This article describes the specific incorporation of ¹⁵N into the N7 and amino positions of adenosine (Basic Protocol 1), and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions (Basic Protocol 2). Two variations of the procedures are also presented that include either ¹²C or ¹³C at the C8 position of adenosine, and ¹³C at either the C8 or C2 position of guanosine. These ¹³C tags permit the incorporation of two ¹⁵N-labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C-N coupling. While the major application of these specifically ¹⁵N-labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. The Support Protocol describes the synthesis of 7-methylguanosine. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1 : Syntheses of [7,NH₂-¹⁵N₂]- and [8-¹³C-7,NH₂-¹⁵N₂]adenosine

Support Protocol : Synthesis of 7-methylguanosine

Basic Protocol 2 : Synthesis of [2-¹³C-1,7,NH₂-¹⁵N₃]- and [8-¹³C-1,7,NH₂-¹⁵N₃]guanosine

As shown in Figure 1, the procedures described here (Pagano, Lajewski, & Jones, 1995; Shallop & Jones, 2000; Zhao et al., 1997) start with the inexpensive pyrimidine 4-amino-6-hydroxy-2-mercaptopyrimidine (1). The first ¹⁵N label is introduced by a direct nitrosation/reduction to give 2. This is followed by ring closure using either diethoxymethyl acetate in dimethylformamide (DMF) to give 3a with a ¹²C at the C8 position, or [¹³C]sodium ethyl xanthate to give 3b with a ¹³CSH at the C8 position. Removing the thiol group(s) with Raney nickel forms hypoxanthine (4a/b), which can readily be converted to 6-chloropurine (5a/b), which are excellent substrates for enzymatic transglycosylation. The second ¹⁵N label is then introduced into the nucleoside by displacement of the chloride by ¹⁵NH₃, which is generated in situ to give the labeled adenosines 7a/b.

CAUTION : The procedures in this article use a number of highly toxic and dangerous reagents. Raney nickel is pyrophoric when dry and may burst into flames if not kept wet. Phosphorous oxychloride (POCl₃) is very reactive and hydrolyzes to hydrochloric and phosphoric acids, which are both highly corrosive to skin and tissue. Cyanogen bromide is very dangerous; improper use of this reagent can cause death. All reactions must be done with great care in an appropriate chemical fume hood.

Materials

• 4-Amino-6-hydroxy-2-mercaptopyrimidine monohydrate (1 ; also called 6-amino-2-thioxo-1,2-dihydro-4(3 H)-pyrimidinone; MilliporeSigma)

• 1 N HCl

• [¹⁵N]Sodium nitrite ([¹⁵N]NaNO₂; Isotec or Cambridge Isotope Laboratories)

• 2:98 to 40:60 (v/v) gradient of acetonitrile/0.1 M triethylammonium acetate (TEAA), pH 6.8

• 95% (v/v) ethanol, 4°C

• Acetone, 4°C

• Phosphorous pentoxide (P₂O₅)

• Saturated aqueous NaHCO₃

• Sodium hydrosulfite (Na₂S₂O₄)

• Glacial acetic acid

• 96% (v/v) formic acid

• Nitrogen gas source

• Dimethylformamide (DMF), anhydrous

• Diethoxymethyl acetate (DEMA), for ¹²C synthesis only

• Acetonitrile, room temperature and 4°C

• [¹³C]Sodium ethyl xanthate ([¹³C]NaSCSOEt; see recipe), for ¹³C synthesis only

• NaOH

• 50% aqueous Raney 2800 nickel (RaNi) slurry (MilliporeSigma)

• Dipotassium salt of EDTA

• Phosphorous oxychloride (POCl₃)

• N,N -Dimethylaniline

• 5% (v/v) NH₃ (diluted with water from 30% concentrated aqueous ammonia)

• Ethyl acetate

• Ethyl ether

• 1 M HCl

• 7-Methylguanosine (see Support Protocol)

• 0.02 M K₂HPO₄

• 3 M NaOH

• Purine nucleoside phosphorylase (MilliporeSigma)

• [¹⁵N]Ammonium chloride ([¹⁵N]NH₄Cl; Isotec or Cambridge Isotope Laboratories)

• Dimethylsulfoxide (DMSO), anhydrous

• KHCO₃, anhydrous

• 100- and 250-ml round-bottom flasks

• Small glass vials

• 1-, 3-, 10-, and 20-ml syringes

• Vacuum desiccator

• Rubber septa fitted with large-bore vent needles

• Condenser

• Rotary evaporator, connected to water aspirator and a vacuum pump, the latter with a dry-ice trap

