current protocols in nucleic acid chemistry || synthesis of triazole-nucleoside phosphoramidites and...

UNIT 4.57Synthesis of Triazole-NucleosidePhosphoramidites and Their Use inSolid-Phase Oligonucleotide Synthesis

Brandon J. Peel,1 Tim C. Efthymiou,1 and Jean-Paul Desaulniers1

1Faculty of Science, University of Ontario Institute of Technology, Oshawa, Ontario, Canada

ABSTRACT

Triazole-backbone oligonucleotides are macromolecules that have one or more triazoleunits that are acting as a backbone mimic. Triazoles within the backbone have been usedwithin oligonucleotides for a variety of applications. This unit describes the preparationand synthesis of two triazole-nucleoside phosphoramidites [uracil-triazole-uracil (UtU)and cytosine-triazole-uracil (CtU)] based on a PNA-like scaffold, and their incorporationwithin oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 55:4.57.1-4.57.38. C© 2013by John Wiley & Sons, Inc.

Keywords: triazole-linkage � cycloaddition � phosphoramidite � solid-phaseoligonucleotide synthesis

INTRODUCTION

Triazoles are versatile functional groups that have unique properties and have been usedas a backbone mimic for a variety of biomolecular molecules (Kolb and Sharpless, 2003;Efthymiou et al., 2012a). In addition, they have been utilized as a bioorthogonal functionalgroup to install a variety of labels on to a desired molecule (Gramlich et al., 2008; Doddet al., 2010; Ingale and Seela, 2013). A variety of different protocols have been publishedthat utilize different approaches for synthesizing different triazole molecules (Huisgenet al., 1967; Rostovtsev et al., 2002; Boren et al., 2008; Meldal and Tornoe, 2008). Severalof the methods reported today involve the use of the copper(I)-catalyzed cycloadditionbetween an azide and a terminal alkyne.

This unit describes the synthesis of both a dimethoxytrityl (DMT)–protected uracil-triazole-uracil (UtU) phosphoramidite and a DMT-protected cytosine-triazole-uracil(CtU) phosphoramidite. The general strategies employed to synthesize these phospho-ramidites involve the preparation of an azide monomer and an alkyne monomer. Withthese two monomers, copper(I)-assisted cycloaddition affords the desired 1,4-triazoledimer. For these units to be incorporated within oligonucleotides, DMT-phosphoramiditechemistry is used (Caruthers, 1985). Methodological differences described in this unitare small between the synthesis of the UtU and CtU phosphoramidite.

Finally, the incorporation of the triazole-backbone derivatives within oligonucleotides isdescribed, using solid-phase controlled pore glass (CPG) supports. The triazole-modifiedoligonucleotide can be purified by conventional methods such as denaturing PAGE(UNIT 10.4) or HPLC (UNIT 10.5).

BASICPROTOCOL 1

PREPARATION OF URACIL AND CYTOSINE PYRIMIDINE BASES

In order to utilize either uracil or cytosine as the base for the triazole-phosphoramidite,the respective N1-substituted acetic acids of the bases are synthesized. These precursorsare synthesized based on methods from Leng and coworkers (Liu et al., 2000) and Nielsen

Current Protocols in Nucleic Acid Chemistry 4.57.1-4.57.38, December 2013Published online December 2013 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142700.nc0457s55Copyright C© 2013 John Wiley & Sons, Inc.

Synthesis ofModifiedOligonucleotidesand Conjugates

4.57.1

Supplement 55

Figure 4.57.1 General scheme for the conversion of uracil into uracil-1-yl acetic acid (2) andcytosine into (N4-(benzoyl)cytosine-1-yl)acetic acid (5).

and coworkers (Christensen et al., 1998; Schwergold et al., 2002). For the synthesis ofuracil-1-yl acetic acid (2), uracil reacts with sodium hydroxide and bromoacetic acid inwater at a temperature of 45°C. Upon completion, the pH is lowered to precipitate theproduct, and the product is isolated as a pure recrystallized white powder. For the synthesisof the cytosine acid monomer, NaH reacts with cytosine, which is then alkylated withmethyl bromoacetate. Protection of the exocyclic amine (-NH2) with benzoyl chloride,followed by saponification of the ester, affords the desired protected monomer (5) as arecrystallized product. See Figure 4.57.1.

Materials

Sodium hydroxide (NaOH), �98%Uracil (1), �99% pureBromoacetic acid, 97% pureMethanol (MeOH), ACS gradeDichloromethane (CH2Cl2), ACS gradeConcentrated hydrochloric acid (HCl), ACS gradeCytosine (3), �99% pureDimethylformamide (DMF), anhydrous, 99.8% pureNitrogen (or argon) gasSodium hydride (NaH), 60% dispersion in mineral oilHexane, ACS gradeMethyl bromoacetate, 97% purePyridine, anhydrous, �99% pureBenzoyl chloride, 99% pure

Analytical balanceWeighing paper50- and 100-mL and 1-L round-bottom flask

Synthesis and Useof Triazole-Nucleoside

Phosphoramidites

4.57.2

Supplement 55 Current Protocols in Nucleic Acid Chemistry

Stir barHot plate magnetic stirrer45°C water bathSeptaDisposable syringes and needlesTLC plates (250-μm thick; Silicycle; cat. no. TLG-R10011B-2020,

http://www.silicycle.com/)Short-wave UV lamppH meterVacuum pumpFilter paper, grade P5Buchner funnels150-, 250-, and 500-mL Buchner flasksOvenRotary evaporator

Additional reagents and equipment for thin-layer chromatography (TLC,APPENDIX 3D)

N1-Alkylation of uracil (1)

1a. Weigh 4.60 g (115 mmol) of NaOH pellets and add to a 100-mL round-bottomflask. Dissolve the pellets in 20 mL of water with magnetic stirring.

2a. Add 3.36 g (30 mmol) of 1 to the alkaline solution. Warm the mixture to 45°C ina water bath. When all of the contents dissolve, seal the flask with a septum andcontinue incubating until needed.

3a. In a separate 50-mL round-bottom flask, add 6.25 g (45 mmol) of bromoaceticacid to 10 mL of water. Stopper the flask with a septum and stir vigorously untilbromoacetic acid is dissolved.

CAUTION: Bromoacetic acid may be fatal if absorbed through the skin. Work in thefume hood and wear acid resistant protective gloves.

4a. Using a syringe and needle, remove the solution from step 3a through the septumand add it dropwise to the solution in step 2a over the course of 30 min.

5a. Monitor the reaction by TLC (APPENDIX 3D) using 10% MeOH in CH2Cl2 as amobile phase. View the TLC plate under a short-wave UV lamp (APPENDIX 3D)

The starting material 1 (Rf = 0.24) appears as a streak and disappears after 4 hr. Theproduct spot will reside on the baseline.

6a. Remove the flask from the warm water bath and allow it to cool to room temperature.

Before continuing, calibrate a pH meter with at least three standard buffer solutions(pH = 4, pH = 7 and pH = 10).

7a. Adjust the pH of the reaction mixture through the dropwise addition of concentratedHCl until the pH = 5.5. Monitor the change in pH with a calibrated pH meter.

8a. Cool the solution in a freezer (–20°C) for 2 hr.

9a. Remove any precipitate (unreacted uracil and undesirable salts) by vacuum filtra-tion through filter paper (grade P5) and collect the filtrate in a 150-mL Buchnerflask. Rinse the inside of the round-bottom flask with chilled water to collect anyremaining residue.

10a. Further acidify the filtrate with concentrated HCl until pH is equal to 2 and placethe collection flask in a freezer (–20°C) for another 2 hr.


4.57.3

Current Protocols in Nucleic Acid Chemistry Supplement 55

11a. Collect the precipitate (2) by vacuum filtration through filter paper (grade P5) anddry the white powder in an oven (40°C) overnight.

Compound 2 is stable for at least 12 months during storage at ambient temperature.

12a. Characterize the compound by 1H/13C NMR and ESI-MS.

2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (2). Yield 4.6 g (91%). 1HNMR (400 MHz, DMSO-d6): δ 4.40 (s, 2H) 5.57 (d, 1H) 7.59 (d, 1H,) 11.33 (s, 1H);13C (100 MHz, DMSO-d6): 49.1, 101.3, 146.5, 151.4, 164.3, 170.0; ESI-MS (ES-) m/zC6H6N2O4: 168.8 [M-H]+.

N1-Alkylation, saponification, and N4-protection of cytosine (3)

1b. Add 10 g (90 mmol) of cytosine (3) to a 1-L round-bottom flask containing400 mL of anhydrous DMF. Apply a dry nitrogen (or argon) atmosphere anddissolve the residue with magnetic stirring at ambient temperature.

Before proceeding, place 2.2 g of NaH (60% in mineral oil) in a 100-mL round-bottomflask equipped with a stir bar. Seal the flask with a septum and run a slow flow ofnitrogen (or argon) through the flask. With a syringe, add some hexanes and let themixture stir until the oil begins to dissolve. Once dissolved, stop stirring and removethe hexanes with a syringe without pulling up any NaH (precipitate at the bottom of theflask). Repeat the washing procedure three more times to remove all of the mineral oil.Dry off any residual hexanes with a stream of N2.

2b. Place the solution from step 1b in an ice-water bath and add 2.2 g (90 mmol) ofthe NaH, prepared as in step 1b, over the course of 10 min.

CAUTION: NaH vigorously reacts with water to produce an exothermic reaction. KeepNaH as dry as possible and under inert atmosphere.

3b. Continue stirring for 2 hr at room temperature, then, with a syringe, add 15.1 g(99 mmol) of methyl bromoacetate dropwise over a period of 15 min to the reactionmixture.

CAUTION: Methyl bromoacetate is an irritant to the eyes and skin. Perform the drop-wise addition in a fume hood and wear reagent-impermeable protective gloves.

4b. Allow the reaction to stir for 48 hr at room temperature.

5b. Dry the crude residue by evaporating with 400 mL of anhydrous DMF on a rotaryevaporator.

6b. Triturate the crude sludge with 200 mL of 4°C water. Store the flask in a refrigerator(4°C) to maximize precipitate formation.

7b. Collect the precipitate in a 500-mL Buchner flask using vacuum filtration throughfilter paper (grade P5), and wash the round-bottom flask with minimal amounts of4°C water to recover any precipitate trapped within the flask.

8b. Dissolve the precipitate in 40°C MeOH/water (1:1), let the solution cool to roomtemperature, and let it further cool in a refrigerator (4°C).

9b. Once cooled, collect the light pink powder (4) using vacuum filtration through filterpaper (grade P5).


10b. Characterize the compound by 1H/13C NMR and ESI-MS.

Methyl 2-(4-amino-2-oxopyrimidin-1(2H)-yl)acetate (4). Yield 9.9 g (60%). 1H NMR(200 MHz, DMSO-d6): δ 3.70 (s, 3H), 4.60 (s, 3H), 6.00 (d, 1H), 7.90 (d, 1H), 7.75 (bs,


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4.57.4


1H), 8.40 (bs, 1H); 13C NMR (200 MHz, DMSO-d6): δ 49.5, 52.0, 93.5, 147.0, 154.0,164.5, 168.5; ESI-MS (ES+) m/z C7H9N3O3: 184.2 [M+H]+.

11b. Place 5.0 g (27.3 mmol) of 4 into a 100-mL round-bottom flask equipped with a stirbar. Dissolve 4 in 50 mL of anhydrous pyridine under N2 with vigorous stirring.

12b. Add 4.6 g (32.8 mmol) of benzoyl chloride and stir the reaction overnight at roomtemperature.

CAUTION: Benzoyl chloride is a lachrymator. Handle this liquid in a fume hood.

13b. Evaporate the solution to dryness using a rotary evaporator and dissolve the cruderemains in 1 M NaOH with stirring for 3 hr.

Before continuing, calibrate a pH meter with at least three standard buffer solutions(pH = 4, pH = 7 and pH = 10).

14b. Using concentrated HCl, adjust the pH of the solution to 5.5. Monitor the changein pH with the calibrated pH meter.

15b. Cool the solution in a freezer (–20°C) for 2 hr.

16b. Remove any undesirable salts that form using vacuum filtration through filter paper(grade P5) and collect the filtrate in a 250-mL Buchner flask. Rinse the inside ofthe flask with chilled water to collect any product clinging to the walls of the flask.

17b. Further acidify the filtrate in the collecting flask to pH 2 and filter the solution asecond time as described in step 16b.

18b. Cool the solution in a freezer (–20°C) for 2 hr.

19b. Collect the light pink powder (5) by vacuum filtration through filter paper (gradeP5) and let it air dry overnight until a constant weight is obtained.


20b. Characterize the compound by 1H/13C NMR.

2-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)acetic acid (5). Yield 2.5 g (53%). 1H NMR(DMSO-d6): δ 4.59 (s, 2H), 7.31 (d, 1H), 7.5–8.2 (aromatics, 7H); 13C NMR (DMSO-d6): δ 50.7, 96.0, 128.5, 133.3, 150.8, 155.2, 163.6, 167.5, 169.4.

BASICPROTOCOL 2

PREPARATION OF ALKYNE AND AZIDE LINKERS

Before proceeding with the copper(I)-assisted cyclization, both the azide and alkynelinkers must be synthesized (see Fig. 4.57.2). The alkyne linker described herein is usedfor both UtU and CtU phosphoramidites. The synthesis of this linker involves usingethanolamine and reacting this with tert-butyldimethylsilyl chloride to form compound7. Compound 7 is an oil and reacts with limiting amounts of propargyl bromide to afford8 after column chromatography.

For the synthesis involving the UtU phosphoramidite, azide 11 is derivatized from 2-bromoethylamine. 2-Bromoethylamine reacts with sodium azide in water, and extractionfrom a basic solution affords 2-azidoethanamine (10; Mayer and Maier, 2007). Com-pound 10 is a liquid which reacts with limiting amounts of ethyl bromoacetate to affordcompound 11 following flash column chromatography.

For the synthesis of the CtU phosphoramidite, an alternate azide linker is employed.In this instance, bromoethanol is TBS-protected in the presence of imidazole to affordcompound 13 as a liquid. This liquid reacts with compound 10 for 2 days to affordazide linker 14 after purification. The uracil and cytosine azide monomers are sensitive


4.57.5


Figure 4.57.2 General scheme for the preparation of alkyne linker 8 and azide linkers 11 and 14.

to different future conditions, and this will be discussed during the reduction step inFigure 4.57.4.

