SPPS lends itself well to library synthesis and there are dozens of different machines capable of making thousands of compounds in a fully automated, high throughput manner.  In keeping with the “limited budget” format of this blog I’ll be glossing over these high tech machines and instead covering manual solid phase synthesis.  These methods are useful for making 5-50 different sequences, with most of my experience in the range of ten to sixteen samples.

First, a little theory.  These procedures are best suited to a basic “mix and split” approach.  A large amount of beads containing the core peptide are produced, then divvied up into smaller containers for final modification.  This leads to sequences like XXXY, where the first three residues are the same for all compounds but the last residue is a wildcard.  It’s also possible to make sequences like XXYX using a tea bag approach (pdf), but that requires a setup I don’t have too much experience with.

Your first purchase should be a reaction vessel large enough to hold all your beads.  In theory this could be a simple sintered funnel, but I highly recommend picking up a custom built reactor.  The two I use are both from Chemglass, items CG-1859-22 (discontinued) and CG-1866-12 (coarse frit).  The larger vessel is for bulk quantities of resin (~1g) and the smaller for when I only need to make one or two different compounds.

The primary advantage of using custom built glassware is the ability for constant agitation.  Solid phase synthesis takes place primarily on the liquid/solid boundary, so thorough mixing is required for good reaction rates and high yields.  Insufficient stirring can lead to partial reactions, which cause (at best) truncated products that can be very difficult to separate later on (this becomes especially relevant in the 12-20 AA range, where the growing peptides can form β-sheets, drastically reducing yields).

The resin I’m most familiar with is Rink Amide MBHA, with standard Fmoc/Boc protecting groups.  A basic coupling scheme below:

1)       Swell the resin in DMF for 30min prior to use to open up some of the more internal coupling sites.  In this and all subsequent steps solvent should be added to three times the height of the beads.  A constant stream of nitrogen gas is used to provide gentle agitation [1].

2)      Cleave the Fmoc protecting groups with 4:1 DMF:Piperidine.  The cleavage should be repeated twice, approximately twenty minutes each time.  The success of the cleavage can be measured by tracking the concentration of cleaved Fmoc in the solvent with UV, if you’re feeling gung-ho.

3)      Wash the resin three times with DMF, then three times with DCM, then a final three times with DMF.  Yes, this is overkill.

4)      Verify cleavage with a chloranil test [2].

5)      Prepare the first amino acid.  Three equivalents of the amino acid, three equivalents of the coupling agent (TBTU in my case), and eight equivalents of diisopropylethylamine (DIPEA/DIEA/Hunig’s Base) should be mixed in a vial ten minutes prior to addition.  In theory the premixing cuts down on potential amino acid racemization.  Following addition of the coupling mix the beads should be gently agitated for ~2hr (longer for proline residues, YMMV.  Repeat treatments may be required in the 12-20 AA range).

6)      Wash the resin again, as in step 3.

7)      Cap any unreacted amines [2].

8)      Go to 2.  Repeat this process as needed.  Once all of your amino acids have been added (XXX in our sample XXXY case), draw off a small amount of beads and cleave them in 95:2.5:2.5 TFA:H2O:TMS.  The cleaved product should then be analyzed via mass spec for truncated chains and overall reaction success.  The bulk of the beads can then be washed with DCM and dried under vacuum.

And that’s solid phase peptide synthesis in a nutshell.  Next up is some of the secondary techniques, and then I’ll get into how to (efficiently) add peptide Y.


[1] This agitation can be (partly) replaced by putting in a small stir bar and moving it around every few minutes with a magnetic wand.  I don’t recommend this though, as the stir bar may crush some of the more delicate beads against the wall of the flask.

[2] Next post!