Peptide class

1. Classical solid-phase synthesis of peptides
Solid-phase synthesis of peptides is achieved by fixing amino acids on solid-phase support, and the solid-phase support is usually polymer resin, such as polystyrene-ethylene glycol cross-linked resin, polyacrylamide-polyethylene glycol resin and biological. These resins require the introduction of reactive groups in order to form covalent bonds with amino acids.
2. Feeding and resin treatment:
The resin was added to a solid-phase synthesizer, swelled with an appropriate solvent such as DCM and then drained. The resin was washed with a solvent such as DMF to remove impurities and drained after washing for later use
3. Condensation reaction:
The first amino acid (usually starting from the C-terminus) was dissolved with a volume of DMF and activated by the addition of a reducing agent (such as DCC, HBTU, etc.). The activated amino acid solution was put into a solid phase synthesizer to replenish DMF to the appropriate reaction concentration, and the condensation reaction was carried out by stirring.
4. Remove the protective tomb:
The degree of reaction was detected using Kaiser reagent, etc., and the solvent was drained when the reaction was over.
The resin was washed with DMF, and then an appropriate alkaline solution (such as PIPDMF solution) was added to remove the protective group at the N-terminal of the amino acid (such as Fmoc group). The degree of deprotection reaction was detected by Kaiser reagent again. After the reaction, the solvent was drained and washed with DMF, ready to add the next amino acid.

5. Condensation cycle:
According to the preset amino acid sequence on the resin, the above steps of “deprotection → washing → activated amino acids → feeding → condensation – washing” were repeated in turn, and the condensation cycle was carried out until all the amino acids were connected.
Discharge and tree wax drying:
At the end of synthesis, resin shrinkage was completed by cross-washing the resin with appropriate solvents, such as IPA and DCM. The resin is discharged into containers such as stainless steel trays.
The resin was dried in a vacuum drying oven at room temperature and weighed after drying and the yield was calculated.
6. Resin lysis and peptide purification
Resin lysis: The lysate (usually containing strong acids such as TFA) is prepared and stored in the freezer before extraction. The peptide resin is added to the reaction kettle, the pre-cooled lysate is added, and the reaction is stirred to split the peptide off the resin. At the end of lysis, the reaction solution was released, the resin was removed by suction and washed with solvents such as TFA.
Peptide purification: The lysate was transferred into a rotary evaporator and concentrated to a small volume at room temperature. Peptides were precipitated using appropriate precipitants such as methyl tert-butyl ether, and crude peptides were obtained by centrifugation, washing, and other steps. The crude peptides were purified using high performance liquid chromatography (HPLC) and other methods to remove impurities and separate the target peptides.
7. Resin lysis and peptide purification
“Concentration, filtration, and freeze-drying: The purified peptide solution was spin-steamed to remove the organic solvent and to obtain a concentrated peptide solution.” The concentrated peptide solution was aseptically filtered. The filtered peptide solution was placed in a lyophilizer and the appropriate heating program was set for lyophilization. After freeze-drying, the peptide products were removed for subsequent operations such as packaging and storage.