This article describes high-yield procedures for protection of purine ribonucleosides based on a reaction that allows highly regioselective 2′-silylation. Each protocol makes use of two transient protection steps. In the case of tritylation of the 5′ hydroxyl, the 2′,3′-diol is protected by reaction with N,N -dimethylformamide dimethylacetal (Zemlicka, 1963) to prevent the small, but potentially troublesome, tritylation of the 2′-hydroxyl that otherwise accompanies tritylation of the 5′-hydroxyl (Zhang et al., 1997). The phenoxyacetylation of the amino group is carried out after transient hydroxyl and guanine O ⁶ protection with trimethylchlorosilane using the hydroxybenzotriazole active ester of phenoxyacetic acid. These protocols give overall yields that are three times the best yields available by conventional procedures for adenosine and guanosine, but offer no advantage for cytidine or uridine. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1 : Synthesis of 5′-O -(4,4′-dimethoxytrityl)-2′-O -tert -butyldimethylsilyl-6-N -acyladenosine

Basic Protocol 2 : Synthesis of 5′-O -(4,4′-dimethoxytrityl)-2′-O -tert -butyldimethysilyl-2-N -acylguanosine

The first step in this protocol is transient protection of the 2′,3′-diol moiety by reaction with N,N -dimethylformamide dimethylacetal (Zemlicka, 1963), followed by tritylation using 4,4′-dimethoxytritylchloride to give 2 (Fig. 1). The 2′,3′-O- dimethylaminomethylene group and the N -dimethylaminomethylene group are cleaved by treatment with aqueous ammonia to give 3.

Phenoxyacetylation is carried out using the hydroxybenzotriazole active ester of phenoxyacetic acid after transient hydroxyl protection with trimethylchlorosilane to give 4 , which is hydrolyzed to 5.

The regioselective silylation is carried out by treatment with a mixture of phenyl-H -phosphonate, tert -butyldimethylchlorosilane, and DBU to generate a mixture of diesters (6). Subsequent transfer of the tert -butyldimethylsilyl (TBDMS) group predominantly to the more acidic 2′-hydroxyl gives 7 along with 10% to 15% of the 3′-O -TBDMS isomer.

The H -phosphonate moiety is removed by reaction with glycerol. The extraordinarily facile transesterification of H -phosphonate diesters in the presence of a vicinal hydroxyl group affects the conversion to 8 quantitatively within minutes.

After careful purification, 8 is converted to the phosphoramidite 9 by reaction with 2-cyanoethyl tetraisopropylphosphorodiamidite using diisopropylammonium tetrazolide as a catalyst. A short silica gel column removes the excess reagent.

Materials

• Adenosine

• Pyridine (reagent grade or better)

• Dimethylformamide dimethyl acetal (e.g., Aldrich or Fisher)

• Nitrogen source

• 4,4′-Dimethoxytrityl chloride (DMTr-Cl)

• Acetonitrile (anhydrous, dried over 3 Å molecular sieves)

• 0.1 M triethylammonium acetate (TEAA)

• Methanol

• Dichloromethane

• Hydrochloric acid

• Sodium bicarbonate

• Concentrated aqueous ammonium hydroxide

• N -Methylmorpholine (e.g., Aldrich or Fisher)

• Trimethylchlorosilane

• Adenosine phenoxyacetylating reagent (see recipe)

• Ammonium phenyl-H -phosphonate (see recipe)

• 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)

• tert -Butyldimethylsilyl chloride (TBDMS∙Cl)

• 0.5 M potassium phosphate buffer, pH 7.0 (see Current Protocols article: Moore, 1996)

• Glycerol

• 1-Adamantanecarbonyl chloride (Aldrich, 117722)

• Ethyl acetate

• Petroleum ether

• Diisopropylammonium tetrazolide (see recipe)

• Argon source

• 2-Cyanoethyl tetraisopropylphosphorodiamidite (Aldrich, 305995)

• Methylene chloride (anhydrous)

• Triethylamine (anhydrous)

• 250-ml and 25-ml round-bottom flask

• Magnetic stirrer and stir bar

• Rotary evaporator

• Vacuum pump

• Septum Vent needles

• Syringes

• Silica gel 60F TLC plates (Merck)

