BLBS102-c36 BLBS102-Simpson March 21, 2012 18:47 Trim: 276mm X 219mm Printer Name: Yet to Come
696 Part 6: Health/Functional FoodsO
O OFmoc-Cl Fmoc-L-AlanineL-Valine BOC-L-valineBOC anhydrideL-Alaninea
H 2 NNH 2OHHONHNH OO
OOOOHO OO HOOOOO++Figure 36.2.Protecting groups commonly used in solid phase peptide synthesis.and so on, by adding acid or by the anaerobic production of lactic
acid by bacteria. Whereas the pepsins are used for gentle bio-
processing of certain fishery products, the peptides occurred by
autolysis may be valuable immune stimulants (Gildberg 2004).
In vitro and in vivo studies have shown that certain peptide
fractions in fish protein hydrolysates may stimulate the nonspe-
cific immune defense system. Both fish sauce and fish silage are
protein hydrolysates with immune stimulating properties. Gen-
erally, fish sauce is regarded as a typical Asian product made
from tropical fish species, but ancient literature reveals that fish
sauce was a common food product in Southern Europe more
than 2000 years ago. Recent studies have shown that it can be
made also from cold water species.Chemical Peptide SynthesisOn the basis of the peptide sequences available in all published
sources and existing peptide databases (Liu et al. 2008), for
example, the EROP-Moscow oligopeptide database, SwePep
database, peptide of interest can be obtained through chemical
synthesis with appreciable purity and reasonable price. Peptide
synthesis is a chemical process of coupling of the carboxyl group
of one amino acid to the amino group of another amino acid.
Usually, chemical techniques are used to synthesize peptides of
up to 30–40 amino acids length. Peptide synthesis process can
be classified on the basis of used techniques and type of the final
product. Liquid-phase peptide synthesis can be divided into two
types, step-by-step peptide synthesis with subsequent adding of
one amino acid at ones fromC-terminal toN-terminal and block-
synthesis with coupling of polypeptide fragments. Liquid-phase
peptide synthesis is used in large-scale peptide production forindustry. In contrast, solid phase peptide synthesis (SPPS) is a
process during which the polypeptide chain is covalently bound
via linker to the porous insoluble bed particles (Merrifield 1963).
SPPS involves two stages in which the first stage of the tech-
nique consists of peptide chain assembly with protected amino
acid derivatives on a polymeric support. The second stage of the
technique is the cleavage of the peptide from the resin support
with the concurrent cleavage of all side chain protecting groups
to give the crude free peptides. Currently, two protecting groups
are commonly used in solid phase peptide synthesis—Fmoc
or 9-fluorenylmethyl carbamate and t-Boc or di-tert-butyl di-
carbonate (Fig. 36.2). Fmoc chemistry is known for generat-
ing peptides of higher quality and in greater yield than t-Boc
chemistry. Impurities in t-Boc-synthesized peptides are mostly
attributed to cleavage problems, dehydration, and t-butylation.
The advantage of Fmoc is that it is cleaved under very mild basic
conditions (e.g., piperidine), but stable under acidic conditions.
After base treatment, the nascent peptide is typically washed and
then a mixture including an activated amino acid and coupling
reagents is placed in contact with the nascent peptide to couple
the next amino acid. After coupling, noncoupled reagents can be
washed away and then the protecting group on theN-terminus of
the nascent peptide can be removed, allowing additional amino
acids or peptide material to be added to the nascent peptide in
a similar fashion. After cleavage from the resin, peptides are
usually purified by reverse phase high-performance liquid chro-
matography using columns such as C-18, C-8, and C-4. The
primary advantage of SPPS is its high yield. With modern SPPS
instrumentation, coupling and deprotection yields greater than
99.99%, giving an overall yield of greater than 99% for a 50
amino acid peptide.