Peptide Synthesis: 101

At the computer go to: Start > Programs > Pioneer Workstation

            File > New > Notebook

Near the top of the window select:

Protocol: \Template                  Chemistry: \OH                        Scale: 0.1 mmol

Fmoc: off                                  First AA: On support                C-term: acid

Enter sequence name as the actual sequence ending with the support amino acid. (i.e. DWSFYLLYYTEFT, would be entered for the DT strand, where T is the Threonine on the Wang resin, or resin).  The first AA entered is the C-terminus and the last AA entered is the N-terminus in the resin.

Now click the “Seq” button, at the bottom enter the sequence EXACTLY as you did before.  The program will automatically show the three letter abbreviation for each amino acid (i.e. V, will show as Val).

Click the button to the left of “Seq” and you should be back to the first notebook page.  Go to File > Properties, or click the button at the top with the finger and paper, this is the properties shortcut.  In the bottom chart make the proper cycle selection for each AA (generally standard or capping).

Once you are in the properties page click the scale tab.  The top selection is “Support substitution”.  Enter the meq/g number on the bottle of the appropriate resin (i.e. Thr-resin 0.61 meq/g).  Press ok.  The program adjusts several measurements to account for the resin being used.

Now click the button to the right of “Seq”, it is the resource page.  Check the printer switch ( above UV Vis machine)  and put it on “Peptide Synthesizer”.  Make sure the sequence appears to be entered correctly and the meq/g number is correct.  Now print the resource page.  Hurray, you are done at the computer for now…

Generally we will use medium size (16 × 100 mm) test tubes for our synthesis.  At the end of the resource page you will see the list of amino acids needed, the amount to weigh out (which is shown in 4x quantity), and which number tubes to put them in.  Label the tubes with a bit of lab tape a little more than a centimeter from the top of the tube (this is also used as a guide for the machine during the synthesis).  Number and fill the tubes accordingly.  It is not overly critical that the amino acids are room temperature, but the closer the powders are the more confidence there will be in the results.

If you will not be starting the synthesis at this point, parafilm the tops of each tube and put them in the refrigerator.

Now time to swell the resin.  It is critical that the resin be up to room temperature before weighing the amount marked on the resource sheet.  This number can be used quantitatively later to figure out exactly how much product was produced.

While you wait for the resin to get to room temperature take some time to clean the synthesis column.  If it is not already disassembled, remove each end by unscrewing the large white nut at either end and pulling the lower filter (short stem) and the top filter out of the clear column tube.  Rinse each filter (brown disc that does NOT come out) by alternating dH₂O and acetone.  Finally, rinse the lower filter and the tube with DMF (since this is the main solvent for the synthesis) and put the two pieces together.

Parafilm the lower end and make sure it is to the point that no solvent will be able to leak out.  Pour the weighed out resin (every bit that you can) into the column.  Use DMF to rinse the sides of the column and wet the resin.  While holding the lower parafilmed portion of the column, put the top filter into the column, a couple of centimeters should be fine.  Place the column in an upright position in a beaker (putting a paper towel in the bottom of the beaker is a good way to keep it clean in case the column leaks).

The resin should be left to swell for no less than 30 minutes.  In the mean time check the levels of all the solutions in the synthesizer.  Activators one (Act 1*) and two (Act 2*) will need to be changed.   Make sure that the other solutions have more than what is recommended on the resource page.

By the time you have made the two activators and refilled anything else that needed it, you will want to inform the machine.  At the synthesizer screen (little blue panel), select Tools > Bottle, then reset whichever solutions you refilled, especially Act 1 and 2, as this will cause a pause in the synthesis if the machine thinks it is out of solution.

FYI:     Act 1: TBTU/HOBT in DMF                Act 2: DIEA in DMF                Act 3:

            Aux 1: MeOH                          Aux 2: Neat DIEA                    Aux 3: Capping

            Wash: DMF                             Deblock*: 20% piperidine in DMF

To place the amino acids properly see that the red tube holder is in the rack and has an empty tube in the blank position (the space furthest from you).  Follow the diagram on the right door for proper placement of successive tube holders and how to place the tubes in numerical order.

Finally, with all solutions and dry goods in place it is time to go back to the computer.  If you closed it earlier, open the notebook you created.  Towards the top of the page there is a button that looks like a piece of paper and has a red arrow.  Click this button to send the sequence to the synthesizer.  Choose which column you prepared and click “OK”.

Back at the blue panel on the synthesizer, go to Prime > System prep > [column #].  Follow the directions that the panel prompts you with.

            Summary: With union in place the system will run all the solutions.  Then with the column in place it will “swell” the column.  Here is where you make final adjustments so that the fluid pressure inside the column does not get too high during synthesis.  Then it will say it is done, press Cancel at the panel (Total time ~10 min.).

Press Strt1 or Strt2, to start the appropriate column.

*

Act 1			Act 2	1M DIEA in DMF	Deblock	20% pip. in DMF
TBTU g	HOBT g	DMF ml	DMF ml	DIEA ml	DMF ml	piperidine ml
2.167	0.912	15	42	8.73	80	20
4.3345	1.824	30	84	17.46	160	40
6.502	2.736	45	168	35	240	60