Print
Email
Solid‐Phase Synthesis of 2′‐Deoxy‐2′‐fluoro‐ β‐D‐Oligoarabinonucleotides (2′F‐ANA) and Their Phosphorothioate Derivatives
Publication Name:
Current Protocols in Nucleic Acid Chemistry
Unit Number:
UNIT 4.15
DOI:
10.1002/0471142700.nc0415s10
Online Posting Date:
November, 2002 Abstract
This unit describes the chemical synthesis of 2'-deoxy-2'-fluoro-b-D-oligoarabinonucleotides (2'F-ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.
Table of Contents
- Unit Introduction
- Basic Protocol 1: Preparation of araF Phosphoramidites
- Basic Protocol 2: Solid-Phase Assembly of Protected araF Phosphoramidites
- Basic Protocol 3: Deprotection and Purification of araF Oligonucleotides
- Reagents and Solutions
- Commentary
- Bibliography
- Figures
- Tables
Materials
Basic Protocol 1: Preparation of araF Phosphoramidites
Materials
- Protected araF nucleosides (unit
- N
6 -Benzoyl-9-[2-deoxy-2-fluoro-5-O-(4-methoxytrityl)--d-arabinofuranosyl] adenine (S.1a) - N
2 -Isobutyryl-9-[2-deoxy-2-fluoro-5-O-(4-methoxytrityl)--d-arabinofuranosyl]guanine (S.1b) - N
4 -Benzoyl-1-[2-deoxy-2-fluoro-5-O-(4-methoxytrityl)--d-arabinofuranosyl] cytosine (S.1c) - 1-[2-Deoxy-2-fluoro-5-O- (4-methoxytrityl)--d-arabinofuranosyl]thymine (S.1d)
- THF, anhydrous (see
- N-Ethyl-N,N-diisopropylamine (DIPEA; Aldrich), double distilled
- 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (Chem Genes)
- Dichloromethane
- 1:9 and 1:19 (v/v) methanol/dichloromethane
- Saturated sodium bicarbonate solution
- Magnesium sulfate, anhydrous
- Solvent system:
- 1:99 (v/v) triethylamine/chloroform (for araF-T)
- 1:9:10 (v/v) triethylamine/dichloromethane/hexanes (for araF-A and araF-C)
- 1:99 (v/v) triethylamine/dichloromethane (for araF-G)
- Silica gel (230 to 400 mesh)
- Diethyl ether
50-mL round-bottom flask equipped with stir bar and rubber septum
Syringe
TLC Merck silica plates (Kieselgel 60 F-254)
254-nm UV lamp
500-mL separatory funnel
Vacuum evaporator (e.g., Savant Speedvac)
3-cm-diameter chromatography column
Additional reagents and equipment for TLC (appendix 3D ) and column chromatography (appendix 3E )
Basic Protocol 2: Solid-Phase Assembly of Protected araF Phosphoramidites
Materials
- AraF phosphoramidites (see
- Argon gas
- Acetonitrile, anhydrous (see
- Liquid Reagent kit for the Expedite 8909 instrument (PerSeptive Biosystems):
- Acetonitrile wash and amidite diluent: anhydrous acetonitrile
- Activator solution: dissolve 1.8 g sublimed tetrazole (0.5 M) in 50 mL
- acetonitrile; store up to 2 wks at room temperature.
