Synthesis of Fluorescence Turn-On DNA Hybridization Probe using the ^DEAtC 2’-deoxycytidine Analogue

Abstract

^DEAtC is a tricyclic 2’-deoxycytidine analogue that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with guanine base in a DNA–DNA duplex. The synthesis of ^DEAtC begins with 5-amino-2-methylbenzothiazole and provides the ^DEAtC nucleobase analogue over four synthetic steps. This nucleobase analogue is then silylated using BSA and conjugated to Hoffer’s chlorosugar to provide the protected ^DEAtC nucleoside in good yield. Following protective group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offered the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the ^DEAtC amidite and a short deprotection time are used to maximize efficiency. By following the protocol described in this unit, the ^DEAtC fluorescent probe can be synthesized and incorporated into any desired synthetic DNA oligonucleotide.

INTRODUCTION

Fluorescent nucleoside analogues have found widespread application in biophysics because of their capacity to report on nucleic acid conformation, recognition, structure, and sequence.(Sinkeldam et al., 2010; Wilhelmsson, 2010; Tainaka et al., 2007; Shin et al., 2011; Rovira et al., 2015; Börjesson et al., 2009; Koga et al., 2011; Cservenyi et al., 2016; Mata et al., 2016) Conventional fluorophores such as fluorescein can be tethered to nucleosides (Zhu et al., 1994) or appended to either terminus of an oligonucleotide, as is widely done. The imprecision inherent to probe tethering limits the opportunity to study precise mechanistic details of DNA/RNA metabolism (Stengel et al., 2007) and sequence specificity is limited to differential hybridization affinity (Riahi et al., 2011). In contrast, the use of fluorescent nucleobase analogues affords the capacity for detailed observations of processes such as base flipping (Roy, 2003), G quadruplex structure and dynamics (Dumas and Luedtke, 2010; Tanpure and Srivatsan, 2015), and optical discrimination of sequence (Suzuki et al., 2014). Our group has recently developed 8-diethylamino-tC (^DEAtC), a new member of the tricyclic cytidine family (Burns et al., 2017; Preus et al., 2010; Rodgers et al., 2014). ^DEAtC has little fluorescence as a free nucleoside and is modestly brighter in single-stranded DNA, but it has a substantial increase in fluorescence intensity upon DNA duplex formation when correctly base-paired with guanine residue in the complementary strand (Figure 1). This fluorescence turn-on has some dependence on the identity of neighboring bases, an aspect that should be considering when planning applications (for a detailed discussion, see Commentary).

Figure 1.

^DEAtC is a 2’-deoxycytidine analogue capable of Watson–Crick hydrogen bonding that has a fluorescence turn-on response to matched DNA duplex formation.

The Protocol described herein conveys detailed information on the synthesis of the ^DEAtC probe and its incorporation into synthetic DNA oligonucleotides using the conventional phosphoramidite method of solid-phase synthesis. Details are provided for the conversion of 5-amino-2-methylbenzothiazole into the ^DEAtC nucleobase analogue, which is then conjugated to Hoffer’s chlorosugar to provide the 2′-deoxyribonucleoside in five synthetic steps (see Basic Protocol 1). Protective group removal, dimethoxytritylation, and phosphoramidite synthesis furnished the probe for incorporation during solid-phase DNA synthesis. Solid-phase DNA synthesis is performed on a 0.2 μmol scale using simple modifications to standard conditions that account for ^DEAtC’s properties. Like other modified phosphoramidites, extended coupling time, capping, and oxidation is recommended.

BASIC PROTOCOL 1

PREPARATION OF THE ^DEAtC PHOSPHORAMIDITE

This protocol describes the synthesis of the ^DEAtC nucleoside 6 and its dimethoxytrityl-protected phosphoramidite 8, which is the standard material used in solid-phase DNA synthesis (Figure 2). The synthesis is related to methods for synthesizing other tC derivatives (Lin et al., 1995; Sandin et al., 2007; Rodgers et al., 2014). The protocol begins with the commercially available starting material 5-amino-2-methylbenzothiazole, which is first doubly ethylated at the amino group to provide the diethylamino-derivative. The thiazole ring is then opened using hydrazine as a nucleophile, and the resulting thiol is oxidized aerobically by exposure to the air to provide the disulfide 2, which facilitates purification and handling. When the synthetic chemist is ready to perform the next step, the disulfide is conveniently reduced using triethylphosphine and conjugated to 5-bromouracil in a one-pot procedure to give thioether 3. Acid-catalyzed condensation completes the stable ^DEAtC nucleobase 4. This nucleobase is glycosylated using Silyl-Hilbert-Johnson conditions (Niedballa and Vorbrüggen, 1974) and commercially available Hoffer’s chlorosugar (Hoffer, 1960) to provide the protected nucleoside 5 as a mixture of anomers, which are easier to separate after deprotection to give 6. Standard conditions for dimethoxytrityl group installation and phosphoramidite synthesis provide 8, which is then purified and dried in preparation for solid-phase oligonucleotide synthesis (Basic Protocol 2).

Figure 2.

Synthetic scheme for the ^DEAtC phosphoramidite 8.

