Peptide Synthesis Steps

There are usually two methods of peptide synthesis: solid-phase synthesis and liquid-phase synthesis, and the following are the general steps of solid-phase synthesis:

Preparation stage
Selection of resin: Select the appropriate solid carrier resin, such as Wang resin, for the peptides synthesized according to the need, and pretreat them to swell sufficiently to provide sufficient reaction sites.
Prepare amino acids: Determine the amino acid sequence of the target peptide and prepare the corresponding protective amino acids. Commonly used protecting groups are FMOC (9-fluorene methoxycarbonyl) and BOC (tert-butoxycarbonyl), among others. At the same time, prepare activators, condensation agents, deprotectants and other reagents, such as DIC(N,N'-diisopropylcarbodiimide), HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate), HOBt (1-hydroxybenzotriazole), piperidine, etc.

Synthesis cycle stage
Ligation of the first amino acid: The first amino acid at the C-terminus is ligated to the resin. Amino acids, condensants, and an appropriate amount of base are added to the swollen resin at room temperature and reacted in an organic solvent such as DMF for several hours to allow the amino acids to be covalently bonded to the resin. After the reaction is complete, the resin is washed with a solvent to remove unreacted reagents and impurities.

Removal of the FMOC protecting group: An alkaline solution (e.g., 20% piperidine DMF solution) is used to remove the FMOC protecting group from the first amino acid, exposing the amino group for ligation of the next amino acid. Generally, it is necessary to react under nitrogen stirring for a certain time, then filter to remove the solution, and then wash the resin with solvent. The effect of removal can be detected with ninhydrin detection reagent, and if it is dark blue, it is a positive reaction, indicating that the removal is complete.

Condensation reaction: weigh an appropriate amount of the next amino acid, activator and base, add it to the reaction tube, dissolve it with solvent, and then add it to the deprotected resin, and react at room temperature for a certain time to condense the activated amino acid and the amino group on the resin to form a peptide bond. At the end of the reaction, the resin is washed with a solvent to remove the unreacted reagent.

Repeat the condensation cycle: follow the above steps to remove the protective group in turn, carry out the condensation reaction, and repeat the operation until all the required amino acids are attached to the resin to synthesize the target polypeptide chain.
Lysis and purification phases

Resin shedding and pure product separation detection: The peptide is cleaved from the resin using a lysate (e.g., 95% TFA:2% TIS:2% EDT:1% H2O) to obtain a solution of the peptide. The lysate was dried with nitrogen or concentrated by rotary evaporation, and then precipitated, centrifuged, and washed with ether to obtain crude polypeptides. Finally, the crude peptides were purified by high-performance liquid chromatography (HPLC) and other methods to remove impurities and isolate the target peptides, which were then concentrated, filtered and lyophilized to obtain high-purity peptide products.

The steps of liquid-phase synthesis are similar to those of solid-phase synthesis, but the reaction takes place in solution as follows:

Preparation stage
Selection of protecting groups: Protection of the α-amino and side-chain functional groups of amino acids, usually using protecting groups such as BOC or FMOC.

Prepare reagents: Choose the appropriate activation reagents, such as DCC, HATU, etc., as well as solvents, such as DMF, DCM, etc.

Synthesis stage
Amino acid activation: The protected amino acids are reacted with the activation reagent in an appropriate solvent to activate the carboxyl group to form active intermediates such as active esters or acid chloride.

Condensation reaction: The activated amino acid is condensed with another protected amino acid in the presence of a base to form a dipeptide. The reaction is usually carried out at low temperatures to reduce the occurrence of side reactions.

Repeated condensation: According to the desired length of the peptide sequence, the condensation reaction is repeated to gradually expand the length of the polypeptide chain. After each step of the condensation reaction, separation and purification are required to remove unreacted reagents and impurities.

Post-processing stage
Removal of protecting groups: After the synthesis of peptides is completed, reagents such as acids or bases are used to remove the protecting groups and restore the free functional groups of amino acids.

Purification and analysis: Chromatography techniques (such as reverse phase chromatography, gel filtration, etc.) are used to purify the synthesized peptides to separate and remove impurities. Then, mass spectrometry, nuclear magnetic resonance, and other methods were used for structural analysis and confirmation to ensure that the structure and purity of the peptide met the requirements.

Keywords: Mobelbiochem, peptide synthesis service