Materials

Ethanolamine (6), 99% pureDichloromethane (CH2Cl2), ACS gradeImidazole, �99.5% pureTert-butyldimethylsilyl chloride (TBS-Cl), >95% pureMethanol (MeOH), ACS gradePotassium permanganate (KMnO4) stain (see recipe)Saturated aqueous solution of sodium bicarbonate (NaHCO3)Sodium sulfate (Na2SO4), ACS grade2-((tert-butyldimethylsilyl)oxy)ethanamine (7)Nitrogen (or argon) gasDistilled N,N-Diisopropylethylamine (DIPEA), 99% pure80% (w/v) 3-bromoprop-1-yne (Sigma-Aldrich, cat. no. p51001) in tolueneEthyl acetate (EtOAc), ACS gradeHexane, ACS gradeSilica gel: 40 to 63 µm (230 to 400 mesh)Sodium azide (NaN3), �99.5%Sodium hydroxide (NaOH), �98%2-bromoethylamine hydrobromide (9; Sigma-Aldrich, cat. no. 06670)Diethyl ether (Et2O), ACS gradeDimethylformamide (DMF), anhydrous, 99.8% pureNitrogen (or argon) gasDistilled triethylamine (TEA), �99% pureEthyl 2-bromoacetate, 98% (Sigma-Aldrich, cat. no. 133973)2-Bromoethanol (12; Sigma-Aldrich, cat. no. B65586)(2-bromoethoxy)(tert-butyl)dimethylsilane, 99% pure (13; Sigma-Aldrich, cat. no.

428426)Saturated solution of NaCl (brine)

50-, 100-, 250- and 500-mL round-bottom flasksStir barHot plate magnetic stirrer


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4.57.6


TLC plates (250-μm thick; Silicycle; cat. no. TLG-R10011B-2020,http://www.silicycle.com/)

Short-wave UV lamp250-, 500-, and 1-L separatory funnels100-, 250-, and, 500-mL Erlenmeyer flasksRotary evaporator7 × 25–cm glass chromatography columnReflux condenser

Additional reagents and equipment for thin-layer chromatography (TLC, APPENDIX

3D) and column chromatography (APPENDIX 3E)

Alkyne linker (8)

1a. Add 9.6 mL (159 mmol) of 6 to a 250-mL round-bottom flask containing a stir bar.Add 100 mL of CH2Cl2 and mix on a magnetic stirrer.

2a. Weigh and add 10.8 g (159 mmol) of imidazole to the mixture and stir the solutionin an ice-water bath until imidazole dissolves.

3a. Remove the flask from the ice-water bath and add 24 g (159 mmol) of TBS-Cl.Continue stirring overnight at room temperature.

4a. Check the progress of the reaction by TLC (APPENDIX 3D) using 10% MeOH inCH2Cl2 as a mobile phase. View the migration of the spots with a KMnO4 stain.

The starting material will be completely consumed and the new product, 7 (Rf = 0.43),will have a higher mobility due to the loss of an alcohol functional group.

5a. Pour the mixture into a 500-mL separatory funnel and perform three liquid-liquidextractions, each time using 35 mL of a saturated NaHCO3. Pool the organic layersin a 250-mL Erlenmeyer flask.

6a. Dry the organic fractions by adding Na2SO4 to absorb any water that may haveeluted into the organic fractions. Filter off the Na2SO4 and collect the organic layerin a 250-mL round-bottom flask.

7a. Concentrate in vacuo using a rotary evaporator to afford the product as a clear lightyellow oil (7).

Compound 7 is stable for at least 12 months during storage at room temperature.

8a. Characterize the compound by 1H/13C NMR.

2-((tert-Butyldimethylsilyl)oxy)ethanamine (7). Yield 26.8 g (96%). 1H NMR (400 MHz,CDCl3): δ 0.00 (s, 2H, mi.), 0.02 (s, 4H, ma.), 0.85 (s, 9H), 2.76 (t, J = 5.5 Hz, 2H),3.61 (t, J = 5.5 Hz, 2H), 4.02 (b, 2H); 13C (100 MHz, CDCl3): −5.3, 18.3, 25.9, 44.3,65.2.

9a. Add 10 g (57 mmol) of 7 to a 250-mL round-bottom flask containing a stir bar.Apply a dry nitrogen (or argon) atmosphere and dissolve 7 in 100 mL of anhydrousCH2Cl2 with magnetic stirring.

10a. Begin cooling the solution in an ice-water bath, and, with a syringe, add 3.7 g(28.5 mmol) of distilled DIPEA.

11a. Once cooled to 4°C, add 3.4 g (28.5 mmol) of 3-bromoprop-1-yne dropwise overthe course of 30 min.

CAUTION: 3-Bromoprop-1-yne is poisonous and should be handled in a well-ventilatedhood.

Slow addition is necessary to avoid dialkylation of the molecular species.


4.57.7


12a. Monitor the reaction by TLC (APPENDIX 3D) in 1:1 (v/v) EtOAc/hexane. Visualizethe TLC plate using KMnO4 stain.

The alkyne linker 8 (Rf = 0.64) is generated after �3 hr. The reaction is complete whena dialkylated by-product spot of higher Rf value begins to form.

13a. Pour the solution into a 500-mL separatory funnel and perform three liquid-liquidextractions, each time using 35 mL of saturated NaHCO3. Pool the organic layersin a 250-mL Erlenmeyer flask.

14a. Dry the organic fractions over Na2SO4, filter off the Na2SO4, and collect theorganic layer in a 250-mL round-bottom flask.

15a. Concentrate the crude oil by evaporating it with 100 mL of CH2Cl2 on a rotaryevaporator.

CAUTION: Concentrate the solution in a 30°C water bath to prevent further dialkyla-tion.

16a. Pack a 7 × 25–cm glass column with silica gel in 7:3 (v/v) hexane/EtOAc (APPENDIX

3E). Load the sample and purify by eluting with a step gradient of hexanes/EtOAC[7:3 (v/v), then 5:5 (v/v), and then 3:7 (v/v)].

The minor dialkylated side-product elutes first with 7:3 (v/v) hexanes/EtOAc.

17a. Evaporate the fractions containing the product 8 with a rotary evaporator to affordthe product as a clear yellow oil.

Compound 8 is stable for approximately 3 months while stored at −20°C.

18a. Characterize the compound by 1H/13C NMR and ESI-HRMS.

N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)prop-2-yn-1-amine (8). Yield 2.9 g (71%). 1HNMR (500 MHz, CDCl3): δ 0.07 (s, 6H), 0.90 (s, 9H), 1.67 (br s, 1H), 2.21 (s, 1H),2.80 (t, 2H, J = 5.5 Hz), 3.46 (s, 2H), 3.75 (t, 2H, J = 5.1 Hz); 13C NMR (125 MHz,CDCl3): δ −5.4, −5.2, 18.3, 25.8, 26.0, 38.2, 50.5, 62.3, 71.2, 71.2, 82.2; ESI-HRMS(ES+) m/z calcd for C11H23NOSi: 213.1549, found 214.1626 [M + H]+.

Uracil azide linker (11)

1b. Add 23.8 g (366 mmol) of NaN3 to a 500-mL round-bottom flask containing200 mL of water. Add a stir bar and mix until NaN3 is dissolved.

2b. Add 25 g (122 mmol) of 9 to the solution and reflux the reaction for 24 hr at 75°C.

CAUTION: NaN3 is shock sensitive, and may react explosively with metals. As aprecaution, it is recommended to use a plastic spatula when weighing out NaN3. It isalso recommended to fit a condenser to this reaction to avoid the evaporation of thewater over the course of 24 hr. In addition, NaN3 is a powerful poison and may befatal if it comes in contact with skin or is swallowed. Reagent-impermeable gloves areadvised when handling NaN3.

3b. Cool the solution in an ice-water bath and add 28.5 g (713 mmol) of NaOH to thereaction mixture. Stir until the NaOH is fully dissolved.

CAUTION: The addition of NaOH to water yields an exothermic reaction. If thetemperature of the reaction mixture is not reduced from 75°C to below 34.6°C beforethe extraction (step 5b) is performed, diethyl ether (Et2O; b.p. 34.6°C) will start boilingin the separatory funnel.

4b. Monitor by TLC (APPENDIX 3D) using 10% MeOH in CH2Cl2 as a mobile phase andvisualize the plate using a KMnO4 stain.

The reaction is complete when the starting material 9 (Rf = 0.33) is completely con-sumed.


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4.57.8


5b. Pour the mixture into a 1-L separatory funnel and perform three liquid-liquidextractions, each time by adding 70 mL of Et2O to the aqueous solution. Collectthe ether fractions in a 500-mL Erlenmeyer flask.

6b. Dry the organic fractions over Na2SO4, filter off the Na2SO4 and collect the organiclayer in a 500-mL round-bottom flask.

7b. Concentrate the organic layer in vacuo using a rotary evaporator to afford theproduct as a clear oil (10).

Compound 10 is stable for approximately 6 months while stored between 5° and 8°C.Avoid exposure to light.

8b. Characterize the compound by 1H/13C NMR and ESI-HRMS.

2-Azidoethanamine (10). Yield 6.7 g (64%). 1H NMR (400 MHz, CDCl3): δ 1.27 (s, 2H), 2.80–2.84 (m, 2 H), 3.30 (t, J = 5.7 Hz, 2 H,); 13C NMR (100 MHz, CDCl3): δ 41.2,54.6; ESI-HRMS (ES+) m/z calcd for C2H6N4: 87.0671, found: 87.0660 [M+H]+.

9b. In a 250-mL round-bottom flask, weigh and add 5 g (58.1 mmol) of 10 to100 mL of anhydrous DMF. Apply a dry nitrogen (or argon) atmosphere andmix the solution with magnetic stirring. When 10 dissolves, add 5.9 g (58.1 mmol)of distilled triethylamine.

10b. Over the course of 15 min, add 5.8 g (34.9 mmol) of ethyl 2-bromoacetate dropwiseto the solution and let it stir for an additional 4 hr at room temperature.

CAUTION: Ethyl 2-bromoacetate is a lachrymator and can be fatal if in contact withskin or inhaled. Wear reagent-impermeable gloves and work within the fume hood.

Adding ethyl 2-bromoacetate to a solution that is chilled in an ice bath may furtherprevent dialkylation.

11b. Monitor the progress of the reaction by TLC (APPENDIX 3D) using a mobile phase of1:1 (v/v) EtOAc/hexanes.

The azide linker 11 (Rf = 0.51) is produced after �4 hr. The reaction is complete whena dialkylated by-product spot of higher Rf value begins to form.

12b. Transfer the reaction mixture into a 500-mL separatory funnel and extract theorganic layer with 100 mL of EtOAc. Wash the organic layer three times, eachtime with 35 mL of water, and collect the EtOAc fraction in a 250-mL Erlenmeyerflask.

13b. Dry the EtOAc fraction over Na2SO4, filter off the Na2SO4 and collect the crudeproduct in a 250-mL round-bottom flask.

14b. Evaporate the solution under reduced pressure with a rotary evaporator to affordthe crude product as an orange oil.

15b. Pack a 7 × 25–cm glass column with silica gel in 5:5 (v/v) hexanes/EtOAc(APPENDIX 3E). Load the oil directly onto a silica column for purification and elutethe product with a step gradient of hexanes/EtOAc [5:5 (v/v), then 3:7 (v/v), andthen 100% EtOAc].

The minor dialkylated side product elutes first.

16b. Collect all fractions containing 11 and concentrate on the rotary evaporator toafford a clear light yellow oil.



4.57.9



Ethyl 2-(2-azidoethylamino)acetate (11). Yield 5.0 g (83%). 1H NMR (400 MHz,CDCl3): δ 1.25 (t, 3H, J = 7.1 Hz), 1.98 (br s, 1H), 2.80 (t, 2H, J = 5.7 Hz), 3.39(t, 2H, J = 5.9 Hz), 3.40 (s, 2H), 4.17 (q, 2H, J = 7.4 Hz); 13C NMR (100 MHz,CDCl3): δ 14.09, 48.01, 50.50, 51.33, 60.74, 172.07; ESI-HRMS (ES+) m/z calcd forC6H12N4O2: 172.0960, found 173.1034 [M + H]+.

Cytosine azide linker (14)

1c. In a 50-mL round-bottom flask containing a stir bar, dissolve 11.6 g (77 mmol) ofTBS-Cl in 25 mL of anhydrous DMF. Apply a dry nitrogen (or argon) atmosphere.

2c. Stir and add 6.2 g (91 mmol) of imidazole, followed by the dropwise addition of8.7 g (70 mmol) of 12 to the reaction mixture.

3c. Let the mixture continue to stir overnight for 12 hr at room temperature.

4c. Check the progress of the reaction with TLC (APPENDIX 3D) in 1:9 (v/v)EtOAc/hexanes. Visualize with a KMnO4 stain.

The reaction is complete when the starting material 12 (Rf = 0.43) is consumed.

5c. Add the reaction mixture to a 250-mL separatory funnel and extract 13 with 25 mLof EtOAc. Wash the EtOAc layer three times, each time with 10 mL of water, andcollect the organic fractions in a 100-mL Erlenmeyer flask.

6c. Soak up any excess water with Na2SO4, filter off any Na2SO4, and collect theproduct in a 100-mL round-bottom flask.

7c. Concentrate the organic layer in vacuo using a rotary evaporator to afford theproduct as a clear colorless oil.

Compound 13 is stable for at least 12 months at ambient temperature.

8c. Characterize the compound by 1H NMR and 13C NMR.

(2-Bromoethoxy)(tert-butyl)dimethylsilane (13). Yield 15.3 g (92%). lH-NMR (400 MHz,CDCl3): 0.00 (s, 6H), 0.84 (s, 9H), 3.27 (t, J = 7 Hz, 2H), 3.82 (t, J = 7 Hz, 2H); 13CNMR (100 MHz, CDCl3): −5.32, 18.26, 25.78, 33.12, 63.47.

9c. Add 2.1 g (24.4 mmol) of 10 (see step 7b to 8b) and 6.7 g (51.8 mmol) of distilledDIPEA to a 250-mL round-bottom flask equipped with a stir bar. Apply a nitrogen(or argon) atmosphere and add 100 mL of dry DMF. Stir the mixture until thesolution is homogeneous.

10c. Place the reaction mixture in an ice bath and add 5.8 g (24.4 mmol) of 13 (see step7c to 8c) dropwise to the mixture.

11c. Let the solution gradually warm to room temperature and let it stir for an additional48 hr.

12c. Check the progress of the reaction by TLC (APPENDIX 3D) using a mobile phase of3% MeOH in CH2Cl2. Visualize the TLC plate using a KMnO4 stain.

The product 14 (Rf = 0.26) should appear more intense than either of the starting-material spots.

13c. Add the mixture to a 500-mL separatory funnel and extract the crude productwith 100 mL of EtOAc. Wash the organic layer first with 35 mL of water, thenthree times with 35 mL of brine, and collect the organic fractions in a 100-mLErlenmeyer flask.


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4.57.10


14c. Dry the EtOAc layer over Na2SO4, filter off the Na2SO4, and collect the organiclayer in a 100-mL round-bottom flask.

