Читать книгу Enzyme-Based Organic Synthesis - Cheanyeh Cheng - Страница 33

Chiral alcohols are important building blocks and among the most valuable key intermediates for the production of pharmaceuticals and fine chemicals [140–143]. As an alternative to chemical processes, an efficient and powerful method to prepare enantiopure alcohols is the use of NAD(P)H‐dependent ADHs to perform the asymmetric hydrogenation of prochiral ketones without protective groups that are common in traditional organic synthesis [144, 145]. Although asymmetric reduction of ketones to optically pure alcohols has become quite mature in asymmetric synthesis during the last decade, it is still the most interesting strategy in preparing single enantiomers of alcohols by recent advancement in genetic engineering, coupled enzyme reaction, reaction design, and the availability of a variety of ADH. At least 150 different ADHs are available from various commercial sources, which allow the most suitable ADH to be selected for a specific substrate to access the desired (S)‐ or (R)‐enantiomer. The following are a concise introduction of the synthetic strategies used for the preparation of optically pure alcohol recently.

Since serine‐threonine kinase, a polo‐like kinase 1 (PLK1), is a key regulator of mitotic progression and cell division in eukaryotes and is highly expressed in tumor cells, it is considered a potential target for cancer therapy. To synthesize the most promising PLK1 inhibitor candidates, thiophene‐benzimidazole and imidazopyridine, for chemotherapy, enantioselective reduction of o‐chloroacetophenone is preferred to produce the chiral key intermediate, (S)‐1‐(o‐chlorophenyl)‐ethanol, for their synthesis (Scheme 2.34) by whole‐cell catalyst of recombining E. coli and recombining S. cerevisiae [146]. The recombinant xylose reductase was from Candida tenuis.

Scheme 2.34 Asymmetric reduction of ketone precursor o‐chloroacetophenone with recombining microorganism toward chiral alcohol product.

Aprepitant (Emend^®), a trichiral compound, is a neurokinin‐1 (NK‐1) receptor antagonist that has been approved by the United States and Europe to treat moderately and highly emetogenic chemotherapy for the prevention of acute and delayed chemotherapy‐induced nausea and vomiting. According to its structure (Figure 2.2), 3,5‐bis(trifluoromethyl) acetophenone was used as the starting material to synthesize asymmetrically the key intermediate, chiral (1R)‐[3,5‐bis(trifluoromethyl)phenyl] ethanol, of aprepitant (Scheme 2.35) [147–149]. In the synthesis, a novel bacterial strain Leifsonia xyli HS0904 was isolated from soil that exhibits R‐stereospecific carbonyl reductase. Under the optimal conditions, the best yield of 62% and an enantiomeric excess of 99.4% for the product was obtained with resting cells.

An intermediate of amphetamine and amphetaminil, (S)‐1‐phenyl‐2‐propanol, has been prepared from the simple ketone, 1‐phenyl‐2‐propanone, via bioreduction with 99% e.e. but low productivity through growing cells of R. erythropolis. The substrate inhibition of the biotransformation with growing cells was solved by stepwise feeding, while product inhibition was solved by repeated removal of product using methods such as centrifugation, absorption with resin, and second phase. Higher productivity can be obtained for the reduction of 1‐phenyl‐2‐propanone with resting cells through cofactor regeneration and recycling by the addition of glucose and permeabilized cells of B. subtilis [150].

Figure 2.2 Structure of aprepitant.

Scheme 2.35 The asymmetric synthesis of 3,5‐bis(trifluoromethyl) acetophenone to (1R)‐[3,5‐bis(trifluoromethyl)phenyl] ethanol.

The family of imidazole derivatives, including miconazole, econazole, isoconazole, ketoconazole, sertaconazole, and sulconazole, is well known for antifungal imidazolium compounds. Miconazole and econazole are usually employed in the treatment of vaginal diseases and several fungal infections in the skin of both human and animals by interfering with ergosterol biosynthesis of fungal organisms. Since the therapeutic efficacies of their enantiomers were usually different, the demand for synthesis of optically pure compounds rather than their racemates is required. However, the synthesis of miconazole and econazole single enantiomers with asymmetric chemical transformations [151–154] was rarely reported. Recently, a simple and novel bioreduction of prochiral ketones using ADHs has been applied for the synthesis of the precursors of miconazole and econazole single enantiomers. Then the target fungicides miconazole and econazole were produced from corresponding enantiomeric pure precursors by a series of chemical modifications. The best results were the synthesis of enantiopure precursor, (R)‐2‐chloro‐1‐(2,4‐dichlorophenyl)ethanol, from 2‐chloro‐1‐(2,4‐dichlorophenyl)ethanone using screened ADHs under very mild conditions (Scheme 2.36) [155].

