1. Chemical resolution method

With DL unnatural amino acids as substrates, tartaric acid, DBTA, camphor sulfonic acid, mandelic acid and other chiral resolving agents were used for separation. The chemical resolution of this kind of resolution agent will have natural disadvantages, and the resolution efficiency can only reach 50%. The crystallization conditions of the process are relatively harsh, and the control is not good. The chirality is easy to exceed the standard, and it is relatively difficult to reach the chirality purity of more than 99%

2. Acylation hydrolase resolution

With n-acetyl-dl-amino acid as the substrate, the unnatural amino acid is hydrolyzed or separated by l-acylase or d-acylase through chemical means. In the resolution of amino acids by acylation hydrolase, d-acylation hydrolase is not universal. Many unnatural amino acids can only use l-acylation hydrolase, and l-acylation hydrolase needs to be acidulated by chemical means to realize the synthesis of D-amino acids. During acidolysis, amino acids will be destroyed, resulting in low overall yield, so the industrial value is getting lower and lower

3. Asymmetric transformation method

Asymmetric transformation is a process of dynamic resolution through solubility differences, using single configuration amino acids as raw materials, organic acids as solvents, aldehydes as racemates, and chiral resolution agents. At present, this method is widely used in industry, but unnatural amino acids are a large class of products, and the solubility of double salts in each product is different, so it cannot be applied to special products

4. Chemical synthesis

Chemical synthesis takes a chiral compound as the substrate and adopts chemical means to synthesize. R1 can be a halogen series, and this route can only be used for the synthesis of a few products. Because it is difficult to obtain chiral precursors, this route cannot be applied on a large scale, and there is chiral inversion in the ammoniation process, and the chiral amino acids obtained are prone to insufficient chiral purity

5. Dehydrogenase method

Dehydrogenase method is to use transaminase or amino acid dehydrogenase as ketone substrate to provide a suitable ammonia donor, add coenzyme NAD, and carry out enzyme catalysis to obtain chiral unnatural amino acids. The synthesis of l-aminobutyric acid and l-tertiary leucine is all based on this method

6. Induced resolution method

With the mixed non natural amino acids as the substrate, through the supersaturated solution, add crystal seeds, control the cooling speed and stirring speed, crystallize slowly, and achieve the process of resolution and separation through the solubility difference. Mother liquor can be used repeatedly, and it can not be used again after being enriched to a certain extent. Fmoc-OSu The other half of unnatural amino acids can be recovered by concentrating and crystallizing. D-glutamic acid and D-Threonine are synthesized in this way.

7. Oxidase method

The oxidase method takes the mixed rotating unnatural amino acids as the substrate, oxidizes the L-amino acids or D  amino acids into ketoacids according to the needs through the amino acid oxidase of engineering bacteria, retains the required L-amino acids or D-amino acids, and never obtains the required chiral unnatural amino acid products

8. Hydantoin enzyme method

The hydantoin enzyme method was first applied to the synthesis of p-hydroxyphenylglycine, including the synthesis of amino acid hydantoin precursor. The hydantoin enzyme method has better achieved the synthesis of unnatural amino acids, and the yield and conversion have reached a relatively high level. Each substituted hydantoin has different properties, CBZ-OSu and the spontaneous racemization power of hydantoin substrate is also different, and the temperature tolerance of hydantoin enzyme is limited, This also leads to the fact that hydantoin enzyme method cannot achieve the synthesis purpose of individual amino acid conversion and hydrolysis. Therefore, some scholars have studied hydantoin racemase and completely solved the problem of synthesizing unnatural amino acids by the hydantoin enzyme method

Other methods for synthesizing unnatural amino acids:

There are many ways to synthesize unnatural amino acids, including membrane separation, electrophoresis, chromatography and so on

The several routes discussed in detail in this paper are all industrialized routes, and they are also the methods used in the development history of unnatural amino acids. Among them, asymmetric transformation method, dehydrogenase method and Hein method are mainly used in industrial production. Other methods cannot realize the synthesis of unnatural amino acids with expert purity due to cost problems or separation disadvantages. N-Fmoc-7-methyl-L-tryptophan Among them, asymmetric transformation method is a typical special case of chemical synthesis, while dehydrogenase method and Heine method are typical special cases of biological methods for the synthesis of unnatural amino acids.

With the development of society and the strengthening of environmental protection awareness, the pressure of chemical method is increasing. In the future, the synthesis of unnatural amino acids by biological method will inevitably replace chemical method. If biological method wants to develop for a long time, it also needs to reduce emissions and fermentation volume and reduce the difficulty of COD treatment. Therefore, the biotransformation of immobilized enzymes should be taken as a new research direction on the existing basis, or more efficient engineering bacteria should be obtained to reduce the amount of enzymes.

As a non protein amino acid, unnatural amino acid developed late and was not found in human body. Insufficient attention was paid in the early stage, and the domestic synthesis of unnatural amino acid started late. However, with the development of chiral drugs, researchers pay more and more attention to the synthesis of unnatural amino acids. The rapid development of molecular biology has led to the development of human genome project, directed evolution, synthetic biology and other disciplines, laying a strong foundation for the synthesis of unnatural amino acids