3.4.1 Chemical Synthesis
The main application of chemical synthesis of peptides in the laboratory or at a
small scale is their use as therapeutic drugs or for diagnosis purposes. There are two
variants in the chemical synthesis of peptides, in a liquid phase and a solid phase.
Although both protocols are well established for peptide synthesis, this technology
has several disadvantages. These disadvantages include (i) racemization of the
resulting peptides after the reaction; (ii) the requirement of previous steps to protect
of the residues or lateral side-chain of the most sensitive amino acids to maintain
peptide functionality; (iii) low overall efficiency and generation of sub-products; iv)
toxicity of solvents and protection and deprotection agents, as well as of chemical
catalysts used in the reaction synthesis [59].
Peptide synthesis in solid phase is used for the generation of sequences of 10–
100 amino acids long, with excellent potential for the development of multifunctional peptides [60]. This strategy requires a solid support to anchor the initial
amino acid or small peptide sequence. The chain grows by extension of the
N-terminal or C-terminal, step by step, by the sequential incorporation of a new
amino acid or small peptide fragment until the synthesis of the desired peptide. The
peptide synthesis in solid-phase approaches facilitates cyclic coupling, protection,
and deprotection steps of more sensible residues of amino acids, as well as the
intermediate purification steps needed until achieving the peptide synthesis with
longer sequences. This rational strategy was initially used for active peptide synthesis. Examples are the synthesis of YGLF and YGG identified as fragment
sequences f(50–53) and f(18–20), respectively, released from a-LA [61]. The great
advantage of this strategy is to implement the automation of the different steps of
the global process with increase income and decrease of consumption of reagents
[59]. On the other hand, peptide synthesis in the liquid phase is the preferred
method for large-scale synthesis of relatively short-chain peptides by direct condensation reaction between different amino acids or smaller peptide fragments using
strategies similar to those used in solid-phase reaction.
3.4.2 Recombinant DNA Technology
Another alternative method, according to the rational approach, is based on
recombinant DNA technology. This technology approach requires an intense and
costly phase of research and development. However, once established a recombinant biosynthesis system of the desired peptide, usually characterized by a long
chain, it is possible to obtain it in large quantities via a fermentation process from
relatively inexpensive nutrients. Despite significant advances, short sequence
biosynthesis remains impractical due to low expression efficiencies and difficulties
encountered in the extraction and purification of the biosynthesized peptide [62].
Besides, recombinant DNA expression systems and biosynthesis of the desired
peptide typically in bacterial hosts cannot be used for the production of aminated
peptides, since these microorganisms do not have the necessary peptidylglycine
a-amidating enzyme (a-AE). This disadvantage represents a significant limitation
considering the widespread occurrence of amidation processes and their role often
crucial in the expression of biological activity by a specific peptide. Another
484
A. F. A. Salvado et al.
The main application of chemical synthesis of peptides in the laboratory or at a
small scale is their use as therapeutic drugs or for diagnosis purposes. There are two
variants in the chemical synthesis of peptides, in a liquid phase and a solid phase.
Although both protocols are well established for peptide synthesis, this technology
has several disadvantages. These disadvantages include (i) racemization of the
resulting peptides after the reaction; (ii) the requirement of previous steps to protect
of the residues or lateral side-chain of the most sensitive amino acids to maintain
peptide functionality; (iii) low overall efficiency and generation of sub-products; iv)
toxicity of solvents and protection and deprotection agents, as well as of chemical
catalysts used in the reaction synthesis [59].
Peptide synthesis in solid phase is used for the generation of sequences of 10–
100 amino acids long, with excellent potential for the development of multifunctional peptides [60]. This strategy requires a solid support to anchor the initial
amino acid or small peptide sequence. The chain grows by extension of the
N-terminal or C-terminal, step by step, by the sequential incorporation of a new
amino acid or small peptide fragment until the synthesis of the desired peptide. The
peptide synthesis in solid-phase approaches facilitates cyclic coupling, protection,
and deprotection steps of more sensible residues of amino acids, as well as the
intermediate purification steps needed until achieving the peptide synthesis with
longer sequences. This rational strategy was initially used for active peptide synthesis. Examples are the synthesis of YGLF and YGG identified as fragment
sequences f(50–53) and f(18–20), respectively, released from a-LA [61]. The great
advantage of this strategy is to implement the automation of the different steps of
the global process with increase income and decrease of consumption of reagents
[59]. On the other hand, peptide synthesis in the liquid phase is the preferred
method for large-scale synthesis of relatively short-chain peptides by direct condensation reaction between different amino acids or smaller peptide fragments using
strategies similar to those used in solid-phase reaction.
3.4.2 Recombinant DNA Technology
Another alternative method, according to the rational approach, is based on
recombinant DNA technology. This technology approach requires an intense and
costly phase of research and development. However, once established a recombinant biosynthesis system of the desired peptide, usually characterized by a long
chain, it is possible to obtain it in large quantities via a fermentation process from
relatively inexpensive nutrients. Despite significant advances, short sequence
biosynthesis remains impractical due to low expression efficiencies and difficulties
encountered in the extraction and purification of the biosynthesized peptide [62].
Besides, recombinant DNA expression systems and biosynthesis of the desired
peptide typically in bacterial hosts cannot be used for the production of aminated
peptides, since these microorganisms do not have the necessary peptidylglycine
a-amidating enzyme (a-AE). This disadvantage represents a significant limitation
considering the widespread occurrence of amidation processes and their role often
crucial in the expression of biological activity by a specific peptide. Another
484
A. F. A. Salvado et al.