• Waters XTerra 2.5-μm C18 chromatography column

• Separatory funnels

• Desiccator with P₂O₅

• Small glass column containing silica gel (Meyers, 2001)

• Additional reagents and equipment for TLC (see Current Protocols article: Meyers & Meyers, 2008), column chromatography (see Current Protocols article: Meyers, 2001), and HPLC (see Current Protocols article Andrus & Kuimelis, 2001)

Tritylate adenosine (1 to 3)

1.Suspend 1.34 g (5 mmol) adenosine (1) in 10 ml pyridine in a 250-ml round-bottom flask with a magnetic stir bar and concentrate to dryness using a rotary evaporator and vacuum pump. Repeat this azeotropic drying process two times with 10-ml portions of pyridine.

2.Suspend the dry adenosine in 60 ml pyridine, reduce the volume to ∼50 ml, and add 2.6 ml (20 mmol) dimethylformamide dimethyl acetal.

3.Seal the flask with a septum and displace the air with nitrogen through a vent needle in the septum. After a few minutes, remove the nitrogen line and vent needle. Allow to sit for 1 hr.

4.Concentrate to an oil, dissolve the oil in 60 ml pyridine, and concentrate to ∼50 ml.

5.Add 2.03 g (6 mmol) 4,4′-dimethoxytrityl chloride and stir 2 to 3 hr.

6.Check the reaction by HPLC (Andrus & Kuimelis, 2001) using a gradient of 2:98 to 80:20 acetonitrile:0.1 M TEAA, pH 6.8, on a C18 column, or by TLC (Meyers & Meyers, 2008) on 60F silica gel plates using 5% methanol in dichloromethane (see Table 1 for compound mobility). Examine the plate under UV light, and then hold it over an open container of fresh aqueous HCl to observe trityl-containing spots.

7.Add 10 ml methanol to quench the excess reagent, wait 5 min, and then pour the solution into 100 ml water containing 1 g (12 mmol) sodium bicarbonate.

8.Extract the solution two times with 80-ml portions of dichloromethane, concentrate the combined organic layers, dissolve the residue in 25 ml pyridine, and add 25 ml concentrated aqueous ammonium hydroxide. Seal the flask tightly and heat 5 hr at 60°C.

9.Cool to room temperature, open the flask carefully, and check the mixture by HPLC or TLC to have a record of the retention time or R _f value of 3 (Table 1).

10.Concentrate the mixture with frequent additions of pyridine so that the water is removed azeotropically to give 3 as an oil. Dry by evaporation of pyridine as in step 1, leaving dry 3 in ∼50 ml pyridine.

Phenoxyacetylate (3 to 5)

11.To the solution of 3 in 50 ml dry pyridine, add 5.5 ml (50 mmol) N -methylmorpholine. Seal the flask with a septum and displace the air with nitrogen through a vent needle in the septum. Keep the nitrogen flowing slowly.

12.Cool this mixture in an ice bath and add 3.2 ml (25 mmol) trimethylchlorosilane over 3 to 5 min.

13.Remove the flask from the ice bath and maintain for 1 hr at room temperature.

14.Add all of the freshly prepared adenosine phenoxyacetylating reagent using a syringe. Remove the nitrogen line and vent needle and stir 12 to 18 hr.

15.Check the reaction by HPLC or TLC.

16.Pour the mixture into 100 ml water containing 2.5 g (30 mmol) sodium bicarbonate, extract two times with 100-ml portions of dichloromethane, and concentrate the combined organic layers to dryness.

17.Dissolve the residue in 50 ml pyridine, add 25 ml water, and stir 12 to 18 hr.

18.Concentrate the solution and check the mixture by HPLC or TLC.

19.Purify the residue by column chromatography (Meyers, 2001) on silica gel using 0:100 to 15:85 (v/v) methanol/dichloromethane to give pure 5 in yields of up to ∼90%.

Silylate (5 to 7)

20.To 2.63 g (15 mmol) of ammonium phenyl-H -phosphonate, add 2.3 ml (15 mmol) DBU and co-evaporate with 50 ml pyridine.

21.Dissolve the residue in 120 ml pyridine, concentrate to ∼100 ml, add 2.26 g (15 mmol) TBDMS-Cl, and mix. Place 3.52 g (5 mmol) 5 in a dry 250-ml round-bottom flask and dry by evaporation of pyridine as in step 1.Using a syringe, add the mixture prepared in step 21 to 5 , followed by 3.8 ml (25 mmol) DBU.

