The technology of long peptide synthesis

At present, solid-phase synthesis is the most commonly used and simplest method in protein chemical synthesis research. This method is also effective in synthesizing long-chain peptides, but as the peptide chain lengthens, the reaction gradually becomes more difficult (such as greater than 150 amino acids). As the sequence increases, the steric hindrance of the condensation reaction increases, and in order to complete the reaction, the reaction time needs to be increased; But as the reaction time prolongs, the more side reactions it produces, and some target peptides with missing residues are also produced.

These peptide chains with missing residues are also the main impurities generated during the synthesis of long peptides. Therefore, the main difficulty we need to overcome in the synthesis of long peptides is to explore better reaction conditions and methods, so as to make the amino acid condensation reaction more complete and thorough. At the same time, shorten the reaction time, because the longer the reaction time, the more uncontrollable side reactions there will be, and the more complex the by-products will be.

After years of exploration and accumulation, we have summarized the following experience:

(1) Using microwave synthesis method: For some amino acids that are difficult to condense during the synthesis process, we use microwave method for synthesis. This method has significant effects, greatly shortens the reaction time, and reduces the production of two main by-products.

(2) Using fragment synthesis method: When certain peptides are difficult to synthesize and purify using conventional synthesis methods, we also use a method of condensing several amino acids in a certain segment of the peptide as a whole onto the peptide chain. This method can also solve many synthesis problems.

(3) Using the hydrazide synthesis method: The method of synthesizing peptides using the hydrazide method involves a chemical selective reaction between the N-terminal Cys peptide synthesized in solid phase and the C-terminal peptide hydrazide to form an amide bond and achieve peptide linkage. This method divides the entire peptide chain into multiple sequences based on the position of Cys in the peptide chain, and synthesizes them separately. Finally, the target peptide is obtained through liquid-phase condensation reaction. This method not only greatly shortens the synthesis time of long peptides, but also significantly improves the purity of the final product, making it widely applicable for the synthesis of long-chain peptides containing Cys.