• Oil bath (silicone oil), 130°C

• Separatory funnel

• Continuous extraction apparatus for solvents lighter than water (MilliporeSigma)

• 30°C oven with shaker

• 50-ml bomb with Teflon liner (Parr Instrument)

• 80°C oven

• Additional reagents and equipment for analytical and preparative reversed-phase high-performance liquid chromatography (HPLC; Current Protocols article: Sinha & Jung, 2015)

Synthesize [5-¹⁵N]-5,6-diamino-2-thioxo-1,2-dihydro-4(3H)-pyrimidinone (2)

1.Weigh 0.805 g (5.0 mmol) 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate (1) into a 100-ml round-bottom flask with a stir bar.

2.Add 25 ml of 1 N HCl and chill suspension 10 min in an ice bath.

3.Weigh 0.385 g [5.5 mmol, 1.1 equivalents (eq)] [¹⁵N]NaNO₂ into a small glass vial, dissolve in ∼1 ml water, and draw solution into a 3-ml syringe.

4.Slowly add the sodium nitrite to the reaction mixture over ∼5 min.

5.Stir mixture ∼7 hr in the ice bath, while monitoring the reaction for completeness by reversed-phase HPLC with a gradient of 2:98 to 40:60 acetonitrile/0.1 M TEAA over 5 min.

6.Collect solid by vacuum filtration and wash it with 5 ml cold water, then 5 ml cold 95% ethanol, and finally 5 ml cold acetone.

7.Without removing it from the funnel, dry over P₂O₅ in a vacuum desiccator overnight.

8.Scrape out most of the solid from the funnel into a 100-ml round-bottom flask. Rinse funnel with portions of saturated aqueous NaHCO₃ and add them to the flask to give a final volume of 40 ml.

9.Add a stir bar and place mixture in an ice bath for 10 min. Stir gently.

10.Quickly weigh 2.61 g (15 mmol, 3 eq) Na₂S₂O₄ into a small beaker. Using a spatula, gradually add it in portions to the reaction over 20 min.

11.Insert a rubber septum containing a large-bore vent needle.

12.Stir mixture ∼7 hr in the ice bath, while monitoring the reaction for completeness by HPLC as described (step 5).

13.Slowly add 1.6 ml glacial acetic acid over ∼5 min to neutralize the NaHCO₃ and stir another 5 min.

14.Collect product by vacuum filtration and wash twice with 5 ml cold water and then twice with 5 ml cold 95% ethanol.

15.Without removing it from the funnel, dry 2 over P₂O₅ in a vacuum desiccator overnight.

Perform ring closure

For [7-¹⁵N]-2-thioxohypoxanthine (3a)

16a. Scrape out most of 2 from the funnel into a 100-ml round-bottom flask. Rinse funnel with portions of 96% formic acid and add them to the flask to give a final volume of 25 ml.

17a. Add a stir bar, attach a condenser, and reflux the solution 1 hr to make the formate salt.

18a. Concentrate to dryness using a rotary evaporator and scrape down the sides of the flask with a spatula, if necessary.

19a. Insert a rubber septum and displace the air with nitrogen.

20a. Use syringes to add the following through the septum:

• 20 ml anhydrous DMF;
• 1.63 ml DEMA (10 mmol, 2 eq);
• 0.24 ml of 96% formic acid (6 mmol, 1.2 eq).

21a. Heat mixture 3 hr in an oil bath set at 130°C. Follow reaction by HPLC.

22a. Cool flask, concentrate solution to a solid using a rotary evaporator, and loosen it with a spatula if necessary.

23a. Add 15 ml acetonitrile to the flask, attach a condenser, and reflux 10 min using the 130°C oil bath.

24a. Cool flask to room temperature, add 10 ml acetonitrile, and then chill in an ice bath.

25a. Collect 3a by vacuum filtration and wash twice with 5 ml cold acetonitrile.

26a. Without removing it from the funnel, dry 3a over P₂O₅ in the vacuum desiccator overnight. Proceed to step 27.

For [8-¹³C-7-¹⁵N]-2,8-dithioxohypoxanthine (3b)

16b. Scrape out most of 2 from the funnel into a 100-ml round-bottom flask and add 0.80 g (5.5 mmol) [¹³C]NaSCSOEt.

