[Resins] [Set-up] [Resin Loading] [Standard Coupling] [Fmoc Removal] [Resin Cleavage]
Notice – This information was developed by Dr. Chris Leitheiser and Frank Schmidtmann while at the University of Virginia. Please do not copy/publish the following methods without proper acknowledgements/consent.
The
most common resin used the McGarvey lab is the Wang Resin. This is an
inexpensive and versatile resin that is standard for solid phase peptide
synthesis. Here are examples of other resins that have been used in the
McGarvey Lab:
One of the most useful aspects of solid phase synthesis is the ease in which reagents can be removed. Therefore it is important to have a simple way to remove the reagents and wash the resin. The method illustrated below is an effective and inexpensive way to run solid phase reactions.
The Mixer - used to agitate reaction without destroying the beads
The Reaction Vessel - as an alternative to glass containers with non-replaceable filters, these empty syringe cartridges can be fit with a filter, a stopcock, and a plastic cap. These materials are compatible with all the reagents involved with solid phase peptide synthesis and the total cost of any size set-up (up to 50ml) is less than $1.
The Set-up - the resin can be added to the reaction vessel and clamped to the mixer. An inert gas line can added by inserting a needle through the plastic cap. The reagents can simply be added and shaken until the reaction is complete.
The Removal of Reagents - a piece of PFA tubing (like tubing used for HPLC) can be attached to the stopcock and a bent vacuum adapter (this can be done by forcing a beveled piece of tubing through a rubber septum). The adapter is then hooked to a vacuum source and a round bottom. By opening the stopcock, the solvent, reagents, and subsequent washes can be collected in the round bottom. The resin is then ready to be dried or taken on to the next reaction.
To
place the first amino acid on the resin, the symmetrical anhydride method is
most often employed. This involves dissolving 10 eq (relative to the
resin loading) of the amine protected amino acid in dry DCM (a few drops of DMF
may be added if needed to dissolve the amino acid). To this solution
stirring at 0 , 5 eq of DIC is added and stirred for 20 minutes. The
mixture is then concentrated in vacuo and redissolved in DMF. The
resulting solution is added to the resin and shaken until the reaction is
complete (usually 1 h to 5 h - catalytic DMAP may be added). The
mixture is filtered, rinsed several times with DMF and DCM, and dried.
Addition
of Fmoc protected amino acid residues can involve many different activators
(such as HBtU, PyBOP, or HATU). Standard procedures for these coupling
reagents are as follows:
The Fmoc amino acid to
be added (5 eq) is dissolved in a minimal amount of DMF with PyBOP, HBtU, or
HATU (4.9 eq of any one) and HOBt (5 eq for PyBOP or HBtU - 5 eq of HOAt is
used for HATU). DIPEA (10 eq) is added and the solution is added
immediately to the free amine resin in the set-up
described above. The reaction is shaken for 30 minutes to 2 h (Kaiser
test may be used to determine if the coupling is >95% complete) and
drained. The resin is washed three times with DMF, three times with DCM,
and dried in vacuo.
To
remove the terminal Fmoc group, piperidine in DMF is most commonly used.
The standard procedure is as follows:
A fresh solution of 20%
piperidine in DMF is added to the resin and shaken for 5 minutes. The
resin is drained, piperidine solution is added agian, and the mixture is shaken
for 5 minutes. The process is completed one more time and the solution is
removed. The resin is washed three times with DMF, three times with DCM,
and dried in vacuo.
Cleavage Procedure (acid labile
resins)
Removal
of the peptide from most acid sensitive resins (such as the Wang resin)
involves triflouroacetic acid and an acid scavanger. The standard
procedure is as follows:
To the resin in the
previously mentioned reaction vessel, a 10% solution of
TES in DCM is added and shaken for 15 minutes. To this mixture an equal
volume of TFA is added and shaken for an additional 1.5 h. The resin is
filtered and washed (DCM:TFA 1:1) twice, collecting the filtrate. The organic
solution is concentrated in vacuo and purified.