3.1.1 Synthesis of the
Adenosine-5
0 -
Phosphoimidazolide (ImpA)
All steps should be performed under a fume hood.
1. Prepare two 25 mL clean and dry round-bottom glass flasks
with two rims and one stirring bar each inside. Flasks are dried
in a drying oven at 140
C overnight.
2. Use a needle and a syringe to fetch 5 mL anhydrous DMF from
a sealed bottle to a clean flask (flask A); and then resuspend
174 mg (0.5 mM) 5
0 -AMP free acid into the anhydrous DMF
in flask A. The flask should be kept closed with a rubber
septum. The AMP will not dissolve completely. Label this as
solution A. Take a 20 μL aliquot for quality control step.
3. Prepare 262 mg (1 mM) triphenylphosphine, 220 mg (1 mM)
2,2
0 -dipyridyldisulfide, and 170 mg (2.5 mM) imidazole in the
other flask (flask B) with 10 mL DMF and 0.9 mL (2.5 mM)
triethylamine. Stir vigorously. The solution shows a yellowgreen color. Keep the flask closed with a rubber septum.
Label this as solution B. Take a 20 μL aliquot for quality
control step.
4. Connect a nitrogen-filled balloon with the flask of solution B
by a needle inserted through the rubber septum (Fig. 2a). A
second needle is inserted into the septum to replace the humid
air in flask B with nitrogen. Remove the second needle once the
switch finishes (this process takes about 1 min).
5. Use a needle and a syringe to fetch all the solution A and insert
the needle through the rubber septum of flask B. Add solution
A dropwise to flask B. Remove the needle and stir the mixture
to react for 1.5 h at room temperature; keep the flask
connected with nitrogen balloon through the reaction
(Fig. 2b).
6. Add 1.1 g (9 mM) sodium perchlorate, 110 mL acetone, and
55 mL anhydrous diethyl ether to a clean 500 mL beaker. Keep
the beaker on ice and stir vigorously. Add the reaction mixture
above dropwise to this beaker. As the yellow-green AMP solution is added, you will expect to observe the mixture turns
cloudier.
7. After adding all the reaction solution, stop the stirrer. Keep the
beaker on ice for 20 min. The cloudy precipitation starts to sink
to the bottom. Decant as much as possible supernatant out.
8. Resuspend the pellet with the residual solution and transfer the
mixture into two 50 mL Cortex tubes evenly. Centrifuge at
4470 Â g for 2 min. Wash pellet with 20 mL acetone and
centrifuge again. Pour off the supernatant. Repeat the washing
step until the supernatant is colorless clear. The pellet should be
white.
Identification and Quantification of sRNAs
235
Adenosine-5
0 -
Phosphoimidazolide (ImpA)
All steps should be performed under a fume hood.
1. Prepare two 25 mL clean and dry round-bottom glass flasks
with two rims and one stirring bar each inside. Flasks are dried
in a drying oven at 140
C overnight.
2. Use a needle and a syringe to fetch 5 mL anhydrous DMF from
a sealed bottle to a clean flask (flask A); and then resuspend
174 mg (0.5 mM) 5
0 -AMP free acid into the anhydrous DMF
in flask A. The flask should be kept closed with a rubber
septum. The AMP will not dissolve completely. Label this as
solution A. Take a 20 μL aliquot for quality control step.
3. Prepare 262 mg (1 mM) triphenylphosphine, 220 mg (1 mM)
2,2
0 -dipyridyldisulfide, and 170 mg (2.5 mM) imidazole in the
other flask (flask B) with 10 mL DMF and 0.9 mL (2.5 mM)
triethylamine. Stir vigorously. The solution shows a yellowgreen color. Keep the flask closed with a rubber septum.
Label this as solution B. Take a 20 μL aliquot for quality
control step.
4. Connect a nitrogen-filled balloon with the flask of solution B
by a needle inserted through the rubber septum (Fig. 2a). A
second needle is inserted into the septum to replace the humid
air in flask B with nitrogen. Remove the second needle once the
switch finishes (this process takes about 1 min).
5. Use a needle and a syringe to fetch all the solution A and insert
the needle through the rubber septum of flask B. Add solution
A dropwise to flask B. Remove the needle and stir the mixture
to react for 1.5 h at room temperature; keep the flask
connected with nitrogen balloon through the reaction
(Fig. 2b).
6. Add 1.1 g (9 mM) sodium perchlorate, 110 mL acetone, and
55 mL anhydrous diethyl ether to a clean 500 mL beaker. Keep
the beaker on ice and stir vigorously. Add the reaction mixture
above dropwise to this beaker. As the yellow-green AMP solution is added, you will expect to observe the mixture turns
cloudier.
7. After adding all the reaction solution, stop the stirrer. Keep the
beaker on ice for 20 min. The cloudy precipitation starts to sink
to the bottom. Decant as much as possible supernatant out.
8. Resuspend the pellet with the residual solution and transfer the
mixture into two 50 mL Cortex tubes evenly. Centrifuge at
4470 Â g for 2 min. Wash pellet with 20 mL acetone and
centrifuge again. Pour off the supernatant. Repeat the washing
step until the supernatant is colorless clear. The pellet should be
white.
Identification and Quantification of sRNAs
235