Enzyme-catalyzed kinetic resolution process

In an enzyme-catalyzed kinetic resolution process of a racemic substrate, one enantiomer is converted preferentially. For an irreversible conversion of a substrate S into a product P starting from a racemic substrate with = C, the enantiomeric ratio depends on conversion x according to ... 

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. 

More than 60% of all biotransformations using isolated enzymes are hydrolase-dependent processes. Several reviews have summarized the achievements in this field [33-37]. From the numerous examples published, two will be discussed in order to highlight the potential of enzyme-catalyzed kinetic resolution for the production of chiral pharmaceuticals. 

The dynamic kinetic resolution (DKR) of secondary alcohols and amines (Scheme 11.11) is a prominent, industrially relevant, example of chemo-enzymatic chemistry in which a racemic mixture is converted into one enantiomer in essentially 100% yield and in high ee. This is in sharp contrast to enzyme-catalyzed kinetic resolutions that afford the desired end-product in a yield of at most 50%, while 50% of the starting material remains unreacted. In DKR processes, hydrolases are typically employed as the enantioselective acylation catalyst (which can be either R or S selective) while a concurrent racemization process racemizes the remaining substrate via an optically inactive intermediate. This ensures that all starting material is converted into the desired end-product. The importance of optically pure secondary alcohols and amines for the pharmaceutical industry triggered the development of a number of approaches that enable the racemization of sec-alcohols and amines via their corresponding ketones and imines, respectively [42],... 

The success of an enzyme-catalyzed kinetic resolution is limited by the maximum chemical yield of 50% for each enantiomer. However, this drawback can be overcome by a process called dynamic kinetic resolution. The key idea of this principle is to racemize the slow reacting enantiomer continuously reproducing the faster one. In an ideal case at the end of the conversion one enantiomer is formed in 100% yield with 100% of enantiomeric excess[13S 1371. The kinetic requirements for a dynamic kinetic resolution are shown in Scheme 11.1-16[8bl. 

The main drawback of the processes catalyzed by oxidoreductases in comparison with hydrolytic reactions mediated by lipases resides in the necessity of cofactors for this reason microorganisms are normally used instead of isolated enzymes. However, they present a very important advantage-the possibility to obtain only one enantiomer in the reaction-so that higher yields can be achieved than in normal kinetic resolution processes catalyzed by lipases. 

In the case of biocatalysis, enzymes [3] and catalytic antibodies [4] have attracted most attention. Since enzymes are inherently the more active catalysts, they have been used most often. Indeed, many industrial processes for the enantioselective production of certain chiral intermediates are based on the application of enzymes, as in the lipase-catalyzed kinetic resolution of an epoxy-ester used in the production of the anti-hypertensive therapeutic Diltiazem [5]. Recently, it has been noted that there seems to be a trend in industry to use enzymes more often than in the past... 

In addition, reviews dealing with aspects of enzyme-catalyzed dynamic resolution and related processes such as stereoinversion and deracemisation have also been published[4 71. Details of the kinetic principles of dynamic kinetic resolution reactions have also been reported,7 9). Interestingly, a dynamic kinetic resolution reaction can provide a product with higher enantiomeric excess than the corresponding kinetic resolution. In a conventional kinetic resolution, the enantiomeric excess of the product often decreases as a function of conversion. This happens because as the reaction proceeds, the proportion of the preferred enantiomer of substrate decreases. Unless the enzyme is able to discriminate perfectly between the substrate enantiomers, it will catalyze the reaction of the less preferred enantiomer of substrate (the proportion of which grows as the reaction proceeds). However, in a dynamic kinetic resolution where the substrate enantiomers are interconverting rapidly, the ratio of substrate enantiomers will be constant at 1 1. Consequently, the enantiomeric excess of the product will not decrease as the reaction proceeds. 

Chiral 6-substituted 5,6-dihydro-2H-p3ran-2-ones (a,p-unsaturated y-lactones) are key structural intermediates of a variety of natural products tiiat exhibit antifungal and antitumor activity [209]. Hasse and Schneider [210] prepared both enantiomeric series of a variety of optically pure 6-alkylated y-lactones via an enzyme-mediated route. The key step in the process was the ring opening of enantiomerically pure alkyloxiranes 117, accessible via the corresponding a-hydroxythioethers 118, which were obtained enantiomerically pure by the lipase-catalyzed kinetic resolutions (Fig. 41). 

