Bitan:Microwave-assistant peptide synthesis
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<p class=MsoNormal style='tab-stops:87.2pt'><b><span style="mso-spacerun: yes"> </span><span style="mso-spacerun: yes"> </span><span style="mso-spacerun: yes"> </span><span style="mso-spacerun: yes"> </span><a href="http://openwetware.org/wiki/Bitan:todo">[Back to Bitan: todo page]</a><o:p></o:p></b></p>
<p class=MsoNormal style='tab-stops:87.2pt'><![if !supportEmptyParas]> <![endif]><o:p></o:p></p>
<table border=0 cellspacing=0 cellpadding=0 bgcolor="#e6e6e6" style='background:
#E6E6E6;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'> <tr> <td width=935 valign=top style='width:935.1pt;padding:0in 5.4pt 0in 5.4pt'> <p class=MsoNormal align=center style='text-align:center'><span style='font-size:16.0pt;color:#993300'><b>Microwave-assistant peptide synthesis</b></span><span style='font-size:16.0pt;color:#993300;mso-fareast-language: ZH-CN'> <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Instrument Type: Discover SPS - Manual Peptide Synthesis </span><b><o:p></o:p></b></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>(The method is modified from the CEM discover SPS installation instruction)<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>1. Install the instruments as manual book.<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b><![if !supportEmptyParas]> <![endif]><o:p></o:p></b></span></p> <p class=MsoNormal style='tab-stops:432.8pt'><span style='mso-fareast-language: ZH-CN'><b>2. Create a microwave method.<span style='mso-tab-count:1'> </span></b></span><span style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Press the Open Folder Button.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Select “New method”.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Press the right arrow key until “ Mode = Discover SPS”. Then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Set power then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) Set maximum temperature then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Set Run time, then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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).<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>8) Set stirring to OFF, then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>9) Set cooling to OFF press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>10) Set “next stage = (N)” then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>11) Set “save method = (Y)” then press ENTER.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>12) Create method name using the arrow keys and highlight “ Exit” and press ENTER when done.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>3. Recommended parameters for Fmoc Solid Phase Synthesis<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Fmoc deprotection<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Power = 20 W; Temperature = 75 °C; Time = 3 min; Delta Temp. = 5°C<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Coupling <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Power = 20 W; Temperature = 75 °C; Time = 5 min; Delta Temp. = 5°C<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) TFA cleavage<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Power = 20 W; Temperature = 38 °C; Time = 18 min; Delta Temp. = 5°C<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>4. Prepare reaction vessel, dry solvents (DMF, DCM, and MeOH), resin, and activation reagents et al.<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>5. Synthesis steps<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Wash the resin with DMF (5×). Allow the liquid to drain and turn off the vacuum pump.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Remove the reaction vessel from the vacuum manifold and reattach the lure plug. The resin is ready to use now. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) If the resin is pre-protected by Fmoc, remove Fmoc with deprotection solvent (deprotection solvent:<span style="mso-spacerun: yes"> </span>20% piperidine + DMF). Add the deprotection solvent to resin. Shake for 20 min in room temperature. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>7) First amino acid coupling: <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>ii) Insert the fiber-optic probe into the thermowell. Insert the thermowell into the clip, and attach the clip to the reaction vessel.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iii) Place the reaction vessel into the holder and insert into the microwave cavity.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iv) Press the play key on the panel and the loaded coupling method will run.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>8) After the method running is done, cool down the solution to the set temperature. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>9) Wash the resin with DMF (5×). Allow the liquid to drain and turn off the vacuum pump.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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). <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>12) Repeat coupling and deprotection Fmoc steps to the end of the sequence. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>6. Cleavage<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Prepare the resin: Wash the resin completely with DMF and then DCM. Dry the resin in vacuum until it is completely dry. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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<i> </i></span><span style='mso-fareast-language: ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Add the cleavage solution to resin. Be sure you wear protection (gloves and goggles) when you handle TFA. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) Collect the product after cleavage:<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>i) Insert a 15 mL centrifuge tube inside the 250 mL bottle and lid is secure.