22.Stir 5 to 8 hr and check the reaction by HPLC or TLC.

23.Pour the mixture into 100 ml of 0.5 M potassium phosphate buffer, pH 7.0, and extract two times with 100-ml portions of dichloromethane. Concentrate the combined organic layers to dryness to give 7.

Dephosphonylate (7 to 8)

24.Add 1.38 g (15 mmol) glycerol to the residue (7) and dry the mixture by co-evaporation with 50 ml pyridine.

25.Dissolve the residue in 60 ml pyridine, concentrate to ∼50 ml, and add 2.98 g (15 mmol) of 1-adamantanecarbonyl chloride. Stir 10 min.

26.Pour the solution into 100 ml of 0.5 M aqueous potassium phosphate buffer and extract two times with 100-ml portions of dichloromethane. Concentrate the combined organic layers to dryness.

27.Check the mixture by HPLC or TLC and purify the residue by column chromatography in silica gel using 60:40 to 100:0 (v/v) ethyl acetate:petroleum ether to give 8 in yields of ∼65% from 5.

Phosphitylate (8 to 9)

28.In an oven-dried 25-ml round-bottom flask, place 3.05 g (3.0 mmol) of pure 8 and 0.28 g (1.5 mmol) of diisopropylammonium tetrazolide. Dry the flask and a rubber septum (not inserted) in an evacuated desiccator over P₂O₅ overnight.

29.Open the desiccator under argon and immediately insert the septum. Displace any air with argon through a vent needle in the septum. Add 15 ml of dry dichloromethane through the septum and swirl 5 to 10 min to dissolve the solids completely.

30.Cool the flask in an ice bath at 0° and add 1.00 ml (3.0 mmol) of 2-cyanoethyl tetraisopropylphosphorodiamidite. Keep the mixture at 0° for 1 hr and swirl it every 15 min or so.

31.Remove a small sample carefully with a syringe with an oven-dried needle to check by HPLC or TLC. Normally, the reaction will be about 75% to 85% done.

32.Remove the flask from the ice bath and keep it at room temperature. Add another 0.5 ml (1.5 mmol) of the phosphitylating reagent and allow the reaction to proceed for several more hours. Check by HPLC or TLC no more than once per hour, each time using a dry syringe needle.

33.Prepare a small glass column containing about 10 cm of silica gel packed in 98:2 dry methylene chloride:triethylamine.

34.Place the reaction mixture directly onto this column and load it using nitrogen pressure. Wash the column using nitrogen pressure with about 30 ml of 98:2 dry methylene chloride:triethylamine, followed by 49:1:50 dry methylene chloride:triethylamine:dry acetonitrile.

35.Combine the fractions containing pure 9 and evaporate to a foam. Dry in a desiccator over P₂O₅.

36.Check for purity by ³¹P NMR (Table 2).

In this protocol, guanosine is first amino protected because the N -phenoxyacetyl derivative is crystalline and therefore easy to isolate (Fig. 2). Silylation, dephosphorylation, and phosphitylation are performed following the procedures for adenosine.

Materials

• Guanosine

• Pyridine

• Nitrogen source

• Trimethylchlorosilane

• Guanosine phenoxyacetylating reagent (see recipe)

• 2-Propanol

• Acetonitrile (anhydrous, dried over 3 Å molecular sieves)

• 0.1 M triethylammonium acetate (TEAA)

• Methanol

• Dichloromethane

• Dimethylformamide dimethyl acetal (e.g., Aldrich or Fisher)

• 4,4′-Dimethoxytrityl chloride (DMTr-Cl)

• Hydrochloric acid (HCl)

• Sodium bicarbonate

• Ammonium phenyl-H -phosphonate (see recipe)

• 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU; e.g., Aldrich or Fisher)

• tert -Butyldimethylsilyl chloride (TBDMS-Cl)

• 0.5 M potassium phosphate buffer, pH 7.0 (Moore, 2001)

• Glycerol

• 1-Adamantanecarbonyl chloride (e.g., Aldrich or Fisher)

• 5:95 to 15:85 (v/v) acetone/dichloromethane (optional; for phosphoramidite synthesis)