The use of S. cerevisiae (baker’s yeast) for the reduction of ketones or aldehydes may result in mixtures of products due to the existence of a variety of reductases possessing overlapping substrate specificity and mixed stereoselectivity [156]. Therefore, S. cerevisiae and the gram‐negative bacterium E. coli were overexpressed with gene encoding the NADPH‐dependent aldo‐keto reductase YPR1 and compared for producing optically active alcohols through whole‐cell bioreduction. The NADPH‐dependent carbonyl reduction of bicycle[2.2.2]octane‐2,6‐dione to produce optically pure (−)‐(1R,4S,6S)‐6‐hydroxy‐bicyclo[2.2.2]octane‐2‐one was used as a model reaction (Scheme 2.37). High purity of the (−)‐keto alcohol (>99% e.e., 97–98% de) was obtained for both engineered microorganisms. However, E. coli had higher initial rate but S. cerevisiae continued the reaction longer to give a higher substrate conversion (95%). S. cerevisiae also demonstrated higher viability during reaction than E. coli [157].

Scheme 2.36 Bioreduction of α‐haloketones in aqueous medium using different alcohol dehydrogenase followed by a series of chemical modifications to optically pure miconazole or econazole.

Scheme 2.37 E. coli or S. cerevisiae catalyzed reduction of bicycle[2.2.2]octane‐2,6‐dione to (−)‐(1R,4S,6S)‐6‐hydroxy‐bicyclo[2.2.2]octane‐2‐one and (+)‐(1S,4R,6S)‐6‐hydroxy‐bicyclo[2.2.2]octane‐2‐one.

The asymmetric reduction of β‐ketoesters mediated by microorganisms has become a standard method for the synthesis of chiral β‐hydroxyesters [158, 159]. Recently, the immobilization of ADH from permeabilized brewer’s yeast on derived attapulgite nanofibers through glutaraldehyde covalent binding for the bioreduction of ethyl 3‐oxobutyrate (EOB) to ethyl (S)‐3‐hydroxybutyrate ((S)‐EHB) was investigated. The effect of immobilization on ADH activity for the bioreduction shows that the immobilized ADH retained higher activity over wider ranges of pH and temperature than those of the free enzyme. The optimum temperature and pH of the immobilized ADH were 7.5 and 35 °C, respectively. Under the optimum conditions, the immobilized enzyme retained 58% of the original activity after 32 hours of incubation. The conversion of substrate (EOB) and the enantiomeric excess value of (S)‐product reached 88 and 99.2%, respectively, within two hours. The immobilized ADH retained about 42% of the initial activity after eight cycles [160]. Also, although the yields and the enantioselectivity are low to moderate, the enantioselective bioreduction of ethyl benzoylacetate and their p‐nitro and p‐methoxy substituted derivatives to form corresponding chiral ethyl 3‐hydroxy‐3‐phenylpropionate and substituted derivatives (Scheme 2.38) has long been of interest in pharmaceutical industry for synthesizing the key chiral building blocks of many compounds such as fluoxetine [161], chloramphenicol [162], and diltiazem [163]. However, the coupling of simple screening procedures and reaction engineering strategy can increase the (S)‐enantioselectivity to 99% e.e. and shows a significant improvement in the yields to around 85%. In this way, yeasts Pichia kluyveri, Pichia stipites, and Candida utilis were screened and better yields and e.e.’s for ethyl benzoylacetate, p‐nitrobenzoylacetate, and p‐methoxybenzoylacetate can be achieved by the addition of glucose, α‐chloroacetophenone as inhibitor, and immobilization of the yeast in alginate beads, respectively. These processes can also be implemented on a preparative scale and still maintain the same yield and e.e. [164].

Scheme 2.38 Yeast mediated enantioselective reduction of ethyl benzoylacetate and substituted derivatives.

