Resolutions ester formation with enzymes

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

Kinetic resolutions by means of the selective formation or hydrolysis of an ester group in enzyme-catalyzed reactions proved to be a successful strategy in the enantioseparation of 1,3-oxazine derivatives. Hydrolysis of the racemic laurate ester 275 in the presence of lipase QL resulted in formation of the enantiomerically pure alcohol derivative 276 besides the (23, 3R)-enantiomer of the unreacted ester 275 (Equation 25) <1996TA1241 >. The porcine pancreatic lipase-catalyzed acylation of 3-(tu-hydroxyalkyl)-4-substituted-3,4-dihydro-2/7-l,3-oxazines with vinyl acetate in tetrahydrofuran (THF) took place in an enantioselective fashion, despite the considerable distance of the acylated hydroxy group and the asymmetric center of the molecule <2001PAC167, 2003IJB1958>. 

Esters are widespread in fruits and especially those with a relatively low molecular weight usually impart a characteristic fruity note to many foods, e.g. fermented beverages [49]. From the industrial viewpoint, esterases and lipases play an important role in synthetic chemistry, especially for stereoselective ester formations and kinetic resolutions of racemic alcohols [78]. These enzymes are very often easily available as cheap bulk reagents and usually remain active in organic reaction media. Therefore they are the preferred biocatalysts for the production of natural flavour esters, e.g. from short-chain aliphatic and terpenyl alcohols [7, 8], but also to provide enantiopure novel flavour and fragrance compounds for analytical and sensory evaluation purposes [12]. Enantioselectivity is an impor-... 

A very straightforward approach as compared with the existing one (Fig. 3) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [30]) by enantioselective aminolysis of ester 2 with histidine methylester 4 or even racemic histidine ester, as it would resolve the objectives of resolution and coupling in one step. Orientating experiments in which 20 proteases adsorbed on porous glass beads (SIKUG 041/02/120/A, Schott) were in contact with EtOH solutions of 2 and 4 with various water contents, however, did not reveal any reaction. 

By far the commonest reaction used in kinetic resolution by enzymes is ester formation or hydrolysis. Normally one enantiomer of the ester is formed or hydrolysed leaving the other untouched so one has the easy job of separating an ester from either an acid or an alcohol. There are broadly two kinds of enzymes that do this job. Lipases hydrolyse esters of chiral alcohols with achiral acids such as 119 while esterases hydrolyse esters of chiral acids and achiral alcohols such as 122. Be warned this definition is by no mans hard and fast If the unreacted component (120 or 123) is wanted, the reaction is run to just over 50% completion, to ensure complete destruction of the unwanted enantiomer, while if the reacted component (121 or 124) is wanted it is best to stop short of 50% completion so that little of the unwanted enantiomer reacts. 

Returning to the resolution of amines, an enzymatic acylation of racemic amines in aqueous solution by a penicillin acylase (enzymes used in industry in the synthesis of penicillins) from Alicaligenes faecalis has recently been reported.40 The best acylating agent is the amide 161 of phenylacetic acid. There is a big advantage here. Unlike the ester formations and hydrolysis we discussed earlier, no amide exchange occurs with simple amides like 161. The amide (/ )-162 was formed in 45% yield and 98.5% ee. Once it has been separated from free (S)-2, it can be hydrolysed to free (R)-2 with the same enzyme This automatically perfects the ee. 

Dynamic Kinetic Resolution. Another typical acid-catalysed reaction is the racemisation of chiral alcohols, due to inversion at the chiral carbon. This can actually be made use of in the formation of enantiopure compounds, by dynamic kinetic resolution using an enzyme, such as a lipase, that catalyses enantioseleetive esterification in an organic medium. By coupling zeolite Beta-catalysed intereonversion of benzylic alcohol enantiomers with enzyme-catalysed esterifieation of only one of the enantiomeric alcohols, almost complete eon version to enantiopure ester ean be achieved. ... 

In a lipase-catalyzed reaction, the acyl group of the ester is transferred to the hydroxyl group of the serine residue to form the acylated enzyme. The acyl group is then transferred to an external nucleophile with the return of the enzyme to its preacylated state to restart the catalytic cycle. A variety of nucleophiles can participate in this process. For example, reaction in the presence of water results in hydrolysis, reaction in alcohol results in esterification or transesterification, and reaction in amine results in amination. Kirchner et al.3 reported that it was possible to use hydrolytic enzymes under conditions of limited moisture to catalyze the formation of esters, and this is now becoming very popular for the resolution of alcohols.4... 

A completely different enzyme-catalyzed synthesis of cyanohydrins is the lipase-catalyzed dynamic kinetic resolution (see also Chapter 6). The normally undesired, racemic base-catalyzed cyanohydrin formation is used to establish a dynamic equilibrium. This is combined with an irreversible enantioselective kinetic resolution via acylation. For the acylation, lipases are the catalysts of choice. The overall combination of a dynamic carbon-carbon bond forming equilibrium and a kinetic resolution in one pot gives the desired cyanohydrins protected as esters with 100% yield [19-22]. 

