Methods for protecting and removing amino, carboxyl and side chains in peptide synthesis

In peptide synthesis, it is not only necessary to protect the amino and carboxyl groups that do not participate in the formation of amide bonds temporarily, but also need to protect the active genes on the side chains of amino acids. After the reaction is completed, the protection genes are removed, so that the peptide with a specific amino acid sequence can be successfully synthesized.

Because racemization is not easy to occur, no matter solid phase synthesis or liquid phase synthesis, α The protective group of amino group is alkoxycarbonyl type. Benzyloxycarbonyl was first used. However, since strong acidolysis conditions are required for its removal, tert butyloxycarbonyl substitution is used for protection, and TFA is used for deprotection. However, it is not applicable to the synthesis of peptides containing tryptophan and other acids that are unstable.

Fmoc was adopted in 1978 by changMeinlofer and Atherton et al α The amino protecting group and Fmoc group are very stable to acids and can be removed by piperidine-CH2CL2 or piperidine-DMF. In recent years, the application of Fmoc synthesis method is more and more extensive.

Peptide synthesis

Carboxyl groups are generally protected by forming ester groups. Methyl ester and ethyl ester are most commonly used in the process of gradually synthesizing and protecting carboxyl groups, because saponification can remove them. In the process of peptide synthesis, in order to avoid side chain functional groups from side chain reactions, appropriate protective groups are usually used to temporarily protect the side chain groups of amino acids with side chain functional groups such as cysteine, histidine, arginine, etc.

The selection of protective group should not only make the side chain group not participate in the reaction of forming amide, but also ensure that the protective group is not damaged during peptide synthesis, and then the protective group is removed when peptide chain is cracked. For example, arginine is protected by adamantoxycarbonyl (Adoc) and removed by cold trifluoroacetic acid; The imidazole ring of histidine is protected by 2,2,2-trifluoro-1-benzyloxycarbonyl and 2,2,2-trifluoro-1-tert-butyloxycarbonyl ethyl, and then removed by catalytic hydrogenation or cold trifluoroacetic acid.

The principle of peptide binding reaction in solid phase and liquid phase is basically the same. Two corresponding amino protected and carboxyl protected amino acids are put into the solution, and no peptide bond is formed. For the formation of amide bond, strong loss agent (such as carbodiimide) is generally used to form symmetric anhydride or activate carboxyl group to form mixed anhydride, active ester, acyl chloride, etc. Among them, the most widely used are the activated ester method and the symmetrical anhydride method using DCC, HOBT or HOBT/DCC.

The purification BOC method of peptide chain splitting and synthesis is to use TFA+HF to split and remove the side chain protection group. The FMOC method uses TFA directly. Due to different conditions, other deprotection methods such as fluorine ion and hydrogenolysis, photolysis, and alkali are also used in some cases. Generally, GX liquid chromatography, affinity chromatography, capillary electrophoresis and other further refining, separation, and purification are used to synthesize peptide chains.