Peptide Synthesis
The Protein Facility offers both large and small scale peptide synthesis, utilizing an Advanced Chemtech Model 396 Multiple Peptide Synthesizer and an Applied Biosystems Model 432A Peptide synthesizer, both performing FMOC synthesis. Below is a general description of the method used in our laboratory. With peptide synthesis, it is a good idea to contact us before the synthesis is performed to discuss your peptide and the probablility of success in the synthesis.
Below are some links that may be helpful in peptide design:
Design of Useful Peptide Antigens by Ruth Hogue Angeletti
Solid phase peptide synthesis, developed by R. B. Merrifield, was
a major breakthrough allowing for the chemical synthesis of peptides
and small proteins. The general scheme of solid phase peptide
synthesis is illustrated in Figure 1.
An insoluble polymer support (resin) is used to anchor the peptide
chain as each additional alpha-amino acid is attached. This polymer
support is constructed of 20-50 µm diameter particles which are
chemically inert to the reagents and solvents used in solid phase
peptide synthesis. These particles swell extensively in solvents,
which makes the linker arms more accessible.
Organic linkers attached to the polymer support activate the resin
sites and strengthen the bond between the (-amino acid and the
polymer support. Chloromethyl linkers, which were developed first,
have been found to be unsatisfactory for longer peptides due to a
decrease in step yields. The PAM (phenylacetamidomethyl) resin,
because of the electron withdrawing power of the acid amide group on
the phenylene ring, provides a much more stable bond than the
classical resin. Another alternative resin for peptides under typical
peptide synthesis conditions is the Wang resin. This resin is
generally used with the FMOC labile protecting group.
A labile group protects the alpha-amino group of the amino acid. This
group should be easily removed after each coupling reaction so that
the next alpha-amino protected amino acid may be added. Typical
labile protecting groups include t-Boc (tert-butyloxycarbonyl) and
FMOC (9-flourenylmethloxycarbonyl). t-Boc is a very satisfactory
labile group which is stable at room temperature and easily removed
with dilute solutions of trifluoroacetic acid (TFA) and
dichloromethane. FMOC is a base labile protecting group which is
easily removed by concentrated solutions of amines (usually 20-55%
piperidine in N-methylpyrrolidone). When using FMOC alpha-amino
acids, an acid labile (or base stable) resin, such as an ether resin,
is desired.
The stable blocking group protects the reactive functional group of
an amino acid and prevents formation of complicated secondary chains.
This blocking group must remain attached throughout the synthesis and
may be removed after completion of synthesis. When choosing a stable
blocking group, the labile protecting group and the cleavage
procedure to be used should be considered.
After generation of the resin bound synthetic peptie, the stable
blocknig groups are removed and the peptide is cleaved from the resin
to produce a "free" peptide. In general, the stable blocking groups
and organic linkers are labile to strong acids such as TFA. After the
peptide is cleaved from teh resin, the resin is washed away and the
peptide is extracted with ether to remove unwanted materials such as
the scavengers usedin the cleavage reaction. The peptide is then
frozen and lyophilized to produce the solid peptide. This is then
characterized by HPLC and MALDI before being used by the
investigator. It may also be necessary to purify the peptide by HPLC
if higher purity is required by the investigator.