15c. Evaporate the solution under reduced pressure to furnish the crude product as anorange oil.

16c. Pack a 7 × 25–cm glass column with silica gel in CH2Cl2 (APPENDIX 3E). Load theoil directly onto the column and elute with an MeOH step gradient (2%, then 6%,then 10% v/v) in CH2Cl2.

17c. Combine all fractions retaining compound 14 and concentrate in vacuo using arotary evaporator to afford a clear yellow oil.


18c. Characterize the compound by 1H/13C NMR and ESI-HRMS.

2-Azido-N-(2-(tert-butyldimethylsilyloxy)ethyl)ethanamine (14). Yield 2.3 g (39%). 1HNMR (400 MHz, CDCl3): δ 0.07 (s, 6H), 0.91 (s, 9H), 1.67 (br s, 1H), 2.74 (t, 2H, J =5.1 Hz), 2.83 (t, 2H, J = 5.5 Hz), 3.43 (t, 2H, J = 5.7 Hz), 3.72 (t, 2H, J = 5.3 Hz); 13CNMR (100MHz, CDCl3): δ −5.4, 18.3, 25.9, 48.4, 51.4, 51.6, 62.3; ESI-HRMS (ES+)m/z calcd for [C10H24N4OSi + H]+: 245.1792, found 245.1797 [M+H]+.

BASICPROTOCOL 3

PREPARATION OF ALKYNE AND AZIDE MONOMERS

For the UtU synthesis, the alkyne linker (8) is used and the amine is amide-bond coupledto 2 with EDC-Cl. After purification, the alkyne monomer 15 is obtained as a whitepowder. For the azide monomer, azide linker (11) is used and is amide-bond coupled to2 with DCC and HOBt in DMF to afford compound 16 after filtration and purificationby column chromatography.

For the synthesis of the CtU dimer, the identical alkyne monomer is used (15). However,for the cytosine azide monomer, an alternative azide linker is used to ensure that thebenzoyl group bound to cytosine is not affected in subsequent downstream reactions(the cytosine base is sensitive to reduction outlined in Fig. 4.57.4). For this reason, thebenzoyl-protected cytosine carboxylic acid 5 is amide-bond coupled with azide linker14 to afford compound 17 as a white solid following purification. The TBS group ofcompound 17 is deprotected to afford the monoalcohol 18 as a white powder, and thenthis compound is reacted with DMT chloride to afford compound 19 as a white solidafter column chromatography. These azide and alkyne monomers, are now prepared forthe cycloaddition (see Fig. 4.57.3)

Materials

Uracil-1-yl acetic acid (2; Basic Protocol 1)N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)prop-2-yn-1-amine (8; Basic Protocol 2)Dimethylformamide (DMF), anhydrous, 99.8% pureNitrogen (or argon) gas1-Ethyl-2-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-Cl;

Protochem, cat. no. c1100), �99% pureHexane, ACS gradeEthyl acetate (EtOAc), ACS gradeSaturated solution of NaCl (brine)Sodium sulfate (Na2SO4), ACS gradeSilica gel: 40 to 63 µm (230 to 400 mesh)N,N′-Dicyclohexylcarbodiimide (DCC), 99% pure1-Hydroxybenzotrizole (HOBt), � 99% pure


4.57.11


Figure 4.57.3 General scheme for the preparation of alkyne monomer 15 and azide monomers 16 and 19.

Ethyl 2-(2-azidoethylamino)acetate (11; Basic Protocol 2)(N4-(Benzoyl)cytosine-1-yl)acetic acid (5; Basic Protocol 1)Dimethylsulfoxide (DMSO), minimum 99.5% GC2-Azido-N-(2-(tert-butyldimethylsilyloxy)ethyl)ethanamine (14; Basic Protocol 2)Methanol (MeOH), ACS gradeDichloromethane (CH2Cl2), ACS gradeTriethylamine trihydrofluoride (3HF/TEA; Sigma, cat. no. 344648), 98% purePyridine, dryDimethoxytrityl chloride (DMT-Cl), 95% pureSaturated aqueous sodium bicarbonate (NaHCO3)

50-, 100-, 250- and 500-mL round-bottom flasksStir barHot plate magnetic stirrerTLC plates (250-μm thick; Silicycle; cat. no. TLG-R10011B-2020,

http://www.silicycle.com/)Short-wave UV lamp250-, 500-, and 1-L separatory funnels100-, 250-, and, 500-mL Erlenmeyer flasksRotary evaporator7 × 25–cm glass chromatography column250-mL Buchner flask3.5 × 25–cm glass chromatography columnAluminum foil



Uracil alkyne monomer (15)

Remove EDC-Cl from the refrigerator and warm to room temperature to prevent anymoisture from entering into the bottle upon opening.


Phosphoramidites

4.57.12


1a. Add 4.0 g (23.5 mmol) of 2 and 4.5 g (21.1 mmol) of 8 to a 250-mL round-bottomflask containing a stir bar and 100 mL of anhydrous DMF. Stir the solution undera nitrogen (or argon) atmosphere.

2a. Place the flask in an ice-water bath and add 9.0 g (46.9 mmol) of EDC-Cl in orderto activate the coupling.

3a. Continue stirring the solution for 30 min in ice and then let the reaction mixturestir for 12 hr at room temperature.

4a. Monitor the progress of the reaction by TLC (APPENDIX 3D) using 7:3 (v/v) hex-anes/EtOAc as a mobile phase. View the TLC plate under a short-wave UV lamp.

The product (15) will have an Rf value of 0.40.

The complete consumption of the limiting material (8) can also be screened as a meansto monitor the reaction, and this is also described in Basic Protocol 2.

5a. Transfer the solution to a 500-mL separatory funnel and extract the crude productwith 100 mL of EtOAc. Wash the organic layer first with 35 mL of water, and thenthree times, each time with 35 mL of brine. Collect the EtOAc layer in a 250-mLErlenmeyer flask.

6a. Dry the organic layer over Na2SO4, filter off the Na2SO4 and collect the filtrate ina 250-mL round-bottom flask.

7a. Concentrate the crude product under reduced pressure with a rotary evaporator.

8a. Pack a 7 × 25–cm glass column with silica gel in 7:3 (v/v) hexanes/EtOAc(APPENDIX 3E). Load the oil directly onto the column and elute the product witha step gradient of hexanes/EtOAc [7:3 (v/v), then 5.5% (v/v), then 3:7 (v/v), then100% EtOAc].

9a. Combine all fractions containing 15 and concentrate with a rotary evaporator toafford a white solid.

Compound 15 is stable for at least 12 months at room temperature.

10a. Characterize the compound by 1H/13C NMR and ESI-HRMS.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-(prop-2-ynyl)acetamide (15).Yield 5.0 g (65%). 1H NMR (500 MHz, CDCl3): δ 0.04 (s, 2H, mi.), 0.08 (s, 4H, ma.),0.88 (s, 3H, mi.), 0.89 (s, 6H, ma.), 2.25 (br s, 0.66H, ma.), 2.41 (br s, 0.33H, mi.),3.58 (t, 0.6H, J = 5.3 Hz, mi.), 3.65 (t, 1.4H, J = 5.2 Hz, ma.), 3.76 (t, 0.6H, J =5.3 Hz, mi.), 3.85 (t, 1.4H, J = 5.0 Hz, ma.), 4.27 (2s, 2H), 4.68 (m, 2H), 5.71-5.74(m, 1H), 7.13 (d, 0.66H, J = 7.9 Hz, ma.), 7.17 (d, 0.33H, J = 7.9 Hz, mi.), 9.43 (brs, 0.66H, ma.), 9.48 (br s, 0.33H, mi.); 13C NMR (125 MHz, CDCl3): δ −5.6, −5.4,−3.7, −3.5, 17.9, 18.1, 18.3, 25.5, 25.7, 25.8, 25.9, 26.0, 35.2, 38.7, 48.0, 48.1, 48.8,49.6, 60.6, 61.7, 72.7, 73.6, 77.9, 78.1, 102.1, 102.2, 145.0, 145.1, 150.9, 163.6, 166.3,166.7; 166.7; ESI-HRMS (ES+) m/z calcd for [C17H27N3O4Si + H]+: 366.1844, found366.1845 [M + H]+.

Uracil azide monomer (16)

1b. Add 1.1 g (6.4 mmol) of 2 to a 250-mL round-bottom flask containing a stir barand 100 mL of anhydrous DMF. Stir the solution under a nitrogen (or argon)atmosphere.

2b. Add 1.3 g (6.4 mmol) DCC and 1.0 g (6.4 mmol) HOBt to the reaction mixture.

3b. Stir the solution for 15 min in an ice-water bath and let the solution continue to stirfor 1 hr at ambient temperature.


4.57.13


4b. Add 1.0 g (5.8 mmol) of 11 dropwise over the course of 10 min and stir the reactionmixture for 24 hr.

5b. Monitor the reaction by TLC (APPENDIX 3D) using 100% EtOAc as the mobile phase.View the TLC plate under a short-wave UV lamp.

The new product 16 spot will have an Rf of 0.32.

The complete consumption of the limiting material (11) can also be screened as a wayof monitoring reaction progress as described in Basic Protocol 2.

6b. Remove any precipitate through vacuum filtration. Collect the filtrate in a 250-mLBuchner flask and transfer all of its contents to a new 250-mL round-bottom flask.

7b. Dry down the crude product in vacuo.

8b. Dissolve the concentrated crude in 100 mL EtOAc and transfer the solution toa 250-mL separatory funnel. Wash the organic layer first with 35 mL of water,and then three times, each with 35 mL of brine. Collect the organic fractions in a250-mL Erlenmeyer flask.

9b. Dry the organic fractions with Na2SO4, filter off the Na2SO4, and collect theorganic layer in a 250-mL round-bottom flask.

10b. Concentrate the solution under reduced pressure with a rotary evaporator.

11b. Redissolve the crude product in a minimal amount of CH2Cl2 and pack a3.5 × 25–cm glass column with silica gel in hexanes/EtOAc [5:5 (v/v)]. Loadthe crude sample and purify by flash column chromatography (APPENDIX 3E) with astep gradient of hexanes/EtOAc [5:5 (v/v), then 3:7 (v/v), then 100% EtOAc)].

12b. Evaporate fractions containing 16 with a rotary evaporator to afford a white solid.

Compound 16 is stable for at least 12 months at ambient temperature. Avoid prolongedexposure to light.


Ethyl 2-(N-(2-azidoethyl)-uracil-1-yl-acetamido)acetate (16). Yield 1.2 g (62%). 1HNMR (500 MHz, CDCl3): δ 1.28 (t, 1.5H, J = 7.1 Hz), 1.33 (t, 1.5H, J = 7.1 Hz), 3.55(s, 2H), 3.59 (t, 1H, J = 5.6 Hz), 3.66 (t, 1H, J = 5.4 Hz), 4.12 (s, 1H), 4.21 (q, 1H,J = 7.1 Hz), 4.26 (s, 1H), 4.28 (q, 1H, J = 7.1 Hz), 4.49 (s, 1H), 4.72 (s, 1H), 5.74(d, 0.5H, J = 6.0 Hz), 5.75 (d, 0.5H, J = 5.6 Hz), 7.20 (d, 0.5H, J = 2.0 Hz), 7.21(d, 0.5H, J = 2.0 Hz), 9.08 (br s, 1H); 13C NMR (125 MHz, CDCl3): δ 47.75, 47.82,48.05, 48.31, 48.86, 49.84, 49.92, 50.95, 61.67, 62.31, 102.30, 102.38, 145.02, 145.14,150.90, 150.92, 163.44, 167.16, 167.46, 168.64, 169.03; ESI-HRMS (ES+) m/z calcdfor C12H16N6O5: 324.1182, found 325.1262 [M + H]+.

Cytosine azide monomer (19)

Remove EDC-Cl from the refrigerator and warm to room temperature to prevent anymoisture from entering into the bottle upon opening.

1c. Weigh and add 2.6 g (9.5 mmol) of 5 to a 250-mL round-bottom flask containinga stir bar. Add 100 mL of anhydrous DMF and 5 mL of anhydrous DMSO to theflask, and then apply a dry nitrogen (or argon) atmosphere. Place the flask in an icebath and stir the solution.

DMSO further solubilizes the starting material.

2c. When the temperature of the flask reaches 4°C, add 2.3 g (9.5 mmol) of 14 and3.7 g (19.3 mmol) of EDC-Cl. Keep the flask at 4°C for 30 min before removingthe ice bath. Let the mixture stir overnight at room temperature.


Phosphoramidites

4.57.14


3c. Monitor the formation of 17 by TLC (APPENDIX 3D) using 2% MeOH in CH2Cl2.

The product 17 will have an Rf value of 0.33.

The complete consumption of the limiting material (14) can also be screened as a meansto monitor the reaction and this is also described in Basic Protocol 2.

4c. Transfer the mixture to a 500-mL separatory funnel and extract the crude productwith 100 mL EtOAc. Wash the organic fractions with 35 mL of water, then threetimes, each time with 35 mL of brine, and collect the EtOAc layers in a 250-mLErlenmeyer flask

5c. Dry the EtOAc layer over Na2SO4, filter off the Na2SO4, and collect the organiclayer in a 250-mL round-bottom flask.

6c. Concentrate the crude product in vacuo using a rotary evaporator to furnish an oil.

7c. Pack a 7 × 25–cm glass column with silica gel in CH2Cl2. Load the crude oil ontothe column and purify by flash column chromatography (APPENDIX 3E) with a stepgradient of MeOH (1%, then 3%, then 5%) in CH2Cl2.

8c. Combine all fractions containing compound 17 and concentrate on a rotary evap-orator to afford a white powder.

Compound 17 is stable for at least 12 months at ambient temperature. Avoid prolongedexposure to light.


N-(1-(2-((2-Azidoethyl)(2-(tert-butyldimethylsilyloxy)ethyl)amino)-2-oxoethyl)-N4-(benzoyl)cytosin-1-yl) (17). Yield 2.5 g (53%). 1H NMR (400 MHz, CDCl3): δ 0.06(s, 2H), 0.11 (s, 4H), 0.90 (s, 3H), 0.92 (s, 6H), 3.50-3.55 (m, 4H), 3.66 (t, 1H, J =5.1), 3.71-3.74 (m, 1H), 3.79 (t, 0.5H, J = 5.3 Hz), 3.84 (t, 1.5H, J = 5.1 Hz), 4.83 (s,0.5H), 4.85 (s, 1.5H), 7.53 (t, 3H, J = 7.62 Hz), 7.61-7.64 (m, 1.5H), 7.72 (d, 0.5H, J= 6.25 Hz), 7.90 (d, 2H, J = 7.03 Hz), 8.64 (br s, 1H); 13C NMR (100 MHz, CDCl3): δ

−5.5, 11.6, 18.2, 18.3, 25.9, 46.2, 46.7, 48.3, 49.0, 49.6, 49.7, 49.9, 51.0, 61.1, 61.3,96.7, 127.5, 129.0, 133.1, 149.9, 150.1, 155.5, 162.5, 162.6, 166.7, 167.3; ESI-HRMS(ES+) m/z calcd for [C23H33N7O4Si + H]+: 500.2436, found 500.2441 [M+H]+.