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>ii) Turn on the vacuum pump.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>iii) Place the reaction vessel in the well located in front of the 250 mL bottle immediately after removing the luer plug. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>v) Turn off the vacuum pump.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>vi) Remove the reaction vessel and discard.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Take the 15 mL tube with the collected peptide solution out of the bottle.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>7. Precipitation<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1) Concentrate the collected peptide solution with a strain of high pure N<sub>2</sub> gas to 1–2 mL.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2) Add cold ether to the concentrated solution and sit the tube in ice or freezer. The crude peptide will precipitate. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3) Wait couple of hours or just leave the tube in freezer overnight. Centrifuge the solution and collect the precipitate. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>4) Add cold ether and centrifuge again to remove the soluble impurities. Repeat 2–3 times. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>5) Collect the precipitate and dry it under vacuum.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>6) Now you have your crude peptide! <o:p></o:p></span></p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><span style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p> </td> </tr>
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<table border=0 cellspacing=0 cellpadding=0 bgcolor="#ffff99" style='background:
#FFFF99;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'> <tr> <td width=611 valign=top style='width:611.1pt;padding:0in 5.4pt 0in 5.4pt'> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Procedure 1: Fmoc-quantitation<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The cuvette 1 is filled with 3 ml of the sample solution (Fmoc deprotection solution with a certain dilution). <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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). <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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)<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><span style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p> </td> </tr>
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<table border=0 cellspacing=0 cellpadding=0 bgcolor="#ffff99" style='background:
#FFFF99;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'> <tr> <td width=611 valign=top style='width:611.1pt;padding:0in 5.4pt 0in 5.4pt'> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Procedure 2: TNBS test<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 1: 5% DIPEA in DMF<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 2: 1% aqueous TNBS<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>A few resin beads are placed in a small test tube and 1-3 drops of each solution are added. <o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The test is positive when the resin beads turn yellow or red within 10 min and negative when the beads remain colorless.<o:p></o:p></span></p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><span style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p> </td> </tr>
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<table border=0 cellspacing=0 cellpadding=0 bgcolor="#ffff99" style='background:
#FFFF99;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'> <tr> <td width=611 valign=top style='width:611.1pt;padding:0in 5.4pt 0in 5.4pt'> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Procedure 3: Kaiser Test<o:p></o:p></b></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 1: 5 g ninhydrin in 100 mL ethanol<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 2: 80 g phenol in 20 ml ethanol<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>Solution 3: 2 mL 0.001 M aqueous KCN in 98 mL pyridine<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>A few resin beads are placed in a small test tube and 2-5 drops of each solution are added.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The tube is placed in an oven and the reaction left to develop for 5 min at 100°C.<o:p></o:p></span></p> <p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>The test is positive when the resin and solution turn blue and negative when the beads remain colorless.<o:p></o:p></span></p> <p class=MsoNormal><![if !supportEmptyParas]> <![endif]><span style='mso-fareast-language:ZH-CN'><o:p></o:p></span></p> </td> </tr>
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<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'><b>Recommended reading:<o:p></o:p></b></span></p>
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<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>1. The microwave-assistant peptide synthesizer used in our lab:<span style="mso-spacerun: yes"> </span><a href="http://www.cem.com/page11.html">http://www.cem.com/page11.html</a><o:p></o:p></span></p>
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>2. Stacey A. Palasek, Zachary J. Cox, Jonathan M. Collins. <i>J. Pept. Sci. </i></span><span style='mso-fareast-language:ZH-CN'>2007; <b>13</b></span><span style='mso-fareast-language:ZH-CN'>: 143–148<o:p></o:p></span></p>
<p class=MsoNormal><span style='mso-fareast-language:ZH-CN'>3. Introduction of Fmoc solid phase peptide synthesis method and procedures </span><a href="http://www.chempep.com/ChemPep-Fmoc-Solid-Phase-Peptide-Synthesis.htm">http://www.chempep.com/ChemPep-Fmoc-Solid-Phase-Peptide-Synthesis.htm</a></p>
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<p class=MsoNormal><b><a href="http://openwetware.org/wiki/Bitan:todo">[Back to Bitan: todo page]</a><o:p></o:p></b></p>
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