Alkenes epoxide hydrolases

As with several other functional groups considered earlier, epoxides are most commonly found in alkaloid metabolites rather than the original compound. These epoxides may arise via oxidation of alkenes or aromatic hydrocarbons. Epoxide hydrolase catalyzes hydrolysis of epoxides to the more hydrophilic diol. As seen in Scheme 6, this is usually a stereospecific reaction that always yields a... 

This chapter begins, thus, with a short introduction to the chemical reactivity of epoxides. We continue with a description of the epoxides hydrolases and their biochemistry, and devote most of its length to a systematic discussion of the substrates hydrated by these enzymes. Arene oxides and diol epoxides will be presented first, followed by a large variety of alkene and cy-cloalkene oxides. 

The microsomal epoxide hydrolases (microsomal EH, mEH), predominantly found in the endoplasmic reticulum, regio- and stereoselectively catalyze the hydration of both alkene and arene oxides, including oxides of polycyclic aromatic hydrocarbons. These enzymes have been purified to homogeneity from various species and tissues [22] [41 - 46], The human microsomal EH contains 455 amino acids (Mr 52.5 kDa) and is the product of the EPHX1 gene [47] (also known as HYL1 [48]). 

The overall reaction catalyzed by epoxide hydrolases is the addition of a H20 molecule to an epoxide. Alkene oxides, thus, yield diols (Fig. 10.5), whereas arene oxides yield dihydrodiols (cf. Fig. 10.8). In earlier studies, it had been postulated that epoxide hydrolases act by enhancing the nucleo-philicity of a H20 molecule and directing it to attack an epoxide, as pictured in Fig. 10.5, a [59] [60], Further evidence such as the lack of incorporation of 180 from H2180 into the substrate, the isolation of an ester intermediate, and the effects of group-selective reagents and carefully designed inhibitors led to a more-elaborate model [59][61 - 67]. As pictured in Fig. 10.5,b, nucleophilic attack of the substrate is mediated by a carboxylate group in the catalytic site to form an ester intermediate. In a second step, an activated H20... 

Together with glutathione conjugation, hydration is a major pathway in the inactivation and detoxification of arene oxides. Exceptions to this rule will be treated when discussing polycyclic aromatic hydrocarbons. Arene oxides are good substrates for microsomal EH, as evidenced in Table 10.1, where hydration of selected arene oxides, alkene oxides, and cy-cloalkene oxides by purified rat liver epoxide hydrolase is compared. The hy- ... 

The data in Table 10.1 suggest that the reactivity of epoxide hydrolase toward alkene oxides is highly variable and appears to depend, among other things, on the size of the substrate (compare epoxybutane to epoxyoctane), steric features (compare epoxyoctane to cycloalkene oxides), and electronic factors (see the chlorinated epoxides). In fact, comprehensive structure-metabolism relationships have not been reported for substrates of EH, in contrast to some narrow relationships that are valid for closely related series of substrates. A group of arene oxides, along with two alkene oxides to be discussed below (epoxyoctane and styrene oxide), are compared as substrates of human liver EH in Table 10.2 [119]. Clearly, the two alkene oxides are among the better substrates for the human enzyme, as they are for the rat enzyme (Table 10.1). 

We also note that some 2,2-disubstituted oxiranes have toxicological significance, as exemplified by 2,2-dimethyloxirane (2-methyl-l, 2-epoxypropane, 10.43, R = Me). This compound is the toxic metabolite of 2-methyl-prop-1-ene (isobutene), a gaseous alkene widely used as a monomer in the industrial production of adhesives, plastics, and other polymers. Interestingly, detoxification of this epoxide catalyzed by liver epoxide hydrolase was high in the human, intermediate in the rat, and low in the mouse [125], These activities were inversely correlated with the epoxide levels measured in vitro in liver tissue of these species. 

W. Levin, D. P. Michaud, P. E. Thomas, D. M. Jerina, Distinct Rat Hepatic Microsomal Epoxide Hydrolases Catalyze the Hydration of Cholesterol 5,6a-Oxide and Certain Xenobiotic Alkene and Arene Oxides , Arch. Biochem. Biophys. 1983, 220, 485 - 494. 

