Читать книгу Enzyme-Based Organic Synthesis - Cheanyeh Cheng - Страница 36

The use of transaminases, or aminotransferases, as a catalyst for the organic synthesis of chiral amino compounds from the corresponding keto acids, ketones, or aldehydes has been known a very powerful method since last decade. Their immense potential in the industrial applications can be due to not only their concise reaction, excellent enantioselectivity and environmental friendliness but also their easy combination with other existing enzymatic or chemical methods. The catalytic process for the transfer of an amino group from the amino donor to the amino group acceptor in general involves two steps: (i) the release of an amino group from the amino donor (deamination) and (ii) the addition of the amino group to the amino acceptor (amination). For this kind of transamination reaction, a vitamin B₆‐based pyridoxal 5′‐phosphate (PLP) is required as a cofactor for transaminase to act as an intermediate amine acceptor and electron sink. Since the cofactor releases the amino group and restores its initial state at the end of the transamination, there is no need to carry out additional cofactor regeneration for the enzyme [1, 2]. However, the use of transaminases in organic synthesis still suffers from the problem of unfavorable reversible reaction equilibrium. This problem is especially serious when an amino acid is used as amino donor in the asymmetric synthesis of amines.

To shift the reaction equilibrium to the product side of the transamination reactions catalyzed by transaminase, a conventional strategy that has been commonly used is to provide an excessive amount of the amino donor. Scheme 3.1 illustrates an example in which an excess amount of isopropyl amine was applied to push the transamination of acetophenone to 1‐phenylethylamine toward complete conversion and an enantiomeric excess of >99% [3]. In addition to the method of using excessive cosubstrate for shifting the equilibrium toward the product side, other methods such as removal of product or coproduct by vaporization, extraction, autodegrading smart coproducts, and biochemical cascade reactions were also utilized in many cases [1].

Scheme 3.1 The transamination between acetophenone and excessive isopropyl amine with transaminase.

Source: Truppo et al. [3].

Another smart synthesis strategy is the combination of transaminase with other biocatalysts to form enzymatic cascade reactions that have been used to efficiently produce unnatural amino acids, optically pure amines, imines, secondary amines, and amides, or for the amination on the –OH or –CH₃ functional group, or for the amines with two stereocenters. The enzymatic cascade reactions in organic synthesis give the advantages of shortening reaction routes, avoiding unstable or toxic intermediate, increasing the atom efficiency, and reducing the amount of wastes [1]. Most importantly, the enzymatic cascade reactions involving transaminases show prominent potential for many industrial applications [2].

There have been many examples to demonstrate the use of ω‐transaminase (ω‐TA) for producing unnatural amino acids such as L‐tert‐leucine and L‐phosphinothricine [4]. One of the many examples employing coupled enzyme reactions for synthesizing the unnatural amino acids was the production of the unnatural L‐homoalanine from L‐threonine by combining a threonine deaminase (TD) with the ω‐TA and using an one‐pot reaction process (Scheme 3.2) that resulted a 91% conversion [5]. The source of TD was from a cloned and overexpressed Escherichia coli and the (S)‐specific ω‐TA was from Paracoccus denitrificans. The formation of N‐benzylacetamide from benzaldehyde through an efficient one‐pot biocatalytic amine transaminase/acyl transferase cascade using methyl acetate as acyl donor is shown in Scheme 3.3 [6].

The conversion of the amide product in this enzymatic cascade reaction was up to 97% with an acyl transferase (AcT) from Mycobacterium smegmatis and an amine transaminase from Silicibacter pomeroyi.

Scheme 3.2 Enzyme cascade reactions for the conversion of L‐threonine to L‐homoalanine using a one‐pot reaction process.

Source: Modified from Park et al. [5].

Scheme 3.3 The formation of N‐benzylacetamide from benzaldehyde through an efficient one‐pot biocatalytic amine transaminase/acyl transferase cascade.

Source: Based on Land et al. [6].

Instead of compounds with one stereocenter, chiral amino alcohols with two stereocenters can be synthesized by the enzymatic cascade reactions without using protection step for the first stereocenter before the production of the second stereocenter. Scheme 3.4 shows an example of the enzymatic cascade route toward all four diastereomers of 4‐amino‐1‐phenylpentane‐2‐ol started from a racemic 1,3‐hydroxy ketone [7].

Scheme 3.4 The enzymatic cascade route toward all four diastereomers of 4‐amino‐1‐phenylpentane‐2‐ol from a compound with two stereocenters.

Source: Modified from Kohls et al. [7].

The first synthesis step of these four 1,3‐amino alcohol diastereomers was through the kinetic resolution for a racemic 1,3‐hydroxy ketone by applying using an (S)‐selective keto reductase (KRED) to regioselectively provide the optically pure (R)‐hydroxyl ketone (86% e.e.) and the corresponding diketone. Further transamination of the (R)‐hydroxyl ketone using either an (R)‐ or an (S)‐selective TA yields the (2R,4R)‐ and (2R,4S)‐1,3‐amino alcohol diastereomers.

