Hydrolase-catalyzed reaction compounds using

Enantiomeric purity. In order to assess the efficiency of an enantioselective hydrolase-catalyzed reaction, it is imperative that one can accurately measure at least the conversion and the enantiomeric excesses of either the substrate or the product (see equations Equation 1, Equation 2, and Equation 3). Although optical rotation is sometimes used to assess enantiomeric excess, it is not recommended. Much better alternatives are various chromatographic methods. For volatile compounds, capillary gas chromatography on a chiral liquid phase is probably the most convenient method. Numerous commercial suppliers offer a large variety of columns with different chiral liquid phases. Hence it is often easy to find suitable conditions for enantioselective GC-separations that yield ee-values in excess of... 

Three routes to enantiopure compounds using hydrolase-catalyzed reactions, (a) Kinetic resolution starts with racemic substrate and converts one enantiomer into product. This separation yields one enantiomer as the product alcohol and one as the starting acetate, both with a maximum yield of 50%. (b) Desymmetrization of a prochiral compound transforms one of prochiral groups to yield a chiral product with a maximum yield of 100%. (c) A dynamic kinetic resolution combines rapid racemization of racemic starting material with a hydrolase catalyzed acylation of one enantiomer. The maximum yield is 100%. 

The 8,9- and 10,11-dihydrodiols formed in the metabolism of BA and DMBA respectively are all highly enriched (>90%) in R,R enantiomers (Table III). Labeling experiments using molecular oxygen-18 in the in vitro metabolism of the respective parent compounds and subsequent mass spectral analyses of dihydrodiol metabolites and their acid-catalyzed dehydration products indicated that microsomal epoxide hydrolase-catalyzed hydration reactions occurred exclusively at the nonbenzylic carbons of the metabolically formed epoxide intermediates (unpublished results). These findings indicate that the 8,9- and 10,11-epoxide intermediates, formed in the metabolism of BA and DMBA respectively, contain predominantly the 8R,9S and 10S,11R enantiomer, respectively. These stereoselective epoxidation reactions are relatively insensitive to the cytochrome P-450 isozyme contents of different rat liver microsomal preparations (Table III). 

Hydrolases catalyze the addition of a molecule of water to a variety of functional moieties. Thus, epoxide hydrolase hydrates epoxides to yield trans-dihydrodiols (reaction 1-B in Fig. 13.5). This reaction is documented for many arene oxides, particularly metabolites of aromatic compounds, and epoxides of olefins. Here, a molecule of water has been added to the substrate without loss of a molecular fragment, therefore the use of the term "hydration" sometimes found in the literature. 

Hydrolase-catalyzed desymmetrizations start with meso compounds or prochi-ral compounds and yield chiral products in up to 100% yield. This high yield is a big advantage in most cases, but demands a symmetrical substtate, so it does not fit most synthetic problems. In a desymmetrization the enantiomeric purity of the product remains constant as the reaction proceeds and is given by ee = (E - 1)/(E + 1), where E is the enantioselectivity. For example, an enantioselectivity of 50 yields product with 96% ee. Reauangement of this equation gives E = (1 + ee)/(l - ee), useful to calculate the enantioselectivity from the enantiomeric purity of the product. 

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. 

Enzymatic Hydrolysis Reactions of Esters. Xenobiotic compounds containing esters or other acid derivatives in their structures (e.g., amides, carbamates, ureas, etc., see Table 17.3) are often readily hydrolyzed by microorganisms. To understand how enzymatic steps can be used to transform these substances, it is instructive to consider the hydrolases (i.e., enzymes that catalyze hydrolysis reactions) used by organisms to split naturally occurring analogs (e.g., fatty acid esters in lipids or amides in proteins). The same chemical processes, and possibly even some of the same enzymes themselves, are involved in the hydrolysis of xenobiotic substrates. 

Among the various enzymes capable of producing optically-active amino acids, transamination reactions, catalyzed by enzymes known as aminotransferases or transaminases, have broad potential for the synthesis of a wide variety of enantio-merically pure (R)- and (S)-compounds containing amine groups. Indeed, various examples of the use of aminotransferases for the production of d- and L-amino acids, both naturally-occurring and non-natural, have been published17 151. In addition, certain aminotransferases have been found to act on amines, and methods for the production of enantiomerically pure amines by transamination have been described116-211. This method allows for yields of up to 100% whereas routes based on hydrolases require external racemization to reach such yield levels. In this section we will focus on the application of aminotransferases. 

The esterases and lipases are members of a still larger group of enzymes that catalyze acyl transfer, either in the direction of solvolysis or by acylation of the substrate. Both types of enzymes are called hydrolases. In water, hydrolysis occurs, but in the presence of alcohols, transesterification can occur. Reactions in the acylation direction are done in the presence of acyl donors. Esters of enols such as vinyl acetate or isopropenyl acetate are often used as sources of the acyl group. These enol esters are more reactive than alkyl esters, and the enol that is displaced on acyl transfer is converted to acetaldehyde or acetone. To avoid side products arising from these carbonyl compounds, one can use 1-ethoxyvinyl esters, which give ethyl acetate as the by-product. ... 

The aim of this study was the development of conversion system of caffeoylquinic acids to valuable compounds. When an IL, l-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([bmim][NTf2]) was used as a reaction solvent, we found that immobilized chlorogenate hydrolase (Kikkoman) catalyzed the conversion of 5-caffeoylquinic acid to methyl caffeate with methanol (Fig. 1). The immobilized enzyme was prepared with chlorogenate hydrolase using quaternary ammonium sepabeads (Mitsubishi Chemical Co., Tokyo, Japan) (Kurata et al., 2011). To synthesize valuable compounds from caffeoylquinic adds, we attempted to develop a method for the conversion of caffeoylquinic acids to CAPE analogues via methyl caffeate. In section 2, we describe the properties of immobilised chlorogenate hydrolase in ILs. Using various caffeoyl quinic acid prepared from immature coffee beans, we developed a system to produce methyl caffeate. 

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