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Synthesis of Glycerol Nucleic Acid (GNA) UNIT 4.40 Phosphoramidite Monomers and Oligonucleotide Polymers Su Zhang1,2 and John C. Chaput1,2 1Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 2The Biodesign Institute at Arizona State University, Tempe, Arizona ABSTRACT This unit describes a straightforward method for preparing glycerol nucleic acid (GNA) phosphoramidite monomers and oligonucleotide polymers using standard cyanoethyl phosphoramidite chemistry. GNA is an unnatural nucleic acid analog composed of an acyclic three-carbon sugar-phosphate backbone that contains one stereogenic center per repeating unit. GNA has attracted significant attention as a nucleic acid derivative due to its unique ability to form stable Watson-Crick anti-parallel duplex structures with thermal and thermodynamic stabilities rivaling those of natural DNA and RNA. The chemical simplicity of this nucleic acid structure provides access to enantiomerically pure forms of right- and left-handed helical structures that can be used as unnatural building blocks in DNA nanotechnology. Curr. Protoc. Nucleic Acid Chem. 42:4.40.1-4.40.18. C 2010 by John Wiley & Sons, Inc. Keywords: glycerol nucleic acid (GNA) r phosphoramidite r oligonucleotide r chemical synthesis r solid-phase synthesis r thermal stability r nanotechnology INTRODUCTION Acyclic oligonucleotides are experiencing a tremendous resurgence in basic and applied research due to their unique structural and biophysical properties (for a review, see Zhang et al., 2010). This unit contains procedures that describe the chemical synthesis of one type of acyclic nucleic acid polymer commonly referred to as glycerol nucleic acid or GNA. The chemical synthesis and purification of glycerol nucleoside analogs bearing adenine (A), cytosine (C), guanine (G), and thymine (T) as the bases, and of oligonucleotides thereof (Fig. 4.40.1), are described in detail. Unless otherwise stated, all of the procedures start from (R)-(+)-glycidol and yield (S)-GNA. The same chemistry can also be applied to (S)-(–)-glycidol to produce (R)-GNA. Commercially available glycidol or tritylated glycidol is used to obtain the 2,3-dihydroxypropyl derivatives of each nucleobase via an epoxide ring opening reaction (see Basic Protocol 1). The glycerol nucleosides are then converted to their corresponding nucleotide phosphoramidites (see Basic Protocols 2 to 5), which can then be used as building blocks to synthesize GNA oligonucleotides (see Basic Protocol 6). NOTE: For phosphoramidite synthesis (see Basic Protocols 2 to 5), the reaction progress is monitored by TLC and should be stopped as soon as the starting material is consumed. SYNTHESIS OF ENANTIOMERICALLY PURE DIMETHOXYTRITYL-O-(S)- BASIC GLYCIDOL PROTOCOL 1 2,3-Dihydroxypropyl derivatives of nucleobases have been synthesized by several differ- ent methods. The current approach is based on a modified version of Acevedo’s procedure Synthesis of (Acevedo and Andrews, 1996), and involves a direct ring opening of the stable glyci- Modified dol intermediate by nucleophilic attack of one of the four natural nucleobases. In this Oligonucleotides and Conjugates Current Protocols in Nucleic Acid Chemistry 4.40.1-4.40.18, September 2010 4.40.1 Published online September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142700.nc0440s42 Supplement 42 Copyright C 2010 John Wiley & Sons, Inc. B OOP B OOP B OOOP O O O H H O OH(H) O O B OOP O OOP B OOP B O O O O (H)HO H O O H RNA (DNA) (S)-GNA (R)-GNA Figure 4.40.1 Chemical structures of GNA, DNA, and RNA. HO DMTO DMT-Cl, Et3N O DCM O 1 2 (90%) Figure 4.40.2 Preparation of (S)-DMT-O-glycidol (S.2). Abbreviations: DMT-Cl,4,4-dimethoxytri- tylchloride; Et3N, triethylamine; DCM, dichloromethane. protocol, pure (R)-(+)-glycidol is tritylated using 4,4-dimethoxytritylchloride (DMT-Cl) in dichloromethane as illustrated in Figure 4.40.2. The tritylated glycidol is used to make the A, C, and T glycerol nucleoside phosphoramidites (see Basic Protocols 2, 3, and 5, respectively). Synthesis of the G glycerol nucleoside phosphoramidite requires an alternative strategy (see Basic Protocol 4). (R)-(+)-Glycidol substrates provide (S)-GNA phosphoramidite monomers that can be used to make GNA oligonucleotides with the natural right-handed stereoconfiguration. Materials (R)-(+)-Glycidol Dichloromethane (DCM) Triethylamine (Et3N), 99.5% 4,4-Dimethoxytritylchloride (DMT-Cl), 95% Argon source Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO3) Brine (sat. aq. NaCl) Sodium sulfate (Na2SO4) ◦ Silica gel (60 A, 230 to 400 mesh; Whatman) Hexanes Ethyl acetate (AcOEt) 100-mL round-bottomed flasks Magnetic stir plate and stir bar Synthesis of GNA Buchner¨ funnels Phosphoramidite Monomers and 200-mL separatory funnels Oligonucleotide Filter paper Polymers Gas balloon 4.40.2 Supplement 42 Current Protocols in Nucleic Acid Chemistry Rotary evaporator equipped with a vacuum pump 2.5 × 25–cm chromatography column Thin layer chromatography (TLC) plate, EMD silica gel 60 F254 254-nm UV lamp Additional reagents and equipment for thin layer chromatography (TLC; APPENDIX 3D) and column chromatography (APPENDIX 3E) 1. To a 100-mL round-bottomed flask equipped with a magnetic stir bar, add: 780 mg (10.6 mmol) of (R)-(+)-glycidol 24 mL of DCM (freshly distilled over calcium hydride) 3.8 mL of Et3N 4.54 g (13.4 mmol) of DMT-Cl. 2. Stir the reaction overnight at room temperature under an argon atmosphere. 