- Cap A (see
- Cap B (see
- Oxidizer solution (see
- Deblock solution: 15 g trichloroacetic acid in 500 mL dichloromethane; store up to 1 yr at room temperature
- Sulfurization reagent (see
- Amidite column (see
Synthesizer vials with caps
Vacuum desiccator containing phosphorus pentoxide
Syringe
Automated DNA synthesizer (e.g., Expedite 8909, Perseptive Biosystems) with trityl monitor
Additional reagents and equipment for oligonucleotide synthesis (appendix 3C )
Basic Protocol 3: Deprotection and Purification of araF Oligonucleotides
Materials
- Fully protected oligonucleotides, attached to the solid support of a synthesis column (see
- Ethanol, anhydrous
- 29% ammonium hydroxide
- Denaturing acrylamide gel stock solution (see
- Loading buffer (e.g., formamide/dye mix, unit
- Sephadex G-25 column (Amersham Pharmacia Biotech)
- 1 M NaClO
4 - Anhydrous and 25% or 50% (v/v) acetonitrile
1.5 mL microcentrifuge tubes or screw-cap microcentrifuge tubes with O-ring seal
Platform shaker
55°C heating block or water bath (optional)
Vacuum evaporator (e.g., Savant Speedvac) with low and high vacuum sources
UV spectrophotometer and cuvette
0.75- and 1.5-mm-thick gel plates
TLC Merck silica plate (Kieselgel 60 F-254)
Hand-held 254-nm UV lamp
Camera with UV filter (optional)
0.45 µm hydrophilic fluid filter (Creative Medical)
0.22 µm membrane filter (Millipore; optional)
Anion-exchange high-performance liquid chromatograph (HPLC) with:
Gradient maker
0.5 mm × 7.5 cm Protein Pak DEAE 5PW column (Waters)
Column heater
Sep-Pak C18 cartridges (Waters Chromatography)
10 mL syringe
Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (appendix 3B and unit 10.4 )
Looking for materials? Suppliers Guide
Figures
-
Figure 4.15.1Synthesis of the four araF-protected nucleoside phosphoramidites (S.2a-d). MMTr, 4-monomethoxytrityl; i-Pr2 NEt, N-ethyl-N,N-diisopropylamine; i-Pr2 -NP(Cl)OCH2 CH2 CN, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. -
Figure 4.15.2Ion-exchange HPLC analysis of crude S-oligonucleotide ATA TCC TTg TCg TAT CCC (cap letters represent araF nucleotides, DNA residues in lowercase). (A) Sulfurization with Beaucage reagent. The small peak at ~58 min represents S-oligonucleotides with one P-O insertion. The main peak at ~62 min represents full S-FANA. (B) Sulfurization with ADTT. System: Waters 600E Multisolvent Delivery System with Waters 486 Tunable Absorbance detector and oven, driven by Millennium (V 3.20) software; column: Waters 0.5-mm × 7.5-cm Protein Pak DEAE 5PW, 50°C; solvent A: H2 O; solvent B: 1 M NaClO4 ; flow rate: 1 mL/min; detection: 260 nm; gradient conditions: 0% B for 9 min, 0% to 15% B in 3 min, 15% to 50% B in 60 min, 50% to 80% B in 2 min, hold 80% B 10 min, 80% to 0% B in 2 min, hold 0% B 10 min. -
Figure 4.15.3Ion-exchange HPLC analysis of (A) crude and (B) purified S-oligonucleotide CTC TAg cgt ctT AAA (cap letters represent araF nucleotides, lowercase letters represent DNA residues). System: Waters Binary HPLC Pump with Waters 2487 Dual Absorbance detector, equipped with in-line degasser and oven, driven by Breeze (V 3.20) software; column: Waters 0.5-mm × 7.5-cm Protein Pak DEAE 5PW, 50°C; solvent A: H2 O; solvent B: 1 M NaClO4 ; flow rate: 1 mL/min; detection: 260 nm; gradient conditions: 0% B for 9 min, 0% to 15% B in 3 min, 15% to 50% B in 45 min, 50% to 80% B in 2 min, hold 80% B 10 min, 80% to 0% B in 2 min, hold 0% B 10 min. Peaks at <20 min correspond to failure sequences and loss of protecting groups (e.g., benzoyl from A or C). -