10c. In a 250-mL round-bottom flask containing a stir bar, mix 2.5 g (5.0 mmol) of 17into 100 mL of CH2Cl2.

11c. Stir the mixture until 17 dissolves and add 2.8 g (7.5 mmol) of 3HF/TEA to thesolution.

CAUTION: HF can be fatal by skin contact, inhalation, or ingestion. Before workingwith HF, apply calcium gluconate gel onto your hands and wear a heavy set of gloves.

12c. Monitor the reaction by TLC (APPENDIX 3D) using 2% MeOH in CH2Cl2 as a mobilephase. Visualize under short-wavelength UV lamp.

The starting material 17 (Rf = 0.33) disappears after 4 hr, and the product will have alower retention factor.

13c. Concentrate the crude product on a rotary evaporator.

14c. Pack a 3.5 × 25–cm glass column with silica gel quenched in 2% MeOH inCH2Cl2 (APPENDIX 3E). Load the concentrated crude product onto the column andpurify eluting with a step gradient of MeOH (2%, then 3.5%, then 5%) in CH2Cl2.

15c. Combine the fractions of interest and concentrate the product on a rotary evaporatorwith the co-evaporation of hexanes to afford 18 as a white powder.

Compound 18 is stable for at least 12 months at room temperature. Avoid prolongedexposure to light.


4.57.15



N-(1-(2-((2-azidoethyl)(2-hydroxyethyl)amino)-2-oxoethyl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (18). Yield 1.7 g (91%). 1H NMR (400 MHz, CDCl3): δ 3.54 (s,3H), 3.61-3.64 (m, 1.5H), 3.68 (d, 0.5H, J = 4.3 Hz), 3.70 (d, 1H, J = 4.7 Hz),3.79-3.82 (m, 0.5H), 3.83-3.85 (m, 1.5H), 4.50 (br s, 1H), 4.83 (s, 0.5H), 4.94 (s,1.5H), 7.47 (t, 2.5H, J = 7.6 Hz), 7.58 (t, 1.5H, J = 7.4 Hz), 7.70 (d, 0.5H, J =2.3 Hz), 7.72 (d, 0.5H, J = 2.3 Hz), 7.90-7.94 (m, 2H), 9.16 (br s, 1H); 13C NMR(100 MHz, CDCl3): δ 46.6, 48.3, 49.0, 49.7, 50.0, 50.5, 50.9, 51.3, 59.9, 60.3, 97.1,127.7, 127.8, 128.9, 132.9, 133.1, 150.3, 150.4, 156.2, 163.0, 163.2, 167.5, 167.7;ESI-HRMS (ES+) m/z calcd for [C17H19N7O4 + H]+: 386.1571, found 386.1582[M+H]+.

17c. Weigh and add 1.7 g (4.4 mmol) of 18 to 50 mL of dry pyridine under N2 in a100 mL round-bottom flask. Dissolve 18 with magnetic stirring.

18c. Wrap the round-bottom flask with aluminum foil to avoid light penetration.

19c. Add 3.0 g (8.8 mmol) of DMT-Cl and stir the reaction overnight at roomtemperature.

20c. Monitor the progress of the reaction by TLC (APPENDIX 3D) using 100% EtOAc asthe mobile phase. Observe the TLC plate under a short-wavelength UV lamp.

The product (19) will have an Rf value of 0.62 and will have a retention factor higherthan the starting material.

21c. Pour the mixture into a 250-mL separatory funnel and perform a liquid-liquidextraction with 50 mL of EtOAc. Wash the organic layer three times, each timewith 20 mL saturated aqueous NaHCO3 and collect the fractions in a 250-mLErlenmeyer flask.

22c. Dry the EtOAc fractions with Na2SO4, filter off the Na2SO4, and collect the organiclayer in a 250-mL round-bottom flask.

23c. Concentrate the crude product in vacuo using a rotary evaporator.

24c. Pack a 7 × 25–cm glass column with silica gel in hexanes/EtOAc [7:3 (v/v)] andpurify it through the use of flash column chromatography (APPENDIX 3E) with a stepgradient of hexanes/EtOAc (7:3 v/v, then 5:5 v/v, then 3:7 v/v, and then 100%EtOAc).

25c. Evaporate fractions retaining compound 19 with a rotary evaporator to afford awhite solid.

Compound 19 is stable for at least 12 months at ambient temperature. Minimize exposureto light.


N-(1-(2-((2-Azidoethyl)(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)amino)-2-oxoethyl)-N4-(benzoyl)cytosin-1-yl) (19). Yield 2.9 g (99%). 1H NMR (400 MHz,CDCl3): δ 3.35-3.41 (m, 2H), 3.46 (t, 3H, J = 5.3 Hz), 3.55 (t, 0.5H, J = 4.9 Hz), 3.67(t, 2H, J = 4.5 Hz), 3.74 (t, 0.5H, J = 5.9 Hz), 3.82 (s, 1.5H, mi.), 3.83 (s, 4.5H, ma.),4.83 (s, 0.5H, mi.), 4.92 (s, 1.5H, ma.), 6.85-6.90 (m, 4H), 7.24 (t, 0.5H, J = 7.0 Hz),7.28-7.29 (m, 2.5H), 7.30 (s, 3.5H), 7.35 (t, 2H, J = 7.6 Hz), 7.39-7.42 (m, 2.5H), 7.54(t, 2H, J = 7.6 Hz), 7.62-7.66 (m, 1H), 7.71 (d, 0.3H, J = 7.4 Hz, mi.), 7.93 (d, 1.7H,J = 7.4 Hz, ma.), 8.77 (br s, 1H); 13C NMR (100 MHz, CDCl3): δ 13.7, 19.1, 22.6, 30.6,31.9, 46.2, 47.1, 47.7, 48.9, 49.0, 49.4, 49.6, 49.9, 55.2, 55.3, 61.1, 61.8, 64.3, 86.6,87.3, 96.5, 113.2, 113.3, 126.8, 127.2, 127.5, 127.9, 128.0, 128.2, 129.0, 129.9, 130.1,133.1, 135.2, 135.8, 144.2, 144.6, 149.8, 158.5, 158.7, 162.5, 166.6, 167.1; ESI-HRMS(ES+) m/z calcd for [C38H37N7O6 + H]+: 688.2878, found 688.2878 [M+H]+.


Phosphoramidites

4.57.16


BASICPROTOCOL 4

PREPARATION OF URACIL-TRIAZOLE-URACIL PHOSPHORAMIDITE (24)

Alkyne 15 and azide 16 are reacted together in the presence of CuSO4 and sodium ascor-bate to afford the triazole-linked dimer 20. The ester of dimer 20 is selectively reducedwith lithium borohydride to afford the monoalcohol 21. Monoalcohol 21 is reactedwith DMT-Cl to afford compound 22 as a white solid after column chromatography.Deprotection of TBS is performed in order to afford monoalcohol 23 as a white solid.Finally, phosphitylation of 23 to afford the final phosphoramidite 24 is described(Fig. 4.57.4).

Materials

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-(prop-2-ynyl)acetamide (15;Basic Protocol 3)

Ethyl 2-(N-(2-azidoethyl)-uracil-1-yl-acetamido)acetate (16; Basic Protocol 3)Tetrahydrofuran (THF), anhydrous, �99.9% puretert-Butyl alcohol (t-BuOH), ACS grade, �99% pure(+)-Sodium L-ascorbate, �98% pureCopper(II) sulfate (CuSO4) pentahydrate, �99% pureAmmonium hydroxide (NH4OH) solution, ACS grade, 28.0% to 30.0% NH3

basisMethanol (MeOH), ACS gradeDichloromethane (CH2Cl2), ACS gradeSilica gel: 40 to 63 µm (230 to 400 mesh)Nitrogen (or argon) gas2.0 M lithium borohydride (LiBH4) in THFPyridine, dryDimethoxytrityl chloride (DMT-Cl), 95% pureSodium sulfate (Na2SO4)1.0 M tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuranN-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-((1-(2-(uracil-1-yl-N-(2-4-dimethylaminopyridine (DMAP), 99% pureSaturated solution of NaCl (brine)Distilled N,N-Diisopropylethylamine (DIPEA), 99% pure13.5% to 15.5% 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, ClDistilled triethylamine (TEA), �99%Hexane, ACS gradeAcetone, ACS grade


http://www.silicycle.com/)Short-wave UV lamp250-, 500-, and 1-L separatory funnels100-, 250-, and, 500-mL Erlenmeyer flasksFilter paper, grade P53.5 × 25–cm and 2.5 × 25–cm glass chromatography columnsReflux condenserDisposable syringes and needles


3D) and column chromatography (APPENDIX 3E)Synthesis ofModifiedOligonucleotidesand Conjugates

4.57.17


Figure 4.57.4 General scheme for the preparation of the uracil-triazole-uracil dimer phosphoramidite (24).

Uracil-triazole-uracil (UtU) ester (20)

1. Add 677 mg (1.9 mmol) of 15 and 600 mg (1.9 mmol) of 16 to a 50-mL round-bottom flask. Dissolve the reactants with magnetic stirring in 18 mL of THF/t-BuOH/water (1:1:1).

2. Add 733 mg (3.7 mmol) of sodium ascorbate and 231 mg (925 µmol) ofCuSO4·5H2O to the solution and stir the reaction mixture for 24 hr at room tem-perature.

3. Monitor the progress of the reaction by TLC using 10% (10% NH4OH in MeOH)in CH2Cl2 as a mobile phase. Analyze the TLC plate under a short-wave UV lamp.

The cyclized product 20 will have an Rf value of 0.47.

4. Transfer the solution to a 250-mL separatory funnel and extract the crude productwith 18 mL of CH2Cl2. Flush the organic layer three times, each time with 6 mLof water, to wash off any excess copper. Collect the organic layer in a 100-mLErlenmeyer flask.

5. Collect the precipitate in the organic layer by vacuum filtration using filter paper(grade P5) and wash the precipitate, three times, each time with 6 mL of 4°C water,afford the crude product.

6. Redissolve the crude product with a minimal amount of MeOH in CH2Cl2 for 1 hr.Pack a 3.5 × 25–cm glass chromatography column with silica gel in 5% (v/v) of a


Phosphoramidites

4.57.18


solution of 10% NH4OH in MeOH in CH2Cl2. Load the dissolved crude producton the column and purify by flash column chromatography (APPENDIX 3E), elutingwith 5% (v/v) of a solution of 10% NH4OH in MeOH in CH2Cl2 to afford 20 as awhite compound.

7. Combine the fractions of interest and concentrate on a rotary evaporator to afford20 as a white powder.

Compound 20 is stable for at least 12 months at room temperature.

8. Characterize the compound by 1H/13C NMR and ESI-HRMS.

Ethyl 2-(N-(2-(4-((N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-uracil-1-yl-acetamido)acetate (20). Yield 1.3 g (98%).1H NMR (400 MHz, DMSO-d6): δ 0.03 (s, 2H), 0.05 (m, 4H), 0.85 ,0.86 (2s, 9H), 1.18(t, 1.5H, J = 7.2 Hz), 1.23 (t, 1.5H, J = 7.0 Hz), 3.48-3.50 (m, 1H), 3.63-3.91 (m,4H), 4.01-4.03 (m, 1H), 4.08 (q, 1.2H, J = 7.3 Hz), 4.16 (q, 0.8H, J = 7.3 Hz), 4.24,4.29 (2s, 1H), 4.44-4.77 (m, 9H), 5.56 (d, 2H, J = 7.8 Hz), 7.31-7.47 (m, 2H), 7.89,8.05, 8.13, 8.24 (4s, 1H), 11.29 (br m, 2H); 13C NMR (100 MHz, DMSO-d6): δ −5.45,−5.39, 13.99, 14.04, 17.89, 18.04, 25.82, 25.90, 41.05, 47.31, 47.58, 47.91, 48.00,60.18, 60.65, 60.83, 61.25, 100.57, 100.79, 123.70, 124.04, 124.12, 124.61, 143.18,143.34, 143.41, 143.56, 146.12, 146.19, 146.29, 146.50, 146.54, 150.92, 150.98, 151.04,151.07, 151.11, 163.76, 163.80, 163.81, 163.85, 163.88, 166.74, 166.82, 166.88, 166.90,167.43, 167.97, 168.03, 168.85, 169.15; ESI-HRMS (ES+) m/z calcd for C29H43N9O9Si:689.2953, found 690.3030 [M + H]+.

Uracil-triazole-uracil (UtU) phosphoramidite (24)

9. Add 1.2 g (1.74 mmol) of 20 to a 100-mL round-bottom flask containing a stir bar.Under nitrogen (or argon), add 48 mL of dry THF and begin stirring the solution.

10. After 5 to 10 min, begin to add MeOH dropwise until the reactant is completelydissolved.

11. Once dissolved, add 2.18 mL (4.36 mmol) of 2.0 M LiBH4 to the reaction mixtureand reflux.

CAUTION: LiBH4 is a flammable solid that forms hydrogen gas when in contact withmoisture. Handle with care in a fume hood and store under anhydrous conditions.

12. Monitor the reaction by TLC (APPENDIX 3D) using a mobile phase of 15% (v/v) ofa solution of 10% NH4OH in MeOH in CH2Cl2. Observe the TLC plate under ashort-wavelength UV lamp.

The complete consumption of 20 will occur in �1.5 hr during the reaction. The productwill have an Rf value of 0.50.

13. Quench the reaction mixture with MeOH and dry it down using a rotary evaporatorto afford the crude product.

14. Redissolve the crude product in CH2Cl2 with minimal amounts of MeOH and packa 2.5 × 25–cm glass column with silica gel in 5% (v/v) of a solution of 10%NH4OH in MeOH in CH2Cl2. Purify by flash column chromatography (APPENDIX

3E), eluting with a step gradient of 5%, then 7.5%, then 10% (v/v) of a solution of10% NH4OH in MeOH in CH2Cl2.

15. Evaporate all fractions possessing 21 using a rotary evaporator to afford a whitesolid.

Compound 21 is stable for at least 12 months at ambient temperature.



