Introduction To Peptide Synthesis Methods

Peptide synthesis refers to the process of connecting amino acid molecules in a specific order to form a peptide chain through chemical methods. Peptide synthesis methods mainly include solid phase peptide synthesis and liquid phase peptide synthesis.

Solid Phase Peptide Synthesis (SPPS)

Solid phase peptide synthesis was first proposed by R. Bruce Merrifield in 1963. This method allows the sequential addition of amino acids to a growing peptide chain. Because of its convenient and rapid synthesis, easy automation, and suitability for large-scale production, it has become the preferred method for peptide synthesis.

Steps of solid phase peptide synthesis:

Resin and linker selection. Choose and prepare suitable resins and linkers for synthesis. Resins are usually composed of polystyrene resins cross-linked with divinylbenzene. These resins provide the solid support required for the reaction. The choice of linker depends on the desired peptide and conditions for cleavage. Common linkers include Wang resin (acid-labile) for Fmoc chemistry and Merrifield resin for Boc chemistry.
Loading the first amino acid. Attach the first amino acid (amino acid residue) to the resin via its carboxyl group (C-terminus). This is done using coupling reagents which activate the carboxyl group to react with the amino groups present on the resin or linker. Common coupling reagents include DIC (N,N'-diisopropylcarbodiimide) and HOBt (1-hydroxybenzotriazole).
Deprotection. After coupling the first amino acid, the protecting group is removed using conditions specific to the protecting group.
Coupling of subsequent amino acids. Sequential addition of protected amino acids to the growing chain. After each coupling, a deprotection step is performed to free the N-terminus for the next round of amino acid addition.
Capping. After each coupling step, any unreacted amino groups on the peptide are capped with acetic anhydride or another capping reagent to prevent them from participating in future reactions.
Final deprotection. Once the peptide chain is fully assembled, it is cleaved from the resin along with any remaining side chain protecting groups.
Purification. The peptide is purified to remove any side products or unreacted starting materials. Techniques such as HPLC (high-performance liquid chromatography) are commonly used.

Liquid phase peptide synthesis (LPPS)

Liquid phase peptide synthesis is another traditional peptide synthesis method that does not require the fixation of amino acids to a solid phase support. In liquid phase peptide synthesis, all reactions are carried out in solution, and the target peptide is finally purified. This method is suitable for the synthesis of shorter peptide chains.

Steps of liquid phase peptide synthesis:

Protection of functional groups. To avoid side reactions, the functional groups of amino acids must be protected.
Coupling of amino acids. Sequentially add amino acids using coupling reagents and catalysts.
Purification of intermediates. Purify peptide intermediates after each coupling step. This is typically done using techniques like recrystallization, liquid-liquid extraction, or chromatography (e.g., silica gel chromatography).
Deprotection. Remove the protecting groups to proceed to the next coupling reaction.
Repetition of coupling cycles. Repeat coupling, purification and deprotection cycles until the desired peptide sequence is achieved.
Final deprotection and cleavage. Remove all protecting groups and isolate the peptide.
Final purification. Purify the peptide to achieve the desired purity.