Figure 4.15.4Ion-exchange HPLC analysis of (A) crude and (B) purified phosphodiester oligonucleotide ATg TCC TTg TCg gTg Agg TTA GG (cap letters represent araF nucleotides, lowercase for DNA residues). System: Waters Binary HPLC Pump with Waters 2487 Dual Absorbance detector, equipped with in-line degasser and oven, driven by Breeze (V 3.20) software; column: Waters 0.5-mm × 7.5-cm Protein Pak DEAE 5PW, 50°C; solvent A: H2 O; solvent B: 1 M NaClO4 ; flow rate: 1 mL/min; detection: 260 nm; gradient conditions: hold 0% B 2 min, From 0% to 30% B in 40 min, From 30% to 45% B in 2 min, hold 45% B 10 min, 45% to 0% B in 2 min, hold 0% B 10 min. -
Figure 4.15.5(A) DNA and 2¢F-ANA primary structures and backbone chemistries. (B) Illustration of linker-modified DNA or 2¢F-ANA antisense constructs. Panal B reprinted from Mangos and Damha (
Literature Cited
| Literature Cited | |
| Altmann, K.-H., Kesselring, R., Francotte, E., and Rihs, G. 1994a. 4¢,6¢-Methano carbocyclic thymidine: A conformationally constrained building block for oligonucleotides. Tetrahedron Lett. 35:2331-2334. | |
| Altmann, K.-H., Imwinkelried, M., Kesselring, R., and Rihs, G. 1994b. 1¢,6¢-Methano carbocyclic thymidine: Synthesis, X-ray crystal structure, and effect on nucleic acid duplex stability. Tetrahedron Lett. 35:7625-7628. | |
| Berger, I., Tereshko, V., Ikeda, H., Marquez, V.E., and Egli, M. 1998. Crystal structures of B-DNA with incorporated 2¢-deoxy-2¢-fluoro-arabino-furanosyl thymines: Implications of conformational preorganization for duplex stability. Nucl. Acids Res. 26:2473-2480. | |
| Bergot, B.J. and Egan, W. 1992. Separation of synthetic phosphorothioate oligonucleotides from their oxygenated (phosphodiester) defect species by strong-anion-exchange high-performance liquid chromatography. J. Chromatogr. 599:35-42. | |
| Christensen, N.K., Petersen, M., Nielson, P., Jacobsen, J.P., Olsen, C.E., and Wengel, J. 1998. A novel class of oligonucleotide analogues containing 2¢-O,3¢-C-linked [3.2.0]bicycloarabinonucleoside monomers: Synthesis, thermal affinity studies and molecular modeling. J. Am. Chem. Soc. 120:5458-5463. | |
| Cook, P.D. 1998. Second generation antisense oligonucleotides: 2¢-modifications. Annu. Rep. Med. Chem. 33:313-325. | |
| Crouch, R.J. and Toulmé, J.J. (eds.) 1998. Ribonucleases H. INSERM, Paris. | |
| Damha, M.J., Meng, B., Yannopoulos, C.G., Wang, D., and Just, G. 1995. Structural basis for the RNA selectivity of oligonucleotides containing alkylsulfide internucleoside linkages and 2¢-O-substituted 3¢-deoxyribose. Nucl. Acids Res. 19:3967-3973. | |
| Damha, M.J., Wilds, C.J., Noronha, A., Brukner, I., Borkow, G., Arion, D., and Parniak, M.A. 1998. Hybrids of RNA and arabinonucleic acids (ANA and 2¢F-ANA) are substrates of ribonuclease H. J. Am. Chem. Soc. 120:12976-12977. | |
| Damha, M.J., Noronha, A.M., Wilds, C.J., Trempe, J.-F., Denisov, A., and Gehring, K. 2001. Properties of arabinonucleic acids (ANA & 2¢F-ANA): Implications for the design of antisense therapeutics that invoke RNase H cleavage of RNA. Nucleosides Nucleotides 20:429-440. | |
| Giannaris, P.A. and Damha, M.J. 1994. Hybridization properties of oligoarabinonucleotides. Can. J. Chem. 72:909-918. | |
| Iyer, R.P., Phillips, L.R., Egan, W., Regan, J.B., and Beaucage, S.L. 1990. The automated synthesis of sulfur-containing oligodeoxyribonucleotides using 3H-1,2-benzodithiol-3-one 1,1-dioxide as a sulfur-transfer reagent. J. Org. Chem. 55:4693-4699. | |
