Synthesis of O6-alkylated preQ1 derivatives

A naturally occurring riboswitch can utilize 7-aminomethyl-O6-methyl-7-deazaguanine (m6preQ1) as cofactor for methyl group transfer resulting in cytosine methylation. This recently discovered riboswitch-ribozyme activity opens new avenues for the development of RNA labeling tools based on tailored O6-alkylated preQ1 derivatives. Here, we report a robust synthesis for this class of pyrrolo[2,3-d]pyrimidines starting from readily accessible N2-pivaloyl-protected 6-chloro-7-cyano-7-deazaguanine. Substitution of the 6-chloro atom with the alcoholate of interest proceeds straightforward. The transformation of the 7-cyano substituent into the required aminomethyl group turned out to be challenging and was solved by a hydration reaction sequence on a well-soluble dimethoxytritylated precursor via in situ oxime formation. The synthetic path now provides a solid foundation to access O6-alkylated 7-aminomethyl-7-deazaguanines for the development of RNA labeling tools based on the preQ1 class-I riboswitch scaffold.


O 6 -Ethyl preQ 0 (1a)
Sodium (2.22 mmol, 51 mg) was dissolved in ethanol (4.0 ml) and compound 3 (0.77 mmol, 213 mg) was added shortly after. The reaction mixture was stirred in a pressure tube for 24 h at 120 °C, was cooled to room temperature and was neutralized by the addition of acetic acid. Solvent was removed in vacuo and the powdery residue was adsorbed on silica. Flash chromatography (100% EtOAc) afforded compound 1a (

7-Formyl-N 2 ,9-bis(4,4'-dimethoxytrityl)-O 6 -ethyl-7-deazaguanine (5a)
Compound 4a (395 mg, 0.49 mmol) was dissolved in dichloromethane (3.1 mL) and the solution was cooled to -78 °C. Diisobutylaluminium hydride (0.62 mL, 1 M in CH 2 Cl 2 , 0.63 mmol) was added dropwise over 30 minutes and stirring was continued for another three hours at -78 °C. The reaction was quenched by the addition of ethyl acetate (2.0 mL) and was subsequently allowed to warm to S12 ambient temperature. Half-saturated potassium sodium tartrate solution (2.0 mL) was added and the reaction mixture was stirred vigorously for one hour until satisfactory phase separation was achieved. The phases were separated and the aqueous layer was washed three times with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate and purified via column chromatography on silica gel (10-30% ethyl acetate, 1% triethylamine in cyclohexane) to give 379 mg of compound 5a as a white foam (96%

7-Aminomethyl-N 2 ,9-bis(4,4'-dimethoxytrityl)-O 6 -ethyl-7-deazaguanine (6a)
Hydroxylamine hydrochloride (0.70 mmol, 48 mg) was added to a suspension of compound 5a (470 mg, 0.58 mmol) in 7 M methanolic ammonia (13.9 mL). The solution was stirred at room temperature for two hours and became colorless. Tetrahydrofuran (9.2 mL) and Raney Nickel (approximately 500 mg) were added and the reaction mixture was stirred under H 2 atmosphere (1 atm, balloon). Additional Raney nickel (approx. 500 mg) was added after three hours. The reaction was deemed complete after 4 h and was then filtered over a pad of Celite. The reaction mixture was concentrated and subjected to column chromatography on silica gel (0-2% MeOH in CH 2 Cl 2 + 1% Et 3 N) to give 367 mg of compound 6a

O 6 -Ethyl preQ 1 (trifluoroacetate salt) (2a)
A solution of compound 6a (105 mg, 0.13 mmol) in dichloromethane (1.0 ml) was treated with a mixture of trifluoroacetic acid (52 µl) and water (9 µl). The reaction was quenched by the addition of methanol (1.0 ml) after 15 min. Solvent was evaporated and the orange oil was coevaporated trice with methanol and dichloromethane respectively. The orange oil was further dried 2 h under high vacuum. The residue was then dissolved in dichloromethane and was left standing at ambient temperature overnight whereas the crude product precipitates as a white powder. The precipitate was collected by centrifugation and was purified by reversed phase chromatography (Lichoprep 0-20% ACN in water) to give 17 mg of compound 2a as a white powder (41%