An elegant way to avoid the low yields and the need for recycling half of the material in the case of kinetic resolutions is a dynamic kinetic resolution (DKR). The dynamic stands for the dynamic equilibrium between the two enantiomers that are kinetically resolved (Scheme 6.6A). This fast racemisation ensures that the enzyme is constantly confronted with an (almost) racemic substrate. At the end of the reaction an enantiopure compound is obtained in 100% yield from racemic starting material. Mathematical models describing this type of reaction have been published and applied to improve this important reaction [32, 33]. There are several examples, in which the reaction was performed in water (see below). In most cases the reaction is performed in organic solvents and the hydrolase-catalysed reaction is the irreversible formation of an ester (for example see Figs. 9.3, 9.4, 9.6, 9.12) or amide (for example see Figs. 9.13, 9.14, 9.16). 

Applications of low temperature work in structural studies have been described in section 3(b). Application to enzyme action is best exemplified by the pioneering work of Fink and Ahmed [221] and Alber etal. [222] on elastase. JV-Carbobenzoxy-L-alanyl-p-nitrophenol ester was selected for study at — 55°C in a 70% methanol-water mixture. Kinetic studies in the presence of cryoprotectant enabled conditions for formation and stabilisation of the acyl-enzyme intermediate to be established. By monitoring changes in intensity of certain reflections as substrate flowed past the crystal at — 55°C, it was possible to show that the rate of formation of the acyl-enzyme was comparable to that obtained by monitoring p-nitrophenol release spectroscopically. The difference electron density map at 3.5 A resolution showed a peak consistent with the formation of an acyl-enzyme intermediate, but a detailed mechanistic interpretation requires higher resolution data. When the crystal was warmed to — 10°C and the data recollected, the peak in the difference synthesis disappeared, indicating that deacylation had occurred, consistent with the predictions from kinetic studies. 

A very straightforward approach in route C (Fig. 2) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [18]) by enantioselective aminolysis of diester 9 with (S)-tert-leucine methylamide 13 or even racemic 13. This would combine three synthetic objectives the resolution of (rac)-9, the resolution of (roc)-13 and the coupling step. In orientating experiments monoester 10 was tested as a model substrate. It was contacted with an equal amount of (S)-amine 13 in the presence and absence of an organic solvent. Solid or liquid subtilisin Carlsberg preparations (Alcalase 2.0 T or Alcalase 2.5 L, respectively) were used as the catalyst. Only with the liquid enzyme preparation was the formation of minor amounts of one of two possible diastereoisomeric peptides observed [19], whereas most of the ester was hydrolyzed to the acid. Likewise, a few selected lipases also provided negative results. 

The crystal structures of several complexes of the metallo enzyme, carboxypeptidase A (CPA)(EC 3.4.17.1), have been examined in considerable detail. The structure of the complex with glycyl tryosine (Gly-Tyr) as been refined to 2.0 A resolution and reveals inter alia interactions between the amide carbonyl oxygen and the catalytically essential zinc, and between the amide nitrogen and the hydroxyl of tryosine-248 (Tyr-248)(Fig. 11). The proposed mechanisms for hydrolysis of peptide and ester bonds by CPA have relied heavily on these crystal structures, but a clear distinction between the possible roles of glutamate-270 (Glu-270) in nucleophilic attack either by general base catalysis (Fig. 11 A) or by covalent any hydride formation (Fig. IIB) remains a major unresolved problem. Indeed, it is not yet certain whether esters and amides are hydrolyzed by CPA via identical mechanisms. 

The recycling of the undesired enantiomer from the enzymatic resolution is of crucial importance particularly on an industrial scale [107]. The classical chemical method consists of the thermal racemization of an amino acid ester at about 150-170°C. Milder conditions can be employed for the racemization of the corresponding amides via intermediate formation of Schiff bases with aromatic aldehydes such as benzaldehyde or salicylaldehyde (Scheme 2.14). More recently, intense research has been devoted to the use of isomerase enzymes (such as amino acid racemases [108]) aiming at the development of dynamic resolution processes. 

Another approach, called kinetic resoiution, depends on the different rates of reaction of two enantiomers with a chiral reagent. A very effective form of kinetic resolution uses enzymes as chiral biocatalysts to selectively bring about the reaction of one enantiomer of a racemic mixture (enzymatic resoiution). Lipases, or esterases—enzymes that catalyze ester hydrolysis and formation—have been successfully used in many kinetic resolutions. In a representative procedure, one enantiomer of an ester undergoes hydrolysis and the other is left unchanged. 

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