17b. Insert a condenser into the flask, attach a nitrogen line and vent needle, and displace air for 5 min.

18b. Add 15 ml DMF and reflux mixture under nitrogen ∼3 hr, using HPLC to monitor the reaction.

19b. Cool mixture in an ice bath and add 50 ml cold acetonitrile to precipitate 3b.

20b. Collect the solid 3b by vacuum filtration and wash twice with 5 ml cold acetonitrile. Save filtrate and both washes.

21b. Concentrate filtrate and washes, and purify by preparative reversed-phase chromatography.

22b. Dry the combined portions of 3b over P₂O₅ in a vacuum desiccator overnight. Continue with step 27.

Synthesize [7-¹⁵N]hypoxanthine and [8-¹³C-7-¹⁵N]hypoxanthine (4a/b)

27.Scrape out most of 3a/b from the funnel into a 100-ml round-bottom flask.

28.Rinse funnel with portions of water and add them to the flask to give a final volume of 30 ml.

29.Add a stir bar and 2 ml of 96% formic acid.

30.Weigh 4.5 g of 50% aqueous RaNi slurry into a small glass vial or beaker.

31.Using a dropper, transfer the RaNi suspension over 5 min to the reaction flask and add 1.5 g dipotassium salt of EDTA.

32.Connect a condenser to the flask. Using the 130°C oil bath, reflux ∼2 hr while monitoring the reaction for completeness by HPLC.

33.Remove flask from the oil bath and allow it to cool only briefly.

34.Remove condenser from the flask and carefully filter the hot reaction mixture to remove the RaNi. Rinse the flask and then the funnel with three 10-ml portions of boiling water, adding these washes to the filtrate.

35.Concentrate filtrate and washes to dryness in a 100-ml round-bottom flask using the rotary evaporator.

36.Dry 4a/b over P₂O₅ in the vacuum desiccator overnight.

Synthesize [7-¹⁵N]- and [8-¹³C-7-¹⁵N]-6-chloropurine (5a/b)

37.Weigh 0.69 g (5.0 mmol) 4a/b into a very dry 100-ml round-bottom flask.

38.Add 20 ml (215 mmol, 43 eq) POCl₃ and 2 ml (16 mmol) N,N -dimethylaniline.

39.Attach a condenser and reflux 20 min under nitrogen.

40.Monitor reaction for completeness by HPLC and continue refluxing the mixture for ≤30 min more.

41.Concentrate mixture to a very small volume using a rotary evaporator, first with an aspirator and then with a vacuum pump protected with a dry-ice trap. Add 10 ml N,N -dimethylaniline and continue the evaporation.

42.Cool flask in an ice bath and very slowly add 30 ml of 5% NH₃ to dissolve the black gum.

43.Make sure the pH of the solution is >10 (add more NH₃ if necessary) and then pour it into a separatory funnel. Wash it first with 30 ml ethyl acetate and then twice with 30 ml ethyl ether. Check the layers by HPLC.

44.Combine all organic layers that contain traces of product in a separatory funnel, backwash with 5% NH₃, and add this aqueous layer to the main reaction mixture.

45.Concentrate this aqueous solution to dryness to remove all the NH₃.

46.Add 20 ml water and chill the flask in an ice bath. Slowly acidify the solution to pH 2 using 1 M HCl.

47.Set up a continuous extraction apparatus for solvents lighter than water. Pour the aqueous layer into the extractor and add ethyl ether until the level is just under the side arm.

48.Fill a 250-ml round-bottom flask with ethyl ether, add a stir bar, connect to the extractor, and place it in an oil bath. Attach a condenser to the top of the extractor and start heating the oil bath to 45°C.

49.Continue the extraction for 3 to 4 days, using fresh ether each day. Check both the aqueous and ether layers each day by HPLC. Verify that the pH of the aqueous layer is still <2 and, if it is not, adjust it with 1 M HCl.

50.Concentrate the ether layers to a small volume, whereupon a significant amount of 5a/b should crystallize out. Collect 5a/b by vacuum filtration and check it for purity by HPLC. Save the filtrate.

51.Concentrate filtrate to dryness and dissolve it in 10 ml water. Purify by reversed-phase preparative chromatography.

52.Concentrate fractions containing pure product to dryness. Dry both portions of 5a/b in a vacuum desiccator over P₂O₅ overnight.