4.57.19


N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-((1-(2-(uracil-1-yl-N-(2-hydroxyethyl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methyl)acetamide (21). Yield 852 mg (76%).1H NMR (500 MHz, DMSO-d6): δ 0.03 (s, 2H), 0.05 (s, 4H), 0.85, 0.87 (2s, 9H),3.16-3.27 (m, 2H), 3.43-3.54 (m, 4H), 3.63-3.74 (m, 3H), 3.80-3.87 (m, 2H), 4.36-4.38(m, 1H), 4.46 (t, 1.2H, J = 6.3 Hz), 4.51 (t, 0.8H, J = 6.3 Hz), 4.56-4.81 (m, 6H),4.94-5.00 (m, 1H), 5.54-5.58 (m, 2H), 7.35-7.47 (m, 2H), 7.87, 8.04, 8.13, 8.23 (4s,1H), 11.30 (br s, 2H); 13C NMR (125 MHz, DMSO-d6): δ −5.44, −5.37, 17.91, 18.05,25.83, 25.92, 30.74, 41.04, 46.24, 46.55, 46.71, 46.92, 48.29, 48.32, 48.51, 49.07,49.26, 58.49, 58.81, 58.85, 60.21, 60.85, 100.57, 100.63, 123.76, 124.11, 124.55,143.23, 143.39, 143.43, 143.58, 146.48, 146.52, 146.58, 151.04, 151.08, 151.11,163.84, 163.87, 166.69, 166.74, 166.81, 167.43, 167.54; ESI-HRMS (ES+) m/z calcdfor C27H41N9O8Si: 647.2847, found 648.2918 [M + H]+.

17. Add 1.06 g (1.64 mmol) of 21 to a 50-mL round-bottom flask. Add a stir bar andapply a dry nitrogen (or argon) atmosphere. With a syringe, add 10 mL of drypyridine to the flask.

18. When 21 has completely dissolved, add 1.66 g (4.91 mmol) of DMT-Cl and stirthe reaction mixture overnight at room temperature.

19. On the following day, monitor the progress of the reaction by TLC (APPENDIX 3D)using 10% MeOH in CH2Cl2.

The product will have a significant increase in Rf due to the addition of a large hy-drophobic protecting group.

20. Transfer the mixture to a 250-mL separatory funnel and extract the crude productwith 10 mL CH2Cl2. Wash the organic layer twice, each time with 5 mL of water,and collect the organic fractions in a 100-mL Erlenmeyer flask.

21. Dry the fractions over Na2SO4, filter off the Na2SO4, and collect the filtrate in a100-mL round-bottom flask.

22. Concentrate the crude product using a rotary evaporator.

23. Redissolve the crude product in CH2Cl2 with minimal amounts of MeOH and packa 3 × 25–cm glass column with silica gel in 100% CH2Cl2. Purify the mixtureby flash column chromatography (APPENDIX 3E), eluting with a step gradient of 5%,then 10%, then 15% MeOH in CH2Cl2.

24. Combine the fractions of interest and concentrate the product on a rotary evaporatorto afford 22 as a white powder.



N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(uracil-1-yl)-N-(2-(4-((2-(Uracil-1-yl)-N-(2-((2,3,3-trimethylbutan-2-yl)oxy)ethyl)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)acetamide (22). Yield 1.22 g (79%). 1H NMR (400 MHz, CDCl3): δ 0.03 (s,2H), 0.06 (s, 4H), 0.86, 0.88 (2s, 9H), 2.87-2.94 (m, 1.5H), 3.03-3.08 (m, 0.5H), 3.20(t, 1.5H, J = 4.7 Hz), 3.26 (t, 0.5H, J = 4.5 Hz), 3.29-3.31 (m, 0.2H), 3.36-3.43 (m,0.3H), 3.46-3.55 (m, 2.5H), 3.60-3.67 (m, 2H), 3.72-3.75 (m, 0.75H), 3.76, 3.77 (2s,7H), 3.80 (t, 1.25H, J = 4.9 Hz), 4.51 (t, 1.5H, J = 5.3 Hz), 4.55 (t, 0.5H, J = 5.3Hz), 4.59 (s, 1H), 4.63-4.64 (m, 1H), 4.67 (s, 3H), 4.73 (d, 1H, J = 7.4 Hz), 5.51-5.55(m, 1H), 5.62 (d, 0.1H, J = 1.6 Hz), 5.64 (d, 0.1H, J = 1.6 Hz), 5.64-5.68 (m, 0.8H),6.57 (d, 0.1H, J = 7.8 Hz), 6.66 (d, 0.7H, J = 7.8 Hz), 6.80-6.84 (m, 4.3H), 7.06-7.09(m, 0.8H), 7.17-7.36 (m, 7.2H), 7.71, 7.81, 7.88, 7.94 (4s, 1H), 10.11 (br s, 2H); 13CNMR (100 MHz, CDCl3): δ −5.44, −5.16, 18.13, 18.29, 25.61, 25.83, 25.90, 41.33,44.08, 47.64, 47.74, 48.27, 48.50, 48.62, 48.87, 48.93, 49.09, 55.23, 60.41, 60.66,60.79, 60.98, 87.24, 87.39, 102.02, 102.21, 102.25, 113.15, 113.25, 124.37, 125.24,


Phosphoramidites

4.57.20


127.18, 127.24, 127.86, 127.98, 128.15, 129.91, 130.08, 135.03, 135.09, 135.70,143.49, 143.76, 144.01, 144.05, 144.65, 145.00, 145.10, 145.15, 145.21, 145.54,151.26, 151.32, 151.38, 151.46, 158.42, 158.67, 158.71, 163.91, 164.00, 164.08,166.48, 167.25, 167.67; ESI-HRMS (ES+) m/z calcd for C48H59N9O10Si 949.4154,found 972.4035 [M + Na]+.

26. Dissolve 1.12 g (1.18 mmol) of 22 in a 50-mL round-bottom flask with 26 mL ofdry THF. Apply a dry nitrogen (or argon) atmosphere and stir the solution withmagnetic stirring.

27. When the starting material dissolves, add 3.54 mL (3.54 mmol) of 1.0 M TBAF tothe mixture and continue stirring for 12 hr at room temperature.

28. Monitor the reaction by TLC using 5% MeOH in CH2Cl2 as a mobile phase.

The new alcohol functionality will cause the product 23 to have a low Rf compared tothe starting material 22.

29. Transfer the reaction mixture to a 250-mL separatory funnel and perform a liquid-liquid extraction with 26 mL of CH2Cl2. Wash the organic layer first with 13 mLof water and then twice each time with 13 mL of brine, and collect the organicfractions in a 100-mL Erlenmeyer flask.

30. Dry the fractions over Na2SO4, filter off the Na2SO4, and collect the organic layerin a 100-mL round-bottom flask.

31. Concentrate the crude product under reduced pressure with a rotary evaporator.

32. Redissolve the crude in CH2Cl2 using a minimal amount of MeOH and pack a 2.5× 25–cm glass column with silica gel in 100% CH2Cl2. First, flush the column withCH2Cl2 to remove trace amount, of TBAF. Elute 23 with 100% CH2Cl2 followedby a step gradient of MeOH (5%, then 10%, then 15%) in CH2Cl2.

33. Collect the fractions containing 23 and concentrate on a rotary evaporator to afforda yellow solid.



N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(uracil-1-yl)-N-((1-(2-(2-(uracil-1-yl)-N-(2-hydroxyethyl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methyl)acetamide (23). Yield840 mg (85%). 1H NMR (400 MHz, CDCl3): δ 2.49 (br s, 1H), 2.98 (br s, 1H),3.16-3.20 (br m, 2H), 3.27-3.31 (m, 3H), 3.45-3.57 (m, 4H), 3.68-3.81 (m, 8H),4.45-4.80 (m, 8H), 5.50-5.53 (m, 0.8H), 5.56-5.60 (m, 1.2H), 6.63 (d, 0.15H, J = 8.2Hz), 6.66 (d, 0.35H, J = 7.8 Hz), 6.78-6.83 (m, 4H), 7.17-7.31 (m, 8.5H), 7.69, 7.88,7.90, 8.09 (4s, 1H), 10.20-10.42 (br m, 2H); 13C NMR (100 MHz, CDCl3): δ 22.13,27.75, 29.14, 29.64, 42.18, 47.37, 48.29, 49.13, 50.35, 55.24, 59.15, 60.78, 68.46,87.23, 87.32, 101.65, 101.91, 107.85, 113.16, 113.26, 124.97, 127.16, 127.99, 128.17,129.92, 130.09, 135.15, 135.73, 143.56, 144.11, 144.28, 145.47, 146.36, 151.41,151.47, 158.40, 158.64, 164.30, 164.39, 164.56, 167.63, 167.68; ESI-HRMS (ES+) m/zcalcd for C42H45N9O10: 835.3289, found 858.3217 [M + Na]+.

Remove 2-cyanoethyl N, N-diisopropylchlorophosphoramidite from the refrigerator andwarm to room temperature before opening to prevent any moisture from entering intothe reagent bottle.

35. Add 230 mg (275 µmol) of 23 to a 10-mL round-bottom flask equipped with a stirbar. Apply a nitrogen (or argon) atmosphere and, with a syringe, add 6 mL of dryCH2Cl2.


4.57.21


36. Add 196 mg (1.51 mmol) of distilled DIPEA, 16.8 mg (138 µmol) of DMAP,and 195 mg (825 µmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite,in that order, to the stirring reaction mixture.

37. Monitor the reaction by TLC using 2:1 (v/v) acetone:hexane in 2% triethylamineas a mobile phase.

The starting material 23 will be completely consumed within 6 hr. The product 24 willhave a higher retention factor due to the loss of a free monoalcohol group.

Triethylamine is used in the mobile phase to prevent acid hydrolysis of the phospho-ramidite from the slightly acidic silica on the TLC plate.

38. Dry the reaction mixture in vacuo using a rotary evaporator to afford the crudeproduct.

39. Dissolve the crude in minimal amounts of 2% triethylamine in 1:1 hexanes/acetoneand pack a 2.5 × 25–cm glass column with silica gel in 2% triethylamine in hexanes.Purify by flash column chromatography (APPENDIX 3E), eluting with a step gradientof 2% triethylamine in acetone/hexanes (1:1, v/v, then 2:1 v/v, then 4:1 v/v).

40. Combine the fractions of interest and concentrate with a rotary evaporator to afford24 as a white powder.

The phosphoramidite 24 is stable for 3 months stored at −20°C under vacuum or undernitrogen. The compound is extremely sensitive to moisture.

41. Characterize the compound by 1H/13C/31P NMR and ESI-HRMS.

2-(N-(2-(4-((N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(uracil-1-)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(uracil-1-yl)acetamido)ethyl (2-cyanoethyl)diisopropylphosphoramidite (24). Yield 180 mg (63%). 1H NMR (400 MHz, CDCl3):δ 1.14-1.19 (m, 12H), 2.62-2.66 (m, 3.25H), 2.92 (t, 1H, J = 3.9 Hz), 3.07 (t, 0.75H,J = 4.3 Hz), 3.21 (t, 1.25H, J = 4.7 Hz), 3.26 (t, 0.5H, J = 4.3 Hz), 3.29-3.37 (m,0.25H), 3.54-3.61 (m, 5.5H), 3.70-3.77 (m, 2H), 3.78 (s, 6.25H), 3.81-3.90 (m, 2.25H),4.52 (t, 1H, J = 5.3 Hz), 4.55 (t, 0.5H, J = 5.5 Hz), 4.61-4.65 (m, 2H), 4.70 (s, 2.5H),4.74-4.77 (m, 1H), 5.52-5.55 (m, 1H), 5.66-5.70 (m, 1H), 6.56 (d, 0.33H, J = 7.8 Hz),6.63 (d, 0.66H, J = 7.8 Hz), 6.81-6.84 (m, 4H), 7.17-7.31 (m, 12H), 7.70, 7.76, 7.89,7.95 (4s, 1H); 13C NMR (100 MHz, CDCl3): δ 14.10, 14.84, 20.45, 20.52, 22.66, 23.34,24.58, 24.62, 24.65, 24.69, 24.77, 29.24, 29.34, 29.66, 31.72, 31.89, 33.80, 34.43,41.51, 43.02, 43.14, 45.82, 47.67, 47.80, 48.29, 48.60, 48.71, 48.93, 53.76, 55.26,58.08, 58.30, 60.53, 60.70, 60.80, 69.48, 87.29, 87.42, 102.08, 102.20, 102.25, 113.27,117.90, 118.12, 125.13, 127.24, 128.01, 128.20, 130.12, 135.05, 135.09, 143.39,143.82, 144.05, 145.07, 145.46, 151.24, 151.34, 151.45, 158.71, 158.75, 163.63,163.87, 163.91, 166.67, 167.41, 167.74; 31P NMR (167 MHz, CDCl3): δ 149.6 and150.8; ESI-HRMS (ES+) m/z calcd for C51H62N11O11P: 1035.4368, found 1058.4298[M + Na]+.

BASICPROTOCOL 5

PREPARATION OF CYTOSINE-TRIAZOLE-URACIL PHOSPHORAMIDITE

Alkyne 15 and azide 19 are reacted together in the presence of CuSO4 and sodiumascorbate to afford the triazole-linked dimer 25. Deprotection of TBS is performed toafford monoalcohol 26 as a white solid. Finally, phosphitylation of 26 to afford the finalphosphoramidite 27 is described (Fig. 4.57.5). An ester azide with a cytosine derivativewas cyclized and reduction of the ester was attempted with a variety of reducing agents.In all cases, the cytosine base was reduced. Therefore, the strategy utilizing the azidelinker 14 for synthesizing the CtU phosphoramidite avoids the need to reduce theester.


Phosphoramidites

4.57.22


Figure 4.57.5 General scheme for the preparation of the cytosine-triazole-uracil dimer phosphoramidite (27).

Materials

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-uracil-1-yl-N-(prop-2-ynyl)acetamide (15;Basic Protocol 3)

N-(1-(2-((2-Azidoethyl)(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)amino)-2-oxoethyl)-N4-(benzoyl)cytosin-1-yl) (19; Basic Protocol 3)

Tetrahydrofuran (THF), anhydrous, �99.9% pure(+)-Sodium L-ascorbate, �98% pureCopper(II) sulfate (CuSO4) pentahydrate, �99% pureMethanol (MeOH), ACS gradeDichloromethane (CH2Cl2), ACS gradeEthyl acetate (EtOAc), ACS gradeSodium sulfate (Na2SO4)Silica gel: 40 to 63 µm (230 to 400 mesh)1.0 M tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuranDistilled N,N-Diisopropylethylamine (DIPEA), 99% pure13.5% to 15.5% 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, ClDistilled triethylamine (TEA), �99% pureAcetone, ACS gradeHexane, ACS grade


http://www.silicycle.com/)Short-wave UV lamp250-, 500-, and 1-L separatory funnels100-, 250-, and, 500-mL Erlenmeyer flasks2.5 × 25–cm and 3.5 × 25–cm glass chromatography columnRotary evaporator


4.57.23




Cytosine-triazole-uracil (CtU) dimer (25)

1. Add 570 mg (1.6 mmol) of 15 and 1.2 g (1.7 mmol) of 19 to 70 mL of THF/water(3:2) in a 250-mL round-bottom flask with a stir bar.