Materials

5-Amino-2-methylbenzothiazole, 99% (Alfa Aesar)

Anhydrous dimethyl sulfoxide (DMSO), 99.9% (Sigma-Aldrich)

Anhydrous potassium carbonate (K₂CO₃), 99% (Fisher Scientific)

Bromoethane, 98% (Alfa Aesar)

Nitrogen (N₂)

Methanol (MeOH), (Fisher Scientific)

Dichloromethane (Methylene chloride, DCM), (Fisher Scientific)

Anhydrous sodium sulfate, granular (Na₂SO₄), 99% (Fisher Scientific)

Ethyl acetate (EtOAc), (Fisher Scientific)

Hexane, (Fisher Scientific)

Silica gel, (Fluka Analytical)

Hydrazine hydrate solution, 64% in water (Sigma Aldrich)

Triethylphosphine solution (PEt₃), 1 M in tetrahydrofuran (THF) (Aldrich)

Anhydrous dimethylformamide (DMF), 99.9% (Sigma-Aldrich)

5-bromouracil, 98% (Alfa Aesar)

Anhydrous sodium carbonate (Na₂CO₃), 99.5% (Acros Organics)

Anhydrous diethylene glycol dimethyl ether (Diglyme), 99.5% (Sigma Aldrich)

Anhydrous ethyl ether (Et₂O), 99.9% (Fisher Scientific)

Anhydrous 1-butanol (1-BuOH), 99% (Acros Organics)

Concentrated hydrochloric acid (HCl), 36.5% (Fisher Scientific)

Ammonium hydroxide solution (NH₄OH), 25% in water (Acros Organics)

Anhydrous acetonitrile (MeCN), 99.9% (Fisher Scientific)

N,O-Bis(trimethylsilyl)acetamide (BSA), 95% (Acros Organics)

3’,5’-di-O-(p-toluoyl)-2’-deoxy-α-D-ribofuranosyl chloride (Hoffer’s chlorosugar), 95% (Accel Pharmtech)

Anhydrous tin (IV) chloride (SnCl₄), (Acros Organics)

Sodium bicarbonate (NaHCO₃), (Fisher Scientific)

Anhydrous methyl alcohol (MeOH), 99.9% (Acros Organics)

Sodium methoxide (NaOMe), 30% in MeOH (Acros Organics)

Glacial acetic acid (AcOH), 99.9% (Fisher Scientific)

Ethanol (EtOH), 95% (Fisher Scientific)

Chloroform (CHCl₃), 99.8% (Fisher Scientific)

Anhydrous pyridine, 99.5% (Acros Organics)

Triethylamine (TEA), 99.5% (Fisher Scientific)

4,4-dimethoxytrityl chloride (DMTrCl), 98% (Acros Organics)

Alumina, basic, 60–325 mesh, Brockman Activity I (Fisher Chemical)

N,N-Diisopropylethylamine, 99% (TCI)

2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, 97% (Acros Organics)

250- and 100-mL three-neck round-bottom flasks

50-mL round-bottom flask

25-mL Schlenk tube

50-mL Schlenk tube

25-mL side-armed round-bottom flask

24-, 12-, and 4-g flash chromatography silica gel columns [RediSep® Rf or equivalent]

Magnetic stir plate with hotplate function and Teflon-coated magnetic stir bars

Vacuum pump

TLC plates (SiliaPlate TLC Plates, Aluminum-Backed, Silica, 200 μm, UV indicator or equivalent)

• 1.Dissolve 5.6 g (29 mmol) of 5-Amino-2-methylbenzothiazole in 15 mL of anhydrous dimethylsulfoxide in a 250-mL three-necked round-bottom flask using magnetic stirring under nitrogen gas.
• 2.Sequentially add 8.0 g (58 mmol) of anhydrous potassium carbonate and inject 8.6 mL (116 mmol) of bromoethane.
• 3.Heat the reaction at 80 °C in an oil bath and allowed to proceed for 72 hours under nitrogen gas. Monitor progress by TLC (Meyers et al., 2008) and disappearance of starting material using 10% methanol in dichloromethane. Product R_f = 0.94.
• 4.When reaction is finished, remove flask from oil bath and let it cool to 25 °C.
• 5.Add 150 mL of deionized water and 150 mL of ethyl acetate to extract the crude product. Shake thoroughly in a 500-mL separatory funnel.
• 6.Let the phases separate before dispensing the aqueous phase into a glass vessel, then dispense the organic phase and set aside in a separate vessel. Caution as entire mixture will be turbid.
• 7.Return aqueous phase to separatory funnel and add 150 mL of ethyl acetate to further extract remaining product.
• 8.Set aside aqueous phase and combine dispensed organic phase with initial extraction. Repeat extraction process once more with aqueous phase and 150 mL of ethyl acetate.
• 9.Combine and dry organic phases over anhydrous sodium sulfate prior to removing solvent by rotary evaporation.
• 10.Purify crude product using flash column chromatography with 0–25% ethyl acetate gradient in hexane. Pure product will appear as yellow oil (5.1 g, 80%).
• 11.Characterize product 1 by ¹H NMR and ¹³C NMR:

¹H NMR (400 MHz, CDCl₃): δ 7.44 (d, J=8.9 Hz, 1H), 7.16 (d, J=2.5 Hz, 1H), 6.66 (dd, J=2.5, 8.9 Hz, 1H), 3.25 (q, J=7.1 Hz, 4H), 2.68 (s, 3H), 1.50 (t, J=7.0 Hz, 6H) ¹³C NMR (100 MHz, CDCl₃): δ 173.2, 148.7, 148.4, 121.8, 115.2, 113.8, 101.2, 44.9, 19.0, 12.2.