Synthesize [7-¹⁵N]- and [8-¹³C-7-¹⁵N]-6-chloro-9-(β-D-erythropentofuranosyl) purine (6a/b)

53.Place 2.12 g (7.5 mmol, 1.5 eq) 7-methylguanosine and 0.78 g (5 mmol) 5a/b into a 100-ml round-bottom flask and add 20 ml of 0.02 M K₂HPO₄. Using pH paper, adjust the pH to 7.4 with 3 M NaOH, if necessary.

54.Add 250 units of purine nucleoside phosphorylase. Insert a septum and heat the mixture ∼3 days in an oven at 30°C with gentle agitation. Monitor reaction for completeness each day by HPLC.

55.Pour mixture into 10 ml DMF and stir ∼1 hr at room temperature.

56.Filter suspension to remove most of the solid 7-methylguanine. Suspend solid in 5 ml fresh DMF, stir 15 min, and filter.

57.Concentrate combined filtrates to a small volume using a rotary evaporator and add 15 ml water (more if necessary to dissolve the solid).

58.Purify the crude 6a/b by preparative reversed-phase HPLC and dry it over P₂O₅ in the vacuum desiccator overnight.

Synthesize [7,NH₂-¹⁵N₂]- and [8-¹³C-7,NH₂-¹⁵N₂]adenosine (7a/b)

59.Place 1.44 g (5 mmol) 6a/b into a clean Teflon liner of a bomb and add 0.54 g (10 mmol, 2 eq) of [¹⁵N]NH₄Cl and 7 ml anhydrous DMSO.

60.Add 1.5 g (15 mmol, 3 eq) KHCO₃ and immediately seal the bomb.

61.Heat the bomb 3 days in an 80°C oven, swirling the mixture once or twice each day.

62.Cool the bomb to room temperature and then to –20°C for ≥30 min. Open it carefully and dilute mixture with 10 ml water. Adjust the pH to 7 with glacial acetic acid.

63.Check the reaction by HPLC.

64.Purify 7a/b by preparative reversed-phase HPLC and dry over P₂O₅ in the vacuum desiccator overnight.

Although 7-methylguanosine can be purchased from MilliporeSigma, it is quite easy to make. Commercial 7-methylguanosine is very expensive and the purity may not be as high.

Materials

• Guanosine

• N,N -Dimethylacetamide

• Nitrogen gas source

• Dimethyl sulfate

• Concentrated aqueous NH₃

• Acetone, 4°C

• 95% (v/v) ethanol

• Ethyl ether

• 250-ml round-bottom flask

• Rubber septum

• 10-ml syringes

CAUTION : Dimethyl sulfate is very dangerous because it is a potent alkylating agent; wear gloves and use caution.

Synthesize 7-methylguanosine

1.Place 9.91 g (35 mmol) guanosine in a 250-ml round-bottom flask along with a stir bar.

2.Add 80 ml N,N -dimethylacetamide as a solvent, insert a rubber septum, and displace the air with nitrogen gas.

3.Carefully withdraw 7.0 ml (74 mmol, 2.1 eq) dimethyl sulfate with a 10-ml syringe and add it to the suspension.

4.Stir mixture ∼6 hr and monitor reaction for completeness by HPLC.

5.Slowly add 10 ml concentrated aqueous NH₃ and then check the pH using pH paper. Continue to add more NH₃ slowly, frequently checking the pH, until the mixture is pH 10.

6.Slowly add the mixture to 300 ml cold acetone in an ice bath to precipitate the product.

7.Collect the white precipitate by vacuum filtration.

8.Check the acetone filtrate by HPLC for product. If there is a significant amount, concentrate the filtrate to a small volume, chill, and collect the additional product by vacuum filtration. Combine it with the rest.

Purify 7-methylguanosine

9.Suspend solid crude product in 300 ml of 95% ethanol, stir 5 min, and collect by filtration.

10.Suspend solid product in 300 ml ethyl ether, stir 5 min, and collect by filtration.

11.Dry pure product over P₂O₅ in a vacuum desiccator overnight.