• Argon source

• Diisopropylammonium tetrazolide (see recipe)

• 2-Cyanoethyl tetraisopropylphosphorodiamidite (Aldrich, 305995)

• Methylene chloride (anhydrous)

• Triethylamine (anhydrous)

• Nitrogen source

• 250-ml and 25-ml round-bottom flasks

• Magnetic stirrer

• Magnetic stir bar

• Vacuum pump

• Rotary evaporator

• Septum

• Vent needle

• Syringes

• Fritted glass filter

• Silica gel 60F TLC plates (Merck)

• Waters Atlantis, XTerra 2.5-μm C18 chromatography column, or similar

• UV source

• Desiccator with P₂O₅

• Silica gel and small glass column (Meyers, 2001)

• Additional reagents and equipment for TLC (see Current Protocols article: Meyers & Meyers, 2008), column chromatography (see Current Protocols article: Meyers, 2001), and HPLC (see Current Protocols article Andrus & Kuimelis, 2001)

Phenoxyacetylate guanosine (10 to 12)

1.Suspend 1.42 g (5 mmol) guanosine (10) in 10 ml pyridine in a 250-ml round-bottom flask with a magnetic stir bar and concentrate to dryness using a rotary evaporator and vacuum pump. Repeat this azeotropic drying process two times with 10-ml portions of pyridine.

2.Suspend the dry guanosine in 60 ml pyridine and concentrate to ∼50 ml.

3.Seal the flask with a septum and displace the air with nitrogen through a vent needle in the septum. Keep the nitrogen flowing slowly.

4.Cool this mixture in an ice bath and add 3.8 ml (30 mmol) trimethylchlorosilane over 3 to 5 min.

5.Remove the flask from the ice bath and maintain 1 hr at room temperature.

6.Add all the freshly prepared guanosine phenoxyacetylating reagent using a syringe. Remove the nitrogen line and vent needle and stir 36 hr.

7.Add 30 ml water, concentrate the solution, and co-evaporate two times with 30-ml portions of water to a final volume of ∼15 ml.

8.Filter the slurry using a fritted glass filter funnel.

9.To remove the 1-hydroxybenzotriazole, shake the solid thoroughly with a 50-ml portion of water and filter. Repeat with 20 ml water followed by three 20-ml portions of 2-propanol to give 12 as a colorless solid in yields of up to 95%.

10.Check the product by HPLC (Andrus & Kuimelis, 2001) using a gradient of 2:98 to 80:20 acetonitrile: 0.1 M TEAA pH 6.8 on a C18 column or TLC (Meyers & Meyers, 2008) on silica gel 60F plates using the appropriate concentration of methanol in dichloromethane (Table 1).

11.Dry the product in a desiccator over P₂O₅ at least overnight and check that the yield is <100%.

Tritylate (12 to 14)

12.Dissolve the dry 12 in 60 ml pyridine, concentrate to ∼50 ml, and add 0.8 ml (6 mmol) dimethylformamide dimethyl acetal.

13.Seal the flask with a septum and displace the air with nitrogen through a vent needle in the septum. After a few minutes, remove the nitrogen line and vent needle. Allow to sit for 1 hr.

14.Concentrate to an oil, dissolve the oil in 60 ml pyridine, and concentrate to ∼50 ml.

15.Add 2.03 g (6 mmol) 4,4′-dimethoxytrityl chloride and stir 2 to 3 hr.

16.Check the reaction by HPLC or TLC. Examine the plate under UV light, and then hold it over an open container of fresh aqueous HCl to observe trityl-containing spots.

17.Add 10 ml methanol to quench the excess reagent, wait 5 min, and then pour the solution into 100 ml water containing 1 g (12 mmol) sodium bicarbonate.

18.Extract the solution two times with 80-ml portions of dichloromethane and concentrate the combined organic layers to dryness.

19.Check the mixture by HPLC or TLC. Purify the residue by column chromatography (Meyers, 2001) on silica gel using 0:100 to 10:90 (v/v) methanol/dichloromethane to give pure 14 in yields of up to ∼90%.

Silylate (14 to 16)

20.To 2.63 g (15 mmol) ammonium phenyl-H -phosphonate, add 2.3 ml (15 mmol) DBU, and co-evaporate with 50 ml pyridine.

