Liquid-phase synthesis is the classical method of peptide synthesis, which generally adopts stepwise synthesis or fragment condensation method. The stepwise synthesis method usually starts from the C’ terminal amino acid of the chain and adds a single α-amino protected amino acid repeatedly to the increasing amino acid fraction.

Fragment condensation generally starts with rational segmentation of the target sequence into fragments, followed by stepwise synthesis of the individual fragments, and finally condensation of the individual fragments according to the sequence requirements. The advantages of liquid-phase synthesis are that the intermediates can be purified at each step, the physicochemical constants of the intermediates can be obtained, non-amino acid modifications can be made at will, amino acid deletions can be avoided, and the disadvantages are that it is more time-consuming and labor-intensive, etc.

In solid phase synthesis, the carboxyl group of the first natural hyaluronic acid of the target peptide is covalently bonded to the solid phase carrier (resin), and then the amino group of this amino acid is used as the starting point for synthesis, and it is acylated with the carboxyl group of the adjacent amino acid (amino protected) to form a peptide bond.

Then, the amino group of the resin peptide containing these two amino acids is deprotected and reacted with the carboxyl group of the next amino acid, and the process is repeated until the target peptide is formed.

The advantages are that it simplifies the post-processing operation of each reaction step, avoids the loss due to manual operation and material transfer, has a higher yield and can be automated, etc.; the disadvantages are that the intermediate products of each step cannot be purified, a larger amino acid overfeed must be used, the purity of the crude product is not as good as that of the liquid-phase synthesis, and must be purified by reliable separation means, etc.

Liquid-phase synthesis and solid-phase synthesis have their own advantages and disadvantages, and a suitable process should be selected according to the actual needs of the synthesis. Generally speaking, liquid-phase synthesis is more suitable for the synthesis of short peptides; solid-phase synthesis is more suitable for the synthesis of medium and long peptides. Of course, it is also possible to combine the two methods, such as using the liquid phase method to synthesize short peptide fragments and then applying the fragments to the solid phase synthesis.

Both the liquid-phase and solid-phase synthesis are based on the designed amino acid sequence, and the target molecule is obtained by the directional amide bond formation method. Theoretically, this is not complicated, but there are still many factors to be considered for implementation.

In the case of simple amide bond formation between carboxylic acid and amine, the carboxyl group is usually converted into a lively carboxyl derivative (e.g., chloride or anhydride) before interacting with the amine, or a condensation agent is added to the reaction system. However, the formation of amide bonds between amino acids is much more complicated, because each amino acid contains both amino and carboxyl groups.

If the carboxyl group of one amino acid is activated, it can react with the amino group of the same or another amino acid molecule; if several amino acids are mixed together and a condensation agent is added, only a mixture of peptides with many different amino acid orders can be obtained. Therefore, the research of peptide synthesis needs to pay attention not only to the activation method, coupling method, etc. but also to the choice of protection or deprotection strategy.

In the process of peptide synthesis, some heteropeptides with a similar structure to the target peptide will be generated, such as diastereoisomers due to amino acid racemization, deletion peptides due to some amino acids not being attached, and broken peptides due to peptide bond breakage.

Therefore, it is necessary to consider the selection of reliable separation and purification methods to make the purity of the peptides as required. The purification of peptide drugs is usually performed by chromatographic methods, but in some cases, purification methods commonly used in organic synthesis (e.g. recrystallization) may also be applicable.

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