As shown in Figure 1, in the adenosine to guanosine transformation (Shallop & Jones, 2000; Zhao et al., 1997), the adenosine amino group becomes the guanosine N1, while the guanosine amino and C2 come from potassium cyanide. The first step is the oxidation of adenosine (7a/b) to the N1 oxide (8a/b), which is followed by a one-flask set of reactions without purification to give 12c-f. Labeled cyanogen bromide is generated in situ from labeled potassium cyanide and bromine, and its reaction with 8a/b gives the oxadiazolidines 9c-f. Treatment with triethylamine opens the oxadiazolidine ring, allowing the N1 oxide to be methylated by methyl iodide to give 10c-f. Aqueous sodium hydroxide then opens the pyrimidine ring, which allows rotation around the C5-C6 bond of 11c-f. This rotation brings the cyano group near the deformylated 3-amino group to facilitate ring closure upon neutralization and heating to give 12c-f. Enzymatic deamination then gives the final products, 13c-f. The reagent combinations to convert labeled adenosines to labeled guanosines are summarized in Figure 2.

Materials

• [7,NH₂-¹⁵N₂]Adenosine (7a) or [8-¹³C-7,NH₂-¹⁵N₂]adenosine (7b ; Basic Protocol 1)

• 50% (v/v) methanol

• 3-Chloroperoxybenzoic acid (MCPBA), purified (see recipe)

• Ethyl ether

• Phosphorous pentoxide (P₂O₅)

• [¹³C,¹⁵N]Cyanogen bromide or [¹⁵N]cyanogen bromide, freshly prepared (see recipe)

• 0.1 M potassium phosphate (KH₂PO₄), pH 7.5

• Dimethyl formamide (DMF), anhydrous

• Acetonitrile

• Triethylamine

• Nitrogen gas source

• Methyl iodide

• 0.1 M NaOH

• 1 M HCl

• 95% (v/v) ethanol

• Adenosine deaminase (ADA; MilliporeSigma)

• 100-ml round-bottom flasks

• Rotary evaporator

• Vacuum desiccator

• Oil bath (silicone oil), 60°C

• Additional reagents and equipment for analytical and preparative reversed-phase high-performance liquid chromatography (HPLC; Current Protocols article: Sinha & Jung, 2015)

Synthesize [7,NH₂-¹⁵N₂]- and [8-¹³C-7,NH₂-¹⁵N₂]adenosine-N1-oxide (8a/b)

1.Weigh 1.35 g (5.0 mmol) of [7,NH₂-¹⁵N₂]adenosine (7a) or [8-¹³C-7,NH₂-¹⁵N₂]adenosine (7b) into a 100-ml round-bottom flask. Add a stir bar and 50 ml of 50% methanol.

2.Add 1.72 g (10 mmol, 2 eq) purified MCPBA, cover the flask with aluminum foil, stir 3 to 4 hr, and monitor the reaction for completeness by HPLC.

3.Dilute solution with 25 ml water and wash with three 50-ml portions of ethyl ether.

4.Use a rotary evaporator to concentrate the aqueous solution to a small volume, purify 8a/b by preparative reversed-phase HPLC, and dry it over P₂O₅ in a vacuum desiccator overnight in a 100-ml round-bottom flask.

Synthesize labeled 2-amino-6-(methoxyamino)-9-(β-D-ribofuranosyl)purines (12c-f)

5.Dissolve 1.43 g (5.0 mmol) 8a/b in 40 ml water.

6.Add 7.5 mmol freshly prepared [¹³C,¹⁵N]cyanogen bromide or [¹⁵N]cyanogen bromide, as appropriate for the desired labeling pattern, stir 2 hr, and monitor reaction for completeness by HPLC.

7.Concentrate solution to a very small volume using a rotary evaporator, add 10 ml anhydrous DMF and 10 ml acetonitrile, and concentrate again. Repeat this drying process two more times.

8.Add 25 ml anhydrous DMF and 2.8 ml (20 mmol, 4 eq) triethylamine under nitrogen gas.

9.Stir 45 min and then slowly add 2.5 ml (40 mmol, 8 eq) methyl iodide.

10.Cover flask with aluminum foil and stir 3 to 4 hr while monitoring the reaction for completeness by HPLC.

11.Concentrate solution to a yellow oil and add 85 ml of 0.1 M NaOH.

12.Stir 20 min and then adjust pH to 7.4 with 1 M HCl.

13.Add 80 ml of 95% ethanol, attach a condenser, and heat solution in an oil bath at 60°C for 4 hr while monitoring the reaction for completeness by HPLC.

14.Concentrate solution to a small volume using a rotary evaporator, purify 12c-f by reversed-phase HPLC, and dry over P₂O₅ in a vacuum desiccator overnight in a 100-ml round-bottom flask.

