Bitan:Microwave-assisted peptide synthesis
Microwave-assistant peptide synthesis
Instrument Type: Discover SPS - Manual Peptide Synthesis
(The method is modified from the CEM discover SPS installation instruction)
1. Install the instruments as manual book.
2. Create a microwave method.
1) Press the Open Folder Button.
2) Select “New method”.
3) Press the right arrow key until “ Mode = Discover SPS”. Then press ENTER.
4) Set power then press ENTER.
5) Set maximum temperature then press ENTER.
6) Set Run time, then press ENTER.
7) Set delta temperature then press ENTER. (Delta temperature is the minimum value the temperature must drop below the maximum temperature before the power will be re-applied. Defult = 5°C).
8) Set stirring to OFF, then press ENTER.
9) Set cooling to OFF press ENTER.
10) Set “next stage = (N)” then press ENTER.
11) Set “save method = (Y)” then press ENTER.
12) Create method name using the arrow keys and highlight “ Exit” and press ENTER when done.
13) The method is now saved in the software. To load different methods, press Open Folder Button and use the arrow keys to scroll through available methods.
3. Recommended parameters for Fmoc Solid Phase Synthesis
1) Fmoc deprotection
Power = 20 W; Temperature = 75 °C; Time = 3 min; Delta Temp. = 5°C
Power = 20 W; Temperature = 75 °C; Time = 5 min; Delta Temp. = 5°C
3) TFA cleavage
Power = 20 W; Temperature = 38 °C; Time = 18 min; Delta Temp. = 5°C
4. Prepare reaction vessel, dry solvents (DMF, DCM, and MeOH), resin, and activation reagents et al.
5. Synthesis steps
1) Place the luer plug on the bottom of reaction vessel and swell the resin in reaction vessel in DMF for around 1 hour before experiment.
2) Turn on the vacuum pump. Place the reaction vessel in front of the waste bottle immediately after removing the luer plug. The liquid waste should drain into the 1L bottle.
3) Wash the resin with DMF (5×). Allow the liquid to drain and turn off the vacuum pump.
4) Remove the reaction vessel from the vacuum manifold and reattach the lure plug. The resin is ready to use now.
5) If the resin is pre-protected by Fmoc, remove Fmoc with deprotection solvent (deprotection solvent: 20% piperidine + DMF). Add the deprotection solvent to resin. Shake for 20 min in room temperature.
6) Turn on the vacuum pump. Place the reaction vessel in front of the waste bottle immediately after removing the luer plug. Wash the resin with DMF (5×). Allow the liquid to drain and turn off the vacuum pump.
7) First amino acid coupling:
i) Prepare the coupling amino acid activation solution. (Recommend amino acid/HBTU/DIEA method for normal peptide synthesis). Equimolar amounts of protected amino acid and HBTU are dissolved in DMF (usually a 3-fold excess). 6-fold excess of base (DIPEA) is added after the solution is stirred for 10 minutes, and then added to the reaction vessel.
ii) Insert the fiber-optic probe into the thermowell. Insert the thermowell into the clip, and attach the clip to the reaction vessel.
iii) Place the reaction vessel into the holder and insert into the microwave cavity.
iv) Press the play key on the panel and the loaded coupling method will run.
8) After the method running is done, cool down the solution to the set temperature.
9) Wash the resin with DMF (5×). Allow the liquid to drain and turn off the vacuum pump.
10) Test the coupling efficiency. (For difficult sequence, the method of test cleavage detected by Mass spectrometer or Fmoc UV detection (see procedure 1 below) is recommended. For normal sequence TNBS test or Kaiser test is recommended (see procedure 2 and 3 below).
11) If the coupling is efficient, deprotect the Fmoc group as step 5.5. If the coupling is not complete, repeat the coupling step with fresh prepared the amino acid activation solution.
12) Repeat coupling and deprotection Fmoc steps to the end of the sequence.
1) Prepare the resin: Wash the resin completely with DMF and then DCM. Dry the resin in vacuum until it is completely dry.
2) Prepare the cleavage cocktail. Different sequence or resin need different cleavage cocktail. Reagents R (TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5)) and B (TFA/phenol/water/TIPS (88/5/5/2)) are compatible with most sequences, and are strongly recommended for sequences containing Trp, His, Met, Cys, Arg, Gln, or Asn, as well as for peptides constructed on a PAL or Rink Amide resin. For other special resin, please look up the resin instruction from the company.
3) Add the cleavage solution to resin. Be sure you wear protection (gloves and goggles) when you handle TFA.
4) Since our microwave system installed with open vessel, we cannot do cleavage under microwave in our system. They have new cleavage system available in CEM Company, but we would like to do this step in room temperature. After add the cleavage solution to the dry resin, seal the vessel and shake for less than 2 hours.
5) Collect the product after cleavage:
i) Insert a 15 mL centrifuge tube inside the 250 mL bottle and lid is secure.
ii) Turn on the vacuum pump.
iii) Place the reaction vessel in the well located in front of the 250 mL bottle immediately after removing the luer plug.
iv) Wash the resin with a small amount of cleavage solution or pure TFA to ensure all cleaved peptide has been transferred to the centrifuge tube.
v) Turn off the vacuum pump.
vi) Remove the reaction vessel and discard.
6) Take the 15 mL tube with the collected peptide solution out of the bottle.
1) Concentrate the collected peptide solution with a strain of high pure N2 gas to 1–2 mL.
2) Add cold ether to the concentrated solution and sit the tube in ice or freezer. The crude peptide will precipitate.
3) Wait couple of hours or just leave the tube in freezer overnight. Centrifuge the solution and collect the precipitate.
4) Add cold ether and centrifuge again to remove the soluble impurities. Repeat 2–3 times.
5) Collect the precipitate and dry it under vacuum.
6) Now you have your crude peptide!
Procedure 1: Fmoc-quantitation
The cuvette 1 is filled with 3 ml of the sample solution (Fmoc deprotection solution with a certain dilution).
The cuvette 2 is filled with 3 ml of the blank solution (blank solution is prepared in the same manner but without addition of the resin).
The cuvette 3 is filled with 3 ml of the reference solution (reference solution is prepared in the same manner but with resin known loading rate)
A UV spectrometer is set to zero at 290 nm on the blank solution, and the optical density of the sample and reference solution measured.
The cuvettes are emptied and cleaned and the measurement repeated twice with fresh solutions. The loading is calculated by compare the UV absorption of sample solution and reference solution.
Procedure 2: TNBS test
Solution 1: 5% DIPEA in DMF
Solution 2: 1% aqueous TNBS
A few resin beads are placed in a small test tube and 1-3 drops of each solution are added.
The test is positive when the resin beads turn yellow or red within 10 min and negative when the beads remain colorless.
Procedure 3: Kaiser Test
Solution 1: 5 g ninhydrin in 100 mL ethanol
Solution 2: 80 g phenol in 20 ml ethanol
Solution 3: 2 mL 0.001 M aqueous KCN in 98 mL pyridine
A few resin beads are placed in a small test tube and 2-5 drops of each solution are added.
The tube is placed in an oven and the reaction left to develop for 5 min at 100°C.
The test is positive when the resin and solution turn blue and negative when the beads remain colorless.
1. The microwave-assistant peptide synthesizer used in our lab: http://www.cem.com/page11.html
2. Stacey A. Palasek, Zachary J. Cox, Jonathan M. Collins. J. Pept. Sci. 2007; 13: 143–148
3. Introduction of Fmoc solid phase peptide synthesis method and procedures http://www.chempep.com/ChemPep-Fmoc-Solid-Phase-Peptide-Synthesis.htm