An astute synthesis of locked nucleic acid monomers

Abstract

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 [3], 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 [6]; there is hardly any synthetic oligonucleotide (ON) based therapeutic strategy which has not been allured by their unique features [4,5,7,8].

Figure 1

Structure and conformations of DNA, RNA and LNA; B = nucleobase.

Seminal papers on LNA were independently instigated by Wengel [9,10] and Imanishi [11] groups. It is well known that the B-form DNA duplex possesses C_2′-endo (S-type) and the A-form RNA duplex has C_3′-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 [14]. 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) [17]. If its phase III trial looks anything like its phase II, Miravirsen could be the first LNA-based drug to get FDA approval.

Table 1 Current clinical trials of oligonucleotides modified with LNA [18]

Figure 2

Mechanism of action of chemically modified drug Miravirsen. (LNA monomers in oligonucleotide are shown red).

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. [11] for the synthesis of LNA-U monomer 1a with uridine (2) as the starting material (Scheme 1).

Scheme 1

Linear synthesis of LNA-U monomer [ 11 ]. Reagents (% yields): (i) Cyclohexanone, PTSA (quantitative); (ii) 2-iodobenzoic acid, CH₃CN (76%); (iii) (a) 37% HCHO, 2N NaOH, 1,4-dioxane; (b) NaBH₄ (38%); (iv) (a) TsCl, Pyridine; (b) TFA, water (34%); (v) PhCHO, ZnCl₂, (80%); (vi) NaBH₃CN, TiCl₄, CH₃CN (75%); (vii) NaHMDS, THF (61%); (viii) 10% Pd-C, H₂, MeOH (quantitative).

Following similar strategy, Koshkin et al. [19] 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. [10] 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).

Scheme 2

Convergent synthesis of LNA monomers [ 10 ]. Reagents (% yields): (i) BnBr, NaH, DMF (71%); (ii) (a) Ac₂O, Pyridine; (b) 80% AcOH; (c) Ac₂O, Pyridine (77%); (iii) nucleobase, N,O-bis(trimethylsilyl)acetamide, TMS-triflate, CH₃CN or 1,2-dichloroethane (13a, 75%; 13b, 76%; 13c, 52%; 13d, 74%); (iv) NaOMe, MeOH (14a, 95%; 14b, 97%; 14c, 73%; 14d, 54%); (v) (a) TsCl, Pyridine; (b) NaH, DMF (15a, 30%; 15b, 42%; 15c, 44%; 15d, 51%); (vi) 20% Pd(OH)₂-C, H₂, EtOH (1a, 78%; 1b, 98%); BCl₃, DCM (1c, 84%); 10% Pd-C, 1,4-cyclohexadiene, MeOH (1d, 36%).

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 [10]. Hence, in order to avoid the regioselective transformation on the furanose diol 10, an alternate convergent synthesis was optimized by Koshkin et al. [20] (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.

Scheme 3

Improved convergent synthesis of LNA monomers [ 20 , 21 ]. Reagents (% yields): (i) MsCl, pyridine, CH₂Cl₂ (98%); (ii) Ac₂O, AcOH, conc. H₂SO₄ (97%); (iii) nucleobase, N,O-bis(trimethylsilyl)acetamide, TMS-triflate, CH₃CN or 1,2-dichloroethane (18a, 90%; 18b, 88%; 18c, 68%; 18d, 82%); (iv) aq. NaOH, THF or dioxane (19a, 97%; 19b, 94%; 19c, 78%; 19d, 87%); (v) NaOBz, DMF (20a, 97%; 20b, 86%; 20c, 88%; 20d, 93%); (vi) aq. NaOH, THF (9a, 95%; 9b, 91%); NH₄OH, MeOH (9c, 86%); (a) 20% Pd(OH)₂-C, HCO₂NH₄, MeOH; (b) NH₄OH (1d, 77%); (vii) 20% Pd(OH)₂-C, 88% HCOOH, THF/MeOH (9:1) (1a, 91%); 20% Pd(OH)₂-C, HCO₂NH₄, MeOH or EtOH (1b, 83%; 1c, 91%).

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 [24]. 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).

Scheme 4

Novozyme®-435 mediated monoacetylation of diol 10 [ 24 ].

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).

Figure 3

Study of recyclability of lipase Novozyme®-435 for acetylation reaction on diol 10. (All these reactions were performed on 100 mg batch size w.r.t. 10).

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).

Scheme 5

Chemo-enzymatic convergent synthesis of 2'-O ,4'-C-methylene-ribonucleosides (LNA monomers) 1a-d [ 24 ]. Reagents & conditions (% yields): (i) TsCl, pyridine, CH₂Cl₂, 0°C to rt (98%); (ii) Ac₂O, AcOH, H₂SO₄ (100:10:0.1), 0°C to rt (97%); (iii) nucleobase, N,O-bis(trimethylsilyl) acetamide, TMS-triflate, acetonitrile or 1,2-dichloroethane, 80°C (24a, 89%; 24b, 88%; 24c, 71%; 24d, 78%); (iv) aq. NaOH, 1,4-dioxane, rt (+NH₄OH for 9c) (9a, 95%; 9b, 93%; 9c, 89%; 9d, 94%); (v) 20% Pd(OH)₂-C, HCOOH, THF:MeOH (9:1), reflux (1a, 91%); 20% Pd(OH)₂-C, HCO₂NH₄, MeOH or EtOH, reflux (1b, 83%; 1c, 91%; 1d, 81%).

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).

Table 2 Overall yields of LNA monomers for the reported classical chemical and chemo-enzymatic convergent synthesis

Conclusion

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.