In eukaryotes, such as mammals and fungi, epoxide hydrolases play a key role in the metabolism of xenobiotics, in particular of aromatic systems [30,31 ]. On the other hand, in prokaryotes (e.g. bacteria) these enzymes are essential for the utilization of alkenes as carbon-source. In general, aromatics can be metabolized via two different pathways (Scheme 5) (i) dioxetane formation via dioxyge-... 

Scheme 5. Involvement of epoxide hydrolases in the biodegradation of aromatics and alkenes...

It is noteworthy that, in contrast to mammalian systems, the majority of bacterial strains exhibited sufficient activity even when the cells were grown under non-optimized conditions. Since enzyme induction is still a largely empirical task, cells were grown on standard media in the absence of inducers. Furthermore, all attempts to induce epoxide hydrolase activity in Pseudomonas aeruginosa NCIMB 9571 and Pseudomonas oleovorans ATCC 29347 by growing the cells on an alkane (decane) or alkene (1-octene) as the sole carbon source failed [27]. 

Epoxide rings of alkene and arene compounds are hydrated by enzymes known as epoxide hydrolases, the animal enzyme forming the corresponding trans-diols, although bacterial hydrolases are known that form d.v-diols. Although, in general, the hydration... 

The body oxidizes the alkene components of drugs and other substances to epoxides, which are then hydrolyzed to diols by an epoxide hydrolase enzyme. The more reactive epoxides are rapidly converted to water-soluble diols and eliminated in the urine. Epoxide hydrolase enzymes are sometimes used in organic synthesis to produce chiral diols. 

Epoxide rings of certain alkene and arene compounds are hydrated enzymatically by epoxide hydrolases to form the corresponding iram-dihydrodiols (Figure 10.11). The epoxide hydrolases are a family of enzymes known to exist both in the endoplasmic reticulum and in the cytosol. In earlier studies they were named epoxide hydratase, epoxide hydrase, or epoxide hydrolase. Epoxide hydrolase, however, has been recommended by the International Union of Biochemists Nomenclature Committee and is now in general use. 

The microsomal epoxide hydrolase converts arene and alkene oxides to vicinal dihydrodiols by hydrolytic cleavage of the oxirane ring. This is a detoxication reaction in that it converts generally highly reactive electrophilic oxirane species to less reactive nonelectrophilic dihydrodiols. It should be borne in mind, however, that some epoxides are unreactive, both toward macromolecules and toward epoxide hydrolase (e.g., dieldrin), and that dihydrodiols may be further epoxidized to produce metabolites that are even more reactive, as is the case with... 

Epoxide rings of alkene and arene compounds are hydrated to form trans-diols. The enzymes that catalyze the addition of a molecule of water to an epoxide ring to yield diols are called epoxide hydrolases (also known as epoxide hydrases). Epoxide hydrolase activity has been detected in numerous species of insects. Enzymatic epoxide hydration of certain cyclodiene insecticides and their analogs has been demonstrated in the housefly, blowfly (Calliphora erythrocephala), yellow mealworm (Tenebrio molitor), Madagascar cockroach (Gromphadorhina portentosa), southern army worm (Spodoptera eridania), and red flour beetle (Tribolium castaneum). Epoxide hydrolase is also important in the metabolism of juvenile... 

Metabolic epoxidation of carbon-carbon double bonds in alkenes and arenes is a fundamentally important biotransformation of foreign compounds. The primary epoxide (oxirane) metabolites formed generally undergo further biotransformation to more polar and readily excreted metabolites via conjugation with glutathione or epoxide-hydrolase-mediated hydrolysis to diols. Thus, epoxidation can be considered the first step in a metabolic detoxification scheme. On the other hand, epoxidation is also a... 

In spite of the considerable value of epoxide hydrolases for fine chemical synthesis, it was only recently that a detailed search for epoxide hydrolases from microbial sources was undertaken by the groups of Furstoss185, 901 and Faber123, 79, 911, bearing in mind that the use of microbial enzymes allows an (almost) unlimited supply of biocatalyst. The screening was based along the following considerations on the one hand, the catabolism of alkenes often implies the hydrolysis of an epoxide inter-... 