3. Remove the precipitate by vacuum filtration and wash the residue with DCM. 4. Wash the filtrate sequentially with 50 mL each of sat. aq. NaHCO3, water, and brine. 5. Dry the organic layer over Na2SO4 10 min and filter. 6. Evaporate to dryness using a rotary evaporator equipped with a vacuum pump and cooling trap. 7. Purify the oily residue by column chromatography (APPENDIX 3E) on 20 g of silica gel in a 5 × 25–cm column. Deactivate the column with 97:3 (v/v) hexanes/Et3Nand elute the column using a step-wise gradient of 99:1 (v/v) hexanes/Et3N to 18:1:1 (v/v/v) hexanes/AcOEt/Et3N. 8. Determine products by TLC. Combine the product fractions and evaporate to dryness. The resulting product, DMT-O-(S)-glycidol (S.2), should be obtained in a 90% yield 1 (3.56 g, 9.47 mmol) as colorless oil. TLC (hexanes/AcOEt 10:1): Rf = 0.12. HNMR (300 MHz, CDCl3): δ=2.61 (dd, J=2.4, 5.1 Hz, 1 H), 2.76 (m, 1 H), 3.12 (m, 2 H), 3.29 (m, 1 H), 3.79 (s, 6 H), 6.83 (m, 4 H), 7.05-7.40 (m, 7 H), 7.45 (m, 2 H). SYNTHESIS OF 2 -O-(2-CYANOETHOXY)(DIISOPROPYLAMINO)- BASIC PHOSPHINO-3 -O-(4,4 -DIMETHOXYTRIPHENYL)METHYL-N6-BENZOYL- PROTOCOL 2 (S)-9-(2,3-DIHYDROXYPROPYL)ADENINE This protocol describes the synthesis of N6-benzoyl-protected (S)-GNA adenosine nucleoside phosphoramidite S.5 from S.2 (Fig. 4.40.3). Materials Adenine 60% sodium hydride in mineral oil (NaH) Dimethylformamide (DMF), anhydrous Argon source Benzoyl chloride (BzCl) DMT-O-(S)-glycidol (S.2; Basic Protocol 1) Ethyl acetate (AcOEt) ◦ Silica gel (60 A, 230 to 400 mesh) Dichloromethane (DCM) Triethylamine (Et N) 3 Synthesis of Methanol (MeOH) Modified Pyridine, anhydrous Oligonucleotides and Conjugates 4.40.3 Current Protocols in Nucleic Acid Chemistry Supplement 42 NH2 N NH2 N N TMS-Cl N N N 2, NaH DMTO BzCl N DMF Py N H HO 3 (48%) NHBz Cl N NHBz N CN N P N (i-Pr) N O N N 2 DMTO N N DIPEA DMTO O DCM HO P CN (i-Pr)2N O 4 (78%) 5 (82%) Figure 4.40.3 Preparation of S.5. Abbreviations: NaH, sodium hydride; DMF, dimethylformamide; TMS-Cl, trimethylsilyl chloride; BzCl, benzoyl chloride; Py, pyridine; DIPEA, diisopropylethylamine; DCM, dichloromethane. Trimethylsilyl chloride (TMS-Cl) Ammonium hydroxide (concentrated NH4OH) Hexanes Diisopropylethylamine, redistilled (DIPEA) Chloro(2-cyanoethoxy)-(diisopropylamino)phosphine 50- and 100-mL round-bottomed flasks Magnetic stir plate and stir bar Graham condenser Buchner¨ funnel Filter paper Rotary evaporator equipped with a vacuum pump 6.4 × 45–cm and 1.3 × 30–cm chromatography columns TLC plate, EMD silica gel 60 F254 254-nm UV lamp Additional reagents and equipment for TLC (APPENDIX 3D)andcolumn chromatography (APPENDIX 3E) Prepare S.3 1. Add 0.71 g (5.25 mmol) of adenine and 43 mg (1.07 mmol) of 60% NaH in mineral oil in 8 mL of DMF to a 50-mL round-bottomed flask. 2. Stir reaction 2 hr at room temperature under an argon atmosphere. 3. Dissolve 1.86 g (4.95 mmol) of S.2 in 13.5 mL of DMF in a 50-mL round-bottomed flask. Synthesis of GNA Phosphoramidite 4. Add the solution from step 3 into the 50-mL round-bottomed flask from step 1, attach Monomers and a condenser to the flask, and stir the reaction mixture 15 hr at 105◦C under an argon Oligonucleotide Polymers atmosphere. 4.40.4 Supplement 42 Current Protocols in Nucleic Acid Chemistry 5. Allow the solution to cool to room temperature and remove the precipitate by vacuum filtration. Wash the residue with 50 mL AcOEt. 6. Evaporate the organic solvent from step 5 to dryness using a rotary evaporator equipped with a vacuum pump and a cooling trap. 7. Purify the residue by column chromatography (APPENDIX 3E) on 50 g of silica gel in a 6.4 × 45–cm column. Deactivate the column with 97:3 DCM/Et3N and elute the product using a step-wise gradient of 35:1:0.01 to 25:1:0.01 (v/v) DCM/MeOH/ Et3N. 8. Collect fractions containing the product, determined by TLC, and evaporate to dryness. The resulting product, 3-O-(4,4-dimethoxytriphenyl)methyl-(S)-9-(2,3-dihydroxypropyl) adenine (S.3), should be obtained in a 48% yield (1.21 g, 2.38 mmol) as a colorless foam.
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