Synthesize labeled guanosines (13c-f)

15.Dissolve 1.58 g (5 mmol) 12c-f in 80 ml of 0.1 M KH₂PO₄, pH 7.5.

16.Add 300 units of adenosine deaminase, stopper the flask, and heat 4 days at 37°C with gentle agitation.

17.Cool mixture in an ice bath and collect crude 13c-f by filtration.

18.Purify 13c-f by recrystallization from water and dry it over P₂O₅ in a vacuum desiccator overnight.

3-Chloroperoxybenzoic acid (MCPBA), purified

1. Dissolve 10 g MCPBA in 200 ml ether and wash with three 150-ml portions of 0.1 M aqueous potassium phosphate, pH 7.5. Concentrate to dryness and dry over P₂O₅ in a vacuum desiccator overnight.
2. Store up to 3 months at –20°C in a bottle with a tight cap.

[¹³C,¹⁵N]Cyanogen bromide

1. Place 3 ml water into a small pear-shaped flask and add 1.2 g (0.38 ml, 7.5 mmol) bromine. Chill the flask in an ice bath, slowly add 0.50 g (7.5 mmol, 1 eq) [¹³C,¹⁵N]potassium cyanide dissolved in 20 ml water, and stir 30 min. Draw the solution into a syringe in order to add it to a reaction.

[¹⁵N]Cyanogen bromide

1. Prepare as for [¹³C,¹⁵N]cyanogen bromide (see recipe) but use [¹⁵N]potassium cyanide instead of [¹³C,¹⁵N]potassium cyanide.

[¹³C]Sodium ethyl xanthate ([¹³C]NaSCSOEt)

1. Dissolve 0.40 g (10 mmol) NaOH in 40 ml absolute ethanol. Add 0.77 g (10 mmol, 1 eq) [¹³C]carbon disulfide ([¹³C]CS₂; Isotec or Cambridge Isotope Laboratories). Cover the flask with aluminum foil and stir the solution overnight at room temperature. Concentrate to dryness and dry over P₂O₅ in a vacuum desiccator overnight. Store up to 6 months at –20°C.

Background Information

¹⁵N NMR studies of specifically ¹⁵N-labeled DNA and RNA fragments have provided significant information about local interactions at nitrogen atoms, such as hydrogen bonding, stacking, and protonation (Wang, Gao, Gaffney, & Jones, 1991; Zhang, Gaffney, & Jones, 1997, 1998). The use of one or more multi-labeled nucleosides can provide more information than single-labeled nucleosides but only as long as all signals can be distinguished. The use of ¹³C tags adjacent to different nitrogens in a pair of nucleosides was designed to allow such differentiation (Abad, Shallop, Gaffney, & Jones, 1998; Shallop & Jones, 2000; Zhao et al., 1997). In addition, the ¹³C chemical shifts can provide valuable information. Recent work using these labeled DNA or RNA fragments include Dayie, Olenginski, and Taiwo (2022); Olenginski, Taiwo, Leblanc, and Dayie (2021); Olengiinsky (2021); Goldberga et al. (2019); Becette, Olenginski, and Dayie (2019); Nussbaumer et al. (2017); Dallmann et al. (2016); and Lagoya (2002).

The route to labeled adenosine described here (Pagano et al., 1995) starts with a direct nitrosation, which is more convenient than the azo coupling described earlier (Gaffney, Kung, & Jones, 1990). This route has been designed so as to remove both thiol groups (in the case of 3b) in the same step. The enzymatic coupling is done with 6-chloropurine because the subsequent displacement with [¹⁵N]NH₃ can be done using much milder conditions. Also, the lipophilicity of the 6-chloro group helps in the purification of the nucleoside. The purine nucleoside phosphorylase uses 7-methylguanosine as a sugar donor and both generates the ribose-α-1-phosphate and couples it to the 6-chloropurine. The reaction is driven to completion by precipitation from solution of the very insoluble 7-methylguanine. The final amination has been optimized so that it only requires two equivalents of [¹⁵N]NH₄Cl with KHCO₃. Although not described here, [7,NH₂-¹⁵N₂]adenosine can be converted to [1,7,NH₂-¹⁵N₃]adenosine (Pagano, Zhao, Shallop, & Jones, 1998). Deamination of any of the adenosines to the corresponding labeled inosine is easily effected. In addition, synthesis of the labeled deoxynucleosides using purine nucleoside phosphorylase (PNP) and 7-methyldeoxyguanosine is straightforward (Drenichev et al., 2022).

