Locked Nucleic Acids: Synthesis and Characterization of LNA‐T Diol

Henrik M. Pfundheller1, Christian Lomholt1

1 Exiqon A/S, Vedbaek, Denmark
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.12
DOI:  10.1002/0471142700.nc0412s08
Online Posting Date:  May, 2002
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Abstract

Locked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. LNAs are synthesized from a commercially available sugar, 1,2:5,6‐di‐O‐isopropylidene‐a‐D‐allofuranose. An efficient and simplified procedure is presented for synthesizing a glycol donor that can be used for synthesis of a variety of LNA monomers. Then, as an example, the synthesis of the thymidine analog of LNA from this glycol donor is presented. The protocols give high yields of the desired products and avoid the use of time‐consuming column chromatography.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 3‐O‐Benzyl‐1,2:5,6‐Di‐O‐Isopropylidene‐α‐D‐Allofuranose Using Benzyl Bromide in Tetrahydrofuran
  • Alternate Protocol 1: Synthesis of 3‐O‐Benzyl‐1,2:5,6‐Di‐O‐Isopropylidene‐α‐D‐Allofuranose Usomg Benzyl Bromide in Dimethylformamide
  • Basic Protocol 2: Synthesis of 3‐O‐Benzyl‐4‐C‐Hydroxymethyl‐1,2‐O‐Isopropylidene‐α‐D‐ Erythropentofuranose
  • Basic Protocol 3: Synthesis of 3‐O‐Benzyl‐1,2‐O‐Isopropylidene‐5‐O‐Methanesulfonyl‐4‐C‐ Methanesulfonyloxymethyl‐α‐D‐Erythropentofuranose
  • Basic Protocol 4: Synthesis of 1,2‐Di‐O‐Acetyl‐3‐O‐Benzyl‐5‐O‐Methanesulfonyl‐4‐C‐ Methanesulfonyloxymethyl‐D‐Erythropentofuranose
  • Basic Protocol 5: Synthesis of (1S,3R,4R,7S)‐7‐Hydroxy‐1‐Hydroxymethyl‐3‐(Thymin‐1‐YL)‐2,5‐ Dioxabicyclo[2.2.1]Heptane (LNA‐T Diol)
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 3‐O‐Benzyl‐1,2:5,6‐Di‐O‐Isopropylidene‐α‐D‐Allofuranose Using Benzyl Bromide in Tetrahydrofuran

  Materials
  • 60% (v/v) sodium hydride in mineral oil
  • Hexane (stored over 3A molecular sieves)
  • Nitrogen (N 2) stream (or argon stream)
  • Tetrahydrofuran (THF; stored over 3A molecular sieves)
  • Dimethylformamide (DMF; stored over 3A molecular sieves)
  • 1,2:5,6‐Di‐O‐isopropylidene‐α‐D‐allofuranose (Pfanstiehl Laboratories)
  • Benzyl bromide
  • Brine (saturated aqueous NaCl)
  • MgSO 4
  • 500‐mL three‐neck round‐bottom flask
  • 250‐mL dropping funnel
  • Nitrogen inlet
  • 20‐mL syringe
  • Sintered glass funnel, pore size 3
  • Rotary evaporator connected to vacuum pump

Alternate Protocol 1: Synthesis of 3‐O‐Benzyl‐1,2:5,6‐Di‐O‐Isopropylidene‐α‐D‐Allofuranose Usomg Benzyl Bromide in Dimethylformamide

  Materials
  • 3‐O‐Benzyl‐1,2:5,6‐di‐O‐isopropylidene‐α‐D‐allofuranose (S.2; see protocol 1 or protocol 2)
  • Acetic acid
  • Dichloromethane
  • Ethyl acetate
  • Toluene
  • Tetrahydrofuran (THF)
  • NaIO 4
  • Brine (saturated aqueous NaCl)
  • 1,4‐Dioxane
  • 37% (w/v) aqueous formaldehyde
  • 4 M NaOH
  • MgSO 4
  • Hexane
  • 1‐L round‐bottom flask
  • Rotary evaporator connected to vacuum pump
  • Sintered glass funnel, pore size 3
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: Synthesis of 3‐O‐Benzyl‐4‐C‐Hydroxymethyl‐1,2‐O‐Isopropylidene‐α‐D‐ Erythropentofuranose

