Enzyme-Based Organic Synthesis. Cheanyeh Cheng

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СКАЧАТЬ cereus has also been used for regioselectively converting 2‐phenylenediamine to 2‐aminoacetanilide with a76% molar yield [17]. Other than bacterium, monooxygenase in the phytopathogenic fungi Colletotrichum gloeosporioides and Botrytis cinerea has been found having the ability of regioselective hydroxylation of the C‐H bonds to yield the corresponding diols [18]. Enzyme in cultured plant cells of Phytolacca Americana can reduce, and regioselectively hydroxylate and glucosylate, raspberry ketone and zingerone to their β‐glycosides [19]. Ginkgo biloba cell suspension cultures were used to regio‐ and stereoselectively convert sinenxan A, 2α,5α,10β,14β‐tetra‐acetoxy‐4(20), 11‐taxadiene, a taxoid isolated from callus tissue cultures of Taxus spp., in Taxol^® synthesis [20]. The regioselective oxidation of (–)‐verbenone, an important component of the essential oil from rosemary, to (–)‐10‐hydroxyberbenone with human liver microsomes has been investigated by Miyazawa et al [21]. Although regioselective oxidation of terpenoids is difficult by chemical methods, regioselective oxidation of (+)‐ and (–)‐citronellene was recently performed with Spodoptera litura, a larvae of common cutworm, to (2R,3S)‐3,7‐dimethyl‐6‐octene‐1,2‐diol (yield: 89.7%) and (2S,3R)‐3,7‐dimethyl‐6‐octene‐1,2‐diol (yield: 56.3%) [22]. These examples of enzyme‐catalyzed regiospecificity clearly elucidates that the specific enzyme–substrate binding configuration at the active site allows only one of several similar function groups in different regions of the substrate molecule reacts to produce the product. The number of binding points of enzyme–substrate complex for regioselective molecular recognition must be a “multi‐point” binding case to match the complexity of the substrate molecule.

      1.4.3 Stereospecificity

      The most magnificent specificity of enzyme is its distinguishable ability for only one enantiomeric structure of racemic substrate molecules. The molecular recognition of an enzyme for enantiomeric molecules is called enantiospecificity or stereospecificity. The stereospecificity is an intrinsic property of enzyme which is due to the chirality of active site of the enzyme. Except for a few cases, all enzymes are chiral catalysts because they all made from L‐amino acids, thus the binding of asymmetric substrate at the active site is stereoselective. Since the stereospecificity of enzyme involves enzyme–substrate complex formation with only one enantiomer of a racemate, only one product is formed from the enzyme‐catalyzed reaction. Therefore, enzyme‐catalyzed reactions are in great favor of the organic asymmetric synthesis [23].

The theoretical explanation for the stereospecificity of enzyme was based on the rationale of three‐point attachment rule [24, 25]. This rule suggests that at least three different binding points should occur between enzyme and substrate at the active site to make recognition for the correct stereostructure of substrate molecule as illustrated in Figure 1.2. However, the three‐point attachment is not strict for the stereospecificity of enzyme to asymmetric molecules. As shown in Figure 1.3, the recognition of the stereostructure of asymmetric molecules with enzyme can be accessed by two‐point binding of enzyme–substrate complex, under the situations that the stability of the enzyme–substrate complex is greatly influenced by the formation of two different 3D tetrahedral structure of enzyme–substrate complex or the ability of interaction for the two unbinding groups of the substrate with enzyme at the active site is obviously different. With only two‐point binding of the enzyme–substrate complex, one of the two substrate enantiomers possessing greater stability of the enzyme–substrate complex will have enough time to react to form product, but this situation will not happen for the other substrate enantiomer.

The preparation of enantiopure compounds is highly demanded by industries, particularly, in pharmaceutical and agrochemical applications because of the different biological activities of drug enantiomers. As a result of advances in asymmetric synthesis and separation technologies, there were many cases in using the stereospecificity of enzyme for the production of single enantiomer to allow the study of its pharmacodynamics and pharmacokinetic properties. For example, in the case of total synthesis of D‐biotin, the novel enantioselective synthesis of the optically active (3aS,6aR)‐lactone (the key D‐biotin intermediate) was through kinetic resolution by inexpensive microbial lipase instead of pig liver esterase [26]. In Scheme 1.4, optically active (3aS,6aR)‐lactone 1 was enantioselectively produced with high enantiomeric excess.

Figure 1.2 Three‐point attachment rule shows that only one enantiomer of the asymmetric molecule can successfully bind with enzyme at the active site to produce the stereospecificity of enzyme.

(ee > 98%) and conversion ratio (≥ 40%) by dry microbial cells of Aspergillus oryzae WZ007 on racemic acid 1 (1,3‐dibenzyl‐5‐(hydeoxymethyl)‐2‐oxo‐4‐imidazolidinecarboxylic acid) that was obtained via chemical hydrolysis of racemic lactone 1.

Recently, an indirect strategy was used for the synthesis of (R)‐phenylephrine (an α₁‐adrenergic receptor agonist) that is widely used in over‐the‐counter drugs to treat the common cold. An amino alcohol dehydrogenase gene isolated from Rhodococcus erythropolis BCRC 10909 was expressed in Escherichia coli NovaBlue, which is able to convert 1‐(3‐hydroxyphenyl)‐2‐(methylamino) ethanone (HPMAE) to (S)‐phenylephrine with more than 99% enantiomeric excess (ee) and 78% yield as shown in Scheme 1.5 [27]. The (S)‐phenylephrine was subsequently converted to (R)‐phenylephrine by Walden inversion reaction [28]. Since enantiopure vicinal diols are useful and valuable intermediate for pharmaceutical production, a simple and green method for preparing several enantiopure 1,2‐diols was developed via regio‐ and stereoselective concurrent oxidations of the racemates with microbial cell Sphingomonas sp. HXN‐200.

Figure 1.3 The stereospecificity of enzyme for two substrate enantiomers by two‐point binding.

Scheme 1.4 Enantioselective synthesis of (3aS,6aR)‐lactone.

As shown in Scheme 1.6, concurrent biooxidations of racemic 3‐O‐benylglycerol 1 with resting cells gave (S)‐1 in 99.2% enantiomeric excess (ee) and 32% yield. Similar biooxidations of racemic 1‐(4‐chlorophenyl)‐1,2‐ethanediol 2, 1‐(4‐methylp henyl)‐1,2‐ethanediol 3, and phenyl‐1,2‐ethanediol 4 gave (R)‐2 in 98.4% ee and 48% yield, (R)‐3 in 99.6% ee and 45% yield, and (R)‐4 in 98.7% ee and 36% yield, respectively [29].

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