O 6 -Benzyl preQ 0 (1b)
Sodium (300 mg, 1.14 mmol) was dissolved in 5.5 mL benzyl alcohol at ambient temperature. To this solution compound 3 (300 mg, 1.08 mmol) was added and the mixture was heated to 140 °C for 48 h. Afterwards, the benzyl alcohol was distilled off and the remaining solid was suspended in methanol and dry-loaded onto silica gel. Flash column chromatography (1-5% methanol in dichloromethane) provided 210 mg of compound 1b (73%) as a white solid. TLC: 8% methanol in dichloromethane, R f 0.5.

N 2 ,9-Bis-(4,4'-dimethoxytrityl)-O 6 -benzyl preQ 0 (4b)
Compound 1b (40.0 mg, 151 µmol) was suspended in dichloromethane (500 µL). N,Obis(trimethylsilyl)-acetamide (80 µL, 320 µmol) was slowly added. After a few minutes a homogenous solution was obtained and the mixture was stirred for another one and a half hour at room temperature. The volatiles were removed in vacuo and the residue was dissolved in pyridine (600 µL). 4,4'-dimethoxytrityl chloride (118 mg, 247 µmol) was added and the solution was stirred for 20 h at 40 °C. Aqueous sodium bicarbonate solution (5%, 10 mL) was added and the mixture was extracted three times with ethyl acetate. After drying over magnesium sulfate the crude mixture was purified by flash column chromatography on silica gel (20% ethyl acetate in cyclohexane) to give 129 mg of compound 4b (98%) as a white foam.

7-Formyl-N 2 ,9-bis(4,4'-dimethoxytrityl) O 6 -benzyl-7-deazaguanine (5b)
Compound 4b (100 mg, 115 µmol) was dissolved in dichloromethane (750 µL) and cooled to -78 °C. After addition of diisobutylaluminium hydride (150 µL, 1.0 M solution in dichloromethane) the mixture was stirred for two hours at this temperature. The reaction was quenched upon the addition of ethyl acetate (2 mL) and allowed to warm to room temperature. Half-saturated aqueous sodium potassium tartrate solution (2 mL) was added and vigorously stirred for two hours. The aqueous and organic S23 phase were separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over magnesium sulfate and purified by flash column chromatography on silica gel (20% ethyl acetate in cyclohexane) to give 90 mg of compound 5b (89%) as a white foam.

7-Aminomethyl-N 2 ,9-bis(4,4'-dimethoxytrityl)-O 6 -benzyl-7-deazaguanine (6b)
Compound 5b (300 mg, 345 µmol) was suspended in 1.5 mL 7 M ammonia in methanol, hydroxylamine hydrochloride (29 mg, 410 µmol) and tetrahydrofuran (0.5 mL) were added and the mixture was stirred for one and a half hour at room temperature. Damp Raney-Nickel (approximately 100 mg) was added and hydrogen was bubbled through the solution for half an hour under vigorous stirring. Bubbling was stopped and the reaction was continued for one hour, after which the mixture was filtered over a Celite pad and the filtrate was concentrated in vacuo. Purification via silica gel column chromatography (1-3% methanol in dichloromethane) afforded 218 mg of compound 6b (73%) as a white foam. TLC: 6% methanol in dichloromethane, R f 0.46.

O 6 -Benzyl preQ 1 (trifluoroacetate salt) (2b)
To a solution of compound 6b (197 mg, 225 µmol) in dichloromethane (750 µL) were added trifluoroacetic acid (90 µL, 2.27 mmol) and water (15 µL). After 30 minutes stirring at room temperature the volatiles were removed in vacuo and the residual oil was coevaporated with methanol and dichloromethane. After short drying on high vacuum the crude product was made basic by the addition of triethylamine and purified by flash column chromatography on silica gel (5-15% methanol in dichloromethane). The pure product was dissolved in a small amount of methanol and trifluoroacetic acid (20 µL) was added. After evaporation and trituration with dichloromethane 64 mg of compound 2b (74%) as a beige solid were obtained. TLC: 15% methanol in dichloromethane 1% triethylamine, R f 0.35