Although water is the first choice as solvent for biocatalysis, the low solubility of organic compounds in water, difficult product separation, and potential side reactions caused by other enzymes in the cell have led to alternative solvents being sought. Since optically pure (S)‐1‐(4′‐methoxyphenyl) ethanol ((S)‐MOPE) is a potential synthon for the preparation of cycloalkyl [b] indoles in clinical treatment of general allergic response, whole microbial cells have been used to synthesize for enantiopure (S)‐MOPE from 4′‐methoxyacetophenone (MOAP) in aqueous systems [165]. Recently, the bioreduction of MOAP to (S)‐MOPE has been successfully performed in a hydrophilic ionic liquid (IL) containing cosolvent system using immobilized Rhodotorula sp. AS2.2241 cells. The novel IL 1‐(2′‐hydroxy)ethyl‐3‐methylimidazolium nitrate (C₂OHMIM•NO₃) gave the best results. Under the optimized conditions of pH 8.5, a temperature of 25 °C, and an optimal C₂OHMIM•NO₃ content of 5.0% (v/v), 12 mM, the initial reaction rate, the maximum yield, and the product e.e. were 9.8 μmol h^–1 (g cell)^–1, 98.3%, and >99%, respectively. Also, the cells exhibited excellent operational stability with the cosolvent system. Moreover, the established system has been highly efficiently applied for the reduction of many other aryl ketones [166]. Immobilization of whole cell of Geotrichum candidum onto an ion exchange resin with polyallylamine was used for the enantioselective bioreduction of various ketones, such as acetophenone, ortho‐fluoro and para‐fluoro acetophenone, ortho‐methyl, 2‐phenylethyl methyl ketone, and phenyl trifluoromethane ketone, in aqueous and supercritical CO₂ (scCO₂) solvents. The immobilization of the cell improved not only the enantioselectivity but also the stability and enabled a continuous‐flow reaction in aqueous solution. Recycling of the immobilized cell accompanying scCO₂ depressurization and continuous‐flow reaction was also made when the reaction was performed in scCO₂ [167]. Hydrophobic ketone such as phenyl n‐propyl ketone has been used as a model compound to survey ADH activity in S. cerevisiae. The enantioselectivity of yeast mediated reduction toward the product (R)‐(+)‐ or (S)‐(−)‐1‐phenyl‐1‐butanol was found to depend on the hexane‐to‐water volume percentage of biphasic cell culture and the cofactor zinc ion [167]. Without Zn²⁺ ion the biphasic cultures of middle to high hexane‐to‐water volume percentage possessed (R)‐enantiomeric excess (54% to >99%) and (S)‐enantiomeric excess (15–47%) for low hexane‐to‐water volume percentage. With Zn²⁺ ion in the biphasic cultures, the enantioselectivity was exclusively (S)‐enantiomeric excess (28% to >99%). The bioreduction mediated with the yeast C. utilis of aqueous cultures showed an (S)‐enantiomeric excess of 79–95% [144]. Glycerol is a nontoxic, biodegradable, and recyclable liquid and has a high boiling point and negligible vapor pressure that make it an ideal green reaction medium for many catalytic and non‐catalytic organic syntheses. The high polarity of glycerol favorably facilitates the simple baker’s yeast mediated reduction of benzaldehyde and ethyl acetoacetate because glycerol can dissolve glucose and ethyl acetoacetate and suspend baker’s yeast. The use of glycerol as reaction medium for baker’s yeast mediated reduction produced a high product yield and >99% enantioselectivity with either free or immobilized cells [168].

The ADH obtained from Thermus sp. ATN1 (TADH) is an NAD(H)‐dependent enzyme, which shows a very broad substrate spectrum including aldehydes, aliphatic ketones, cyclic ketones, and double‐ring systems and produces exclusively the (S)‐enantiomer in high enantiomeric excess (>99%) for ketones. TADH can be used in the presence of 10% (v/v) water‐miscible solvents like 2‐propanol or acetone, which plays as sacrificial substrate in substrate‐coupled cofactor regeneration reactions. TADH retained 80% of its activity when water‐insoluble solvent like hexane or octane is used as cosolvent that forms an aqueous/organic biphasic reaction medium to allow the reaction of low‐water‐soluble substrates [169].

Asymmetric reduction of ketones to pure alcohols has been applied for material chemistry. For example, by carefully selecting the right ADH, the enantiomerically pure (S)‐ and the (R)‐monomers were biosynthesized from the p‐vinylacetophenone. Then, varied ratio of (S)‐ and (R)‐monomers can be polymerized to prepare different polymers via free‐radical polymerization. The polymers formed from the (S)‐ and (R)‐monomer mixtures had a number‐average molecular weight (M _n ) of 5000–6000 g mol⁻¹ and a polydispersity of 1.7–2.1. The thermal properties of the polymer material (T _g) can be further fine‐tuned by enantioselective grafting of the (R)‐alcohol groups with vinylacetate by a lipase (Scheme 2.39). Therefore, a decrease of T _g for the acetylation modified polymers was shown by the differential scanning calorimetry (DSC) analysis [170].

Scheme 2.39 Asymmetric reduction of ketones and polymerization of the optically pure monomers for application in material chemistry.