Enzyme-catalyzed hydrolysis kinetic resolution

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). 

Another factor to consider is that the site of enzyme-catalyzed hydrolysis would not be adjacent to the stereocenter but rather /> to it. While there are many examples where reaction enantioselectivity is attenuated by the distance of the stereo-genic carbon from the enzymatic action site [31], resolution of centers up to five bonds distant from the site of enzymatic action is still possible [32]. We hoped that we would be able to reproduce the example of the enzymatic kinetic resolution of 9 but with a thioester, inasmuch as the resolution of 9 had been excellent and the resolved center was located two bonds from the ester. 

An additional strategy employed by Sih and co-workers involved sequential enzyme-catalyzed reactions. Pseudomonas lipases were found to tolerate a wide range of substrates although the enantioselectivity was generally only moderate. However, by first performing a methanolysis of the oxazolinone followed by a separate enzyme-catalyzed hydrolysis under kinetic resolution conditions, a highly enantio-merically enriched product could be obtained, as shown in Fig. 9-211491. 

Figure 9.3 Products of kinetic resolutions of the esters indicated by enzyme-catalyzed hydrolysis in continuous-flow mode [87. 90-95).

Biocatalytic hydrolysis or transesterification of esters is one of the most widely used enzyme-catalyzed reactions. In addition to the kinetic resolution of common esters or amides, attention is also directed toward the reactions of other functional groups such as nitriles, epoxides, and glycosides. It is easy to run these reactions without the need for cofactors, and the commercial availability of many enzymes makes this area quite popular in the laboratory. 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, enzymes that catalyze the hydrolysis of hydantoins 7-11,147). Because synthetic hydantoins are accessible by a variety of chemical syntheses, including Strecker reactions, enan-tioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, because hydantoins are easily racemized chemically or enzymatically by appropriate racemases, dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases have been reported 147). However, if asymmetric induction is poor or if inversion of enantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of L-methionine as part of a whole cell system E. coll) (Figure 22) 148). 

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids 7-11). Enantioselectivity is relevant in the kinetic resolution of racemic nitriles or desymmetrization of prochiral dinitriles. Both versions have been applied successfully to a number of different substrates using one of the known currently available nitrilases. Recently, scientists at Diversa expanded the collection of nitrilases by metagenome panning 150). Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity and limited activity. 

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>. 

Enzyme-based processes for the resolution of chiral amines have been widely reported [2, 3] and are used in the manufacture of pharmaceuticals, for example, BASF s process for chiral benzylic amine intermediates. Scheme 13.1 [4]. The methods used are enantioselective hydrolysis of an amide and enantioselective synthesis of an amide, both of which are kinetic resolutions. For high optical purity products the processes depend upon a large difference in the catalyzed reaction rates of each enantiomer. 

In a different approach, the hydrolase-catalyzed kinetic resolution of chiral acetates was studied using a high-throughput ee assay also based on an enzyme-coupled test, the presence of a fluorogenic moiety not being necessary [16]. The assay is based on the idea that the acetic acid formed by hydrolysis of a chiral acetate can be transformed stoichiometrically into NADH in a series of coupled enzyme reactions using commercially available enzyme kits (Fig. 9.10). The NADH is then... 

Optically pure trans-2-phenylcyclohexanol can also be prepared by resolution of the phthalate esters using brucine to obtain the (-t-)-alcohol and strychnine to obtain the (-)-alcohol (after basic hydrolysis of their respective salts).11 Enzyme-catalyzed kinetic resolution of the acetate esters using pig liver esterase4 and pig liver acetone powder12 has been used to prepare both enantiomers of this chiral auxiliary. The hydroboration of 1-phenylcyclohexene with isopinocampheylborane has been reported to give the chiral auxiliary in 97% enantiomeric excess.13... 

Kurokawa et al81 reported the enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, A-Boc, N-Cbz proline esters and prolinols using protease and Candida antarctica lipase B. The latter was efficient in the enantioselctive hydrolysis of both N-Boc and N-Cbz proline derivatives with E > 100. 

Both enzymes catalyze the hydrolysis of the amino ester 30 enantioselectively (Scheme 6.15). At about 60% substrate conversion, the enantiomeric excess of recovered ester 32 from both reactions exceeds 98%. In addition, the acid product 31 (96-98% ee) was obtained by carrying the hydrolysis of the ester to 40%. The rates of hydrolysis become significantly slower when conversion approaches 50%, allowing a wide window for kinetic control of the resolution process. Both enzymes function well in a concentrated water/substrate (oil) two-phase system while maintaining high enantioselectivity, making this system very attractive for industrial processes. 

