Hydrolases epoxide

The CUps-O epoxide 33-41 and related substrates are currently the only available selective fluorogenic substrates for EH. On the other hand various indirect assays have been reported to detect either the unreacted epoxide [46] or the carbonyl product resulting from periodate cleavage of the 1,2-diol product [26,47, 48], These assays are suitable for fluorescence or colorimetric assays for the hydrolysis of any epoxide of interest 

There are several forms of EHs that have been used to effect enantioselective opening of epoxides. One commonly used form is isolated as a crude microsomal preparation from rodent livers. EH can also be isolated from bacteria, fiingi, and yeasts. The structure of the EH from Agrobacterium radiobacter ADI has been solved by X-ray crystallography. In this enzyme, the catalytic triad involves His-275, Asp-107, and Tyr-152 and/or Tyr-215. The tyrosine functions as a general acid 

Entry 5 is an interesting example that entails both enzymatic and hydrolytic epoxide conversion. In the first step, an enzymatic hydrolysis proceeds with retention of the configuration at the tertiary center. This reaction is selective for the 5-epoxide. The remaining 7 -epoxide is then subjected to acid-catalyzed hydrolysis, which proceeds with inversion at the center of chirality (see p. 186). The combined reactions give an overall product yield of 94%, having 94% e.e.  

Entry 6 is one of several examples demonstrating enantioselectivity for both the cis and trans isomers of heptane-2,3-epoxide. Entry 7 shows the kinetic resolution of an exocyclic cyclohexane epoxide. The two stereoisomeric monomethyl analogs were only partially resolved and the 3-methyl isomer showed no enantioselectivity. This shows that the steric or hydrophobic effect of the dimethyl substiments is critical for selective binding. 

Thus far only three reports regarding the directed evolution of enantioselective EHs have appeared 95,96,143), notwithstanding the fact that these enzymes, even as wild types, constitute important catalysts in synthetic organic chemistry 7-12,144,145). Indeed, since two EHs became commercially available recently, this type of biocatalysis offers exciting prospects for the practicing organic chemist. 

The first two reported studies concern the epoxide hydrolase from Aspergillus niger (ANEH) 95,96). The enzyme had previously been purified to homogeneity, the gene cloned and expressed in E. coli, and the catalytic hydrolysis of epoxides optimized to high substrate concentrations. Initial attempts were made to enhance the enantioselectivity of the ANEH-catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-33). The WT leads to an E value of only 4.6 in favor of (5)-34 96). 

It is clear that K332E and A390E occur at positions remote from the active center, whereas A217 V appears to be rather close. The side chain of the amino acid 

Parallel to these efforts, an efficient and reliable expression system for ANEH was developed (95). Subsequently, two libraries of mutants were generated by epPCR, comprising 3500 and 4 600 clones, respectively. Mutants were discovered displaying an expression level 3.4 times higher than the original WT and a 3.3-fold enhancement of catalytic activity as measured by the hydrolysis of 4-(p-nitrophenoxy)-1,2-epoxybutane (95). The distribution of ANEH activities from the clones of these two libraries is shown in Fig. 21. 

In addition to the above two studies concerning ANEH (95,96), which set the stage for further improvements using the methods of directed evolution, another 

Several libraries of mutant ANEHs were prepared by applying epPCR at various mutation rates and transforming into E. coli BL21 (DE3). About 20 000 clones were screened, the most selective ANEH variant showing a selectivity factor of only E= 10.8 in the kinetic resolution of rac-19 [58]. Thus, this enzyme appeared to be difficult to evolve. 

The ANEH-mutant displaying enhanced enantioselectivity ( =10.8) was sequenced and shown to be characterized by three mutations, A217V near the active site and K332E and A390E both at remote positions [58]. The X-ray crystal structure of the WT ANEH had been analyzed earlier [61], revealing a dimer comprising identical 

Directed Evolution as a Means to Engineer Enantioselective Enzymes 

In another study that appeared prior to the advent of CASTing, the traditional combination of epPCR and DNA shuffling was used to enhance the enantioselectivity of the hydrolytic kinetic resolution of p-nitro phenyl glycidyl ether and other epoxides catalyzed by the EH from Agrobacterium radiobacter [59]. Several mutants were obtained with up to 13-fold improved enantioselectivity. The amino acid exchanges took place around the active site. 

Addition of azide occurs aimost exciusiveiy at the p-position, in contrast to the non-enzyme cataiysed process. 

Strategies for Controlling and Enhancing the Enantioselectivity of Enzyme-Catalysed 

It is well known that various parameters (e.g. solvent, pH, immobilization, chemical modification and temperature) can have an effect on the enantioselectivity of enzyme-catalysed processes. Most studies in this respect have been carried out on hydrolytic enzymes, especially lipases, esterases and proteases [28]. Recent reports, especially those involving non-hydrolytic enzymes, are discussed below. 