2. Add 519 mg (2.6 mmol) of sodium ascorbate and stir until all remnants havedissolved.

3. To this mixture, add 220 mg (880 pmol) of CuSO4 and continue stirring the reactionmixture at room temperature.

4. Monitor the progress of the reaction using 2% MeOH in CH2Cl2 as the mobilephase and stop the reaction when 15 is completely consumed.

The reaction is complete in � 4 hr. The product has a lower Rf value compared to bothstarting materials.

5. Add the mixture to a 250-mL separatory funnel and perform a liquid-liquid extrac-tion with 70 mL of EtOAc. Wash the organic layer three times, each time with 25mL of water, and collect the fractions in a 250-mL Erlenmeyer flask.

6. Dry the EtOAc layer with Na2SO4, filter off all Na2SO4 hydrates, and collect theorganic layer in a 250-mL round-bottom flask.

7. Concentrate the crude product in vacuo using a rotary evaporator.

8. Pack a 3.5 × 25–cm glass column with silica gel in 2% MeOH in CH2Cl2. Load thecrude product directly onto the column and elute the product with a step gradientof MeOH (2%, then 3.5%, then 5%) in CH2Cl2.

The final product 25 is a white solid.

9. Evaporate fractions containing the product 25 using a rotary evaporator to afford awhite solid.



N-(1-(2-((2-(bis(4-Methoxyphenyl)(phenyl)methoxy)ethyl)(2-(4-((N-(2-((tertbutyldime-thylsilyl) oxy)ethyl)-2-(uracil-1-yl)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)amino)-2-oxoethyl)-N4-(benzoyl)cytosin-1-yl). (25). Yield 1.6 g (98%). 1H NMR (400MHz, CDCl3): δ 0.06 (s, 2H), 0.10 (s, 4H), 0.90 (s, 3H), 0.92 (s, 6H), 3.03 (m, 1.5H),3.12 (m, 0.5H), 3.24-3.28 (m, 2.5H), 3.51-3.54 (m, 1H), 3.55-3.57 (m, 1.5H), 3.63-3.69(m, 2H), 3.74-3.78 (m, 2H), 3.81 (s, 1.5H), 3.83 (s, 4.5H), 4.49-4.52 (m, 1.5H),4.56-4.57 (m, 0.5H), 4.72 (br s, 1.5H), 4.74-4.78 (dd, 3.5H, J = 7.0 Hz), 4.84 (d, 0.5H,J = 5.1 Hz), 4.88 (s, 0.5H), 5.59-5.70 (m, 1H), 6.81-6.84 (m, 1H), 6.87 (d, 3H, J =9.0 Hz), 7.23 (d, 3H, J = 2.3 Hz), 7.25 (s, 2H), 7.27-7.29 (m, 2H), 7.31 (s, 1.5H),7.34 (d, 2.5H, J = 4.7 Hz), 7.38-7.42 (m, 0.5H), 7.52 (t, 2.5H, J = 7.6 Hz), 7.60-7.63(m, 1H), 7.70-7.73 (m, 0.25H), 7.75 (s, 0.5H), 7.93-8.02 (m, 2H), 8.07 (d, 0.25H, J =7.4 Hz), 9.66 (br s, 0.5H), 9.85 (br s, 0.5H); 13C NMR (100 MHz, CDCl3): δ −5.5,18.1, 18.3, 25.8, 25.9, 41.8, 44.0, 47.6, 47.7, 47.9, 48.0, 48.4, 48.6, 48.7, 49.0, 49.1,49.2, 50.9, 55.2, 60.7, 60.8, 87.3, 96.8, 101.6, 101.7, 113.1, 113.2, 124.7, 124.9, 127.2,127.9, 128.0, 128.1, 128.7, 128.8, 129.9, 130.1, 132.9, 133.0, 135.1, 135.8, 143.4,144.1, 144.2, 145.7, 146.0, 149.9, 151.1, 155.7, 158.4, 158.7, 163.3, 164.0, 164.1,166.6, 167.4, 167.5, 167.7; ESI-HRMS (ES+) m/z calcd for [C55H64N10O10Si + H]+:1053.4649, found 1053.4643 [M+H]+; for [C55H64N10O10Si + Na]+: 1075.4468,found 1075.4459 [M+Na]+.


Phosphoramidites

4.57.24


Cytosine-triazole-uracil phosphoramidite (27)

11. Weigh and add 1.3 g (1.2 mmol) of 25 to a 50-mL round-bottom flask containinga stir bar and 25 mL of THF.

12. Stir the solution until the starting material fully dissolves and add 1.9 mL (1.9mmol) of TBAF.

13. Monitor the reaction using 10% MeOH in CH2Cl2 as a mobile phase. Visualize theplate under a short-wave UV lamp.

Stop the reaction when 25 is completely consumed and the product 26 (Rf = 0.5)emerges. The reaction should be complete in �2.5 hr.

14. Remove the 25 mL of THF on a rotary evaporator.

15. Pack a 3.5 × 25–cm glass column with silica gel in CH2Cl2. Purify the crudeproduct by flash column chromatography (APPENDIX 3E) with a step gradient ofMeOH (2%, then 6%, then 10%, then 15%) in CH2Cl2.

16. Combine the fractions of interest and concentrate on a rotary evaporator to afford26 as a white powder.



N-(1-(2-((2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-(4-(2-(uracil-1-yl)-N-(2-hydroxyethyl)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)amino)-2-oxoethyl)-N4-(benzoyl)cytosin-1-yl). (26) Yield 1.1 g (99%). 1H NMR (400 MHz, CDCl3): δ 3.09 (brs, 1.5H), 3.15 (br s, 0.5H), 3.25-3.30 (m, 2H), 3.62 (d, 4H, J = 3.1 Hz), 3.73 (br s,2H), 3.81 (s, 1.5H), 3.83 (s, 4.5H), 4.51 (d, 1.5H, J = 4.7 Hz), 4.59-4.61 (m, 0.5H),4.67-4.71 (m, 3.5H), 4.82 (br s, 1.5H), 4.87-4.89 (m, 2H), 5.61 (t, 1H, J = 7.0 Hz),6.88 (d, 4H, 9.0 Hz), 7.24 (s, 3H), 7.26 (s, 2H), 7.28- 7.32 (m, 3H), 7.35 (d, 3.5H, J= 4.7 Hz), 7.38, (br s, 0.5H), 7.50 (t, 2H, J = 7.6 Hz), 7.58-7.61 (m, 1.5H), 7.76 (s,0.5H), 8.00 (d, 1.5H, J = 7.8 Hz), 8.04 (d, 0.5H, J = 10.9 Hz), 9.72-9.85 (m, 2H); 13CNMR (100 MHz, CDCl3): δ 20.1, 43.0, 43,8, 47.4, 48.0, 48.9, 49.2, 48.0, 51.2, 51.3,55.3, 59.6, 60.7, 60.8, 87.3, 87.4, 97.3, 101.6, 113.2, 113.3, 124.9, 125.6, 127.2, 128.0,128.1, 128.2, 128.8, 129.9, 130.1, 132.9, 133.1, 135.1, 135.7, 143.3, 144.1, 144.2,144.5, 146.0, 146.2, 150.0, 151.1, 151.2, 156.2, 158.2, 158.5, 158.7, 163.6, 164.1,164.2, 167.0, 167.6, 167.8; ESIHRMS (ES+) m/z calcd for [C49H50N10O10 + H]+:939.3784, found 939.3776 [M+H]+; for [C49H50N10O10 + Na]+: 961.3604, found961.3591 [M+Na]+.

18. Add 300 mg (320 µmol) of 26 to a 50-mL round-bottom flask containing a stir bar.Under a nitrogen (or argon) atmosphere, add 10 mL of dry CH2Cl2 with a syringe.

19. Subsequently, add 227 mg (1.8 mmol) of distilled DIPEA, 20 mg (160 µmol)of DMAP, and 227 mg (960 µmol) of 2-cyanoethyl diisopropylchlorophospho-ramidite, in that order, to the reaction mixture.

20. Monitor the reaction by TLC (APPENDIX 3E) using a mobile phase of 2% triethylaminein 2:1 (v/v) acetone/hexanes. Visualize the TLC plate under a short-wave UV lamp.

The starting compound 26 will be completely consumed within 3 hr. The product 27 willdecrease in polarity due to the loss of a free monoalcohol group.

21. Concentrate the crude product in vacuo using a rotary evaporator.

22. Pack a 2.5 × 25–cm glass column with silica gel and load the crude product directlyonto the column for purification. Push the crude product through the column with


4.57.25


a step gradient of acetone/hexanes (1:1 v/v, then 2:1 v/v, then 3:1 v/v) containing2% triethylamine.

23. Combine all fractions containing compound 27 and concentrate on a rotary evap-orator to afford a white powder.

The phosphoramidite 27 is stable for 3 months stored at −20°C under vacuum or undernitrogen. The compound is extremely sensitive to moisture.

24. Characterize the compound by 1H/13C/31P NMR and ESI-HRMS.

2-(N-((1-(2-(2-(N4-(Benzoyl)cytosin-1-yl)-N-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(uracil-1-yl)acetamido)ethyl (2-cyanoethyl) diisopropylphosphoramidite. (27). Yield 266 mg (73%). 1H NMR (400MHz, CDCl3): δ 1.15-1.20 (m, 13H), 1.26 (s, 3H), 2.63-2.68 (m, 3H), 3.02 (br s, 1H),3.13-3.18 (m, 1H), 3.21 (t, 1H, J = 4.3 Hz), 3.24-3.28 (m, 1H), 3.55-3.67 (m, 6H),3.75-3.79 (m, 2.5H), 3.80 (s, 1.5H), 3.81 (s, 4.5H), 3.82-3.90 (m, 1.5H), 4.51 (t, 1.5H,J = 4.5 Hz), 4.55 (t, 0.5H, J = 4.7 Hz), 4.70 (s, 2H), 4.75 (d, 2.5H, J = 4.3 Hz),4.79 (d, 0.5H, J = 3.1 Hz), 4.88 (s, 1H), 5.66-5.69 (m, 1H), 6.85 (d, 4H, J = 9.0 Hz),7.21 (d, 2H, J = 2.3 Hz), 7.23 (m, 2H), 7.28-7.29 (m, 0.5H), 7.32 (d, 3.5H, J = 3.9Hz), 7.37-7.40 (m, 1H), 7.52 (t, 2.5H, J = 7.6 Hz), 7.60-7.63 (m, 1H), 7.79 (s, 0.5H),7.95-8.01 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 20.3, 20.4, 24.5, 24.6, 24.7, 26.6,29.2, 29.6, 31.7, 31.8, 41.7, 42.9, 43.0, 43.1, 43.5, 46.0, 47.1, 47.5, 47.8, 48.0, 48.5,48.8, 49.0, 51.0, 53.8, 55.2, 58.1, 58.2, 58.4, 60.6, 60.8, 60.9, 69.4, 87.2, 87.3, 96.8,101.6, 106.5, 113.1, 113.2, 117.9, 118.1, 123.6, 124.7, 124.9, 127.2, 127.8, 127.9,128.0, 128.1, 128.6, 128.8, 129.9, 130.1, 132.9, 133.0, 135.1, 143.3, 144.1, 144.2,146.1, 149.9, 151.1, 155.7, 158.4, 158.7, 163.3, 164.0, 164.2, 166.8, 167.5, 167.6;31P NMR (167 MHz, CDCl3): δ 149.02 and 149.56; ESI-HRMS (ES+) m/z calcd for[C58H67N12O11P + Na]+: 1161.4682, found 1161.4672 [M+Na]+.

BASICPROTOCOL 6

SYNTHESIS, PURIFICATION, AND CHARACTERIZATION OFOLIGONUCLEOTIDES CONTAINING CtU OR UtU UNITS

The triazole phosphoramidites are stable at −20°C as a solid for prolonged storage underinert atmosphere. They are capable of efficient coupling during RNA or DNA synthesis.The dimers do not have complete solubility in acetonitrile; however, using CH2Cl2 as aco-solvent completely solubilizes the phosphoramidites.

Oligonucleotide synthesis can be achieved on any automated DNA/RNA synthesizer.The authors use an Applied Biosystems 394 on both a 0.2-µmol and 1.0-µmol scaleusing standard 2′-deoxyribonucleoside phosphoramidites (for DNA) and 2′-O-TBDMSphosphoramidites (for RNA). A variety of techniques can be employed for isolationand purification of oligonucleotides bearing the triazole modification, such as reversed-phase or ion-exchange HPLC, or gel purification on PAGE gels. Denaturing PAGEpurification is described, and the molecular mass of the oligonucleotides can be verifiedby MALDI-TOF mass spectrometry on a Waters Tofspec-2E or a ESI q-TOF through aZorbax Extend C18 HPLC column in negative mode. Thermal denaturation experimentswith oligonucleotide duplexes were performed on a Jasco-815 spectrophotometer withtemperature control. Evaporation of small volumes was achieved with the use of aSpeedvac evaporation system.

