Chemical Synthesis of ImpA (adenosine 5'-phosphorimidazolide)

2. Dissolve 262 mg (1 mmol) of triphenylphosphine, 220 mg (1 mmol) of 2,2'-dipyridyldisulfide, and 170 mg (2.5 mmol) of imidazole in 15 ml DMF and 0.9 ml (2.5 mmol) triethylamine.  Keep a 50-μl aliquot for TLC analysis.

3. Add the AMP solution dropwise to a vigorously stirred triphenylphosphine containing solution. Cover beaker and stir for 1.5 hr at room temperature under fume hood.

 

4. Precipitate the ImpA by adding the reaction mixture dropwise into a vigorously stirred solution of 1.1 g (9 mmol) sodium perchlorate, 115 ml acetone, and 55 ml anhydrous ethyl ether.

5. Let the precipitate settle to the bottom of the beaker for 1 hr and decant ∼150 ml of the supernatant without perturbing the precipitate.
A large glass pipet connected to a pipetting aid may also be used to aspirate off the supernatant.

6. Once the volume has been reduced to ∼20 ml, resuspend the precipitate with the residual supernatant and transfer the suspension to 30-ml Corex tubes. Transfer the residual precipitate by rinsing the beaker with small volumes (5 ml) of acetone.
Collect the precipitate by centrifuging 10 min at 3000 × g (5000 rpm with a Sorvall SS34 rotor), room temperature.

7. Remove the supernatant and wash the pellet two times by resuspending it with 10 ml acetone and then centrifuge 5 min at 3000 × g, room temperature.

8. Resuspend the pellet in 10 ml anhydrous ethyl ether and centrifuge 20 min at 3000 × g, room temperature. Dry the pellet overnight in a vacuum oven at 40C.

9. Store the dried powder up to 3 weeks at -20C protected from humidity.
The yield of ImpA is ∼80 mg. The molecular weight of ImpA is 396.28 g/mol.

10. Soak cellulose-F TLC plates in 10% saturated aqueous (NH4)2SO4 and dry the TLC plates in open air for 1 hr or blow dry.

11. Dissolve 1 mg ImpA in 50 μl water and spot the sample on pre-treated TLC plates with aliquots of the AMP solution and triphenylphosphine solutions using glass capillaries. Develop the TLC by using 80% ethanol and visualize the spots under 254-nm UV light.
The AMP should have the lowest retention factor (Rf = 0.26), ImpA should run faster (Rf = 0.4), and the components of the triphenylphosphine solution all run near the solvent front.