Formation of disulfide

• 12.Add 5 g (23 mmol) of 1 to a 250-mL round-bottom flask and mix with 30 mL of 64% hydrazine hydrate solution using magnetic stirring under nitrogen gas.
• 13.Reflux under nitrogen gas at 100 °C for 18 hours. Monitor progress by TLC using 10% methanol in dichloromethane. After starting material has disappeared from TLC plate reaction has completed. Product R_f = 0.91.
• 14.Aerate the reaction mixture by stirring vigorously in a large beaker while gently blowing a fume hood airline across the surface of the solution for 45 min. Alternatively, the stream of air may be bubbled into the reaction mixture with vigorous stirring. This step oxidizes the product thiolate to produce the disulfide, which forms as a yellow precipitate as the aerobic oxidation takes place.
• 15.Transfer reaction mixture to a 250 mL separatory funnel and add 20 mL of deionized water and 30 mL of ethyl acetate.
• 16.Shake funnel vigorously and remove aqueous phase. Repeat twice more by adding an equal volume of water to the organic phase and discarding aqueous washings.
• 17.Dry organic phase over anhydrous sodium sulfate and remove solvent by rotary evaporation and vacuum Schlenk line.
• 18.Purify crude product using flash column chromatography with 25% ethyl acetate in dichloromethane.
• 19.Remove solvent impurities by recrystallizing using ethyl acetate and hexane—dissolve material in a minimal amount of ethyl acetate and gradually add hexanes while stirring until precipitate crashes out of solution. Remove solvent by vacuum filtration and dry precipitated product in vacuo for 20 min. Pure product will appear as yellow solid (2.9 g, 79.5%).
• 20.Characterize product 2 by ¹H NMR and ¹³C NMR:

¹H NMR (500 MHz, CDCl₃): δ 7.08 (d, J=8.4 Hz, 2H), 6.00 (m, 4H), 4.25 (s, 4H), 3.32 (q, J=7.1 Hz, 8H), 1.15 (t, J=7.1 Hz, 12H) ¹³C NMR (126 MHz, CDCl₃): δ 150.7, 149.9, 138.6, 106.0, 103.1, 96.9, 44.3, and 12.7.

Coupling to 5-bromouracil and formation of 8-diethylamino-tC nucleobase

• 21.Add 1.0 g (2.5 mmol) of product 2 in a dry 50 mL Schlenk tube under nitrogen gas and dissolve with 2 mL of anhydrous DMF and 3 mL of diethylene glycol dimethyl ether (diglyme) using magnetic stirring. Degas the solution by using a conventional freeze-pump-thaw cycle (3×).
• 22.Add sequentially 5.1 mL (5.1 mmol) of 1 M triethylphosphine solution and 92 μL (5.1 mmol) of deionized water and stir for 10 min at 25 °C under nitrogen gas.
• 23.Complete reduction of disulfide is suggested by a colorless translucent solution. Add more 1 M triethylphosphine solution and water in increments of 0.5 molar equivalents if necessary and let reaction stir 10 min longer. This additional amount of triethylphosphine is more likely to be needed when using an older bottle of reagent that may have some oxidative decomposition.
• 24.Add 1.2 g (6.4 mmol) of 5-bromouracil and 0.79 g (7.5 mmol) of anhydrous sodium carbonate and immerse reaction flask in oil bath at 120 °C.
• 25.Stir reaction under nitrogen gas at 120 °C for 18 hours.
• 26.Monitor progress by TLC and disappearance of starting material using 10% methanol in dichloromethane. Product R_f = 0.64.
• 27.After reaction completion, remove flask from oil bath and allow to cool to room temperature.
• 28.Pour contents of flask with precipitated product onto a filter paper on a Büchner funnel attached to a 250 mL vacuum flask to filter precipitate from liquids.
• 29.Rinse flask thoroughly with water and sonication, transferring the remaining solids to filter paper. Repeat with chloroform to remove residual disulfide.
• 30.Wash filtered precipitate with a total of 100 mL of water and then 20 mL of chloroform. Precipitate should be pale gray or white—wash further if discolored.
• 31.Let precipitate dry in vacuo for 1 hour (0.95 g, 60%).
• 32.Characterize product 3 by ¹H NMR and ¹³C NMR:

¹H NMR (400 MHz, DMSO-d₆): δ 11.27 (s, 1H), 10.91 (s, 1H), 7.14 (s, 1H), 7.11 (d, J= 8.6Hz, 1H), 6.02 (d, J=2.7Hz, 1H), 5.93 (dd, J=8.7, 2.7Hz, 1H), 5.25 (d, J=8.4 Hz, 1H), 3.31 (m, 1H), 3.24 (m, 4H), 1.06 (t, J=6.9Hz, 6H). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.0, 151.3, 150.9, 149.6, 140.9, 137.8, 108.8, 101.8, 98.7, 96.4, 43.6, 12.6.

• 33.Suspend 800 mg (2.6 mmol) of product 3 in a dry 50 mL round-bottom flask with 15 mL of 1-butanol solvent and magnetic stirring.
• 34.Complete dissolution by stirring for 10 min at 120 °C.
• 35.Let flask cool to 25 °C, then add 1.5 mL (15 mmol) of concentrated (36.5%) hydrochloric acid and let stir at 120 °C under N2 for 24 hours.
• 36.Monitor progress by TLC and disappearance of starting material using 10% methanol in dichloromethane. Product R_f = 0.48.
• 37.When reaction is finished, neutralize remaining acid by adding 1 mL of 25% ammonium hydroxide. Remove solvent by rotary evaporation.
• 38.Purify product by flash column chromatography using 0–20% methanol gradient in dichloromethane. Remove solvent from fractions containing product by rotary evaporation to yield yellow solid (0.73 g, 97%).
• 39.Characterize product 4 by ¹H NMR and ¹³C NMR:

¹HNMR (400MHz, DMSO-d₆): δ 10.87 (s, 1H), 9.87 (s, 1H), 7.32 (s, 1H), 6.77 (d, J=8.6 Hz, 1H), 6.41 (d, J=2.4 Hz, 1H), 6.27 (dd, J=8.7, 2.4 Hz, 1H), 3.23 (m, 4H), 1.51 (t, J=6.9 Hz, 6H) ¹³C NMR (100 MHz, DMSO-d₆): δ 161.5, 155.9, 147.3, 137.8, 137.1, 126.9, 107.8, 100.7, 99.7, 94.8, 44.1, 12.6.

