Day 1: Peptide Cleavage Protocol
- All work done under the hood.
1. Before you can start the cleavage reaction, the resin should be dried for 30-60 minutes on the lyophilizer (this can be done at any time after you have completed the synthesis).
2. Weigh the resin.
3. Place the resin in a 50 mL round-bottomed flask (a 24/40 joint makes it easier to get the resin in and out).
4. Be very careful with this step. These compounds are toxic and foul- smelling. If you work neatly, you will hardly notice… • 4.5 mL trifluoracetic acid (TFA) • 0.25 mL thioanisole • 0.15 mL 1,2-ethanedithiol • 0.10 anisole
5. Make sure the deprotection solution is thoroughly mixed (Pasteur pipet) before you add it to the resin. Put a rubber stopper on the rb flask, place it in a plastic beaker and tape I onto the shaker for 2 hrs.
6. While the deprotection reaction is shaking, you can get ready for the workup. Obtain:
• 1 x 60 ML sintered glass funnel (medium) • 1 x 60 mL sintered glass funnel (fine) • 2 x 125 mL vacuum filtering flasks, equipped with sleeves, to go with the above • ca 100 mL diethyl ether on ice • put a small plug of glass-wool in a Pasteur pipet. (During the concentration step described below, you will use a stream of nitrogen or argon that will be passed through the pipette. The plug is a filter to ensure that no solid particles get into the reaction mixture). • 2 x 50 mL plastic centrifuge tubes (blue screw cap)
7. Filter the resin through the medium porosity funnel to remove the resin. Use a lead donut around our filtering flask so that it will no tip over. Your peptide should now be in the filtrate! Wash the resin with a 3 x 5 mL of TFA. To optimize the washing step, break the vacuum (by, for example removing the hose), add the TFA in one portion and mix the slurry with a spatula or glass rod. Apply vacuum (by re-attaching the hose). Repeat twice.
8. Use a stream of nitrogen or argon to concentrate the filtrate to 3-5 mL. We don’t use the rotary evaporator for this step because of the TFA. Connect the wool-plugged Pasteur pipette to the end of a nitrogen or argon line. Secure he Pasteur pipette with a small clamp. The tip of the pipette should be placed right above the surface of the solution. SLOWLY open to the gas. You want the surface to get as much agitation as possible without it splattering. The concentration step may take ca 20min or so.
9. Now to the real exciting step. (Please get me so I can watch!) Add ice-cold ether to the concentrated peptide solution. In the beginning, use a Pasteur pipette. You should get a white precipitate. Add enough ether until no more precipitate is observed.
10. Collect the white precipitate using vacuum filtration and the fine porosity funnel. I will help you with this step. The product should then be dissolved in acetonitrile and dd water in a plastic centrifuge tube, frozen, and placed on the lyophilizer (I will help you with this step).
11. Put all solid waste in solid waste container. Place filters and other glassware in bleach (gets rid of thiols and other toxic chemicals).
12. It usually takes 2 days for the freeze-drying step (samples freeze dried on the lyophilizer) .
13. Record the weight of the peptide and run HPLC trace!
Day 2: Cleanup
1. Remove filters from bleach. Wash well with soap and water.
2. Rinse by filtering methanol (non-halogenated waste), DCM (halogenated waste), and acetone (nonhalogenated waste) into separate flasks. When complete, pour off each filtrate into the correct waste bin.
- To ensure filter is clean, make sure to fill the apparatus approximately halfway with each cleaning agent.
3. When complete, turn flask upside down to ensure that everything dries. Leave filters in the hood to dry.
4. Repeat step 1 (ONLY) for other glassware soaked overnight in bleach. Let dry in the drying rack.