Enzymes kinetic resolution, enzymatic

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

The resolution of racemic ethyl 2-chloropropionate with aliphatic and aromatic amines using Candida cylindracea lipase (CCL) [28] was one of the first examples that showed the possibilities of this kind of processes for the resolution of racemic esters or the preparation of chiral amides in benign conditions. Normally, in these enzymatic aminolysis reactions the enzyme is selective toward the (S)-isomer of the ester. Recently, the resolution ofthis ester has been carried out through a dynamic kinetic resolution (DKR) via aminolysis catalyzed by encapsulated CCL in the presence of triphenylphosphonium chloride immobilized on Merrifield resin (Scheme 7.13). This process has allowed the preparation of (S)-amides with high isolated yields and good enantiomeric excesses [29]. 

Catalytic transformation based on combined enzyme and metal catalysis is described as a new class of methodology for the synthesis of enantiopure compounds. This approach is particularly useful for dynamic kinetic resolution in which enzymatic resolution is coupled with metal-catalyzed racemization for the conversion of a racemic substrate to a single enantiomeric product. 

To outweigh disadvantages of the kinetic resolution presented above, an enzymatic desymmetrization of prochiral sulfinyldiacetates 19 was performed. The use of various enzymes, PLE, a-chymotrypsin (a-CT) ° and PPL," made it... 

In another approach, the alcohol moiety, formed by an enzymatic hydrolysis of an ester, can act as a nucleophile. In their synthesis of pityol (8-37a), a pheromone of the elm bark beetle, Faber and coworkers [17] used an enzyme-triggered reaction of the diastereomeric mixture of ( )-epoxy ester 8-35 employing an immobilized enzyme preparation (Novo SP 409) or whole lyophilized cells of Rhodococcus erythro-polis NCIMB 11540 (Scheme 8.9). As an intermediate, the enantiopure alcohol 8-36 is formed via kinetic resolution as a mixture ofdiastereomers, which leads to the diastereomeric THF derivatives pityol (8-37a) and 8-37b as a separable mixture with a... 

Long, W.S., Kamaruddin, A.H. and Bhatia, S. (2005) Enzyme kinetics of kinetic resolution of racemic ibuprofen ester using enzymatic membrane reactor. Chemical Engineering Science, 60 (18), 4957—1970. 

The procedure shows that it is feasible to combine racemization with the kinetic resolution process (hence the DKR) of R,S)- ethoxyethyl ibuprofen ester. The chemical synthesis of the ester can be applied to any esters, as it is a common procedure. The immobilized lipase preparation procedure can also be used with any enzymes or support of choice. However, the enzyme loading will need to be optimized first. The procedures for the enzymatic kinetic resolution and DKR will need to be adjusted accordingly with different esters. Through this method, the enantiopurity of (5)-ibuprofen was found to be 99.4 % and the conversion was 85 %. It was demonstrated through our work that the synthesis of (5)-ibuprofen via DKR is highly dependent on the suitability of the reaction medium between enzymatic kinetic resolution and the racemization process. This is because the compatibility between both processes is crucial for the success of the DKR. The choice of base catalyst will vary from one reaction to another, but the basic procedures used in this work can be applied. DKRs of other profens have been reported by Lin and Tsai and Chen et al. ... 

Enzymes may be used either directly for chiral synthesis of the desired enantiomer of the amino acid itself or of a derivative from which it can readily be prepared, or for kinetic resolution. Resolution of a racemate may remove the unwanted enantiomer, leaving the intended product untouched, or else the reaction may release the desired enantiomer from a racemic precursor. In either case the apparent disadvantage is that the process on its own can only yield up to 50% of the target compound. However, in a number of processes the enzyme-catalyzed kinetic resolution is combined with a second process that re-racemizes the unwanted enantiomer. This may be chemical or enzymatic, and in the latter case, the combination of two simultaneous enzymatic reactions can produce a smooth dynamic kinetic resolution leading to 100% yield. 

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... 

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. 

In work concerning the directed evolution of enantioselective enzymes, there was a need for fast and efficient ways to determine the enantiomeric purity of chiral alcohols, which can be produced enzymatically either by reduction of prochiral ketones (e.g., 26) using reductases or by kinetic resolution of rac-acetates (e.g., 19) by lipases (111). In both systems, the CD approach is theoretically possible. In the former case, an LC column would have to separate the educt 26 from the product (A)/(J )-20, whereas in the latter, (5)/(J )-20 would have to be separated from (S)/(R)-19. 

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). 