Glycosylation of 8-diethylamino tC nucleobase and formation of β-nucleoside

• 40.Suspend 346 mg (1.2 mmol) of compound 4 in a 25 mL Schlenk tube with 5 mL of anhydrous acetonitrile and magnetic stirring under nitrogen gas.
• 41.Add 0.44 mL (1.8 mmol) of Bis(trimethylsilyl)acetamide and stir for 1 hour at 50 °C in an oil bath. Solution should become transparent. If it does not, see Critical Parameters and Troubleshooting.
• 42.Remove Schlenk tube from oil bath and let cool to 25 °C.
• 43.Add 700 mg (1.8 mmol) of 3’,5’-di-O-(p-toluoyl)-2’-deoxy-α-D-ribofuranosyl chloride (Hoffer’s chlorosugar) and stir at 0 °C in an ice bath, then add 28 μL of anhydrous tin(IV) chloride.
• 44.Remove Schlenk tube from ice bath and let reaction stir at 25 °C for 1 hour.
• 45.Monitor progress by TLC and disappearance of starting material using 10% methanol in dichloromethane. Products R_f = 0.68 for β-anomer, R_f =0.64 for α-anomer.
• 46.After the reaction is finished, transfer liquid contents to 250 mL separatory funnel and add 30 mL of ethyl acetate and 30 mL of saturated sodium bicarbonate solution.
• 47.Discard aqueous phase and wash organic phase with an equal volume of water twice, discarding aqueous washings.
• 48.Dry organic phase over sodium sulfate and remove solvent by rotary evaporation.
• 49.Purify by flash column chromatography using 0–10% methanol gradient in dichloromethane. Remove solvent from fractions containing product by rotary evaporation to yield yellow solid containing α- and β-nucleoside anomers (0.76 g, 86%).
• 50.Dissolve 660 mg (1.03 mmol) of anomeric mixture in 5 mL of anhydrous methyl alcohol in a 25 mL round-bottom flask under nitrogen gas with magnetic stirring.
• 51.Add 0.74 mL (4.12 mmol) of 30% sodium methoxide in methanol solution and let reaction stir under nitrogen gas for 2.5 hours at 25 °C.
• 52.Check for reaction completion by TLC and disappearance of starting material using 10% methanol in dichloromethane. Product R_f = 0.38 for β, R_f = 0.34 for α-anomer.
• 53.When complete, quench reaction with 30 μL (1.68 mmol) of glacial acetic acid.
• 54.Purify β-nucleoside product using column chromatography with 10–12% ethanol in chloroform. The β-nucleoside elutes immediately prior to the α-nucleoside.
• 55.Check by TLC for fractions containing only β-nucleoside with no α-anomer. Pool fractions with product and remove solvent by rotary evaporation to yield yellow solid (168 mg, 39%).
• 56.Characterize product 6 by ¹H NMR and ¹³C NMR:

¹H NMR (400 MHz, CD₃OD): δ 7.88 (s, 1H), 6.77 (d, J=8.7Hz, 1H), 6.35 (dd, J=8.5, 2.6Hz, 1H), 6.32 (d, J=2.5Hz, 1H), 6.19 (t, J=6.4Hz, 1H), 4.36 (m, 1H), 3.92 (q, J=3.5Hz, 1H), 3.82 (dd, J=12.1, 3.1Hz, 1H), 3.72 (dd, J=12.0, 3.6Hz, 1H), 3.34 (m, 4H), 2.35 (m, 1H), 2.14 (m, 1H), 1.14 (t, J=6.9Hz, 6H) ¹³C NMR (100 MHz, CD₃OD): δ 160.6, 156.1, 147.6, 136.7, 134.1, 126.3, 108.3, 100.6, 100.1, 98.7, 87.6, 86.4, 70.3, 61.1, 48.4, 44.0, 11.4.

Synthesis of 5’-O-dimethoxyltrityl-3’-O-phosphoramidite

• 57.In a dry 25 mL Schlenk tube, transfer 130 mg (0.32 mmol) of compound 6 while separately placing 217 mg (0.64 mmol) of dimethoxytrityl chloride in a 50-mL round-bottom flask.
• 58.Dry both flasks with contents under vacuum for 18 hours.
• 59.Flush each flask with nitrogen gas and dissolve compound 6 in 3 mL of anhydrous pyridine with magnetic stirring.
• 60.Add the dimethoxytrityl chloride (DMTrCl) to the reaction mixture swiftly so as to minimize exposure to the outside air.
• 61.Let reaction stir at 25 °C for 90 min monitoring progress by TLC using 10% methanol in dichloromethane and for disappearance of starting material. Product R_f = 0.55.
• 62.When starting material has completely disappeared from TLC, add 5 mL of methanol to quench any remaining DMTrCl and remove solvent by rotary evaporation.
• 63.Purify product by flash column chromatography using 5% ethanol in dichloromethane with 1% trimethylamine. Collect fractions with product and recrystallize by dissolving in a minimal amount of chloroform (filtered through basic alumina to remove acid) before gradually adding hexane until precipitate crashes out of solution.
• 64.Remove solvent by vacuum filtration and rinse with hexane and subsequently water to remove triethylammonium salts.
• 65.Dry in vacuo for 20 min to yield the product as a yellow solid (204 mg, 90%).
• 66.Characterize product 7 by ¹H NMR:

¹H NMR (400 MHz, CDCl₃): δ 7.64 (s, 1H), 7.43 (m, 2H), 7.31 (m, 6H), 7.21 (t, J=7.3,1H), 6.84 (m, 4H), 6.68 (d, J=8.7Hz, 1H), 6.28 (t, J=6.3Hz, 1H), 6.26 (m, 1H), 6.12 (d, J=2.6Hz, 1H), 4.48 (m, 1H), 4.09 (m, 1H), 3.77 (s, 3H), 3.76 (s, 3H), 3.37 (m, 2H), 3.29 (q, J=7. 1Hz, 4H), 2.64 (m, 1H), 2.23 (m, 1H), 1.13 (t, J=7.0Hz, 6H).