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]. 

In an ideal kinetic resolution (common in enzyme-catalyzed processes), one enantiomer of a racemic substrate is converted tvhile the other is unreactive [70]. In such a kinetic resolution of 5-methyl-2-cyclohexenone, even with 1 equivalent of Me2Zn, the reaction should virtually stop after 50% conversion. This near perfect situation is found with ligand 18 (Fig. 7.10) [71]. Kinetic resolutions of 4-methyl-2-cyclohexenone proceed less selectively (s = 10-27), as might be expected from the lower trans selectivity in 1,4-additions to 4-substituted 2-cyclohexenones [69]. 

The reversibility of hydrogen transfer reactions has been exploited for the racemi-zation of alcohols and amines. By coupling the racemization process with an enantioselective enzyme-catalyzed acylation reaction, it has been possible to achieve dynamic kinetic resolution reactions. The combination of lipases or... 

It is worth noting here that with two enzymes displaying opposite enantioselec-tivity it is possible to produce both enantiomers of the ester products. If the remaining alcohols can be continuously and rapidly racemized during the much slower acylation reaction, either the R- or S-esters can be obtained in high yields (>>5096) from reactions catalyzed by two hydrolases that display opposite enantio-preference. The combined process of racemization and simultaneous resolution, dynamic kinetic resolution (DKR), is described in Chapter 6. 

During the past few years great efforts have been made to overcome the 50% threshold of enzyme-catalyzed KRs. Among the methods developed, deracemization processes have attracted considerable attention. Deracemizations are processes during which a racemate is converted into a non-racemic product in 100% theoretical yield without intermediate separation of materials [5]. This chapter aims to provide a summary of chemoenzymatic dynamic kinetic resolutions (DKRs) and chemoenzymatic cyclic deracemizations. 

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. 

A kinetic resolution is a chemical reaction in which one enantiomer of a racemate reacts faster than the other. Most kinetic resolutions of pharmaceutical compounds are catalyzed processes. Catalysts used in a kinetic resolution must be chiral. Binding of a chiral catalyst with a racemic material can form two different diastereomeric complexes. Since the complexes are diastereomers, they have different properties different rates of formation, stabilities, and rates of reaction. The products form from the diastereomeric substrate-catalyst complexes at different rates. Therefore, a chiral catalyst is theoretically able to separate enantiomers by reacting with one enantiomer faster than the other. The catalysts used in kinetic resolutions are often enzymes. Enzymes are constructed from chiral amino acids and often differentiate between enantiomeric substrates. 

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. 

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]. 

The above examples demonstrate the DSR concept as a useful approach to generate and interrogate simultaneously complex systems for different applications. A range of reversible reactions, in particular carbon-carbon bond-formation transformations, was used to demonstrate dynamic system formation in both organic and aqueous solutions. By applying selection pressures, the optimal constituents were subsequently selected and amplified from the dynamic system by irreversible processes under kinetic control. The DSR technique can be used not only for identification purposes, but also for evaluation of the specificities of selection pressures in one-pot processes. The nature of the selection pressure applied leads to two fundamentally different classes external selection pressures, exemplified by enzyme-catalyzed resolution, and internal selection pressures, exemplified by transformation- and/or crystallization-induced resolution. Future endeavors in this area include, for example, the exploration of more complex dynamic systems, multiple resolution schemes, and variable systemic control. 

Kinetic resolution of racemic compounds is by far the most common transformation catalyzed by lipases, in which the enzyme discriminates between the two enantiomeric constituents of a racemic mixture. It is important to note that the maximum yield of a kinetic resolution is restricted to 50% for each enantiomer based on the starting material. The prochiral route and transformations involving meso compounds, the meso-trkk, have the advantage of potentially obtaining a 100% yield of pure enantiomer. A theoretical quantitative analysis of the kinetics involved in the biocatalytic processes described above has been developed. - The enantiomeric ratio ( ), an index of enantioselectivity, can be calculated from the extent of conversion and the corresponding enantiomeric excess (ee) values of either the product or the remaining substrate. The results reveal that for an irreversible process. 

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