Kinetic resolution. Kinetic resolution is a separation process based on the different rates of the transformation of the enantiomers into certain products under the influence of chiral reagents or catalysts. In recent years the use of enzyme-catalyzed enantio-selective hydrolysis of chiral esters has attracted much attention (3). A large rate difference in the transformation of the starting enantiomers is an important criteria for this technology to be of practical use. 

Because of the specificity and the enantioselectivity of some enzyme-catalyzed reactions, the application of enzymes is increasingly important in asymmetric induction and kinetic resolution in organic synthesis. A large number of publications were recently reviewed, focusing on utilization of enzymes and microorganisms to stereospecific hydrolysis and other reactions to produce pure stereoisomers (2,3). However, the use of an enzyme as a catalyst has usually been limited to small-scale experiments in the laboratory. 

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

Lipases are the most frequently used enzymes in organic chemistry, catalyzing the hydrolysis of carboxylic acid esters or the reverse reaction in organic solvents [3,5,34,70]. The first example of directed evolution of an enantioselective enzyme according to the principle outlined in Fig. 11.2 concerns the hydrolytic kinetic resolution of the chiral ester 9 catalyzed by the bacterial lipase from Pseudomonas aeruginosa [8], This enzyme is composed of 285 amino acids [32]. It is an active catalyst for the model reaction, but enantioselectivity is poor (ee 5 % in favor of the (S)-acid 10 at about 50 % conversion) (Fig. 11.10) [71]. The selectivity factor E, which reflects the relative rate of the reactions of the (S)- and (R)-substrates, is only 1.1. 

In order to extend the two-enzyme system to other 2-hydroxy acids, a racemase with a broader activity was found in Lactobacillus paracasei. This was exploited for deracemization of 2-hydroxy-4-phenylbutanoic acid and 3-phenyllactic acid, which are important synthetic intermediates. In addition, in this case the procedure requires a kinetic resolution step and a successive racemization step. O-Acetyl derivatives of the absolute (S)-configuration can be obtained in two successive repeating cycles. Yields are around 60%. Of course the 0-acetyl derivatives of opposite configuration can be obtained when the lipase-catalyzed reaction is apphed in the hydrolysis direction. Obtaining the O-acetyl derivatives of the absolute (R)-configuration requires an additional acetylation step of the initially resolved and racemized (S)-hydroxy acid [12]. 

The kinetic resolution (KR) of racemic isoxazoline 45 catalyzed by enzymes was studied. The best result was obtained with lipase B from Candida antarctica (CALB) that hydrolyzed the ethyl ester function of (-)-45 to the corresponding monoacid (-)-46. The reaction, which was run in 0.1 M phosphate buffer/acetone at rt, spontaneously stopped at 50% conversion to yield monoacid (-)-46 and the residual ester (+)-45 in ee s higher than 99% <04TA3079>. The C-5 epimer of 45 underwent enantioselective hydrolysis (> 99% ee) of the methyl ester linked to C-5 in the presence of the protease proleather (Subtilisin Carlsberg) whereas CALB and other lipases were not able to resolve it. 

When amino acids are synthesized in nature, only the L-enantiomer is formed (Section 5.20). However, when amino acids are synthesized in the laboratory, the product is usually a racemic mixture of D and l enantiomers. If only one isomer is desired, the enantiomers must be separated. They can be separated by means of an enzyme-catalyzed reaction. Because an enzyme is chiral, it will react at a different rate with each of the enantiomers (Section 5.20). For example, pig kidney aminoacylase is an enzyme that catalyzes the hydrolysis of A -acetyl-L-amino acids, but not Ai-acetyl-D-amino acids. Therefore, if the racemic amino acid is converted into a pair of Wacetylamino acids and the A -acetylated mixture is hydrolyzed with pig kidney aminoacylase, the products will be the L-amino acid and A -acetyl-D-amino acid, which are easily separated. Because the resolution (separation) of the enantiomers depends on the difference in the rates of reaction of the enzyme with the two A -acetylated compounds, this technique is known as a kinetic resolution. 

---