Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 409

synthesized. Once the required sequence is synthesized, it is treated with hydro-
fluoric acid to remove the peptide and side protecting groups from the solid resin
support.
Solid supports (polymers/resins): The first solid polymer resin supports to be
introduced were copolymer of styrene and divinyl benzene. The resin is available in
the form of spherical beads that are insoluble in the majority of solvents but have
good swelling index when placed in organic solvent. Until the mid-1970s, simple
benzyl ester was mainly used as a resin; however, later on an acetamidomethyl group
was inserted between benzyl ester and polystyrene resin. This modification resulted
in a manifold increase in the stability of the resin. There was marked reduction in
acid lability of the newly formed resin.
To prevent the premature detachment of the peptides from polymeric support, the
concept of “orthogonal” protection scheme was adopted by Barany and Merrifield
[39] . These techniques mainly involve the use of techniques that remove protecting
groups.
In this method, the amino protecting group removed by treatment with base is
reacted with side chain groups and a peptide resin linkage, followed by photolysis.
The advantage of such a technique is that the structural integrity of the peptide resin
linkage is maintained intact in spite of repetitive application of this technique.
8.6.2.1 Attachment to Resin
The carboxy terminal amino acid of the candidate protein must be covalently
attached to polystyrene resin prior to initiation of the solid-phase synthesis of a pep-
tide. This is achieved by functionalization of polystyrene resin with a chloromethyl
benzyl residue, which is then reacted with a triethylamine salt to form a benzyl ester
linkage. However, the method results in incomplete esterification, leaving residual
chloromethyl benzyl moieties that trigger unwanted side reactions.
Advancement in solid phase peptide synthesis is a major breakthrough in the area
of peptide synthesis. Availability of automated, highly sophisticated equipment has
made the process of peptide synthesis quite fast, saving time. The only limitation
of these modern-day instruments is their inability to separate and purify the protein
from resin.
8.6.3 Enzymatic Synthesis of Peptides
With the dawn of the twenty-first century, landmark developments in biotechnol-
ogy have facilitated a cost-effective technique for protein production using proteo-
lytic enzymes. Endogeneous enzymes like trypsin, chymotrypsin, pepsin, and similar
enzymes have been widely explored to catalyze the synthesis of peptides and pro-
teins. The fascination for enzymatic synthesis of peptides is due to (1) stereospeci-
ficity and stereoselectivity of enzymes; (2) improved probability of getting optically
pure isomers using enzyme catalyzed methods; (3) enzymatic reactions are con-
ducted in aqueous milieu, avoiding the use of potentially toxic organic solvents as
used in former processes; and (4) the realization that protection or masking of side