| Lebedeva, I. and Stein, C.A. 2001. Antisense oligonucleotides: Promise and reality. Annu. Rev. Pharmacol. Toxicol. 41:403-419. | |
| Lima, W.F. and Crooke, S.T. 1997. Binding affinity and specificity of Escherichia coli RNase H1: Impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids. Biochemistry 36:390-398. | |
| Lok, C.-N., Viazovkina, E., Min, K-L., Nagy, E., Wilds, C.J., Damha, M.J., and Parniak, M.A. 2002. Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2¢-deoxy-2¢-fluoro-d-arabinose and 2¢-deoxyribose nucleotides. Biochemistry 41:3457-3467. | |
| Mangos, M.M. and Damha, M.J. 2002. Flexible and frozen sugar-modified nucleic acids: modulation of biological activity through furanose ring dynamics in the antisense strands. Curr. Topics Med. Chem. 2:1145-1169. | |
| Manoharan, M. 1999. 2¢-Carbohydrate modifications in antisense oligonucleotide therapy: Importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1489:117-130. | |
| Minasov, G., Teplova, M., Nielsen, P., Wengel, J., and Egli, M. 2000. Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA. Biochemistry 39:3525-3532. | |
| Myers, N.M. and Dean, K.J. 2000. Sensible use of antisense: How to use oligonucleotides as research tools. TIPS 21:19-23. | |
| Nakamura, H., Oda, Y., Iwai, S., Inoue, H., Ohtsuka, E., Kanaya, S., Kimura, S., Katsuda, C., Katayanagi, K., Morikawa, K., Miyashiro, H., and Ikehara, M. 1991. How does RNase H recognize a DNA:RNA hybrid Proc. Natl. Acad. Sci. U.S.A. 88:11535-11539. | |
| Noronha, A. and Damha, M.J. 1998. Triple helices containing arabinonucleotides in the third (Hoogsteen) strand: Effects of inverted stereochemistry at the 2¢-position of the sugar moiety. Nucl. Acids Res. 26:2665-2671. | |
| Noronha, A.M., Wilds, C.J., Lok, C.-N., Viazovkina, K., Arion, D., Parniak, M.A., and Damha, M.J. 2000. Synthesis and biophysical properties of arabinonucleic acids (ANA): Circular dichroic spectra, melting temperatures and ribonuclease H susceptibility of ANA:RNA hybrid duplexes. Biochemistry 39:7050-7062. | |
| Oda, Y., Iwai, S., Ohtsuka, E., Ishikawa, M., Ikehara, M., and Nakamura, H. 1993. Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy. Nucl. Acids Res. 21:4690-4695. | |
| Plavec, J., Thibaudeau, C., and Chattopadhyaya, J. 1994. How does the 2¢-hydroxy group drive the pseudorotational equilibrium in nucleoside and nucleotide by the tuning of the 3¢-gauche effect J. Am. Chem. Soc. 116:6558-6560. | |
| Sangvhi, Y.S. 1998. Synthesis of nitrogen containing linkers for antisense oligonucleotides. In Carbohydrate Mimics (Y. Chapleur, ed.) pp. 523-536. Wiley-VCH, Germany. | |
| Schmit, C., Bèvierre, M-O., De Mesmaeker, A., and Altmann, K.-H. 1994. The effects of 2¢- and 3¢-alkyl substituents on oligonucleotide hybridization and stability. Bioorg. Med. Chem. Lett. 4:1969-1974. | |
| Shen, L.X., Kandimalla, E.R., and Agrawal, S. 1998. Impact of mixed-backbone oligonucleotides on target binding affinity and target cleaving specificity and selectivity by E. coli RNase H. Bioorg. Med. Chem. 6:1695-1705. | |
| Sørensen, M.D., Kvaernø, L., Bryld, T., Håkansson, A.E., Verbeure, B., Gaubert, G., Herdewijn, P., and Wengel, J. 2002. Alpha-l-ribo-configured locked nucleic acid (alpha-l-LNA): Synthesis and properties. J. Am. Chem. Soc. 124:2164-2176. | |
| Still, W.C., Kahn, M., and Mitra, A. 1978. Rapid chromatographic technique for preparative separation with moderate resolution. J. Org. Chem. 43:2923-2925. | |
| Tang, J.-Y., Han, Y., Tang, J.X., and Zhang, Z. 2000. Large scale synthesis of oligonucleotide phosphorothioates using amino-1,2,4-dithiazoline-5-thione as an efficient sulfur-transfer reagent. Org. Proc. Dev. 4:194-198. | |
