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A peptide is formed by linking two or more amino acids via amide bonds to create an amino acid chain, typically ranging from 2 to 70 amino acids in length. Unlike proteins, peptides can exert their biological activity without undergoing folding. Peptides exist in animals and plants as peptide hormones (such as angiotensin, LHRH, and enkephalins) as well as toxins. They hold great research value both as lead compounds for drug discovery and as therapeutic agents themselves. Additionally, peptides are widely applied in vaccines, biomaterials, and histological probes, and are extensively used as antigens for antibody production.
Peptides are chemically synthesized in either liquid phase or solid phase. The synthesis process involves the regioselective and selective formation of amide bonds between an N-protected amino acid and another amino acid with a free amino group and a protected carboxylic acid group. In solid-phase synthesis, the carboxyl protecting group is bound to a polymeric support. After bond formation, the amino protecting group of the dipeptide is removed, followed by coupling with the next N-protected amino acid.
Solid-Phase Peptide Synthesis (SPPS) has become the most commonly used peptide synthesis method due to its high efficiency, simplicity, rapidity, and ease of parallel processing. SPPS refers to the sequential addition of amino acid residues and side-chain protected amino acid residues to a soluble polymeric support (Figure 1).
For N-α protection, either the acid-labile tert-butyloxycarbonyl group (Boc SPPS) or the base-labile Fmoc group (Fmoc SPPS) can be adopted. Upon deprotection, the next protected amino acid is incorporated using coupling reagents or pre-activated protected amino acid derivatives. The C-terminal amino acid is anchored to the resin via a linker, whose properties determine the conditions required for cleaving the peptide chain from the support after chain elongation. Side-chain protecting groups are generally selected to be cleaved simultaneously with the separation of the peptide from the resin.
Most peptides are prepared by the Fmoc method, as final cleavage and global deprotection can be achieved by treatment with trifluoroacetic acid. In contrast, the Boc method requires the use of highly toxic and corrosive anhydrous hydrogen fluoride in dedicated equipment.
Peptides consisting of up to 50 amino acids can be routinely synthesized, and the synthesis of proteins with more than 100 amino acids is also widely reported. Longer proteins can be prepared by native chemical ligation of fully deprotected peptides in solution. This approach enables the synthesis of natural peptides that are difficult to express in bacteria, incorporation of unnatural or D-amino acids, and the generation of cyclic, branched, labeled, and post-translationally modified peptides.
Traditional liquid-phase peptide synthesis using Boc or Z-amino protection has been largely replaced by solid-phase peptide synthesis, except for existing large-scale peptide synthesis processes for industrial applications.