Materials

Phosphoramidites:

2-(N-(2-(4-((N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(uracil-1-)acetamido)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-(uracil-1-yl)acetami-do)ethyl (2-cyanoethyl) diisopropylphosphoramidite (24; Basic Protocol 4)

2-(N-((1-(2-(2-(N4-(Benzoyl)cytosin-1-yl)-N-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-


Phosphoramidites

4.57.26


2-(uracil-1-yl)acetamido)ethyl (2-cyanoethyl)diisopropylphosphoramidite (27; Basic Protocol 5)

2′-TBDMS guanosine (n-ibu) phosphoramidite, �98% pure (ChemGenes,cat. no. ANP-5673)

2′-TBDMS cytidine (n-bz) phosphoramidite, 97.4% pure (ChemGenes, cat.no. ANP-5672)

2′-TBDMS adenosine (n-bz) phosphoramidite, 99.3% pure (ChemGenes,cat. no. ANP-5671)

2′-TBDMS uridine phosphoramidite, 98.8% pure (ChemGenes, cat. no.ANP-5674)

2′-Deoxyguanosine (n-ibu) phosphoramide, 99% pure (ChemGenes, cat. no.ANP-5553)

2′-Deoxyadenosine (n-bz) phosphoramidite, 99.2% pure (ChemGenes, cat.no. ANP-5551)

2′Deoxycytosine (n-bz) phosphoramidite, 99.5% pure (ChemGenes, cat. no.ANP-5552)

5′-DMT thymidine phosphoramidite, �98.0% pure (ChemGenes, cat. no.ANP-5554)

2-[2-(4,4′-Dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (chemical phosphorylating reagent (ChemGenes, cat. no.CLP-1544), �98.1% pure

Acetonitrile (ACN), anhydrousDichloromethane (CH2Cl2), anhydrous, �99.8%Nitrogen (or argon) gasAcetic anhydride/pyridine/THF (Cap A)16% N-methylimidazole (ChemGenes, cat. no. RN-7776) in THF (Cap B)5-ethylthio tetrazole (activator; ChemGenes, cat. no. RN-1466), 0.25 M in ACN0.02 M iodine/pyridine/H2O/THF (oxidation solution)3% trichloroacetic acid/dichloromethaneEMAM: 1:1 mixture of 40% (w/v) methylamine in H2O and 33% (w/v)

methylamine in ethanolDimethylsulfoxide (DMSO), minimum 99.5% GCTriethylamine trihydrofluoride (3HF/TEA), 98% pureEthanol (EtOH), 95%3 M sodium acetate (NaOAc), pH 5.2Nuclease-free H2O40% acrylamide (see recipe)10× and 0.5× TBE buffer (see recipe)Urea, 99.5% pure25% (w/v) ammonium persulfate (APS) (see recipe)Tetramethylethylenediamine (TEMED)Denaturing loading solution (see recipe)Ethidium bromide, >98.0%Dry ice/95% ethanol bathGel eluting buffer (see recipe)Matrix solution for MALDI-TOF (see recipe)Matrix solution for ESI Q-TOF (see recipe)Sodium phosphate buffer (see recipe)

DNA/RNA synthesizer (e.g., Applied Biosystems 394; see APPENDIX 3C)0.2 µM or 1.0 µM CPG 500 (with desired nucleoside bound)0.2 µM or 1.0 µM Universal III solid supports50-mL conical centrifuge tubes (e.g., BD Falcon)10-mL syringe


4.57.27


1.5-mL screw-cap vialsNeedle to puncture lid of screw-cap vialSpeedvac evaporatorParafilm65°C water bathSpectrophotometer (Thermo Scientific, cat. no. 840-208200)UV-compatible cuvetteGel platesSpacersGasketCombClamps100-mL beakerStir barGel electrophoresis apparatus (see UNIT 10.4 & APPENDIX 3B)SpatulaShakerLarge TLC plateShort-wavelength UV lampCameraScalpelSkinny ScoopulaCentrifuge pestle (Fisher Scientific cat. no. 05-559-26)Shaker0.4-µm syringe filterMWCO 3000 cellulose centrifugal filterMillipore ZipTip C18-column micropipet tips (see UNIT 10.1)Metal sample plate (UNIT 10.1)Zorbax Extend C18 HPLC column (UNIT 10.5)Quartz cuvettes, 1 mm path lengths, Teflon capsSpectropolarimeter (e.g., Jasco)Meltwin software version 3.5 (http://www.meltwin3.com/)

Additional reagents and equipment for automated solid-phase oligonucleotidesynthesis (APPENDIX 3C) and PAGE purification of oligonucleotides (UNIT 10.4 &APPENDIX 3B), MALDI-TOF mass spectrometry (UNIT 10.1), electrospray ionizationmass spectrometry (UNIT 10.2), and HPLC purification of oligonucleotides(UNIT 10.5)

Synthesizer preparation and oligonucleotide synthesis on an ABI 394 synthesizer

1. Dissolve all of the commercial phosphoramidites and the chemical phosphorylationreagent (if required) in anhydrous acetonitrile to a concentration of 0.1 M. Dissolvethe chemically synthesized triazole-modified phosphoramidites to a concentrationof 0.1 M using a 3:1 (v/v) acetonitrile:CH2Cl2 solvent ratio.

2. Turn on the DNA/RNA synthesizer (APPENDIX 3C) and open the nitrogen tank to apressure of 55 psi.

3. Connect the phosphoramidite bottles, capping reagents, activator, I2/water/pyridine, 3% trichloroacetic acid in CH2Cl2, and 2-[2-(4,4′-dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (chemicalphosphorylating reagent) to the synthesizer.

4. Attach a 0.2-μm or 1.0-μm CPG 500 (depending on the amount of modifiedoligonucleotide that needs to be synthesized) to the synthesizer in order to constructan oligonucleotide with a site-specific modification lying outside of the 3′-end.


Phosphoramidites

4.57.28


Sequences containing 3′-modifications need to be synthesized on a 0.2 µmol or 1.0 µmolUniversal III solid support.

5. Enter the order in which the nucleosides will be added to the solid support on thesynthesizer and ensure that the coupling time is set to 16 min for each phospho-ramidite.

Oligonucleotides are synthesized on the solid support in the 3′ to 5′ direction.

When entering in the triazole-nucleoside phosphoramidites, remember that the phos-phoramidites are dimers and will therefore take the place of TWO nucleosides ratherthan one on the instrument.

If a Universal III column is being used, enter in an additional base at the 3′-end ofthe oligonucleotide sequence in order to remove the DMT-protected group from theuniversal column.

Additionally, if the oligonucleotide needs to be phosphorylated at the 5′ end, incorporatethe chemical phosphorylation reagent into the cycle after the last DMT removal.

6. Start the automated solid-phase oligonucleotide synthesis and elongate the desiredoligonucleotide chain.

7. Upon completion, remove the column from the synthesizer and thoroughly dry thesupport in a stream of nitrogen gas. Place the column in a sealed 50-mL conicalcentrifuge tube and store it at −20°C.

Oligonucleotide cleavage from the solid support and deprotection

8. Connect a clean 10-mL syringe to the luer fitting of the column.

Avoid syringes that have a rubber plunger.

9. With a second syringe, take up 1 mL of EMAM. Connect the second syringe to theother luer fitting of the column and gently pass the solution through the columnfour to five times.

10. Push the solution so that it is in full contact with the controlled pore glass and letthe column stand for 30 min at room temperature.

11. Transfer the solution to a clean 1.5-mL screw-cap vial.

12. Rinse the column with an additional 0.5 mL of EMAM and add the solution to thescrew-cap vial, giving a total volume of 1.5 mL.

13. Incubate oligonucleotides in EMAM overnight at room temperature to deprotectthe bases.

14. On the following day, perforate the lid of the screw-cap vial and concentrate theoligonucleotide on a Speedvac evaporator overnight.

15. Once dried, seal the perforated cap with Parafilm and store the sample at 4°C.

If oligonucleotide consists of DNA, the following three steps (16 to 18) are not necessary

16. Resuspend the sample in a solution of DMSO:3HF/TEA (100 µL:125 µL) andtransfer the contents to a new 1.5-mL screw-cap vial. Incubate in a 65°C waterbath for 2.5 hr in order to remove the 2′-O-TBS groups (UNIT 3.21)

17. Perforate the screw cap and dry down the sample on a Speedvac evaporatorovernight.

18. Once dried, seal the cap with Parafilm and store the sample in a refrigerator(4°C).


4.57.29


EtOH precipitation

19. Resuspend the oligonucleotides in 600 µL of EtOH, chilled in dry ice.

CAUTION: Dry ice is extremely cold. Wear heavy gloves or use tongs to handle dryice.

20. Add 25 µL of 3 M NaOAc (pH 5.2).

21. Vortex the sample and place it in dry ice for 1 hr.

22. Microcentrifuge the sample 30 min at 12,000 rpm, 4°C, and remove as muchsupernatant as possible.

23. Resuspend the pellet in 600 µL of cold EtOH and repeat the process two to threetimes without adding any additional NaOAc.

24. After the final removal of the supernatant, dry down the sample using a Speedvacevaporator.

Oligonucleotide quantification

25. Turn on the spectrophotometer and allow a 30-min warm-up period.

26. Redissolve the sample in 200 µL of nuclease-free water.

27. Add 999 µL of nuclease-free water to a 1-mL UV-compatible cuvette and zero thespectrometer at 260 nm.

28. Without removing the cuvette, add 1 µL of the redissolved sample to the 999 µLof nuclease-free water and mix the solution thoroughly.

29. Measure the absorbance of the diluted sample at 260 nm and record the reading.

30. Multiply the absorbance value by the total volume of the stock oligonucleotidesample (200 µL) to obtain an optical density (OD) value.

Prepare a PAGE gel

31. Prepare the gel plates, spacers, ampersal gasket, and comb using the manufacturer′srecommended protocol (UNIT 10.4 & APPENDIX 3B).

32. In a 100 mL beaker equipped with a stir bar, add 30 mL of 40% acrylamide, 6mL of 10× TBE buffer and 25.23 g of urea (7 M final) topped up to 60 mL withnuclease-free water. Stir vigorously until all of the urea dissolves.

33. Add 80 µL of 25% ammonium persulfate (APS) and 80 µL of TEMED and mixfor 30 sec.

Shaking the solution too vigorously will create air bubbles that will stay trapped withinthe matrix when pouring the solution between the gel plates.

34. Pour the solution into the prepared gel plates (0.5 cm from the top) and ensure thatthere are no air bubbles present between the plates.

35. Insert the 12-well comb at the top of the gel and dislodge any air bubbles betweenthe comb and the gel by tapping on the glass plates.

36. Allow the gel to set for 45 min.

The gel can be placed in the refrigerator at 4°C and used the next day.

Running a PAGE gel

37. Remove the gasket surrounding the plates by feeding it through the clamps.

38. Remove the clamps and remove the comb from the polymerized gel.


Phosphoramidites

4.57.30


39. Place the gel plates in the gel electrophoresis apparatus and secure the plates assuggested by the manufacturer.

40. Fill the top and bottom reservoir of the gel apparatus with 0.5× TBE running buffer(25 mL of 10× TBE in 475 mL of water) until the buffer is in contact with boththe anode and cathode.

41. Remove any air bubbles trapped under the gel plates by using a syringe filled with0.5× TBE running buffer from the reservoir.

The same method can be used for air bubbles trapped within the wells of the topreservoir.

42. For visualizing oligonucleotide purity, combine 0.05 to 0.5 OD units of oligonu-cleotide with denaturing loading solution containing bromphenol blue dye for atotal volume of 20 µL and add to each well.

43. Connect the electrophoresis apparatus to the power supply, adjust the voltage to300 V, and let the gel run for 5 hr (or until the bromphenol blue band has traveled75% of the gel′s length).

Visualization of oligonucleotide on gel

44. Upon completion, separate the gel from the glass plates using a spatula.

45. Stain the gel in a dilute solution of ethidium bromide in 0.5× TBE running bufferfor 30 min. Gently stir the solution on a shaker (100 rpm).

46. Place the gel on a fluorescent silica background (e.g., on a large TLC plate) andimage it under a short-wavelength UV lamp.

DNA and RNA absorb UV light and cast a shadow on the fluorescent silica background.

47. Take a picture of the gel and make a record of the migrated distances of the bandsof interest.

Crush and soak oligonucleotide purification

48. For purification, set up and run the gel in the same manner as described above upto and including step 44, with the exception of step 42. Instead of combining 0.05to 0.5 ODs in each well with denaturing loading solution for a total volume of 20µL, add a maximum of 2 ODs of oligonucleotide per well.

49. Place the gel on a fluorescent silica background and image the gel under a short-wavelength UV lamp.

50. Physically excise the bands of interest with a clean scalpel and collect each indi-vidual slice in its own 1.5-mL microcentrifuge tube.

UV light will damage the oligonucleotide, so it is important to excise the bands asquickly (and as safely) as possible.

All bands of interest should be those that had the slowest migratory rate, and shouldtherefore be the bands located closest to the wells at the top of the plate.

51. Freeze the gel pieces in a dry ice/EtOH bath for 5 min.

52. Chop up the pieces as much as possible using a skinny Scoopula.

53. Re-freeze the chopped up pieces in the dry ice/EtOH bath.

54. Suspend the chopped-up pieces in 500 µL of cold gel eluting buffer and quicklychop up the gel pieces even further with a centrifuge pestle as they begin to defrostin the buffer.


4.57.31


A centrifuge pestle works better than a skinny Scoopula to grind the chopped up gelpieces to further increase the surface area of the gel slabs and ultimately give you abetter oligonucleotide yield. The pieces must first be chopped up with a skinny Scoopula(instead of using the centrifuge pestle twice) because the pestle is inefficient in grindingone large gel slab but effective in grinding a gel slab that has been chopped up.

55. Re-freeze the slurry in the dry ice/EtOH bath for 15 min and then leave it to thawfor another 15 min at room temperature.

56. Incubate the slurry for 48 hr at 37°C on a shaker at 200 rpm.

57. Filter the slurry through a 0.4-µm syringe filter and collect the filtrate in a ster-ile microcentrifuge tube. Alternatively, microcentrifuge the slurry for 30 min at12,000 rpm, room temperature, and collect the supernatant in a sterile microcen-trifuge tube.

58. Perforate the caps of the microcentrifuge tubes using a needle and concentrate thesamples in the Speedvac evaporator.

59. Collect all of the samples in one microcentrifuge tube in preparation for EtOHprecipitation. Perform EtOH precipitation as described in steps 19 to 24, above.

Desalting of gel-purified oligonucleotides

60. Dissolve the dried sample from step 59 in 500 µL of nuclease-free water.

61. Transfer the dissolved sample to an MWCO 3000 cellulose centrifugal filter andmicrocentrifuge �10 min at 12,000 rpm at 25°C, or until there is roughly 50 µLof oligonucleotide solution left.

62. Add 400 to 500 µL of nuclease-free water to the remaining amount of oligonu-cleotide solution and re-spin the sample.

63. Repeat the process for a total of three to four times.

64. Collect the remaining 50 µL sample in a microcentrifuge tube with a perforatedcap.

65. Dry down the sample using a Speedvac evaporator.

66. Resuspend the purified sample in 100 µL of nuclease-free water and quantify aspreviously described.

Characterization of oligonucleotides using MALDI-TOF

67. Elute the chemically modified oligonucleotides through Millipore Zip-Tip C18-column micropipet tips to a concentration of 5µM (procedure described in UNIT 10.1).

68. Add 5 µL of matrix solution for MALDI-TOF to each sample vial.

69. Allow the samples to dry for 30 min and run a MALDI-TOF analysis (UNIT 10.1) toconfirm the weight of the modified oligonucleotide.

Characterization of oligonucleotides using ESI Q-TOF

Electrospray ionization (ESI) mass spectrometry is discussed in UNIT 10.2.

70. Elute the chemically modified oligonucleotides through a Zorbax Extend C18HPLC column with matrix solution for ESI Q-TOF, and finally 70% MeOH(UNIT 10.5)

71. Subject the oligonucleotides to ESI-MS (ES-) and produce a raw spectrum ofmultiply charged anions. See UNIT 10.2.

72. Using resolved isotope deconvolution, confirm the molecular weights of the resul-tant neutral oligonucleotides.


Phosphoramidites

4.57.32


Characterization of double-stranded oligonucleotides utilizing UV-monitoredthermal denaturation

73. To a microcentrifuge tube, add equimolar amounts of chemically modified DNAor RNA oligonucleotide (0.5 ODs) to their complementary sequence in 500 µL ofsodium phosphate buffer.