The adenosine to guanosine transformation described here (Shallop & Jones, 2000; Zhao et al., 1997) is based on a previous method (Goswami & Jones, 1991) that was in turn derived from earlier work (Ueda, Miura, & Kasai, 1978).

Critical Parameters

In the chlorination that converts 4a/b to 5a/b , it is essential to remove all excess POCl₃, or heat generated during subsequent neutralization with ammonia will cause the reaction to reverse to some extent. During the one-flask set of reactions for the adenosine to guanosine transformation, the intermediates are not particularly stable, and the reactions should not be left for longer than the stated times. After formation of cyanogen bromide, there should be no excess bromine present (as indicated by no orange color present). The order of addition during the formation of cyanogen bromide is also important. In the final enzymatic deamination to make 13c-f , it is important that 12c-f be purified for the enzymatic reaction to work satisfactorily.

Understanding Results

The early steps in these protocols generally give high yields, even for inexperienced workers. The later steps are more challenging, and, with experience, the stated yields can be obtained. Because most of these compounds are fairly polar, the use of TLC to monitor reactions is not very convenient. Reversed-phase HPLC is much more informative, with UV λ_max data from an HPLC system with a diode array detector and mass data from an LCMS being particularly helpful.

Time Considerations

The total time for Basic Protocol 1 is 2 to 3 weeks and that for Basic Protocol 2 is 1 to 2 weeks. The Support Protocol requires 1 to 2 days.

Acknowledgments

This work was supported by grants from the National Institutes of Health (GM31483 and GM48802).

Author Contributions

Roger A. Jones : Conceptualization, project administration, supervision, writing original draft, writing review and editing; Barbara L. Gaffney : Investigation, project administration, supervision, writing original draft, writing review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

Data sharing not applicable – no new data generated.