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Epoxide-hydrolases as asymmetric catalysts for ring opening of oxiranes 97T15617. 

For a review on epoxide hydrolases and related enzymes in the context of organic synthesis, see Faber, K. Biotransformations in Organic Chemistry, Springer New York 2004. 

Scheme 10.32 Examples of reactions catalyzed by different classes of dehalogenases. HD haloalcohol dehalogenase EH epoxide hydrolase CL p-chlorobenzoyl-CoA ligase CBD p-chlorobenzoyl-CoA dehalogenase.

The metabolism of foreign compounds (xenobiotics) often takes place in two consecutive reactions, classically referred to as phases one and two. Phase I is a functionalization of the lipophilic compound that can be used to attach a conjugate in Phase II. The conjugated product is usually sufficiently water-soluble to be excretable into the urine. The most important biotransformations of Phase I are aromatic and aliphatic hydroxylations catalyzed by cytochromes P450. Other Phase I enzymes are for example epoxide hydrolases or carboxylesterases. Typical Phase II enzymes are UDP-glucuronosyltrans-ferases, sulfotransferases, N-acetyltransferases and methyltransferases e.g. thiopurin S-methyltransferase. 

Figure 2.14 CASTing of the epoxide hydrolase from A. niger (ANEH) based on the X-ray structure of the WT [61]. (a) Defined randomization sites A-E (b) top view of tunnel-like binding pocket showing sites A-E (blue) and the catalytically active D192 (red) [23].

Figure 2.15 Iterative CASTing in the evolution of enantioseiective epoxide hydrolases as catalysts in the hydrolytic kinetic resolution ofrac-19[23].

Furstoss et al. have reported their studies on the use of an epoxide hydrolase with four styrene oxide derivatives (Figure 5.26) [39]. The (R)-diol (43) was obtained in 91% ee at 100% conversion from racemic (42), demonstrating an enantioconvergent... 

Figure 5.25 Enantioconvergent hydrolysis of epoxides (35) to the corresponding diols (36) using mung bean epoxide hydrolase.

Chiral epoxides and their corresponding vicinal diols are very important intermediates in asymmetric synthesis [163]. Chiral nonracemic epoxides can be obtained through asymmetric epoxidation using either chemical catalysts [164] or enzymes [165-167]. Biocatalytic epoxidations require sophisticated techniques and have thus far found limited application. An alternative approach is the asymmetric hydrolysis of racemic or meso-epoxides using transition-metal catalysts [168] or biocatalysts [169-174]. Epoxide hydrolases (EHs) (EC 3.3.2.3) catalyze the conversion of epoxides to their corresponding vicinal diols. EHs are cofactor-independent enzymes that are almost ubiquitous in nature. They are usually employed as whole cells or crude... 

Epoxide hydrolases hydrate epoxides to yield transdihydrodiols without any requirement for cofactors. Examples are given in Figure 2.12. Epoxide hydrolases are... 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

Emphasis is given to the critical role of metabolism, both detoxication and activation, in determining toxicity. The principal enzymes involved are described, including monooxygenases, esterases, epoxide hydrolases, glutathione-5 -transferases, and glucuronyl transferases. Attention is given to the influence of enzyme induction and enzyme inhibition on toxicity. 

In the rabbit, the nonplanar PCB 2,2, 5,5 -tetrachlorobiphenyl (2,2, 5,5 -TCB) is converted into the 3, 4 -epoxide by monooxygenase attack on the meta-para position, and rearrangement yields two monohydroxymetabolites with substitution in the meta and para positions (Sundstrom et al. 1976). The epoxide is also transformed into a dihydrodiol by epoxide hydrolase attack (see Chapter 2, Section 2.3.2.4). This latter conversion is inhibited by 3,3,3-trichloropropene-l,2-oxide (TCPO), thus providing strong confirmatory evidence for the formation of an unstable epoxide in the primary oxidative attack (Forgue et al. 1980). 

Epoxide hydrolase A type of enzyme that converts epoxides to diols by the addition of water. 

McElroy NR, Jurs PC, Morisseau C, Hammock BD. QSAR and classification of murine and human epoxide hydrolase inhibition by urea-like compounds. J Med Chem 2003 46 1066-80. 

Lewis DF, Lake BG, Bird MG. Molecular modelling of human microsomal epoxide hydrolase (EH) by homology with a fungal (Aspergillus niger) EH crystal structure of 1.8 A resolution structure-activity relationships in epoxides inhibiting EH activity. Toxicol In Vitro 2005 19 517-22. 

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