After decades of development and continuous improvement and improvement, polypeptide solid-phase synthesis has been able to synthesize many bioactive polypeptides and proteins, but there are still many problems and defects in it, such as short sequence, long time required, low efficiency and purity of synthesis, and high cost. These problems limit the application of polypeptide solid-phase synthesis.

1. The sequence of synthetic peptide is short

Since bmerrifield invented the solid-phase synthesis method of polypeptides, chemical synthesis of polypeptides has become an effective auxiliary tool for large-scale recombinant expression of proteins Fmoc-Pen(Acm)-OH, which has been widely used in structural biology, immunology, protein engineering and biopharmaceutical research. Although the properties and analysis of peptide chain after synthesis have been greatly improved, when the number of peptide chain residues is more than 30, especially more than 50, the synthesis of peptide chain will be restricted to a certain extent. This is because there are many

In the solid-phase synthesis of peptides, the limitation of polymers is that the concentration of synthetic peptides is low when carboxyl groups are activated. Even if the azide method can effectively prevent racemization, the reaction is still relatively slow. Therefore, the solid-phase synthesis of polypeptides is limited to the synthesis of small polypeptides (less than 30 amino acids, a few less than 50 amino acids). For the synthesis of large polypeptides, genetic engineering and liquid phase synthesis can be applied.

2. Long synthesis time

After more than 40 years of development, the rate of peptide solid-phase synthesis has been greatly improved, but compared with other synthesis methods (such as biosynthesis), the rate of peptide chain synthesis is still very slow. It takes about 20-120 minutes for each amino acid to bind, and the average synthesis time of a nine peptide is more than 5 hours.2,3,5-Trimethylphenol  In addition, when synthesizing polypeptide sequences with complex structures, the binding time of peptide chains is longer.

3. The synthesis efficiency and purity are low, and the cost is high. 

For the solid-phase synthesis of polypeptides rich in disulfide bonds and basic amino acid residues, even within 30 amino acids, it is still difficult, and some polypeptides can not be successfully synthesized. With the increase of the number of amino acids, the synthesis efficiency gradually decreases, the content of non target peptides gradually increases, and the purity of target peptides correspondingly decreases. Subsequent purification and repeatability become more difficult. Raw materials such as resins and protective amino acids for synthesizing polypeptides are expensive, and the cost of polypeptide synthesis is high, which can not fully meet the requirements for commercial production of drugs