4′-C-methyleneuridine (1a), 2′-O,4′-C-methylenethymidine (1b), 2′-O,4′-C-methyleneadenosine (1c) and 2′-O,4′-C-methylenecytidine (1d) in 81-91% yields (Scheme 5).
Starting from the diol 10, the overall yields for developed chemo-enzymatic convergent synthesis of LNA monomers (Scheme 5) have been compared with the literature convergent methodology (Scheme 3). The results revealed that the developed biocatalytic methodology is more efficient in all cases with remarkable improvement for LNA-A (Table 2).
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
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.
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.
De Clercq E. A 40-year journey in search of selective antiviral chemotherapy. Annu Rev Pharmacol Toxicol. 2011;51:1–24.
Watts JK. Locked nucleic acid: tighter is different. Chem Commun. 2013;49:5618–20.
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.
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.
Sharma VK, Rungta P, Prasad AK. Nucleic acid therapeutics: basic concepts and recent developments. RSC Adv. 2014;4:16618–31.
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.
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.
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.
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.
Sanger W. Principles of Nucleic Acid Structures. New York: Springer-Verlag; 1984.
Kaur H, Babu BR, Maiti S. Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem Rev. 2007;107:4672–97.
Veedu RN, Wengel J. Locked nucleic acids: promising nucleic acid analogs for therapeutic applications. Chem Biodivers. 2010;7:536–42.
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.
Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol. 2012;199:407–12.
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.
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.
Kumar TS, Kumar P, Sharma PK, Hrdlicka PJ. Optimized synthesis of LNA uracil nucleosides. Tetrahedron Lett. 2008;49:7168–70.
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.
Youssefyeh RD, Verheyden JPH, Moffatt JG. 4’-Substituted nucleosides. 4. Synthesis of some 4’-hydroxymethyl nucleosides. J Org Chem. 1979;44:1301–09.
Sharma VK, Kumar M, Olsen CE, Prasad AK. Chemoenzymatic convergent synthesis of 2-O,4-C-methyleneribonucleosides. J Org Chem. 2014;79:6336–41.
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.
The authors declare that they have no competing interests.
VKS, PR, VKM and AKP wrote the manuscript, with VKS being the main contributor. All the authors read and approved the final manuscript.
VKS, PR and VKM are research fellows under the supervision of AKP at the Department of Chemistry, University of Delhi, India.
About this article
Cite this article
Sharma, V.K., Rungta, P., Maikhuri, V.K. et al. An astute synthesis of locked nucleic acid monomers. sustain chem process 3, 2 (2015) doi:10.1186/s40508-015-0028-3
- Locked nucleic acid
- Nucleic acid therapeutics
- Modified oligonucleotides
- Linear synthesis
- Convergent synthesis