1. BOC peptide synthesis

  2. Fmoc peptide synthesis

  3. Liquid phase segmented synthesis of polypeptide

Peptide synthesis is a process of repeated addition of amino acids, which is usually synthesized from C-terminal to N-terminal (amino terminal). The principle of solid-phase peptide synthesis is that the C-terminal of the first amino acid of the target peptide is connected with the solid-phase carrier through covalent bond, and then the N-terminal of the amino acid is taken as the starting point of the synthesis, the amino protection group is removed and the excess activated second amino acid is reacted to lengthen the peptide chain and repeat the operation to achieve the ideal synthetic peptide chain length. Finally, the peptide chain is split off from the resin and purified The target peptide was obtained.

  1. BOC peptide synthesis

BOC method is a classical solid-phase peptide synthesis method. BOC is used as the protective group of amino acid α - amino group and benzyl alcohol is used as side chain protection group. The removal of BOC is usually carried out by trifluoroacetic acid (TFA). During peptide synthesis, N - α - amino acids protected by BOC were covalently crosslinked onto the resin. The BOC protecting group was removed by TFA, and the N-terminal was neutralized with weak base.

The peptide chain was extended by DCC activation and coupling, and the target peptide was dissociated from the resin by hydrofluoric acid (HF) or trifluoromethanesulfonic acid (tfmsa). In BOC peptide synthesis method, in order to facilitate the next step of peptide synthesis, acid is repeatedly used for deprotection, and some side effects are brought into the experiment, such as the peptide is easy to be removed from the resin, and the side chain of amino acid is unstable under acidic conditions.

  1. Fmoc peptide synthesis

Carpino and Han developed a new solid phase peptide synthesis method Fmoc based on BOC peptide synthesis.

Fmoc was used as the protective group of amino acid α - amino in Fmoc peptide synthesis. Its advantages are that it is stable under acidic conditions, not affected by TFA and other reagents, and can be protected by mild alkali treatment, so the side chain can be protected by BOC protection group which is easy to be removed by acid.

TFA / dichloromethane (DCM) can be used to quantitatively remove the peptide from the resin, avoiding the use of strong acids. At the same time, compared with BOC method, Fmoc method has mild reaction conditions, fewer side reactions, high yield, and Fmoc group itself has characteristic UV absorption, which is easy to monitor and control the reaction. Fmoc method is more and more widely used in solid phase synthesis of polypeptide.

Liquid phase segmented synthesis of polypeptide

With the development of synthetic methods of polypeptides, the selective peptide synthesis method has become more and more prominent. It can be used for the synthesis of long peptide, and has high purity and easy purification.

The liquid phase synthesis of polypeptides can be divided into natural chemical linkage and stoudinger junction. Natural chemical ligation is the basic method of peptide segmented synthesis. The limitation is that the synthesized peptide must contain Cys residue, so the application scope of natural chemical linking method is limited. The extension of natural chemical linking methods includes chemical region selective joining, removable auxiliary group linking and light sensitive auxiliary base linking.

Staudinger ligation is another basic method of fragment linking, which opens up a broader idea for peptide fragment linking. The orthogonal chemical linking method is an extension of stoudenger method, which can improve the condensation rate between fragments by simplifying the auxiliary group of phosphothioester.