74. Denature the strands by increasing the temperature of the mixture to 90°C for 2min in a hot water bath, and let the mixture slowly cool to room temperature alongwith the bath.

75. Add the 500-µL samples to quartz cuvettes with 1-mm path lengths, sealed withTeflon caps.

76. Place the cuvettes in a spectropolarimeter and set the UV absorbance to 260 nm.

77. Increase the temperature of the samples from 10 to 95°C at a rate of 0.5°C/minwith the absorbance measured at the end of each 0.5°C increment.

78. Adjust the absorbance readings against the baseline absorbance of sodium phos-phate buffer.

79. After averaging the range of absorbance values obtained from three independentexperiments, calculate the Tms using Meltwin software version 3.5 and assume theVan′t Hoff two state model.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stocksolutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Acrylamide, 40%

380 g acrylamide20 g bisacrylamideNuclease-free H2O for total volume of 1 L

Let the solution stir overnight to dissolve all of the added acrylamide; do not apply heat,which can unwantedly aid in polymerization of the gel. Avoid exposure to light duringpreparation. Store in dark bottles at 4°C; stable for several weeks.

Ammonium persulfate (APS), 25%

375 mg ammonium persulfate1.25 mL nuclease-free H2OStore up to 1 month at 4°C

Denaturing loading solution

74 mg EDTA4.8 g urea50 µL 1 M Tris base (pH 7.5 adjusted with concentrated HCl)1.5 mL glycerol25 mg bromphenol blueNuclease-free H2O for a total volume of 10 mLStore up to 1 year at room temperature

Gel eluting buffer

1 mL 3 M sodium acetate (pH 5.2 adjusted with glacial acetic acid; APPENDIX 2A)2.9 mg EDTAAdjust pH to 7.0 with NaOH pelletsAdd nuclease-free H2O for a total volume of 10 mLStore up to 1 year at room temperature


4.57.33


Matrix solution for ESI Q-TOF

Prepare an MeOH/water (5:95) solution containing 200 mM hexafluoroisopropylalcohol (Sigma-Aldrich, cat. no. 105228) and 8.1 mM triethylamine. Store up to 2weeks at 4°C.

Matrix solution for MALDI-TOF

25 mg/mL 3-hydroxypicolinic acid (Sigma-Aldrich, cat. no. 56197)50:50 acetonitrile:5 mg/mL aqueous ammonium citrateStore up to 2 weeks at 4°C

Potassium permanganate (KMnO4) stain

0.75 g of KMnO4

5 g K2CO3

0.625 mL 10% (w/v) NaOHH2O for a total volume of 100 mLStore up to 3 months at 4°C

Sodium phosphate buffer

90 mM NaCl10 mM Na2HPO4

1 mM EDTAAdjust to pH 7 with NaOHStore up to 1 year at 4°C

TBE buffer, 10×108 g Tris base55 g boric acid40 mL 0.5 M EDTAAdjust pH to 8.3 with NaOHNuclease-free H2O for a total volume of 1 LStore up to 6 months at room temperature

COMMENTARY

Background InformationThe protocols described herein are intended

to alter the structure-activity profile of theoligonucleotide through the incorporation of atriazole backbone. Using this strategy in otherstudies, we have synthesized short-interferingRNAs, constructs which offer high activity asgene-silencing agents. Furthermore, we ob-serve a decrease in thermal stability whenthese modifications are incorporated internallywithin a DNA- or RNA-based oligonucleotide,and observe an increase in nuclease stabil-ity when these modifications are placed as3′-overhangs within siRNAs (Efthymiou andDesaulniers, 2011; Efthymiou et al., 2012b).

A number of different triazole-backboneoligonucleotide scaffolds have been devel-oped over the years since the first publica-tion of the robust copper(I)-catalyzed (3+2)cycloaddition (Rostovtsev et al., 2002). Themajority of these systems are DNA based

and share the deoxyribose sugar core; sev-eral of them do not utilize phosphoramiditechemistry as the basic building block to serveas the triazole-backbone core (vonMatt andAltmann, 1997; Chittepu et al., 2008; Isobeet al., 2008; Lucas et al., 2008; Fujino et al.,2009). Work by El-Sagheer and Brown (2009)identified that DNA-based oligomers can becyclized together and are compatible withpolymerase chain reaction protocols. Fujinoand coworkers have designed oligomers of de-oxyribonucleotides through iterative cycload-ditions. Examples of DNA-based triazole-phosphoramidites include the following stud-ies: Chandrasekhar et al. (2010) and Varizhuket al. (2012). Examples of triazole-backboneunits within RNA include the following stud-ies: El-Sagheer and Brown (2010) and Fu-jino et al. (2012). Work by Rozners and co-workers involved the synthesis of a UTRAphosphoramidite in which a 2′-O-propargyl


Phosphoramidites

4.57.34


ribonucleoside and 5′-azido ribonucleoside arecyclized (Mutisya et al., 2011). In their study,the distance between adjacent nucleobases in-troduced through the triazole linkage was 8atoms, whereas our triazole-based linkage pro-duces a distance of 7 atoms between adjacentbases.

Both the UtU and CtU phosphoramiditesyntheses presented here are based on aPNA-type scaffold, and as such the structuredoes not utilize a sugar moiety. This inher-ent flexibility allows the modification to beincorporated within both DNA- and RNA-based oligonucleotides, as indicated by UV-monitored thermal denaturation studies (seeTable 4.57.1). The ability to use a phospho-ramidite allows the user to site-specifically in-corporate the desired modification within a re-spective oligonucleotide. The characteristicsof the RNA duplex with triazole units inter-spersed are consistent with the canonical A-type helical conformation.

Synthesis of the triazole phosphoramiditesPreparation strategies for both carboxylic

acid derivatives of the uracil (2) and cy-tosine (5) pyrimidine bases were adoptedfrom already published protocols and proceedsmoothly with high yield without the require-ment of column chromatography (Christensenet al., 1998; Liu et al., 2000). The alkyneand azide linkers are synthesized from readilyaccessible starting materials. For the synthe-sis of 8 from 7, care must be taken to en-sure that the propargyl bromide is the lim-iting reagent and is added slowly and drop-wise to the solution to minimize dialkylatedproduct. The synthesis of 2-azidoethanaminefrom 2-bromoethylamine proceeds smoothly;however, caution must be exercised when us-ing sodium azide and in the handling of theorganoazide product 10. Alkylation of 10 toform 11 with the excellent electrophile ethyl-bromoacetate proceeds smoothly. Care mustbe taken that ethylbromoacetate is the limit-ing reagent and that it is added dropwise andslowly to the stirring solution of 10 to mini-mize dialkylation.

With both the azide and alkyne linkers andthe respective acids of the bases, monomerpreparation is accomplished. In our attempts,we observed that EDC-Cl was a very effectivecarbodiimide coupling agent for the synthesisof both 15 and 17. The advantages of usingEDC-Cl over other traditional carbodiimidecoupling reagents such as DCC or DIC includehigher yields and easier post-reaction purifica-tion. The urea byproduct derivative of EDC-Cl

is water soluble, and is easily removed dur-ing extraction. The synthesis of 16 was doneby using a combination of DCC/HOBt. Em-ploying this method, DCU is filtered off asa byproduct, and then purification by columnchromatography ensures a pure product. Theyields are in the 53% to 65% range for thesecouplings, which are quite acceptable giventhe steric hindrance of the secondary amineutilized for these reactions.

Cyclization of 15 with 16 to providecompound 20 proceeds smoothly in nearlyquantitative yield using standard conditionsof copper (II) sulfate, ascorbic acid, and aco-solvent mixture of THF/t-BuOH/H2O ina 1:1:1 (v/v/v) ratio. Following this step,selective reduction of the ester in the presenceof the uracil bases proceeds quickly and ingood yield with LiBH4. Care must be takento continuously monitor the reaction by TLC,to avoid reduction of the multiple amidebonds throughout the molecule. Silyl groupdeprotection and DMTr-group installationproceed smoothly, along with phosphitylationto afford the final phosphoramidite 24.

During the cyclization of the cytosine azidemonomer 19 with the uracil alkyne monomer15, we need to adjust the solvent parameters ofthe reaction to optimize the yield. A co-solventratio of THF:H2O of 3:2 (v/v) is used to obtaina near quantitative yield. Interestingly, using t-BuOH has been detrimental to the overall yieldof the reaction for reasons that are not entirelyclear to us. Once cyclization occurs, depro-tection steps and phosphitylation occur as ex-pected, to afford the desired phosphoramidite27.

Synthesis of oligonucleotides containingUtU or CtU residues

Neither of these phosphoramidites are fullysoluble in a solution of anhydrous acetonitrile.Solubility is achieved by using a 3:1 (v/v)ratio of acetonitrile to dichloromethane. Au-tomated solid-phase oligonucleotide synthesisis done on an ABI 394 RNA/DNA synthesizerusing the manufacturer’s protocols. The onlyparameter that is adjusted during the synthesisis the increased coupling time for the modi-fied phosphoramidites, from 10 min for stan-dard RNA synthesis to 16 min. The oligonu-cleotides are synthesized either internally orat the 5′-end with DMTr-OFF mode with stan-dard columns, or at the 3′-end with a Universalsupport. MALDI-TOF mass spectra or q-TOFmeasurements confirm the mass of the respec-tive oligonucleotides.


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Table 4.57.1 Melting Temperatures of Duplexes Containing UtU or CtU Modifications

Duplex Duplex sequence Tm (°C) �Tm (°C)

RNA 1 5′-CUUACGCUGAGUACUUCGAtt-3′ 71.4 —

3′-ttGAAUGCGACUCAUGAAGCU-5′

RNA 2 5′-CtUUACGCUGAGUACUUCGAtt-3′ 71.1 −0.3


RNA 3 5′-CtUUACGCUGAGUACtUUCGAtt -3′ 51.1 −20.3


RNA 4 5′-CUtUACGCUGAGUACUUCGAtt-3′ 67.9 −3.5


RNA 5 5′-CUUACGCtUGAGUACUUCGAtt-3′ 58.4 −13.0


RNA 6 5′-CUUACGCUGAGUACtUUCGAtt-3′ 59.6 −11.4


RNA 7 5′- CUUACGCUGAGUACUtUCGAtt - 3′ 62.9 −8.5


RNA 8 5′-CUUACGCUGAGUACUUCGAUtU-3′ 72.1 +0.7


RNA 9 5′-CUUACGCUGAGUACUUCGAUtU-3′ 72.5 +1.1

3′-UtUGAAUGCGACUCAUGAAGCU-5′

RNA 10 5′-CUtUACGCUGAGUACUtUCGAUtU-3′ 57.3 −14.1


RNA 11 5′-CUUACGCUGAGUACUUCGAtt-3′ 73.1 +1.7

3′-UtUGAAUGCGACUCAUGAAGCU-5′

DNA 1 5′-TTTTTCTCTCTCTT-3′ 40.9 —

3′-AAAAAGAGAGAGAA-5′

DNA 2 5′-TTTTTCTCTCTCUtU-3′ 38.1 −2.8


DNA 3 5′-TTTTTCTCTCTC-3′ 37.1 −3.8


DNA 4 5′-UtUTTTCTCTCTCTT-3′ 36.4 −4.5


DNA 5 5′-TTTCTCTCTCTT-3′ 35.3 −5.6

3′-AAAAAGAGAGAGAA -5′

Effect of triazole-modifications on duplexstability

Melting studies performed with UtU andCtU residues placed within both DNA andRNA duplexes confirmed an overall destabi-lization effect. These observations are sup-ported by other studies which place triazole-modifications at single- or double-specific po-sitions (vonMatt and Altmann, 1997; Mutisyaet al., 2011). The UtU modification onthe 3′-overhang produces duplexes with

slightly elevated thermostabilities, perhapsdue to increased stacking interactions occur-ring between the free dimer and the duplex(Efthymiou et al., 2012b).

One application for these triazole-modifiedoligonucleotides is illustrated in a previouspublication, where we placed these modifi-cations within siRNAs and observed activegene silencing, especially when we positionedthem within the 3′ region of the sense strand.This observation supports the thermodynamic


Phosphoramidites

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asymmetry rule of siRNAs, which relatesheightened siRNA activity to the destabiliz-ing effects caused by modifications within thisregion (Schwarz et al., 2003). In addition toincreases in duplex thermostability, siRNAswith UtU dimers as their 3′-overhangs exhib-ited increase nuclease stability in our previousstudy.

Critical ParametersThe synthesis of both UtU and CtU phos-

phoramidites proceeds using relatively stan-dard materials. However, care must be takenwhen performing steps in basic organic chem-istry. Preparation of the various compoundsrequires some form of prior experience withextraction, TLC, and column chromatography.Purification of oligonucleotides is performedby gel electrophoresis (UNIT 10.4); however,HPLC is also another widely used method ofpurification (UNIT 10.5). Characterization of thesmall molecules and the oligonucleotides by1H, 13C, and 31P NMR, UV, and MALDI-TOFor q-TOF are accomplished in order to confirmthe structures and masses of each compound.General laboratory safety is needed when ex-ercising these reactions; adherence to the out-lined methods is highly recommended.

Anticipated ResultsGood to moderate yields are anticipated

when using the present protocols to gener-ate either the UtU or CtU phosphoramidite.Many of the initial steps are relatively simpleschemes that do not require purification forsubsequent reaction. Automated solid-supportoligonucleotide synthesis on either a 0.2 µmolor 1.0 µmol column proceeds smoothly.

Time ConsiderationsThe synthesis of the UtU phosphoramidite

starting from the basic building blocks can beaccomplished in 2 to 3 weeks. For the synthe-sis of the CtU phosphoramidite, 3 to 4 weeksare required, due to the increase in steps re-quired for cytosine protection and the use ofthe alternative azide linker (14) instead of theazide linker (11) used for the UtU dimer. Inpreparing for both the CtU and UtU phos-phoramidites, the authors suggest that a largeamount of 15 (>2 g) be synthesized due itsrole in both of the UtU and CtU synthesis. Thelongest step of the entire synthetic scheme in-volves the alkylation of 13 with 10 to afford14. This requires 48 hr, so proper planningcan ensure that this step is not a rate-limitingstep. The synthesis, purification and character-ization of oligonucleotides are the same withstandard techniques.

AcknowledgmentsThis work was supported by the Natural

Sciences and Engineering Research Councilof Canada and the Canada Foundation forInnovation.

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Phosphoramidites

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current protocols in nucleic acid chemistry || synthesis of triazole-nucleoside phosphoramidites and...

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