21.Dissolve the residue in 120 ml pyridine, concentrate to ∼100 ml, add 2.26 g (15 mmol) TBDMS-Cl and mix. Place 3.52 g (5 mmol) 14 in a dry 250-ml round-bottom flask and dry by evaporation of pyridine as in step 1.Using a syringe, add the mixture prepared in step 21 to 14 , followed by 3.8 ml (25 mmol) DBU.

22.Stir 5 to 8 hr and check the reaction by HPLC or TLC.

23.Pour the mixture into 100 ml of 0.5 M aqueous potassium phosphate buffer, pH 7.0, and extract two times with 100-ml portions of dichloromethane. Concentrate the combined organic layers to dryness to give 16.

Dephosphonylate (16 to 17)

24.Add 1.38 g (15 mmol) glycerol to the residue (16) and dry the mixture by co-evaporation with 50 ml pyridine.

25.Dissolve the residue in 60 ml pyridine, concentrate to about ∼50 ml, and add 2.98 g (15 mmol) of 1-adamantanecarbonyl chloride. Stir 10 min.

26.Pour the solution into 100 ml of 0.5 M potassium phosphate buffer, pH 7.0, and extract two times with 100-ml portions of dichloromethane. Concentrate the combined organic layers to dryness.

27.Check the mixture by HPLC or TLC and purify the residue by column chromatography on silica gel using 5:95 to 15:85 acetone/dichloromethane to give 17 in yields of ∼65% from 14.

Phosphitylate (17 to 18)

28.In an oven-dried 25-ml round-bottom flask, place 3.05 g (3.0 mmol) of pure 17 and 0.28 g (1.5 mmol) of diisopropylammonium tetrazolide. Dry the flask and a rubber septum (not inserted) in an evacuated desiccator over P₂O₅ overnight.

29.Open the desiccator under argon and immediately insert the septum. Displace any air with argon through a vent needle in the septum. Add 15 ml of dry dichloromethane through the septum and swirl 5 to 10 min to dissolve the solids completely.

30.Cool the flask in an ice bath at 0° and add 1.00 ml (3.0 mmol) of 2-cyanoethyl tetraisopropylphosphorodiamidite. Keep the mixture at 0° for 1 hr and swirl it every 15 min or so.

31.Remove a small sample carefully with a syringe with an oven-dried needle to check by HPLC or TLC. Normally, the reaction will be about 75% to 85% done.

32.Remove the flask from the ice bath and keep it at room temperature. Add another 0.5 ml (1.5 mmol) of the phosphitylating reagent and allow the reaction to proceed for several more hours. Check by HPLC or TLC no more than once per hr, each time using a dry syringe needle.

33.Prepare a small glass column containing about 10 cm of silica gel packed in 98:2 dry methylene chloride:triethylamine.

34.Place the reaction mixture directly onto this column and load it using nitrogen pressure. Wash the column using nitrogen pressure with about 30 ml of 98:2 dry methylene chloride:triethylamine, followed by 49:1:50 dry methylene chloride:triethylamine:dry acetonitrile.

35.Combine the fractions containing pure product and evaporate to a foam. Dry the product (18) in a desiccator over P₂O₅.

36.Check 18 for purity by ³¹P NMR (Table 2).

Adenosine phenoxyacetylating reagent

• Co-evaporate 2.03 g (15 mmol) of 1-hydroxybenzotriazole and 2.2 ml (20 mmol) N -methylmorpholine two times with 20-ml portions of acetonitrile. Dissolve the residue in 50 ml dichloromethane. Add 2.1 ml (15 mmol) phenoxyacetyl chloride and shake for 10 min. Prepare fresh.

Ammonium phenyl-H-phosphonate

• Add 38.3 ml (0.20 mol) diphenyl phosphite over 10 min to 400 ml of 7.4 M aqueous ammonia. Stir for 1 hr. Concentrate to dryness and co-evaporate the residue two times with 100-ml portions of absolute ethanol. Stir the residue with 400 ml ethyl ether for 30 min to give up to 85% of the colorless crystalline product. Store up to 1 month at –20°C.