For the enantioselective preparations of chiral synthons, the most interesting oxidations are the hydroxylations of unactivated saturated carbons or carbon-carbon double bonds in alkene and arene systems, together with the oxidative transformations of various chemical functions. Of special interest is the enzymatic generation of enantiopure epoxides. This can be achieved by epoxidation of double bonds with cytochrome P450 mono-oxygenases, w-hydroxylases, or biotransformation with whole micro-organisms. Alternative approaches include the microbial reduction of a-haloketones, or the use of haloperoxi-dases and halohydrine epoxidases [128]. The enantioselective hydrolysis of several types of epoxides can be achieved with epoxide hydrolases (a relatively new class of enzymes). These enzymes give access to enantiopure epoxides and chiral diols by enantioselective hydrolysis of racemic epoxides or by stereoselective hydrolysis of meso-epoxides [128,129]. 

Biocatalytic asymmetric epoxidation of alkenes catalyzed by monooxygenases cannot be performed on a preparative scale with isolated enzymes due to their complex nature and their dependence on a redox cofactor, such as NAD(P)H. Thus, whole microbial cells are used instead. Although the toxic effects of the epoxide formed, and its further (undesired) metabolism by the cells catalyzed by epoxide hydrolases (Sect. 2.1.5), can be reduced by employing biphasic media, this method is not trivial and requires bioengineering skills [1151]. Alternatively, the aUcene itself can constitute the organic phase into which the product is removed, away from the cells. However, the bulk apolar phase tends to damage the cell membranes, which reduces and eventually abolishes all enzyme activity [1152]. 

Ordinary alkenes are also subject to attack by MFOs to form epoxides. These epoxides, since they are not arene oxides, most commonly undergo hydrolysis catalyzed by epoxide hydrolase to give vicinal diols (Figure 7.10). 

Figure 7.10 Ordinary alkenes also undergo epoxidation mediated by MFO.These have little incentive to rearrange and so they generally serve as substrates for epoxide hydrolase to form vicinal diols. In this example the allylic group of the hypnotic agent secobarbital is epoxidized, but the epoxide is not observed among the isolated metabolites. Instead it is the diol that is found as a major metabolite.

As explained in the Introduction, alkene oxides (10.3) are generally chemically quite stable, indicating reduced reactivity compared to arene oxides. Under physiologically relevant conditions, they have little capacity to undergo rearrangement reactions, one exception being the acid-catalyzed 1,2-shift of a proton observed in some olefin epoxides (see Sect. 10.2.1 and Fig. 10.3). Alkene oxides are also resistant to uncatalyzed hydration, thus, in the absence of hydrolases enzymes, many alkene oxides that are formed as metabolites are stable enough to be isolated. 

In view of the efficient inhibition of a particular crystalline glycan hydrolase by the epoxylbutyl -cellobioside 7 (n = 2), we decided to prepare the deoxy iodo derivative 21 to aid in the X-ray crystallographic analysis. We soon found that, although the alkene 22 was easily available as a direct precursor to our target, the epoxide functionality had to be introduced indirectly using bromo-hydrin 23 technology any direct oxidation of 22 invariably led to some loss of the iodine atom [23]. 

Another early discovery was that CALB accepts H202 as nucleophile to produce peroxycarboxylic acids from esters or carboxylic acids (perhydrolysis activity can also be found in other serine hydrolases) [46, 47]. The in situ formed peracid can subsequently be used to epoxidize an alkene by (non-enzymatic) Prileshajev epoxidation. Hence, oleic acid incubated with CALB and H202 will produce 9,10-epoxyoctadecanoic acid [48]. Other alkenes can be epoxidized by H202 and a catalytic amount of carboxylic acid (and CALB) (Scheme 13.2) [49],... 

D. Hydration Epoxide hydratase (EC 4.2.1.64), also called epoxide hydrase or hydrolase Epoxides (arene and alkene oxides) Epoxides are hydrated to dihydrodiols. Product glycols which are stereochemically fixed by a ring structure invariably have the /ra/i5-configuration... 

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