• 67.Dissolve 155 mg (0.22 mmol) of compound 7 with 2 mL of degassed anhydrous dichloromethane in a dry 25-mL Schlenk tube under nitrogen. Mix with magnetic stirring.
• 68.Add 155 μL (0.89 mmol) of N,N-diisopropylethylamine and stir for 5 min at 25 °C.
• 69.Subsequently add 61.5 μL (0.26 mmol) of 2-cyanoethyl-N,N-diisopropylchlorophosphoroamidite and stir for 40 min at 25 °C.
• 70.Monitor progress by TLC and disappearance of starting material using 5% methanol in dichloromethane. Check for both diastereotopic products (R_f = 0.33, 0.36).
• 71.Remove solvent by rotary evaporation prior to column chromatography purification.
• 72.Purify crude product material by flash column chromatography using 1% ethyl acetate in dichloromethane with 1 % triethylamine.
• 73.Collect fractions with product and remove solvent by rotary evaporation. Remove residual impurities and solvent impurities by dissolving in a minimal amount of DCM and adding ice-cold hexane to precipitate the product.
• 74.Remove solvent by vacuum filtration and rinse with hexane, allow to dry, and rinse with water. Dry product in a vacuum dessicator for at least 12 h and then store at −18 °C. Product appears as a yellow solid (88 mg, 44%) comprising mixture of both diastereomeric products (epimers at phosphorus).
• 75.Characterize the final product, compound 8, by ¹H NMR and ³¹P NMR:

¹H NMR (400 MHz, CDCl₃): δ 7.71 and 7. 65 (s, 1H), 7. 42 (m, 2H), 7. 30 (m, 6H), 7. 21 (m, 1H), 6. 83 (m, 4H), 6.66 (m, 1H), 6.27 (m, 2H), 6.01 (m, 1H), 4.55 (m, 1H), 4.10 (m, 1H), 3.77 and 3.76 (m, 6H), 3.58 (m, 4H), 3.42 and 3.34 (m, 2H), 3.28 (q, J=7.1Hz, 4H), 2.75 and 2.64 (m, 1H), 2.61 and 2.42 (t, J=6.5Hz, 2H), 2.24 (m, 1H), 1.22–1.04 (m, 12H), 1.06 (t, J=7.0Hz, 6H). ³¹P NMR (162MHz, CDCl₃): 150.0, 149.8.

BASIC PROTOCOL 2

SOLID-PHASE SYNTHESIS OF ^DEAtC-CONTAINING ODNs

This protocol describes incorporation of ^DEAtC (Fig. X.XX.1) into oligodeoxyribonucleotides (ODNs) using phosphoramidite 8 (see Basic Protocol 1) and an automated DNA synthesizer. Standard DNA synthesis conditions are used, except 0.1 M phosphoramidite concentrations are used and coupling is extended (10 mins) for the ^DEAtC phosphoramidite. Extended capping (8 pulses, 22 sec) is used during the synthesis. Extended capping is recommended for monomers that may not couple as efficiently as unmodified DNA phosphoramidites (Glen Research).

Materials

5′-O-Dimethoxytrityl-N²-isobutyryl-2′-deoxyguanosine, 3′-O-succinyl-long chain alkylamino-CPG (LCAA-CPG) support (500 Å pore size, 43 μmol/g, Glen Research) Standard DNA phosphoramidites (dried over activated 3Å molecular sieves at least 16 hrs):

• N⁶-Benzoyl-2′-deoxyadenosine phosphoramidite (Bz-dA, ChemGenes)

• N⁴-Acetyl-2′-deoxycytidine phosphoramidite (Ac-dC, Glen Research)

• N²-Isobutyryl-2′-deoxyguanosine phosphoramidite (iBu-dG, ChemGenes)

• Thymidine phosphoramidite (T, Glen Research)

Phosphoramidite 8 (see Basic Protocol 1)

Reagents for solid-phase oligonucleotide synthesis:

• Activator solution: 1H-tetrazole in anhydrous acetonitrile (Glen Research)

• Cap A: Acetic anhydride (Alfa Aesar):2,6-lutidine (Acros):THF (1:1:8)

• Cap B: 16% 1-methylimidazole (Alfa Aesar) in THF (v/v)

• Oxidizing solution: 0.03 M I₂ (Alfa Aesar) in THF/pyridine/H₂O (75:20:2)

• Deblocking Mix: 3% trichloroacetic acid in CH₂Cl₂

• Anhydrous acetonitrile wash (CH₃CN, Glen Research)

Conc. aq. ammonia (28 – 30%, Sigma-Aldrich)

Acetic Acid (Fisher Scientific, HPLC grade)

3 M sodium acetate (EM Science)

5 M sodium perchlorate (GFS Chemicals, HPLC grade)

Acetone (EMD, HPLC grade)

Ethanol (200 Proof, Pharmco-Aaper)

Mobile phase A: 0.05 M triethylammonium acetate (TEAA, Glen Research), pH 7

Mobile phase B: 25% H₂O in acetonitrile

ZipTip_C18 Pipette Tips (Millipore)

