Enzymes in Organic Chemistry:S.M.Roberts
| Contents | Introduction | Hydrolases | Oxidoreductases |
| Carbon Bond Forming Reactions | Biotransformations | References |

Recent Uses of Hydrolase Enzymes

Enzymes are available for the hydrolysis of carboxylic acid esters, phosphate esters, amides, nitriles and epoxides:

Hydrolysis of Racemic Esters

  • Kinetic Resolutions

     The enzyme-catalysed hydrolysis of alkyl carboxylates has been extensively investigated. For example the acetate (1) is easily prepared and is hydrolysed enantioselectively using Mucor miehei lipase or lyophilized yeast cells. The alcohol (2) is obtained [enantiomeric excess (e.e.) 80-100%] and this synthon is utilized in the preparation of 13-HODE (coriolic acid) (3), a naturally occurring, biologically active compound (Chan et al., 1988 and 1990). While this chemo-enzymatic synthesis of 13-HODE is interesting and useful for the preparation of selected analogues, the preparation of the natural product itself is more efficiently accomplished using another enzyme-catalysed reaction namely the conversion of linoleic acid into 13-HODE (in 70% yield) using a lipoxygenase enzyme. We have developed this process to allow the production of gram quantities of the hydroxydienoic acid (vide infra).

    Kinetic resolutions of selected esters have been used in the preparation of a number of important compounds in these laboratories including a synthon for the hypocholestemic delta-lactones (Olivo et al., 1993), anti-AIDS agents (McCague et al., 1994) and the fascinating anti-fungal agent called brefeldin - A (Carnell et al., 1994).

  • Asymmetrization of Meso-Esters

    Meso-diacetates such as the compound (4) are interesting substrates in enzyme-catalysed transformations since "asymmetrization" can be observed. Thus compound (4) is converted into the half-ester (5) using porcine pancreatic lipase. The yield of (5) is almost quantitative and the optical purity is very high (> 98% e.e.). Compound (5) has been converted into the nucleoside analogue (6) and the latter compound is being used in various enzyme studies (Payne and Roberts, 1992).

    Similarly the dimethyl cyclopentane-dicarboxylate (7) is hydrolysed with exquisite selectivity using pig liver esterase to give the mono-ester (8) in a highly pure form (96% yield; 98% e.e.) (Cotterill et al., 1991a). Enzyme-catalysed reactions such as (4) => (5) and (7) => (8) give the possibility of forming optically pure products in quantitative yield from the chosen substrates, a process that is very difficult to emulate using conventional chemical catalysts.

    Esterification of Racemic Acids

    Roberts, 1989). For example, the racemic alcohol (9) in hexane is converted into the optically active ester (11) (90% e.e.) and recovered optically enriched bicyclo[3.2.0]hept-2-en-6endo-ol using cyclohexane carboxylic acid and the catalyst Lipozyme® (Mucor miehei lipase attached to an inert solid support) (Cotterill et al., 1988a).

  • Inter-Esterification

    Interestingly the ester (11) is obtained in even higher optical purity (> 99% e.e.) on employing an inter-esterification reaction involving the acetate (10) and cyclohexane carboxylic acid. The separated esters can be used in syntheses of prostaglandin-F2ã, a naturally occurring material with a plethora of biological activities (Scheme 1) (Macfarlane et al., 1990).

    The inter-esterification process has recently been extended to provide an example of double enantioselection by using the ester (±)-10 and racemic 2-(para-chlorophenoxy)propanoic acid. One of the four possible diastereoisomeric esters, compound (12), is formed almost exclusively (Fowler et al., 1991). Two stereoselective enzyme-catalysed processes are in operation, namely the preferential hydrolysis of the 6(R)-acetate (10), and selective acylation of the product alcohol by 2(-)-(para-chlorophenoxy)-propanoic acid.

  • Dynamic Resolution

    Lipozyme can also operate in organic solvents under almost anhydrous conditions as illustrated by the following example. Conversion of substituted racemic azlactones e.g. (13) to the corresponding optically active amino acid derivatives (14) occurs using toluene as the solvent in the presence of Lipozyme. In this process the azlactone starting material is able to racemise in situ leading to high yield (~ 94%) of the homochiral (99.5% e.e.) product (14). Subsequent two-step hydrolysis of (14) leads to L-(S)-tert-leucine (15), an important amino acid that is used in therapeutic peptides and as a chiral auxilliary (Scheme 2) (Turner, 1995).