The beneficial effect of the hydrophobicity of [BMIM]PFg was shown to extend to other enzymes a remarkably enhanced enantioselectivity was observed for lipases AK and Pseudomonas fluorescens for the kinetic resolution of racemic P-chiral hydroxymethanephosphinates (Scheme 31) (278). The ee values of the recovered alcohols and the acetates were about 80% when the enzymatic reactions were conducted in the hydrophobic [BMIMJPFg. In contrast, there was little enantioselectivity (<5%) observed with the enzymes in hydrophilic [BMIM]BF4. The lack of stereoselectivity in [BMIM]BF4 was attributed to the high miscibility of [BMIM]BF4 with water. The relatively hydrophilic ionic liquid is capable of stripping off the essential water from the enzyme surface, leading to insufficient hydration of the enzyme and a consequently strong influence on its performance (279). 

When the water-miscible ionic liquid [MMIM][MeS04] was used as a neat medium for the enzymatic transformations, however, poorer performance was observed. For the kinetic resolution of mc-l-phenylethanol by transesterification with vinyl acetate with a set of different lipases dispersed in the pure ionic liquid, it was found that [MMIM][MeS04] was among the poorest media for the enzymes (291). It has been recognized that some water-miscible ionic liquids in the pure form are denaturants (27), but, when they are used in the presence of excess water, their tendency to... 

An efficient method to prepare enantiomerically enriched hydroperoxides is the enzymatic kinetic resolution of racemic hydroperoxides using different kinds of enzymes (mainly lipases, chloroperoxidase, horseradish peroxidase). However, the scope of these reactions may be limit by the narrow substrate specificity of the enzyme. 

Rakels, J.L.L., Paffen, H.T., Straathof, A.J.J. and Heijnen, J.J. (1994) Comparison of enzymatic kinetic resolution in a batch reactor and a CSTR. Enzyme Microbial Technology, 16,791-794. 

It should be mentioned that the great majority of dynamic kinetic resolutions reported so far are carried out in organic solvents, whereas all cyclic deracemizations are conducted in aqueous media. Therefore, formally, this latter methodology would not fit the scope of this book, which is focused on the synthetic uses of enzymes in non-aqueous media. However, to fully present and discuss the applications and potentials of chemoenzymatic deracemization processes for the synthesis of enantiopure compounds, chemoenzymatic cyclic de-racemizations will also be briefly treated in this chapter, as well as a small number of other examples of enzymatic DKR performed in water. 

Yeast-mediated reductions predominantly form a single enantiomer and it is often difficult to find conditions which produce the opposite stereoisomer selectively. It has, however, been possible to obtain both enantiomers in 50% yield in 100% via enzymatic optical resolution. Chiral fluorinated secondary alcohols possessing the mono-, di- and/or trifluoromethyl group have been prepared by enzyme-catalyzed kinetic resolutions [27]. 

Enzymatic resolution of racemic secondary alcohols by enantiomer-selective acylation gives optically pure compounds with up to 50% yield [332], When this method is coupled with the principle of dynamic kinetic resolution (see Section 1.4.1.5), the theoretical yield increases to 100%. Thus a reaction system consisting of an achiral transition-metal catalyst for racemization, a suitable enzyme, acetophenone, and an acetyl donor allows the transformation of racemic 1-phenylethanol to the R acetates with an excellent ee (Scheme 1.93) [333]. The presence of one equiv. of acetophenone is necessary to promote the alcohol racemization catalyzed by the... 

Resolution of racemic 1,3/4/6,7,llb-hexahydro-2H-pyrazino[l,2-a]iso-quinolin-4-one into enantiomers was unsuccessful either through the crystallization of diastereomeric chiral salts prepared from enantiopure acids in different solvent mixtures, or with kinetic resolution by an enzymatic acylation using different enzymes (08EJO895). 

Kinetic resolutions in general are regularly applied in organic synthesis. Since enzymes are highly attractive for asymmetric synthesis, various types of biocatalysts have been used in enzymatic (dynamic) kinetic resolutions, but the focus will remain on lipase- and esterase-mediated resolutions as the most common tools in early steps of natural product syntheses. 

Williams, J. M. J. Parker, R. J. Neri, C. Enzymatic kinetic resolution in enzyme catalysis in organic synthesis (2nd Edition) 2002, 1, 287-312. 

Biocatalysts, mainly hydrolytic enzymes and oxidoreductases, have been used for organic reactions due to their excellent enantioselectivities and environmentally friendliness.1 Typical enzymatic reactions used for the organic synthesis are shown in Figure 1. Especially, hydrolytic enzymes for kinetic resolutions of racemates have been utilized widely because of their high stabilities, wide substrate specificities, lack of cofactor requirements and high availabilities. 

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