1963 R B Merrifield founded the solid-phase peptide synthesis method, which overcomes the cumbersome separation and purification process of the classical liquid-phase synthesis method In recent decades, this technology has been widely used and developed, especially the discovery and establishment of Fmoc amino protection method in recent years, which further promotes the development of peptide synthesis The fundamental difference between Fmoc method and BOCP method lies in the use of alkali removal: Fmoc is the protective group of a-amino group, which has special advantages in the synthesis of some polypeptides containing amino residues that are unstable under acidic conditions

However, it is reported that in peptide synthesis, the amino group of glycine will produce dipeptides or even tripeptides when protected by Fmoc. Although this impurity can be detected by effective detection methods, it is difficult to separate from the single glycine protected by Fmoc. Therefore, When protecting glycine, we should choose the protection method to avoid the formation of dipeptides and tripeptides. Here we mainly discuss the synthesis of different Fmoc protective reagents and the amino protection reaction of glycine:

1. Method 1: Fmoc-Cl

Using Fmoc CL as a protective reagent, it reacts with glycine to produce Fmoc Gly in a yield of 83.1%. The product is analyzed by capillary electrophoresis, and it is obvious that the product contains

The two substances, referring to the corresponding literature, are considered to have the production of by-product Fmoc Gly Gly Oh dipeptide. Even if recrystallization is used, it is difficult to remove two of them

Peptides and even Fmoc Gly Gly Oh tripeptide by-products

2. Method 2: Fmoc Cl + TMS-Cl

If trimethylchlorosilane tms-cl is added during the reaction, the Fmoc Gly prepared by adding tms-cl is analyzed by capillary electrophoresis. The results show that the mole fraction of dipeptide has been reduced to less than 0.7%, and the purity has been greatly improved

2.Fmoc-N3

Fmoc CL is not easy to preserve, so a relatively stable fmoc-n3 can be selected as the protective reagent. Only less than 0.5% dipeptide is generated by capillary electrophoresis analysis, which shows that fmoc-n3 is a good protective reagent, but HN3 will be produced during the reaction

4.Fmoc-OSu

The introduction of -osu group on Fmoc group and fmoc-n3 can also prevent the formation of dipeptide "and can speed up the reaction process and improve the reaction rate. The yield of Fmoc Gly prepared by this protective reagent is 93.9%. The molar fraction of this product can be close to 100% by capillary electrophoresis. Therefore, this method is a very good method to introduce Fmoc group on the amino group of amino acids

Comparison of four methods:

Summary:

The yield of Fmoc CL protection is high, but the purity of the product is low, containing dipeptides and tripeptides that are difficult to remove However, TMS CL is used to react with glycine to form an intermediate, and Fmoc CL can not only improve the yield of the reaction, but also greatly improve the purity of the product, which greatly reduces the generation of dipeptide. The dipeptide in the product obtained with fmoc-n3 as the protective reagent is less than 0.5%, and the purity of the product is high, but the yield is low HN3 is generated during the reaction. When Fmoc-OSU is used as the protective reagent, good experimental results are obtained in terms of yield and purity, and the molar fraction of the product is close to 100% by capillary electrophoresis

To sum up, it is the best method to introduce Fmoc group with Fmoc-OSU reagent when Fmoc protection is applied to amino groups of glycine in peptide synthesis