Esterases resolution

These are major disadvantage of the esterase resolution process. Since die optimum pH of die enzymic reaction is generally on the alkaline side, die esters used as substrates are non-enzymatically hydrolysed and die optical purity of die L-amino adds obtained is generally low. Also the substrate has to be protected at the amino group in most cases in order to prevent formation of diketopiperasines. The esterase method is not attractive in practice and to the best of our knowledge is not used on an industrial scale. 

Other similar lipase/esterase resolution processes have been developed such as the use of Bacillus that esterase to produce the substituted propanoic acids that are precursors of non-steroidal anti-inflammatory drags, snch as naproxen and ibuprofen etc., and the formation of chiral amines by Celgene. Other methods start from prochiral precursors and have the advantage that enantioselective synthesis allows the production of particular isomers in yields approaching 100%, rather than the 50% yields characteristic of resolution processes. For instance Hoechst have patented the production of enantiomers using Pseudomonas fluorescens lipase to either acylate diols or hydrolyse diacetate esters. 

One approach called enzymatic resolution, involves treating a racemic mixture with an enzyme that catalyzes the reaction of only one of the enantiomers Some of the most commonly used ones are lipases and esterases enzymes that catalyze the hydrol ysis of esters In a typical procedure one enantiomer of the acetate ester of a racemic alcohol undergoes hydrolysis and the other is left unchanged when hydrolyzed m the presence of an esterase from hog liver... 

Eig. 3. Enzyme-catalyzed resolutions. PLE = pig liver esterase. See text. 

Enzymatic hydrolysis of A/-acylamino acids by amino acylase and amino acid esters by Hpase or carboxy esterase (70) is one kind of kinetic resolution. Kinetic resolution is found in chemical synthesis such as by epoxidation of racemic allyl alcohol and asymmetric hydrogenation (71). New routes for amino acid manufacturing are anticipated. 

Optically Active Alcohols and Esters. In addition to the hydrolysis of esters formed by simple alcohols described above, Hpases and esterases also catalyze the hydrolysis of a wide range of esters based on more complex and synthetically useful cycHc and acycHc alcohols (Table 5). Although the hydrolysis of acetates often gives the desirable resolution, to achieve maximum selectivity and reaction efficiency, comparison of various esters is recommended. 

Both saturated (50) and unsaturated derivatives (51) are easily accepted by lipases and esterases. Lipase P from Amano resolves azide (52) or naphthyl (53) derivatives with good yields and excellent selectivity. PPL-catalyzed resolution of glycidyl esters (54) is of great synthetic utiUty because it provides an alternative to the Sharpless epoxidation route for the synthesis of P-blockers. The optical purity of glycidyl esters strongly depends on the stmcture of the acyl moiety the hydrolysis of propyl and butyl derivatives of epoxy alcohols results ia esters with ee > 95% (30). 

Fipases and esterases are often used for Idnetic resolution of racemates, variously by hydrolysis, esterification, or transesterification of suitable precursors. Scheme 8.3-3 illustrates the principal for the resolution of a secondary alcohol by esterification with vinyl acetate. 

Figure 9.9 Resolution of DL-menthol using an esterase from Rhodotorula minuta var texensis.

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... 

In another study a hyperthermophilic esterase from Aeropyrum pemix K1 (APE1547) was used as a catalyst in the hydrolytic kinetic resolution of rac-3-octanol acetate [53]. Following a single round of epPCR, a mutant displaying a 2.6-fold increase in enantioselectivity was identified having five amino acid substitutions, which were shown to be spatially distal to the catalytic center. 

Figure 14.4 Resolution of methyl-5-chloro-2-isopropyl-4-pentenoate with PharmaPLE, a nonanimal-derived isoform of pig liver esterase. The corresponding 5-ester is used as intermediate for production of Aliskiren...

E. Guibe-Jampel, G. Rousseau, L. Blanco, Enzymatic Resolution of Racemic Bicyclic Lactones by Horse Liver Esterase , Tetrahedron Lett. 1989, 30, 67-68. 

The above-mentioned facts have important consequences on the stereochemical outcome of the kinetic resolution of asymmetrically substituted epoxides. In the majority of kinetic resolutions of esters (e.g. by ester hydrolysis and synthesis using lipases, esterases and proteases) the absolute configuration at the stereogenic centre(s) always remains the same throughout the reaction. In contrast, the enzymatic hydrolysis of epoxides may take place via attack on either carbon of the oxirane ring (Scheme 7) and it is the structure of the substrate and of the enzyme involved which determine the regioselec-tivity of the attack [53, 58-611. As a consequence, the absolute configuration of both the product and substrate from a kinetic resolution of a racemic... 

The synthesis of chiral carboxylic acids by enzymatic resolution of the corresponding racemate is a widely established method, and for this purpose a broad variety of esterases are commercially available. Consequently, this... 

Figure 11, Double kinetic resolution using enzymes with opposite enantioselectivity. PPL, porcine pancreatic lipase PLE, pig liver esterase.

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

There are basically two approaches to the synthesis of enantiomerically pure alcohols (i) kinetic resolution of the racemic alcohol using a hydrolase (lipase, esterase or protease) or (ii) reduction mediated by a ketoreductase (KRED). Both of these processes can be performed as a cascade process. The first approach can be performed as a dynamic kinetic resolution (DKR) by conducting an enzymatic transesterification in the presence of a redox metal [e.g. a Ru(ll) complex] to catalyze in situ racemization of the unreacted alcohol isomer [11] (Scheme 6.1). We shall not discuss this type of process in any detail here since it forms the subject of Chapter 1. 

A.l. Assay for Screening Lipases or Esterases in the Kinetic Resolution of Chiral p-Nitrophenyl Esters... 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

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. 

A typical example that illustrates the method concerns the lipase- or esterase-catalyzed hydrolytic kinetic resolution of rac-1-phenyl ethyl acetate, derived from rac-1-phenyl ethanol (20). However, the acetate of any chiral alcohol or the acetamide of any chiral amine can be used. A 1 1 mixture of labeled and non-labeled compounds (S)- C-19 and (f )-19 is prepared, which simulates a racemate. It is used in the actual catalytic hydrolytic kinetic resolution, which affords a mixture of true enantiomers (5)-20 and (J )-20 as well as labeled and non-labeled acetic acid C-21 and 21, respectively, together with non-reacted starting esters 19. At 50% conversion (or at any other point of the kinetic resolution), the ratio of (5)- C-19 to (1 )-19 correlates with the enantiomeric purity of the non-reacted ester, and the ratio of C-21 to 21 reveals the relative amounts of (5)-20 and (J )-20 (98). 

In a more detailed study, the same esterase P. fluorescens) was again subjected to mutagenesis using the same mutator strain, but also by saturation mutagenesis at selected positions 133a). In addition to 3-phenylbutyric acid ethyl ester (27), 3-bromo-2-methyl-propionic acid methyl ester rac-31) was chosen for the hydrolytic kinetic resolution, with the WT PFE showing an E factor of 12 in favor of the (5)-32. 

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