  Materials
  • 3‐O‐Benzyl‐4‐C‐hydroxymethyl‐1,2‐O‐isopropylidene‐α‐D‐erythropentofuranose (S.5; see protocol 3)
  • Dichloromethane (stored over 3A molecular sieves)
  • Pyridine (stored over 3A molecular sieves)
  • Methanesulfonylchloride (e.g., Aldrich)
  • Brine (saturated aqueous NaCl)
  • 1 M HCl
  • Saturated aqueous NaHCO 3
  • MgSO 4
  • Methanol
  • 500‐mL round‐bottom flask
  • 250‐mL dropping funnel
  • Guard tube/nitrogen inlet
  • Sintered glass funnel, pore size 3
  • Rotary evaporator connected to vacuum pump

Basic Protocol 3: Synthesis of 3‐O‐Benzyl‐1,2‐O‐Isopropylidene‐5‐O‐Methanesulfonyl‐4‐C‐ Methanesulfonyloxymethyl‐α‐D‐Erythropentofuranose

  Materials
  • 3‐O‐Benzyl‐1,2‐O‐isopropylidene‐5‐O‐methanesulfonyl‐4‐C‐methanesulfonyloxymethyl‐α‐D‐erythropentofuranose (S.6; see protocol 4)
  • Acetic acid
  • Acetic anhydride
  • Concentrated H 2SO 4
  • Dichloromethane
  • Saturated aqueous Na 2CO 3
  • Saturated aqueous NaHCO 3
  • MgSO 4, anhydrous
  • 1‐L round‐bottom flask
  • Guard tube/nitrogen inlet
  • Sintered glass funnel, pore size 3
  • Rotary evaporator connected to vacuum pump

Basic Protocol 4: Synthesis of 1,2‐Di‐O‐Acetyl‐3‐O‐Benzyl‐5‐O‐Methanesulfonyl‐4‐C‐ Methanesulfonyloxymethyl‐D‐Erythropentofuranose

  Materials
  • 1,2‐Di‐O‐acetyl‐3‐O‐benzyl‐5‐O‐methanesulfonyl‐4‐C‐methanesulfonyloxymethyl‐D‐erythropentofuranose (S.7; see protocol 5)
  • Acetonitrile (stored over 3A molecular sieves)
  • Thymine
  • N,O‐Bis(trimethylsilyl)acetamide (Fluka)
  • Trimethylsilyl trifluoromethanesulfonate (Fluka)
  • Saturated aqueous NaHCO 3
  • Dichloromethane
  • Brine (saturated aqueous NaCl)
  • Tetrahydrofuran (THF)
  • LiOH⋅H 2O
  • Acetic acid
  • Ethyl acetate
  • MgSO 4, anhydrous
  • Anhydrous dimethylformamide (DMF; stored over 3A molecular sieves)
  • Sodium benzoate
  • Hexane
  • Methanol
  • 20% Pd(OH) 2/C (palladium hydroxide catalyst on carbon; Fluka)
  • Ammonium formate
  • 2‐cm‐thick Celite pad
  • 250‐mL, 500‐mL, and 1‐L round‐bottom flasks
  • Rotary evaporator connected to vacuum pump
  • Condensers
  • Guard tubes/nitrogen inlets
  • Sintered glass funnel, pore size 3
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Figures