Matrix solution for MALDI-MS: 10 mg 2,4,6-trihydroxyacetophenone (THAP, Matrix Scientific) in 1:1 MeCN:H₂O (1 mL), 230-mg ammonium citrate, dibasic (Chem Impex) in 10 mL H₂O, mix 8:1 THAP:ammonium citrate

Expedite 8909 Nucleic Acid Synthesis System

Empty synthesis column, 0.2 μmol scale (Glen Research)

1- and 2- mL plastic syringes

1.5 mL microtube with o-ring cap (Sarstedt)

55 °C incubation block (Eppendorf ThermoStat Plus)

1.7 mL microcentrifuge tubes (Sorenson Bioscience)

Speedvac evaporator (Labconco refrigerated centrivap concentrator, Model 7310021, or equivalent)

Thermo Fisher Vanquish UHPLC system equipped with: Hypersil Gold C18 column (5 μm, 4.6 × 150 mm, Thermo Fisher) Fraction Collector (Dionex UltiMate 3000)

1. Prime the DNA synthesizer, i.e., flush the lines with solvents and reagents using a union or an empty synthesis column.

This step is performed to remove residual moisture from the hoses to improve coupling yields.

2. Weigh out 0.2 μmol solid support (3′-succinoyl-long chain alkylamino-CPG (LCAA-CPG)), add to an empty 0.2 μmol synthesis column and attach to the DNA synthesizer.

3. Elongate the ODNs in the 5′-direction using standard DNA phosphoramidites and synthesis conditions, except the use of extended coupling (10 mins) for ^DEAtC. The final 5′-O-DMTr group is retained on the ODN to simplify RP-HPLC purification.

To ensure consistently high coupling efficiency, the dissolved DNA phosphoramidites (0.10 M in MeCN) should be stored over activated 3Å molecular sieves at least 16 hrs prior to use.

The overall ODN yield was estimated in the following manner: Collect DMT waste after the first coupling step and at the penultimate detritylation step, dilute to 25 mL in a volumetric flask with 0.1 M para-toluenesulfonic acid in acetonitrile and measure the absorbance at 495 nm. Estimate the yield as ODN% = (A495 of penultimate deblocking/A495 of first coupling) x 100.

Coupling efficiency of ^DEAtC monomer was measured using collected DMT waste as described above, i.e., ^DEAtC% = (A495 of ^DEAtC deblocking/A495 of deblocking directly before ^DEAtC coupling) x 100 = 97–99% coupling efficiency.

4. Remove the column from the DNA synthesizer and dry on a high vacuum line (~30 mins). Remove column from vacuum line, attach an empty 2 mL syringe to the column and subsequently attach a second 2 mL syringe containing ~1.2 mL concentrated aqueous ammonia to the other end of the column. Mix gently over 1 hr to cleave the ODN from the solid support.

Push the plungers of the syringes back and forth gently every 10–15 minutes. It is recommended to use 2 mL leur-lock syringes to hold the column in place during the deprotection from solid support.

5. Transfer the ammonia solution to a 1.5 mL microtube with o-ring cap and close the lid tightly.

6. Incubate the solution at 70 °C (45 min) in a temperature-controlled incubator to remove the nucleobase protecting groups.

Note that the use of Bz-dC during the ODN synthesis results in poor quality analytical RP-HPLC chromatograms using NH₄OH deprotection at 55 °C for 16 hours, likely due to incomplete deprotection. Extended deprotection (24–36 h) at 55 °C resulted in degraded crude chromatograms. Likewise, the use of room temperature deprotection for 36 hours results in poor quality ON and low yields. It is recommended to use Ac-dC and shorter deprotection times.

7. Remove the microtube from the incubator and let cool to room temperature before opening. Evaporate the ammonia solution to near dryness at room temperature using a Speedvac evaporator.

Evaporation of the crude ODN to full dryness results in a pellet that is difficult to reconstitute for purification.

8. Purify the crude ODN using a RP-HPLC using the following conditions:

Hypersil Gold C18 column (5 μm, 4.6 × 150 mm)

Flow rate: 1 mL/min

Column Temperature: 60 °C

UV-Vis detection at 260 nm

Buffer A: 0.05 M TEAA, pH 7

Buffer B: 25% H₂O in acetonitrile

Gradient:

Isocratic hold at 100% A for 2 min

Linear gradient to 50% B over 30 min

Linear gradient to 100% B over 0.5 min

Isocratic hold at 100% B for 3 min

Linear gradient to 100% A over 0.5 min

Isocratic hold at 100% A for 2 min

9. Pool and transfer appropriate DMTr-on fractions to a 1.7 mL microcentrifuge tube and evaporate to dryness using a Speedvac evaporator.

10. Add 100 μL of 80% aq. acetic acid. Mix the solution using a vortex mixer and let stand at room temperature for 20 min to cleave the terminal 5′-trityl group.

11. Add 100 μL H₂O, 25 μL 3M sodium acetate, and 15 μL 5M sodium perchlorate and vortex briefly.

12. Add 1000 μL acetone, vortex briefly, and leave at–18 °C overnight.

13. Microcentrifuge (~15,000 rpm) for 5 mins at 4 °C and remove the supernatant.

14. Wash the resulting ODN pellet with 200 μL ethanol, microcentrifuge (~15,000 rpm) for 5 mins at 4 °C and remove supernatant (repeat once more) and let resulting ODN pellet air dry.

15. Dissolve purified DMTr-off ODN in DNase/RNase free H₂O and determine concentration by measuring the absorbance at 260 nm using the following extinction coefficients (OD/μmol): G (12.01), A (15.20), T (8.40), C (7.05), and ^DEAtC (20.0).

16. Verify the purity of the ODNs by performing RP-HPLC on a small aliquot (~1 nmol) using similar conditions as for RP-HPLC purification (step 8).