Diisopropylammonium tetrazolide

• Place 2.00 g (28.5 mmol) of solid tetrazole in a dry 250-ml round-bottom flask containing a dry stir bar and add 130 ml of dry acetonitrile. Stir until dissolved, then add 9.0 ml (63.9 mmol) of freshly distilled diisopropylamine. After 2 min of stirring, collect the white precipitate by filtration in a dry glass funnel and wash it four times with 10-ml portions of dry acetonitrile. Dry the solid in a desiccator over P₂O₅. Store up to 3 months at –20°C.

Guanosine phenoxyacetylating reagent

• Co-evaporate 1.35 g (10 mmol) of 1-hydroxybenzotriazole and 1.1 ml (10 mmol) N -methylmorpholine two times with 20-ml portions of acetonitrile. Dissolve the residue in 25 ml dichloromethane. Add 1.39 ml (10 mmol) phenoxyacetyl chloride and shake for 10 min. Prepare fresh.

Background Information

The method for selective silylation described above was developed based on the hypothesis, which proved to be correct, that a combination of phenyl-H -phosphonate and tert -butyldimethylchlorosilane would give the same selectivity for 2′ silylation of adenosine and guanosine that we had previously observed for reaction of a mixture of the adenosine and guanosine 2'(3') H-phosphonates with tert -butyldimethylchlorosilane and DBU (Zhang et al., 1997). Presumably, the reagent combination reacts to give the mixture of the H-phosphonate diester isomers we had observed earlier starting from the nucleoside H-phosphonates, followed by transfer of the tert -butyldimethylsilyl (TBDMS) group predominantly to the more acidic 2′-hydroxyl. The H -phosphonate monoester group produced in the silylation reaction is then cleaved, without silyl migration (Song et al., 1999; Zhang et al., 1997). The selectivity is 85% to 90%, with pure 2′ isomer readily isolated by silica gel chromatography, ready for phosphitylation to yield the phosphoramidites.

The procedures described here use adenosine and guanosine that are 5′-protected with the 4,4′-dimethoxytriyl (DMTr) group and amino-protected with the labile phenoxyacetyl group (Chaix, Duplaa, Molko, & Téoule, 1989; Sinha, Davis, Schultze, & Upadhya, 1995; Singh & Nahar, 1995; Wu, Ogilvie, & Pon, 1988). They should also be applicable to nucleosides containing most other amino-protecting groups. Amino protection of adenosine with the benzoyl group and guanosine with the isobutyryl group can be carried out by the same transient protection procedure (Fan, Gaffeny, & Jones, 2004; Ti, Gaffney, & Jones,1982), and these derivatives are more easily handled than are the more-labile phenoxyacetylated compounds. A general discussion of amino-protecting groups and literature references is given in Current Protocols article Iyer (2001).

The original procedures for 2′-silylation proceed with only modest regioselectivity (Ogilvie, Beaucage, Schifman, Theriault, & Sadana, 1978, Ogilvie, Schifman, Penney, 1979). Although use of silver nitrate improves the selectivity, the results are variable and do not approach the selectivity of the above protocols (Hakimelahi, Proba, & Ogilvie, 1982). The need for selective RNA protecting groups has been reviewed (Somoza, 2008). A site-specific catalytic approach for silylation is promising, particularly for pyrimidines (Blaisdell, Lee, Kasaplar, Sun, & Tan 2013).

Critical Parameters

The most difficult parts of the above protocols to carry out are the tritylation and phenoxyacetylation steps. Skill and practice are required to achieve high yields on these protection reactions, largely because of the phenoxyacetyl group. In contrast, the regioselective silylation and dephosphonylation reactions work well even in the hands of unskilled researchers.

Anticipated Results

For the reasons discussed above, some experience is required to achieve high yields for the phenoxyacetylation reactions, and initial efforts are likely give more modest yields (e.g., 50%). With experience, yields of ∼90% can be expected. The regioselectivity of the silylation reaction is invariably 85% to 90% regardless of experience.

Time Considerations

The total time for conversion of adenosine or guanosine to the fully protected derivatives 9 and 18 , respectively, is ∼1 week.

Acknowledgments

This work was supported by grants from the National Institutes of Health (EB002809 and GM48802).

Author Contributions

Roger Jones : conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, writing original draft, writing review and editing; Barbara Gaffney : funding acquisition, investigation, methodology, 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 were generated.

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