BACKGROUND

Ⅰ. The applications of L-tert-leucine

L-tert-leucine is an important and attractive chiral animo acid. 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 synthesisin 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 applications of L-tert-leucine

1. Pharmaceutical applications 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 [2].

Figure 2 The estimated number of people infected with HIV during 1993-2014 [2]

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 [2]. Today, the basic structure of HIV-protease inhibitors is phenylnorstatine [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid （Figure 2） [3].

Figure 3 Structure of HIV-protease inhibitor [2]

However, as Figure 3 shows, phenylnorstatine is not enough. In order to optimize protease inhibitors, numerous protected, deprotected and derivatized L-tert-leucines are used to modify phenylnorstatine. Modified compounds could be nice protease inhibitors with considerable antiviral activity.

Today, the most efficient HIV-protease inhibitor is Atazanavir (Figure 4) [4]. Atazanavir is one of the most effective HIV-protease inhibitor confirmed by FDA which is distinguished from other protease inhibitors by reducing the dosage and enhancing the pesticide effect. But Atazanavir is still very expensive, which makes the cure very difficult. What we can see from the structure is that L-tert-leucine plays an important role. L-tert-leucine can stabilize the structure and enhance the effect. If we lower the cost of L-tert-leucine, the cost of Atazanavir production may lower to a certain degree. So production of L-tert-leucine is necessary.

Figure 4 The structure of Atazanavir [4]

As for Hepatitis C, it is also a severe public health issue [5]. In order to cure this disease, we also need a protease inhibitor. And the first option is Telaprevir [6]. The same as Atazanavir, the structure of Telaprevir shows that L-tert-leucine is also an important intermediate.

Figure 5 The structure of Telaprevir [6]

For the treatment of IL-l-induced cartilage degradation in tissue culture, L-tert-leucine plays an important role. Thirty years ago, Roche Company discovered an N-substituted Tle-N-methylamide (Ro 31-9790, Figure 6) to be a potent collagenase inhibitor which could prevent IL-l-induced cartilage degradation [2].

Figure 6 The structure of Ro 31-9790 [2]

L-tert-leucine is essential in many fields so that the large-scale production is indispensible.

2. Asymmetric synthesis by L-tert-leucine and its derivatives

When L-tert-leucine or its derivatives were employed in asymmetric reactions, the results always showed high optical purity [2]. For example, the following reaction is a reported Michael additions of Grignard reagents to α,β-unsaturated aldimines derived from L-tert-leucine (Figure 7). After hydrolysis and hydrogenation, the finalist product shows high optical purity. Owing to the bulky tert-butyl side chain of compound 1, the side of stronger steric hindrance was locked. Grignard reagents could only attack compound 1 from the special side so that the product shows high enantiomerical purity.

Figure 7 A Michael addition of L-tert-leucine derivatives

Enantiomerical pure L-tert-leucine are important in many fields. So the efficient production of it is significant.

Ⅱ. The synthesis of L-tert-leucine

In recent years, many different technologies have been applied in the synthesis of L-tert-leucine. For example, Strecker synthesis, amidocarbonylation and Acetamidomalonic ester synthesis have already been applied in the production of L-tert-leucine (Figure 8)[1]. But from Figure 8, we could know that there are some bugs in these methods. And the most obvious bugs are low efficiency and pool charity of products. In order to get high optical pure products, chemical recemizations should be carried out after the reaction. Chemical recemization processes are sophisticated and costly and some chemical catalysts contain toxic elements. So these methods are gradually abandoned.

Figure 8 Synthesis of recemic amino acid [1]

As a matter of fact, with the development of synthetic biology, enzymes become very efficient and important catalysts in production of L-tert-leucine. What is more, the production of L-tert-leucine was introduced into industrial production by applying enzymatic reductive amination as a method [7].

