Peptide Knowledge Center

Comparison of 2 common methods for peptide solid-phase synthesis

Before the amino acid enters the target peptide, the amino terminal needs a protecting group. According to different protecting groups, solid-phase peptide synthesis methods can be divided into two categories: Boc method and Fmoc method. The synthetic principles of these two methods are basically the same, but due to the difference of the protective groups, a series of reagents and conditions used later have been changed accordingly.


Fmoc (9-fluorenylmethyloxycarbonyl) peptide solid phase synthesis method


RB Merrifield once reported that the incomplete reaction resulted in the accumulation of side reaction products. He looked forward to the further improvement of the solid-phase peptide synthesis method. He had lived up to his expectations. After forty years, the solid-phase peptide synthesis method has been greatly improved.


Example: This Fmoc-Arg(Pbf)-OH is a commonly used fmoc protected amino acid in peptide synthesis.


The Fmoc method is a new method for solid-phase peptide synthesis developed by Carpino and Han on the basis of the Boc method. The protective group of the α-amino group used in this method is Fmoc. Since Fmoc is stable to acid and easy to react with alkalis, the use of basic compounds as deprotecting agents has the advantages of mild reaction conditions, fewer side reactions, and high yield. At the same time, the Fmoc group has a characteristic ultraviolet absorption, which is easy to monitor and control the progress of the reaction. Therefore, it is more and more favored by people.


Boc (tert-Butoxycarbonyl) peptide solid-phase synthesis method


The Boc method is a classic solid-phase peptide synthesis method. When B Merrifield synthesizes bradykinin, Boc is used as the protective group of α-amino acids. After consulting a lot of data, it is found that the Boc method is not used at a high rate. Although the Boc method is gradually being replaced by the Fmoc method, its own advantages cannot be ignored, and some aspects are worthy of our reference and application.


When synthesizing a specific protein, according to the specific amino acid contained in the protein, select the appropriate resin. The binding rate of the first amino acid to the resin will directly affect the final synthesis amount of the target peptide. Therefore, when synthesizing proteins with more complex structures, the Boc method and the Fmoc method can be used in combination. The solid-phase peptide synthesis method can not only synthesize peptides with "difficult sequences", but also synthesize peptides that require chemical modification (such as C-terminal acetylation).


Principles of Peptide Solid Phase Synthesis


First, the amino acids that make up the peptide of interest are modified to make them an amino acid with a protective group at the amino terminus (this amino acid can be purchased directly from the relevant biological company). The carboxyl group of the first amino acid of the target peptide is connected to the solid phase carrier (resin) in the form of a covalent bond, and then the amino group of this amino acid is used as the starting point for synthesis to cause an acylation reaction with the carboxyl group of the adjacent amino acid to form a peptide key. Then let the amino group of the resin peptide containing these two amino acids react with the carboxyl group of the next amino acid, and repeat this process until the target peptide is formed. Then the target peptide is cleaved from the resin and oxidized and folded, so that the crude product of synthetic peptide is obtained. Purification, chemical modification, and finally activity identification are carried out.


The artificially synthesized peptide is linear and inactive at first, and needs to undergo oxidative folding to form the natural conformation of the target peptide before it has physiological activity. Therefore, artificially synthesized peptides (especially target peptides rich in disulfide bonds, such as conotoxin, etc.) form the correct disulfide bond connection and form the correct conformation, which is the key to the synthesis of biologically active peptides.