• Abad, J.-L., Shallop, A. J., Gaffney, B. L., & Jones, R. A. (1998). Use of ¹³C tags with specifically ¹⁵N-labeled DNA and RNA. Biopolymers , 48, 57–63. doi: 10.1002/(SICI)1097-0282(1998)48:1<57::AID-BIP6>3.0.CO;2-B.
• Becette, O., Olenginski, L. T., & Dayie, T. K. (2019). Solid-phase chemical synthesis of stable isotope-labeled RNA to aid structure and dynamics studies by NMR spectroscopy. Molecules , 24, 3476. doi: 10.3390/molecules24193476
• Dayie, T. K., Olenginski, L. T., & Taiwo, K. M. (2022). Isotope labels combined with solution NMR spectroscopy make visible the invisible conformations of small-to-large RNAs. Chemical Reviews , 122(10), 9357–9394. doi: 10.1021/acs.chemrev.1c00845
• Drenichev, M. S., Dorinova, E. O., Varizhuk, I. V., Oslovsky, V. E., Varga, M. A., Esipov, R. S., … Alexeev, C. S. (2022). Synthesis of fluorine-containing analogues of purine deoxynucleosides: Optimization of enzymatic transglycosylation conditions. Doklady Biochemistry and Biophysics , 503(1), 52–58. doi: 10.1134/S1607672922020053
• Dallmann, A., Beribisky, A. V., Gnerlich, F., Rübbelke, M., Schiesser, S., Carell, T., & Sattler, M. (2016). Site-specific isotope-labeling of inosine phosphoramidites and NMR analysis of an inosine-containing RNA duplex. Chemistry Europe , 22(43), 15350–15359. doi: 10.1002/chem.201602784
• Gaffney, B. L., Kung, P.-P., & Jones, R. A. (1990). Nitrogen-15-labeled deoxynucleosides. 2. Synthesis of [7-¹⁵N]-labeled deoxyadenosine, deoxyguanosine, and related deoxynucleosides. Journal of the American Chemical Society , 112, 6748–6749. doi: 10.1021/ja00174a065
• Goldberga, I., Li, R., Chow, W. Y., Reid, D. G., Bashtanova, U., Rajan, R., … Duer, M. J. (2019). Detection of nucleic acids and other low abundance components o native bone and osteosarcoma extracellular matrix by isotope enrichment and DNP-enhanced NMR. RSC Advances , 9(9), 26686–26690. doi: 10.1039/C9RA03198G
• Goswami, B., & Jones, R. A. (1991). Nitrogen-15-labeled deoxynucleosides. 4. Synthesis of [1¹⁵N]-and [2-¹⁵N]-labeled 2′-deoxyguanosines. Journal of the American Chemical Society , 113, 644–647. doi: 10.1021/ja00002a037
• Lagoya, I. M., & Herdewijn, P. (2002). Chemical synthesis of ¹³C and ¹⁵N labeled nucleosides. Synthesis , 3, 301–314. doi: 10.1055/s-2002-20030
• Olenginski, L. T., Taiwo, K. M., Leblanc, R. M., & Dayie, T. K. (2021). Isotope-labeled RNA building blocks for NMR structure and dynamics studies. Molecules , 26(18), 5581. doi: 10.3390/molecules26185581
• Nussbaumer, F., Juen, M. A., Gasser, C., Kremser, J., Müller, T., Tollinger, M., & Kreutz, C. (2017). Synthesis and incorporation of ¹³C-labled DNA building blocks to probe structural dynamics of DNA by NMR. Nucleic Acids Research , 45(15), 9178–9192. doi: 10.1093/nar/gkx592
• Pagano, A. R., Lajewski, W. M., & Jones, R. A. (1995). Synthesis of [6,7-¹⁵N]-adenosine, [6,7-¹⁵N]-2′-deoxyadenosine, and [7-¹⁵N]-hypoxanthine. Journal of the American Chemical Society , 117, 11669–11672. doi: 10.1021/ja00152a006
• Pagano, A. R., Zhao, H., Shallop, A., & Jones, R. A. (1998). Synthesis of [1,7-¹⁵N₂]- and [1,7, NH₂-¹⁵N₃]-adenosine and 2′-deoxyadenosine via an N1-alkoxy mediated Dimroth rearrangement. Journal of Organic Chemistry , 63, 3213–3217. doi: 10.1021/jo9718152
• Shallop, A. J., & Jones, R. A. (2000). Use of a ¹³C “indirect tag” to differentiate two ¹⁵N⁷specifically labeled nucleosides. Middle Atlantic Regional Meeting, American Chemical Society, Newark, DE. Abstract 33:215-ORGN.
• Sinha, N. D., & Jung, K. E. (2015). Analysis and purification of synthetic nucleic acids using HPLC. Current Protocols in Nucleic Acid Chemistry , 61, 10.5.1–10.5.39. doi: 10.1002/0471142700.nc1005s61
• Ueda, T., Miura, K., & Kasai, T. (1978). Synthesis of 6-thioguanine and 2,6-diaminopurine nucleosides and nucleotides from adenine counterparts via a facile rearrangement in the base portion. Chemical and Pharmaceutical Bulletin , 26, 2122–2127. doi: 10.1248/cpb.26.2122
• Wang, C., Gao, H., Gaffney, B. L., & Jones, R. A. (1991). Nitrogen-15-labeled deoxynucleotides. 3. Protonation of the adenine N1 int he A·C and A·G mispairs of the duplexes [d[CG(¹⁵N¹)AGAATTCCCG]}₂ and {d[CGGGAATTC(¹⁵N¹)ACG]}₂. Journal of the American Chemical Society , 113, 5486–5488. doi: 10.1021/ja00014a068
• Zhang, X., Gaffney, B. L., & Jones, R. A. (1997). ¹⁵N NMR of a specifically labeled RNA fragment containing intrahelical GU wobble pairs. Journal of the American Chemical Society , 119, 6432–6433. doi: 10.1021/ja970370w
• Zhang, X., Gaffney, B. L., & Jones, R. A. (1998). ¹⁵N NMR of RNA fragments containing specifically labeled tandem G·A pairs. Journal of the American Chemical Society , 120, 6625–6626. doi: 10.1021/ja9738384
• Zhao, H., Pagano, A. R., Wang, W., Shallop, A., Gaffney, B. L., & Jones, R. A. (1997). Use of a ¹³C atom to differentiate two ¹⁵N-labeled nucleosides: Syntheses of [¹⁵NH₂]-adenosine, [1,NH₂-¹⁵N₂]- and [2-¹³C-1,NH₂-¹⁵N₂]-guanosine, and [1,7,NH₂-¹⁵N₃]-and [2-¹³C-1,7,NH₂-¹⁵N₃]-2′-deoxyguanosine. Journal of Organic Chemistry , 62, 7832–7835. doi: 10.1021/jo971206u