The diketone was then used to prepare the remaining two diastereomers, (2S,4S)‐ and (2S,4R)‐1,3‐ amino alcohol, by first converting the diketone to (S)‐1,3‐hydroxy ketone and subsequently using either an (R)‐ or an (S)‐selective TA to stereoselective amination of the (S)‐1,3‐hydroxy ketone to (2S,4R)‐1,3‐amino alcohol and (2S,4S)‐1,3‐amino alcohol, respectively.

The industrial applications of enzymatic cascade reactions can be demonstrated by the in vitro biosynthesis of the nylon‐6 monomer 6‐aminohexanoic acid (hydrolyzed ε‐caprolactam) from cyclohexanol as illustrated by Scheme 3.5 [8]. In addition to transaminase, this enzymatic cascade route additionally involves a Baeyer–Villiger monooxygenase (BVMO) and an esterase that all these biocatalysts can function independently in a one‐pot synthesis under mild conditions. Two cofactor self‐sufficient cascade modules are involved in this route. One is for the production of ε‐caprolactone from cyclohexanol, the other is the subsequent production of 6‐aminohexanoic acid. This biosynthesis route thus shows advantages over chemical approaches that require sophisticated transition‐metal catalysts and high temperature and pressure.

Generally, the asymmetric amination of prochiral ketones with an amine donor employing ω‐transaminases (ω‐TAs) is performed in aqueous medium. Since most ketones are lipophilic with only moderately soluble in aqueous solution, the transformations involving ω‐TAs require the addition of an organic cosolvent, and enzyme engineering might be necessary to let it stand the harsher reaction conditions. In addition, the amination of prochiral ketones with ω‐TAs is commonly employed at pH < 10; the product amines are generally protonated necessitating the basification of the reaction mixture and the extraction with organic solvents. As a result, it would be desirable to employ ω‐TAs for the transamination exclusively in organic solvents. Mutti et al. employed nine different lyophilized crude cell‐free extracts of (R)‐selective and (S)‐selective ω‐TAs for the asymmetric amination of ketones in organic solvents with 2‐propylamine as amine donor. The best enzyme activity for the transamination was found for methyl tert‐butyl ether (MTBE) at a water activity of 0.6 that allowed excellent stereoselectivity of optically pure amines (e.e. > 99%) and a conversion rate > 99% [9].

Scheme 3.5 The oxidation/transamination cascade reactions for the biosynthesis of 6‐aminohexanoic acid from cyclohexanol.

Source: Based on Sattler et al. [8].

The use of organic solvent MTBE has also been applied for the production of optically pure 1,2‐amino‐alcohols such as valinol from corresponding prochiral hydroxyl ketone using ω‐TAs [9]. Chiral 1,2‐amino‐alcohols are common building blocks embedded in many synthetic and naturally occurring molecules having biological activity, and valinol is a typical example of the versatile vicinal amino alcohols. Thus, the reductive amination of isopropyl methyl alcohol ketone can be performed in MTBE using 2‐propyl amine as amino donor to yield either (R)‐valinol or (S)‐valinol by the choice of (R)‐ and (S)‐selective ω‐TAs. The use of (R)‐selective ω‐TA purified from Bacillus megaterium afforded the (R)‐valinol with an ideal optical purity (>99% e.e.), although a low conversion rate of 15%. The (S)‐selective ω‐TA originating from Arthrobacter sp. can produce the (S)‐valinol with a much better conversion rate (95%) and a perfect stereoselectivity (>99% e.e.) [10].

A pharmaceutical industrial application of the ω‐transaminase was the synthesis of antidiabetic compound sitagliptin. The synthesis protocol was the use of a stable (R)‐selective amine transaminase and DMSO/water system to perform the transamination between prositagliptin ketone and isopropyl amine that gives an excellent stereoselectivity (99.95% e.e.) [11]. Later, the synthesis protocol was modified by immobilizing the transaminase on polymer‐based resins, and the immobilized enzyme activity of the transamination was evaluated in neat organic solvent isopropyl acetate that results 91% sitagliptin production yield and >99% enantioselectivity (Scheme 3.6) [12].

The immobilized enzyme enables the use of flow reaction system to give high throughput, clean production, high enzyme stability, and excellent mass recovery. E. coli cells containing the overexpressed (R)‐selective ω‐TA from Arthrobacter and the cofactor pyridoxal 5′‐phosphate (PLP) were immobilized on methacrylate polymeric resin beads that were used for continuous flow applications to produce chiral amines continuously by asymmetric transamination of ketones using a packed‐bed reactor in organic solvent methy tert‐butyl ether (MTBE). The use of organic solvent helps in the suppression of PLP leaching from the cells. Non‐natural α‐alkoxy‐ and α‐aryl acetones were transformed under flow conditions using isopropyl amine as the amine donor with excellent enantioselectivity (>99% e.e.) [13].

Scheme 3.6 Synthesis of sitagliptin from prositagliptin ketone using immobilized transaminase in organic solvent.

Source: Truppo et al. [12].