Peptide Knowledge Center
Problems and Solutions in Solid Phase Synthesis of Peptides
Although the peptide solid-phase synthesis method has been continuously improved and perfected by later generations, it has been able to synthesize many biologically active peptides and proteins, but it still has many problems. For example, the direct synthesis sequence is short, the synthesis time is long, the synthesis efficiency and purity are low, and the cost is high. The existence of these problems greatly limits,The application range of peptide solid-phase synthesis method is described.
Synthetic peptides have short sequences
Since B Merrifield invented the solid-phase synthesis of peptides, the chemical synthesis of peptides has become an effective complementary means for the recombinant expression of a large number of proteins, and has been widely used in structural biology, immunology, protein engineering and biopharmaceutical research. Although the properties of the peptide chain after synthesis and the analysis after synthesis have been greatly improved, when the peptide chain residues are more than 30, especially more than 50, the synthesis of the peptide chain will be restricted to a certain extent. This is because in the solid-phase synthesis of peptides, the concentration of synthetic peptides is relatively low for the polymer. When carboxyl groups are activated, although the azide method can effectively prevent the occurrence of racemization, the reaction proceeds quite slowly.
Therefore, due to these various reasons, the solid-phase synthesis of peptides is limited to the synthesis of small fragments of peptides (within 30 amino acids, and a few within 50 amino acids). When synthesizing large fragments of peptides, it is best to turn to genetic engineering methods to produce recombinant proteins cheaply.
It takes a long time to synthesize
After more than 30 years of development, the rate of solid-phase synthesis of peptides has been greatly improved during the synthesis of peptides, but compared to other synthesis methods (such as biosynthesis), the rate of chain synthesis is still very slow. It takes 20 to 120 minutes for each amino acid to be combined, and the average synthesis of a nonapeptide takes more than 5 hours, which mainly depends on.For chemical selection and the next synthetic strategy. In addition, when synthesizing difficult peptide sequences, the chain binding time is longer.
Low synthesis efficiency and purity, high cost
Even for the solid-phase synthesis of small peptides within 30 amino acids, the synthesis of peptides rich in disulfide bonds and basic amino acid residues is still relatively difficult, and a considerable number of peptides have not been successfully synthesized. With the increase in the number of amino acids, the synthesis efficiency gradually decreases, the content of non-target peptides gradually increases, and the purity of the target peptide gradually decreases, making subsequent purification and renaturation more difficult. The raw materials such as resins and protective amino acids for the synthesis of peptides are expensive, which makes the cost of peptide synthesis very high, and cannot fully meet the requirements of commercial production as drugs.
Synthetic reagents are highly toxic
Reagents for peptide synthesis are very toxic. Organic chemical reagents such as DCM, DMF, and piperidine need to be reported to the Environmental Protection Agency for disposal after they become waste. Although some instruments can provide a fully enclosed synthesis process, the exhaust gas and liquid discharged from it still pollute the environment and cannot guarantee 100% safety. For those synthetic reactions that are not carried out in a fully enclosed manner, toxic reagents are more harmful to the environment. Therefore, with the wide application of solid-phase peptide synthesis, the development of "green" synthetic reagents is imminent.
In view of the above-mentioned problems in peptide synthesis, scientists have been working hard to solve them. Improve the efficiency of peptide synthesis through the improvement of synthesis methods and equipment. For example, the use of "polypeptide piece connection method" can increase the length of peptide synthesis. Based on the traditional solid-phase peptide synthesis method, firstly synthesize peptide fragments within 50 amino acids, and then these peptide fragments through condensation reaction to form a complete target protein.
It is considered that the "chemical selective ligation" method is the most promising method in solid-phase peptide synthesis, but this method is also limited to 40-200 amino acids. Biron E et al. introduced the use of the Fmoc method to synthesize peptides in three steps, and at the same time the amino acid N-terminus was methylated, which greatly improved the synthesis efficiency. However, there are still thousands of proteins that cannot be directly synthesized by solid phase. The synthesis of these proteins requires new methods for synthesis, or the development of reaction reagents that are not prone to side reactions, or the improvement of reaction conditions for artificial synthesis.
On the other hand, by improving the function of the peptide synthesizer, the synthesis process can be fully automated, thereby improving synthesis efficiency, saving reagents, and reducing costs. The 433A peptide synthesizer is equipped with a conduction detection system, which uses UV to detect when the protective group is removed, generates feedback information, and automatically controls, so that some complex reactions can be carried out smoothly, and the peptide synthesis can be optimized. According to our usage, the existing peptide synthesizer still needs improvement in many areas. For example, the cleavage reaction and the synthesis reaction are combined through a closed pipeline, which helps to avoid the synthetic peptide from contacting air and prevent oxidation.
From the current point of view, there are still many specific conditions that limit the scope of application of peptide solid-phase synthesis, but it is believed that through the joint efforts of scientists all over the world, in the near future, with the improvement of peptide solid-phase synthesis methods and tools, it will be There are more and more types of peptides and proteins that can be produced by solid-phase synthesis, and the synthesis rate and efficiency of synthesis will be improved.
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