| Thibaudeau, C. and Chattopadhyaya, J. 1997. The discovery of intramolecular stereoelectronic forces that drive the sugar conformation in nucleosides and nucleotides. Nucleosides Nucleotides 16:523-529. | |
| Thibaudeau, C., Plavec, J., Garg, N., Papchikhin, A., and Chattopadhyaya, J. 1994. How does the electronegativity of the substituent dictate the strength of the gauche effect J. Am. Chem. Soc. 116:4038-4043. | |
| Trempe, J.F., Wilds, C.J., Denisov, A.Y., Pon, R.T., Damha, M.J., and Gehring, K. 2001. NMR solution structure of an oligonucleotide hairpin with a 2¢F-ANA/RNA stem: Implications for RNase H specificity toward DNA/RNA hybrid duplexes. J. Am. Chem. Soc. 123:4896-4903. | |
| Uhlmann, E. and Peyman, A. 1990. Antisense oligonucleotides: A new therapeutic principle. Chem. Rev. 90:543-584. | |
| Venkateswarlu, D. and Ferguson, D.M. 1999. Effects of C2¢-substitution on arabinonucleic acid structure and conformation. J. Am. Chem. Soc. 121:5609-5610. | |
| Walder, R.Y. and Walder, J.A. 1988. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 85:5011-5015. | |
| Wang, J., Verbeure, B., Luyten, I., Luyten, I., Lescrinier, E., Froeyen, M., Hendrix, C., Rosemeyer, H., Seela, F., Aerschot, A.V., and Herdewijn, P. 2000. Cyclohexene nucleic acids (CeNA): Serum stable oligonucleotides that activate RNase H and increase duplex stability with complementary RNA. J. Am. Chem. Soc. 122:8595-8602. | |
| Wengel, J., Koshkin, A., Singh, S.K., Nielsen, P., Meldgaard, M., Rajwanshi, V.K., Kumar, R., Skouv, J., Nielsen, C.B., Jacobsen, J.P., Jacobsen, N., and Olsen, C.E. 1999. LNA (Locked nucleic acid). Nucleosides Nucleotides 18:1365-1370. | |
| Wilds, C.J. and Damha, M.J. 1999. Duplex recognition by oligonucleotides containing 2¢-Deoxy-2¢-fluoro-d-arabinose and 2¢-deoxy-2¢-fluoro-D-ribose. Intermolecular contacts versus sugar puckering in the stabilization of triple helical complexes. Bioconjug. Chem. 10:299-305. | |
| Wilds, C.J. and Damha, M.J. 2000. 2¢-deoxy-2¢-fluoro--D-arabinonucleosides and oligonucleotides (2¢F-ANA): Synthesis and physicochemical studies. Nucl. Acids Res. 28:3625-3635. | |
| Xu, Q., Musier-Forsyth, K., Hammer, R.P., and Barany, G. 1996. Use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) for synthesis of phosphorothioate-containing oligodeoxyribonucleotides. Nucl. Acids Res. 24:1602-1607. | |
| Key References | |
| Crouch and Toulmé, 1998. See | |
| A comprehensive book on ribonucleases H, their sources, properties, biological utility, and antisense applications. | |
| Damha et al., 2001. See | |
| Highlights the role of the 2¢-sugar position in ANA and 2¢F-ANA conformations, and the origins of RNase H activity. | |
| Freier, S.M. and Altmann, K.-H. 1997. The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA:RNA duplexes. Nucl. Acids Res. 25:4429-4443. | |
| An extensive resource that relates duplex stabilities to nucleotide structure with 197 examples of oligonucleotide modifications. | |
| Kvaernø, L. and Wengel, J. 2001. Antisense molecules and furanose conformations—is it really that simple Chem. Commun. 1419-1424. | |
| A concise review briefly comparing RNA binding versus cleavage with promising antisense candidates. | |
| Lebedeva and Stein, 2001. See | |
| Insightful discussion of antisense design that focuses on in vivo toxicity from nonspecific interactions and potential irrelevant cleavage of nontargeted RNA. | |
Join the Conversation
Post new comment