27. Verify the molecular mass of ^DEAtC ODNs via MALDI-MS after ZipTip desalting (Castleberry et al., 2008), using a 1:1 (v/v) mixture of desalted ODN and matrix solution.

ALTERNATE PROTOCOL 2

SOLID-PHASE SYNTHESIS OF ^DEAtC-CONTAINING ODNs

An alternative deprotection and precipitation procedure is available that replaces steps 10–14 of Basic Protocol 2. This protocol is suggested in the case that Basic Protocol 2 results in less than desired yield or purity of ODNs, but the expected yield is good based on an observed high coupling efficiency.

Materials

Acetic acid (Fisher Scientific, HPLC grade)

Dry ice

Ethyl acetate (EtOAc), (Fisher Scientific)

Ethanol (200 Proof, Pharmco-Aaper)

1 M sodium chloride

Speedvac evaporator (Labconco refrigerated centrivap concentrator, Model 7310021, or equivalent)

Alternative Deprotection and Precipitation of ^DEAtC ODNs

1. Add 200 μL 80% AcOH, vortex, stand at room temperature for 10 mins. Freeze over dry ice and evaporate ODN to dryness in a vacuum centrifuge.

2. Extract 5’-OH-DNA with EtOAc to remove DMT-OH and any other organic impurities. Dissolve ODN in 500 μL H₂O, add 500 μL EtOAc and vortex, Allow the organic layer to separate from the aqueous layer and pipet off the organic layer and discard. Repeat the extraction procedure two more times. During the third and final extraction, centrifuge for 5–10 minutes to separate organic and aqueous phases well enough to spin down particulates in the aqueous portion. Remove the organic layer, then carefully remove the aqueous, DNA-containing layer from any particulates spun to the bottom. Transfer to a clean microcentrifuge tube. Concentrate to dryness in a vacuum centrifuge.

3. Precipitate the ODN with EtOH to remove any TEAA impurities and residual organic impurities. Dissolve the ODN in 300 μL 1.0 M NaCl. Precipitate by adding 1.0 mL absolute EtOH (200 proof) and vortex briefly. Chill on ice for ~30 mins. Centrifuge (15,000 rpm) for 5 mins at 4 °C. Remove supernatant and repeat precipitation once more. Evaporate the aqueous layer to dryness in a SpeedVac evaporator.

COMMENTARY

Background Information

^DEAtC is a tricyclic 2’-deoxycytidine analogue that can be incorporated in place of 2’-deoxycytidine in synthetic oligonucleotides that retain their capacity for stable duplex formation with a matched complementary strand (Burns et al., 2017). The resulting duplexes retain the natural B conformation of double-stranded DNA. The most important application of ^DEAtC is as a fluorescence turn-on probe for duplex formation. Single-stranded oligonucleotides containing ^DEAtC behave largely like natural oligonucleotides and are minimally fluorescent, but become substantially brighter when hybridized to a matched complementary sequence. This fluorescence turn-on effect does not operate when ^DEAtC is mispaired with adenosine, thereby making ^DEAtC-containing oligonucleotides especially selective fluorescence hybridization probes.

Some design considerations are relevant to applications of ^DEAtC fluorescence turn-on oligonucleotides. The fluorescence turn-on property of ^DEAtC has some sequence dependence (Figure 3). Neighboring bases on the 5′ side of ^DEAtC have the most effect on fluorescence properties, but the 3′ neighbor also has some impact. For this reason, the choice of placement of the ^DEAtC probe in a fluorescence turn-on ODN should include consideration of the immediate neighboring bases. The best sequences for fluorescence turn-on, as measured by the increase in fluorescence quantum yield upon hybridization to a matched complement, include 5′-A(^DEAtC)A-3′ (AXA in Figure 3) and 5′-G(^DEAtC)C-3′ (GXC). Fluorescence turn-on is slightly greater for AXA (5-fold as compared with 4-fold), but the GXC sequence gives more overall brightness. For these reasons, it is recommended that ^DEAtC be placed between two A residues or between a G and a C residue, in the 5′ to 3′ direction. Another consideration is that ^DEAtC’s presence can perturb the melting temperature of matched DNA duplexes (for details, see (Burns et al., 2017)). For the AXA sequence described, ΔT_m = −2.6 °C (i.e. the duplex containing ^DEAtC is slightly less stable than the corresponding natural duplex). For the GXC sequence, ΔT_m = −14.5 °C. The greater brightness of the GXC sequence comes at the expense of duplex stability. For this reason, we believe that the AXA sequence motif will be the most broadly useful for fluorescence hybridization turn-on probe design. Last, we recommend considering the possible formation of secondary structure in the probe design, including hairpins and self-dimers. Because both of these secondary structures can allow ^DEAtC-G base pairing in the absence of a complementary target sequence, they may increase intrinsic probe strand fluorescence and accordingly diminish the extent of fluorescence turn-on that is observed when adding a complementary target sequence.

Figure 3.

Sequence-dependent fluorescence turn-on response of ^DEAtC in single-stranded and double-stranded oligonucleotides, measured in 1× PBS buffer, pH 7.2, 296 K. The sequences are 5′-CGC-A__-_TC-G-3′, where __-_ corresponds to the three-base sequences given on the x-axis. The GXC sequence has the greatest quantum yield of fluorescence emission in a duplex oligonucleotide, whereas the AXA sequences has the largest % change upon strand hybridization.

Critical Parameters and Troubleshooting

This protocol should not be attempted by individuals without prior training in a modern synthetic organic chemistry laboratory and without appropriate safety equipment and procedures in place. Prior knowledge of safe reagent handling, vacuum gas manifold operation, reaction work-up procedures, chromatography, and NMR spectroscopy is needed. For Basic Protocol 2, familiarity with standard protocols for solid-phase DNA synthesis is assumed. Specific points of difficulty are discussed below, along with procedural modifications that can overcome problems.

