Facile Access to 5′‐S‐(4,4′‐Dimethoxytrityl)‐2′,5′‐Dideoxyribonucleosides via Stable Disulfide Intermediates

Chandra Shekhar Reddy L.1, Vivek K. Sharma1, Rajesh Kumar1, Ankita Singh1, Virinder S. Parmar1, Yogesh S. Sanghvi2, Ashok K. Prasad1

1 Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, 2 Rasayan Inc, Encinitas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.34
DOI:  10.1002/0471142700.nc0134s62
Online Posting Date:  September, 2015
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Abstract

Thionucleosides represent an important class of modified nucleos(t)ides that have found distinct applications in the chemical biology of synthetic oligonucleotides, but the use of these compounds is substantially lessened by the instability or high reactivity of the sulfhydryl group. This unit describes a protocol for the synthesis of 2′,5′‐dideoxy‐5′‐thioribonucleoside disulfides by utilizing Mitsunobu reaction conditions on 3′‐O‐levulinyl‐2′‐deoxyribonucleosides in the presence of thiobenzoic acid followed by facile hydrolysis and in situ oxidation of the resulting 5′‐thiolated nucleosides using methanolic ammonia. The utility of these disulfides has been demonstrated as stable precursors for the synthesis of 5′‐thio‐modified 2′‐deoxynucleosides. To validate the potential of the methodology, 5′‐S‐(4,4′‐dimethoxytrityl)‐2′,5′‐dideoxythymidine phosphoramidite has been synthesized by in situ cleavage of the disulfide linkage of 2′,5′‐dideoxy‐5′‐thiothymidine disulfide followed by protection with a dimethoxytriphenyl (DMT) group and 3′‐phosphitylation using 2‐cyanoethyl N,N‐diisopropylchlorophosphoramidite. © 2015 by John Wiley & Sons, Inc.

Keywords: thionucleosides; disulfide dinucleosides; Mitsunobu reaction

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of 2′,5′‐Dideoxy‐5′‐Thioribonucleoside Disulfides and 5′‐S‐(4,4′‐Dimethoxytrityl)‐2′,5′‐Dideoxythymidine‐3′‐O‐Phosphoramidite
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 2′,5′‐Dideoxy‐5′‐Thioribonucleoside Disulfides and 5′‐S‐(4,4′‐Dimethoxytrityl)‐2′,5′‐Dideoxythymidine‐3′‐O‐Phosphoramidite

  Materials
  • Argon gas
  • 3′‐O‐levulinyl‐2′‐deoxyribonucleosides 1ad (donated by Rasayan; see García et al., 2006)
  • TLC plates (silica coated with fluorescent indicator 60 F 254)
  • Triphenyl phosphine (PPh 3)
  • Thiobenzoic acid
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • Diisopropyl azodicarboxylate (DIAD)
  • N,N‐dimethylformamide (DMF)
  • Dichloromethane (DCM)
  • Acetonitrile (CH 3CN)
  • Tetrahydrofuran (THF)
  • Pyridine (Py)
  • Methanol (MeOH)
  • Chloroform (CHCl 3)
  • Diisopropyl ethylamine (DIPEA)
  • Triethylamine (Et 3N)
  • Anhydrous dichloroethane
  • Methanolic ammonia (25%, w/v; S D Fine‐Chem Limited)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP)
  • Silica gel (100‐200 mesh)
  • Half‐saturated sodium bicarbonate (NaHCO 3) solution (1:1 saturated sodium bicarbonate solution/water, v/v)
  • Brine
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Cotton (for filtration)
  • 25‐ and 50‐mL round‐bottom flasks
  • 100‐ and 250‐mL separatory funnels
  • 100‐ and 250‐mL conical flasks
  • Glass stopper
  • Glass funnel
  • 1‐, 5‐ and 10‐mL glass syringes with needles
  • 3 × 10 cm and 5 × 7 cm glass columns
  • Rubber septa
  • Whatman filter paper
  • Magnetic stirrer
  • Magnetic stirring bars
  • 254‐nm UV lamp
  • Rotary evaporator
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Figures

Videos

Literature Cited

Literature Cited
  Acevedo‐Acevedo, D., Matta, J., and Meléndez, E. 2011. Synthesis, characterization and cytotoxic studies of water soluble [(η5‐C5H5)2Mo(thionucleobase/thionucleoside)]Cl complexes in breast and colon cancer cell lines. J. Organomet. Chem. 696:1032‐1037.
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  Devi, G., Yuan, Z., Lu, Y., Zhao, Y., and Chen, G. 2014. Incorporation of thio‐pseudoisocytosine into triplex‐forming peptide nucleic acids for enhanced recognition of RNA duplexes. Nucleic Acids Res. 42:4008‐4018.
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  García, J., Díaz‐Rodríguez, A., Fernández, S., Sanghvi, Y.S., Ferrero, M., and Gotor, V. 2006. New concept for the separation of an anomeric mixture of α/β‐D‐nucleosides through regioselective enzymatic acylation or hydrolysis processes. J. Org. Chem. 71:9765‐9771.
  García, J., Fernández, S., Ferrero, M., Sanghvi, Y.S., and Gotor, V. 2002. Building blocks for the solution phase synthesis of oligonucleotides: Regioselective hydrolysis of 3′,5′‐di‐O‐levulinylnucleosides using an enzymatic approach. J. Org. Chem. 67:4513‐4519.
  Jahn‐Hofmann, K. and Engels, J.W. 2004. Efficient solid phase synthesis of cleavable oligodeoxynucleotides based on a novel strategy for the synthesis of 5‐S‐(4,4′‐dimethoxytrityl)‐2′‐deoxy‐5′‐thionucleoside phosphoramidites. Helv. Chim. Acta 87:2812‐2828.
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  Kojima, T., Furukawa, K., Maruyama, H., Inoue, N., Tarashima, N., Matsuda, A., and Minakawa, N. 2013. PCR amplification of 4′‐thioDNA using 2′‐deoxy‐4′‐thionucleoside 5′‐triphosphates. ACS Synth. Biol. 2:529‐536.
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  Ohkubo, A., Nishino, Y., Yokouchi, A., Ito, Y., Noma, Y., Kakishima, Y., Masaki, Y., Tsunoda, H., Seio, K., and Sekine, M. 2011. Stable triplex formation using the strong stacking effect of consecutive thionucleoside moieties. Chem. Commun. 47:12556‐12558.
  Ruano, J.L.G., Parraa, A., and Alemána, J. 2008. Efficient synthesis of disulfides by air oxidation of thiols under sonication. Green Chem. 10:706‐711.
  Sabbagh, G., Fettes, K.J., Gosain, R., O'Neil, I.A., and Cosstick, R. 2004. Synthesis of phosphorothioamidites derived from 3′‐thio‐3′‐deoxythymidine and 3′‐thio‐2′,3′‐dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3′‐S‐phosphorothiolate linkage. Nucleic Acids Res. 32:495‐501.
  Thottassery, J.V., Sambandam, V., Allan, P.W., Maddry, J.A., Maxuitenko, Y.Y., Tiwari, K., Hollingshead, M., and Parker, W.B. 2014. Novel DNA methyltransferase‐1 (DNMT1) depleting anticancer nucleosides, 4′‐thio‐2′‐deoxycytidine and 5‐aza‐4′‐thio‐2′‐deoxycytidine. Cancer Chemother. Pharmacol. 74:291‐302.
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