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BACKGROUND
The Application of L-tert-leucine

Ⅰ. The applications of L-tert-leucine

L-tert-leucine is an important and attractive chiral building block. Owing to its bulky and hydrophobic tert-butyl side chain which would provide particularly great steric hindrance in the process of reaction, this unnatural amino acid is also widely used as chiral auxiliaries and catalysts in asymmetric synthesis in developing chiral pharmaceutically active chemicals ^[1]. What’s more, it also plays an important role in the industry of food additive and cosmetics.

Figure 1 The application of L-tert-leucine

L-tert-leucine can apply in various Pharmaceutical fields. L-tert-leucine was introduced into new and more efficient protease inhibitors of many viral diseases, such as HIV, HCV, IL-l-induced cartilage degradation and so on.

As we can see, AIDS is an awful disease which disturbed humans for many years. Lots of people suffered from AIDS for many years and died in pain. Investigations show that HIV-protease is an aspartic acid protease which is necessary for viral replication. So inhibition of this protease could make HIV non-infectious, which could be a useful approach against AIDS.

Today, the most efficient HIV-protease inhibitor is Atazanavir (Figure 2) ^[2]. Atazanavir is distinguished from other protease inhibitors by reducing the dosage and enhance the pesticide effect. What we can see from the structure is that L-tert-leucine plays an important role. L-tert-leucine can stabilized the structure and enhance the effect. So L-tert-leucine is very important.

Figure 2 The structure of Atazanavir ^[4]

Ⅱ. The synthesis of L-tert-leucine

With its special importance, many methodologies, including chemical and biological resolutions, were developed for its preparation in the past decades. Chemical resolution could be easily carried out on a large scale, however, the complicated process in low yield and the difficulties in the racemization of the opposite enantiomer were also observed ^[3].

Figure 1 Comparison of biological method and chemical method of L-tert-leucine synthesis

Biocatalytic protocols, which can be conducted under mild conditions with high selectivity, usually offer greater benefits than chemical procedures and thus gain more and more attention from organic chemists. However, most of these biological resolution procedures are complicated and possess an inherent 50% yield limit ^[4-7].

Owing to different activity of leucine dehydrogenase and formate dehydrogenase, the NADH consumption rate does not equal to its regeneration. Therefore, it is necessary to add excess NADH. The cofactor-NADH is a pretty expensive raw material, which will make the mass production of L-tert-leucine not cost-effective.

The circuits with LeuDH and with FDH were inserted into two E.coli separately. Then they added different wet biomass of two E.coli ^[8]. They hoped to keep the activity of two enzyme equal through this method. Researchers used isolated enzymes,and find it disadvantageous because enzymes are easily destabilized in the isolation and purification process.

Figure 4 Circuits inserted into two E.coli cells separately

Then researchers planned to use whole-cell biocatalyst to stabilize enzymes and reduce the need of cofactor NADH. They envisaged that a promising strategy for a successful coexpression could be based on the same inducible promoter for both genes but located on two E.coli plasmids with different copy numbers, producing LeuDH and FDH on a different level. FDH was inserted in the plasmid with the higher copy number, while LeuDH was inserted in the medium copy number plasmid. We hope to regulate the copy number of plasmid to ensure the continuous recycling of the cofactor NADH. Presumably, this was achieved by a higher production of FDH compared to LeuDH due to the higher copy number vector for the FDH gene. Furthermore, this LeuDH/FDH-strain is suitable for high-cell density fermentation. Compared with isolated enzymes，whole cell-catalyzed asymmetric process has many advantages, such as simple, efficient, environmentally and economically attractive. However, the trouble is that the activity of two enzymes are both inhibited. Obviously, successful coexpression of two genes is still a challenge for scientists.

Reference:

[1] Shioiri, T., Izawa, K. & Konoike, T. Application of Whole‐Cell Biocatalysts in the Manufacture of Fine Chemicals. Pharmaceutical Process Chemistry.184-205 (2011)
[2] https://en.m.wikipedia.org/wiki/Atazanavir
[3] Jin, Jian-Zhong, Chang, Dong-Liang, Zhang, Jie. Discovery and application of new bacterial strains for asymmetric synthesis of L-tert-butyl leucine in high enantioselectivity. Applied Biochemistry and Biotechnology.2011
[4] Eun young Hong,Minho Cha,Hyungdon Yun,Byung-Gee Kim. Asymmetric synthesis of L-tert-leucine and L-3-hydroxyadamantyglycine using branched chain aminotransferase. Journal of Molecular Catalysis B:Enzymatic 66(2010)228-233
[5] Jing Li, Jiang Pan, Jie Zhang, Jian-He Xu Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum.
Journal of Molecular Catalysis B:Enzymatic 105(2014)11-17
[6] Jian-Zhong, JinDong-Liang, Chang Jie Zhang Discovery and application of new bacterial strains for asymmetric synthesis of L-tert-butyl leucine in high enantioselectivity. Appl Biochem Biotechnol (2011) 164:376-385
[7] Weiming Liu, Jixing Luo, Xiaojian Zhuang, Wenhe Shen, Yang Zhang, SHuang Li, Yi Hu, He Huang Efficient preparation of enantiopure L-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium. Biochemical Engineering Journal 83(2014) 116-120
[8] Menzel, A., Werner, H., Altenbuchner, J., Gröger, H. From enzymes to "designer bugs" in reductive amination: A new process for the synthesis of L-tert-leucine using a whole cell-catalyst. Eng. Life Sci. 4, 573-576, (2004)