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Chemical Synthesis of Peptide Drug with Solid-Phase Methodology

Peptide plays a key role in basic biochemical and physiological processes, and it is essential to many aspects of biomedical research, especially when the pharmaceutical industry continues to apply new drug candidates in the biological field. Therefore, rapid, efficient, and reliable methodology for the chemical synthesis of peptide drugs is of utmost interest in chemical biology research.


In general, peptide drugs are chemically synthesized through coupling the carboxyl group of amino acid to the N-terminus of the growing peptide chain. Due to the complex nature of in vitro protein synthesis, amino acids are added to the growing peptide chain in a gradual and cyclic manner. Although there are some key differences in commonly used peptide synthesis methods, they all follow the same amino acids addition method (step by step method).


Classical methodologies for peptide drug chemical synthesis include liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS). The former method is generally applied for the large-scale synthesis of small peptides such as dipeptides or tripeptides. Step-by-step synthesis and fragments combination are two main strategies scientists use in the LPPS process. Although LPPS has advantages of higher crude purity, lower reagent consumption, and greater ease of scaling, the use of LPPS methodology could be problematic for the synthesis of longer and more complex peptides.


The introduction of solid-phase peptide synthesis made the synthesis of long peptides easier, more efficient, and accessible. It is the most preferable technique for the chemical synthesis of peptides and allows peptide synthesis to be carried out with the application of automation while several posttranslational backbone modifications and insertions of non-natural amino acids are easily achieved. This approach circumvents the comparatively time-consuming isolation of the product peptide from the solution after each reaction step and opens the doors for automation as subsequent couplings could be iteratively performed by a defined set of unit operations. As a result, many challenging peptides have been successfully synthesized using SPPS approach.


SPPS can be divided into Fmoc (9-fluorene methyloxy carboxyl) method and Boc (tert-butoxy carboxyl) method. In Fmoc solid-phase peptide synthesis, the peptide chain is assembled stepwise, one amino acid at a time, while attached to insoluble resin support. This allows the reaction by-products to be removed at each step by simple washing. Compared to the Boc method, the great advantage of Fmoc is that it cracks under moderate basic conditions, but remains stable under acidic conditions. This allows the use of labile mild acid protecting groups, such as Boc and benzyl groups on the side chains of the amino acid residues of the target peptide. Many peptide institutions, therefore, use Fmoc for SPPS.


The advent of solid-phase techniques accelerates peptide drug synthesis in present-day drug discovery and development. The applications of the high-quality chemical synthesized peptides are gaining increasing popularity as a result of the developments in biotechnology and peptide synthesis techniques and for many research purposes including cancer diagnosis and treatment, antibiotic drug development, epitope mapping, production of antibodies, and vaccines.