Videos

Literature Cited

Literature Cited
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   Horton, D. and Tindall, C.G. Jr. 1970. Methylene‐insertion reactions with unsaturated sugar, synthesis of 4‐C‐cyclopropyl‐D‐ribo‐tetrafuranose derivatives. Carbohydr. Res. 15:215‐232.
   Jones, G.H., Taniguchi, M., Tegg, D., and Moffat, J.G. 1979. 4′‐Substituted nucleosides. 5. Hydroxymethylation of nucleoside 5′‐aldehydes. J. Org. Chem. 44:1309‐1317.
   Koshkin, A.A., Rajwanshi, V.K., and Wengel, J. 1998a. Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides. Tetrahedron Lett. 39:4381‐4384.
   Koshkin, A.A., Singh, S.K., Nielsen, P., Rajwanshi, V.K., Kumar, R., Meldgaard, M., Olsen, C.E., and Wengel, J. 1998b. LNA (locked nucleic acids): Synthesis of the adenine, cytosine, guanine, 5‐methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54:3607‐3630.
   Koshkin, A.A., Fensholdt, J., Pfundheller, H.M., and Lomholt, C. 2001. A simplified and efficient route to 2′‐O, 4′‐C‐methylene‐linked bicyclic ribonucleosides (LNA). J. Org. Chem. 66:8504‐8512.
   Leland, D.L. and Kotick, M.P. 1974. Studies on 4‐C‐(hydroxymethyl)pentofuranoses. Synthesis of 9‐[4‐C‐(hydroxymethyl)‐α‐L‐threo‐pentofuranosyl]adenine. Carbohydr. Res. 38:C9‐C11.
   Obika, S., Nanbu, D., Hari, Y., Morio, K., In, Y., Ishida, T., and Imanishi, T. 1997. Synthesis of 2′‐O, 4′‐C‐methyleneuridine and ‐cytidine. Novel bicyclic nucleosides having a fixed C3′‐endo sugar puckering. Tetrahedron Lett. 38:8735‐8738.
   Obika, S., Nanbu, D., Hari, Y., Andoh, J., Morio, K., Doi, T., and Imanishi, T., 1998. Stability and structural features of the duplexes containing nucleoside analogues with a fixed N‐type conformation, 2′‐O,4′‐C‐methyleneribonucleosides. Tetrahedron Lett. 39:5401‐5404.
   Obika, S., Uneda, T., Sugimoto, T., Nanbu, D., Minami, T., Doi, T., and Imanishi, T. 2001. 2′‐O,4′‐C‐Methylene bridged nucleic acid (2′,4′‐BNA): Synthesis and triplex‐forming properties. Bioorg. Med. Chem. 9:1001‐1011.
   Ørum, H., Jakobsen, M.H., Koch, T., Vuust, J., and Borre, M.B. 1999. Detection of the factor V Leiden mutation by direct allele‐specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids. Clin. Chem. 45:1898‐1905.
   Singh, S.K., Nielsen, P., Koshkin, A.A., and Wengel, J. 1998. LNA (locked nucleic acids): Synthesis and high‐affinity nucleic acid recognition. Chem. Commun. (1998):455‐456.
   Sinha, N.D., Biernat, J., and Köster, H. 1983. β‐Cyanoethyl N,N‐dialkylamino/N‐morpholinomonochloro phosphoramidites, new phosphitylating agents facilitating ease of deprotection and work‐up of synthesized oligonucleotides. Tetrahedron Lett. 24:5843‐5846.
   Sowa, W. and Thomas, G.H.S. 1966. The oxidation of 1,2:5,6‐di‐O‐isopropylidene‐D‐glucose by dimethylsulfoxide‐acetic anhydride. Can. J. Chem. 44:836‐838.
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   Waga, T., Nishizaki, T., Miyakawa, I., Ohrui, H., and Meguro, H. 1993. Synthesis of 4′‐C‐methylnucleosides. Biosci. Biotech. Biochem. 57:1433‐1438.
   Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hϕkfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K., Ossipov, M., Koshkin, A., Jakobsen, N., Skouv, J., Oerum, H., Jacobsen, M.H., and Wengel, J. 2000. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl. Acad. Sci. U.S.A. 97:5633‐5638.
   Youssefyeh, R., Tegg, D., Verheyden, J.P.H., Jones, G.H., and Moffat, J.G. 1977. Synthetic routes to 4′‐hydroxymethylnucleosides. Tetrahedron Lett. 5:435‐438.
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Key References
   Koshkin et al., 1998a,b. See above.
   This unit's protocols were developed based on the synthetic results described in these papers.
   Wengel, J. 1999. Development of locked nucleic acid. Acc.Chem. Res. 32:301‐310.
  This article describes the development of LNA.
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