Figure 9 The synthesis of L-tert-leucine

Up to now, the most efficient enzyme is leucine dehydrogenase (LeuDH, from Bacillus sp). It can transform substrate trimethylpyruvate into L-tert-leucine in very good yields and excellent optical purities with the help of cofactor NADH. However, from the Figure 10, we could know that NADH is a rather expensive raw material [8]. As a result, NADH should be regenerated so that this system would commercial attractive. The regeneration of NADH is the so-called cofactor regeneration.

Figure 10 Costs of redox equivalents in US [10]

Cofactor regeneration could be carried out by means of many different enzymes whose cofactors are NAD⁺. As for synthesis of L-tert-leucine, cofactor regeneration is accomplished by formate dehydrogenase (FDH, from Candida boidinii) [1]. This process has been introduced into industrial production in ton scale for many years.

But it is not excellent. There are still some bugs should be amended. And the most interesting aspect is the different activities of LeuDH and FDH. The result caused by different activities is that the different consuming and regenerating rates of NADH. Owing to that the activity of LeuDH is significantly higher than FDH. NADH would be consumed to a low level before the synthesis finished, which results in stopping of production and need excess NADH to support these reactions.

Some scientists used mutational FDH or FDH from other bacterial strains to take the place of FDH from Candida boidinii [9,10]. And some other scientists chose glucose dehydrogenase (GDH) to regenerate NADH (Figure 11)[1,11]. However, in our opinion, none of these methods made a difference. Cofactor regeneration with the FDH from Candida boidinii is a stable system which has been used for many years. Mutational FDH or FDH from other bacterial strains may be unstable and difficult to be introducted into industrial production. And as for the GDH, the substrate is glucose and the product is gluconate. If we apply LeuDH and GDH into industrial production, the cost must be raised owing to the excess substate and purification process.

Figure 11 Choosing glucose dehydrogenase as a co-enzyme of leucine dehydrogenase [12]

This year, what we want to do is providing a method to solve this problem.

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] Bommarius, A., S., Schwarm, M., Stingl, K., Kottenhahn, M., Huthmacherand, K. & Drauz, K. Synthesis and use of enantiomerically pure tert-leucine. Tetrahedron: Asymmetry. 6, 2851-2888 (1995)
[3] Ettmayer, P., Hübner, M., Billich, A., Rosenwirth, B. & Gstach, H. Design and synthesis of potent β-secretase (BACE1) inhibitors with P’₁ carboxylic acid bioisosteres. Bioorg. Med. Chem. Lett. 4, 2851-2856 (1994)
[4] https://en.m.wikipedia.org/wiki/Atazanavir
[5] https://en.m.wikipedia.org/wiki/Hepatitis_C
[6] https://en.m.wikipedia.org/wiki/Telaprevir
[7] Gröger, H., May, O., Werner, H., Menzel, A., & Altenbuchner, J. A “second-generation process” for the synthesis of L-neopentylglycine: asymmetric reductive amination using a recombinant whole cell catalysis. Org. Process Res. & Dev.. 10, 666−669 (2006)
[8] Wandrey, C. Biochemical reaction engineering for redox reactions. Chem. Rec. 4, 254-265 (2004)
[9] Liu, W., Ma, H., Luo, J., Shen, W., Xu, X., Li, S., Hu,Y. & Huang, H. Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli. Biochem. Eng. J. 91, 204–209 (2014)
[10] Bai, J., Song, Y., Luo, X., Yang, H., Du, W., & Zhang, T. Construction of L-tert-Leucine Producing Strain by Expressing Heterologous Leucine Dehydrogenase and Formate Dehydrogenase in Escherichia coli. Advances in Applied Biotechnology. 21-30 (2015)
[11] Li, J., Pan, J., Zhang, J. & Xu, H., J. Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum. J. Mol. Catal. B-Enzym. 105, 11-17 (2014)
[12] Tao, R., Jiang, Y., Zhu, F. & Yang, S. A one-pot system for production of L-2-aminobutyric acid from L-threonine by L-threonine deaminase and a NADH regeneration system based on L-leucine dehydrogenase and formate dehydrogenase. Biotechnol. Lett. 36, 835-546 (2014)