After 5-amino-2-methylbenzathiazole has been converted to compound 1, the work-up may be difficult due to the resulting reaction mixture being opaquely black and the high solvating ability of DMSO. After adding EtOAc and water, there may not be an immediately visible delineation between the two immiscible phases and so dispensing the aqueous phases should be done slowly to mitigate the risk of losing organic phase. As the separatory funnel tappers near the stopcock, the difference between the two phases will be more visible once the aqueous phase ends and organic phase approaches.

Preparation of compound 2 involves the aerobic oxidation of the thiol that is initially formed when the 2-methylbenzothiazole undergoes nucleophilic ring opening by hydrazine. In this protocol, the reaction mixture is stirred vigorously when open to the air or is aerated by bubbling with an air inlet. In both variants, aerobic oxidation forms the disulfide bond. However, extended exposure to O₂ further oxidizes the molecule leading to degradation as observed by a color change from yellow to black. The product may be carried to subsequent steps without recrystallization if consumed within one week, however for optimal reaction results and prolonged storage, recrystallization as described in this Protocol is strongly recommended.

When coupling to 5-bromouracil for the production of compound 3, the first portion of the reaction involves reduction with triethyl phosphine and water to generate a thiophenolate intermediate. The completion of this reduction is indicated by a colorless, translucent solution. When scaling up reaction quantities, the solution may appear as very pale brown-yellow and additional reductant and water may be unnecessary or cause adverse effects to the second step of the reaction, the coupling with 5-bromouracil.

When coupling the ^DEAtC nucleobase 4 to Hoffer’s chlorosugar to give nucleoside 5, the presence of trace amounts of water can lower the yield. Water competes against 4 for reaction with BSA in the first step of this Silyl-Hilbert-Johnson reaction. Because 4 dissolves as it reacts with BSA, the presence of small amounts of water will manifest as incomplete solubilization of the starting nucleobase during heating with BSA. Should this problem arise, a small amount of additional BSA can be added to quench any residual water and complete the silylation of 4. The addition of Hoffer’s chlorosugar and the tin(IV) catalyst may then be performed as normal in the protocol.

During synthesis of the compound 8 there will be two spots on the TLC plate for the two phosphoramidite products. The reaction yields the product as two diastereoisomers on account of the chiral phosphorus atom—both diastereomers are viable for solid phase DNA synthesis. When characterizing 8, Phosphorous(V) impurities may be evident in the ³¹P NMR and the product can be dissolved in minimal DCM and precipitated by the addition of cold hexanes for additional purification. Undesired oxidation during the reaction can be mitigated by degassing the DCM solvent using the freeze-pump-thaw method (x3) prior to usage. The most likely impurity to be seen in the ³¹P NMR is 2-cyanoethyl-N,N-bis(isopropyl)phosphonamidate, which appears as a singlet at 14 ppm, and which remains in the supernatant solution when the amidite 8 is precipitated as described.

Anticipated Results

The anticipated result of following Basic Protocol 1 is the obtainment of approximately 90 mg of the ^DEAtC phosphoramidite 8, which is adequate for 3–4 incorporations into oligodeoxynucleotides. This protocol can be scaled up if a larger amount of 8 is desired. In previous experiences, there has been no adverse effect when scaling up the reaction quantities relative to the quantities detailed in this procedure. The largest amount of 5-amino-2-methylbenzathiazole that we have used in one reaction is 10 g. Subsequent steps can be scaled up accordingly. Certain reactions can be divided into smaller batches depending on reagent supply and experience. For instance, generating 3 from 2 on a smaller scale (100–200 mg batches) is easier in our hands. The amounts of 7 reacted to generate 8 have ranged from 50 mg to 800 mg and, in those experiments, the purity of 7 has be a more influential factor than scale on yield. Because of the challenging, multistep nature of this synthesis, we recommend running the reactions at scales not greater than those given in Basic Protocol 1 on the first use of this Protocol.

Basic Protocol 2 results in oligodeoxynucleotides containing ^DEAtC at any desired position and multiple incorporations are possible. Typical yields range from 70–95 nmol when the synthesis of 10-mer oligodeoxynucleotides including a single ^DEAtC (e.g., sequences in Figure X.XX.3) is conducted on a 0.2 μmol scale. Yields can be expected to be lower for longer oligodeoxynucleotides and for those that include multiple ^DEAtC residues. Alternative Protocol 2 is suggested for cases where yield or purity are less than ideal following deprotection and precipitation.

Time Considerations

Synthesizing ^DEAtC phosphoramidite 8 from 5-amino-2-methylbenzathiazole takes approximately two to three weeks. Product 1 should be used within one week of synthesis because of the potential for oxidative degradation. Products 3 and 4 can be stored at room temperature and are stable for at least one year. Recrystallized products 5–7 can be stored at 0–10 °C, while recrystallized 2 and precipitated 8 should be stored at −20 °C, and all are stable for at least six months. Containment vessels for products (e.g. scintillation vials, flasks) should be flushed with nitrogen or argon gas to mitigate any potential oxidative degradation during product storage. ODN synthesis, purification, and characterization takes approximately an additional one week.

Significance Statement:

^DEAtC is a tricyclic 2’-deoxycytidine analogue that functions as a surrogate for natural cytidine in Watson-Crick C-G base pairs, with the added property of a fluorescence turn-on response to DNA duplex formation. This fluorescence turn-on is specific to correct base-pairing with guanine residue. ^DEAtC can be incorporated into synthetic oligonucleotides using conventional solid-phase synthesis to give a new type of hybridization probe with a sequence-specific fluorescence turn-on function.