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The controlled peptide synthesis requires selective protection and deprotection of the various
functional groups: the amino group, the -carboxyl group or the side chain functional groups. The
side group gives each amino acid its distinctive properties and helps to dictate the folding of the
protein.
There are two peptide synthesis strategies used for the manufacture of peptides. The first system
uses the Fmoc (base labile, Fig. 3) and the second Boc (acid labile, Fig. 4) for the amine terminus
of the amino acid.
Solid phase synthesisconsists of assembling amino acids from the C-terminal to the N-terminal.
SPPS allows efficient removal of excess reagents and soluble byproducts after each reaction
cycle because the peptide remains anchored to an insoluble solid resin support. Resins commonly
used are composed of polystyrene.
The controlled synthesis of peptides and formation of amide bonds requires the use of reversible
ion of the amino group. Three common protection chemistries are: tert-Butoxycarbonyl (tBoc),
9-Fluorenylmethyloxycarbonyl (Fmoc) and N-Allyloxycarbonyl (Alloc).
A kaiser test is needed to confirm the complete coupling. The process is repeated through a cycle
of deprotection, coupling and washing until the peptide is completely synthesized. Then the
synthesized peptide is cleaved from the resin and side chain protection groups are removed. The
synthetic peptide purification is usually including the peptide precipitation from the cleavage
reaction mixture by the addition of diethyl ether. The peptide and resin mixture can be suspended
in water or aqueous acid and filtered to remove the resin. Further purification can be by gel-
filtration, ion exchange chromatography and reversed-phase HPLC.