    Polyesters

    The use of lipases for the preparation of polyesters has been studied at Exeter (Binns et al., 1993) and the condensation of adipic acid and butane-1,4-diol has been developed, by an industrial partner, to form part of a large scale process for the production of polyurethanes.

    Amide Hydrolysis

    The employment of acylases (such as hog kidney acylase) for the cleavage of amide bonds under mild conditions is well known. This type of hydrolysis is commercially important in the preparation of 6-amino-penicillanic acid and in the synthesis of some optically pure amino-acids.

    More recently it has been found that a microbial acylase can effect the enantiospecific hydrolysis of the lactam (16) to give the amino-acid (17) and recovered starting material. A second microorganism can effect the enantio-complementary hydrolysis to give the mirror image of compound (17) and recovered lactam (Taylor et al., 1990). These optically active amino-acids and lactams can be used to prepare the important anti-AIDS agent carbovir (18) in homochiral form. The synthesis of the GABA-inhibitor (19) from both enantiomers of the lactam (16) in an enantio-convergent strategy exemplifies another important application of this useful enzyme-catalysed kinetic resolution (Evans et al., 1991).

    Phosphate Esters

    The synthesis of phosphate esters can be accomplished by kinase enzymes using adenosine triphosphate (ATP) as the phosphate donor. The technique will work with unnatural substrates: for example the racemic nucleoside analogue (20) was converted into the racemic nucleotide (21) using thymidine kinase and ATP. Enantioselective hydrolysis of the (±)-phosphate (21) can be achieved using 5'-nucleotidase from snake venom to give the dextrorotatory carbocyclic nucleoside (+)-(20) which exhibited very powerful anti-Herpes activity (Borthwick et al., 1988 and 1990).

    Nitrile Hydrolysis

    The hydrolysis of nitrile groups using hydratase enzymes is of great interest to many synthetic organic chemists, principally because the hydrolysis takes place under very mild conditions (for a recent review see Crosby et al., 1994) The sequence outlined in Scheme 3 (Cohen et al., 1990) illustrates the selectivity of processes of this type (see also Kakeya et al., 1991).

    Recently it has been shown that this reaction can be carried out in an enantioselective fashion (Kakeya et al., 1991; Cohen et al., 1992) in which the resolution occurs during the amide to acid conversion. For example racemic 2-phenylbutyronitrile yields (R)-2-phenylbutyramide and (S)-2-phenylbutyric acid, both with > 98% e.e. (Scheme 4). The asymmetric hydrolysis of 3-substituted glutaronitrile derivatives has also been achieved (Beard et al., 1993; Kerridge et al., 1994).

    Epoxide Hydrolases

    The substrate selectivity of epoxide hydrolase enzymes is being explored. To date most of the biotransformation systems have been derived from animal sources (e.g. rabbit or rat liver microsomes) and so the methodology is not at all useful to the non-specialist wishing to prepare optically active vic-diols on a reasonable scale.

    Enzyme Models for Use by Non-Specialists.

    The use of hydrolase enzymes by the non-specialist is aided by the availability of models of the active site of enzymes such as pig liver esterase, porcine pancreatic lipase, Pseudomonas fluorescens lipase and Candida cylindracea lipase (Toone et al., 1989; Oberhauser et al., 1989; Santianello et al., 1988).

    Glycosidases

    Finally, glycosidases are a diverse group of enzymes whose natural function is to catalyse the hydrolytic cleavage of glycosides. Recently we have exploited the inherent selectivity of one particular glycosidase, ß-glucuronidase, in the synthesis of the potent analgesic morphine-6-glucuronide (23). Treatment of morphine-3,6-diglucuronide (22) with ß-glucuronidase from Patella vulgata results in regioselective hydrolysis of the more reactive 3-glucuronide (Brown et al., 1995).

    In addition it has long been known that these enzymes can also catalyse 'transglycosylation' reactions resulting in the formation of novel glycosides (Scheme 5). By using certain amino acid derivatives as the nucleophilic alcohol component it is possible to prepare carbohydrate-amino acid linkages (e.g. 24) that are important constituents of many glycoproteins (Turner and Webberley, 1991).

    | Contents | Introduction | Hydrolases | Oxidoreductases |
    | Carbon Bond Forming Reactions | Biotransformations | References |

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