A Simple Introduction To Peptide Synthesis

 

What is peptide: A Simple Introduction of Peptide Synthesis. Peptides and proteins are linear polymers of amino acids linked by amide "peptide bonds" (Fig. 1). The peptide bond is formed by linking an amino group to a caroboxyl group on another amino acid. Amino acids are primary amines that contain an alpha carbon that is connected to an amino (NH3) group, a carboxyl group (COOH), and a variable side group (R). Carboxylic acid and carboxylate groups are normally not very reactive. It requires activating the carboxylic acid for the formation of the amide peptide bond (Fig. 2).

Fig. 1: Peptide Structure(Bond lengths in Angstrom units; Bond angles in degrees)

Fig. 2: Peptide bonds created with peptide coupling agents 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.

Fig. 3: Fmoc Strategy (Wang resin)

 

 

Fig. 4: Boc Strategy (Merifield resin) 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). These represent different protection and deprotection chemistries. It is also necessary to reversibly mask reactive side chain functional groups. After the anchoring of the first amino acid onto the resin, the Fmoc group is removed. To confirm that the Fmoc protecting groups are removed, a kaiser test is performed. Then another Fmoc amino acid is attached by activation of its carboxyl group (Coupling). See examples of the coupling agents at Fig. 5.

Fig. 5: Examples of coupling agents. 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 gelfiltration, ion exchange chromatography and reversed-phase HPLC.