Kinetic chemical resolution

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

The classical chemical kinetics allows resolution of the problem by clas sifying aU of the steps of the stepwise process into two categories fast (i.e., those resulting in partial dynamic equihbria of some of elementary reac tions) and slow (those that are far from their dynamic equilibria). In this case, the overall reaction rate v appears to be a function of parameters kj of the direct reaction only for the slow stages and of parameters Kj for the fast stages. Thus, the slow elementary reactions are considered as kinet icaUy irreversible— that is, only a forward reaction i but not its backward reaction can be considered. 

Biocatalysts are not always immobilized on membranes in bioreactors, though. As enzymes are macromolecules and often differ greatly in size from reactants they can be separated by size exclusion membrane filtration with ultra- or nano-filtration. This is used on an industrial scale in one type of enzyme membrane reactor for the production of enantiopure amino acids by kinetic racemic resolution of chemically derived racemic amino acids. The most prominent example is the production of L-methionine on a scale of 400 t/y (Liese et al, 2006). The advantage of this method over immobilization of the catalyst is that the enzymes are not altered in activity or selectivity as they remain solubilized. This principle can be applied to all macromolecular catalysts which can be separated from the other reactants by means of filtration. So far, only enzymes have been used to a significant extent. 

Supplemental use of racemose enzymes The main disadvantage of kinetic racemate resolution is the limitation of yield to 50%, unless the undesired isomer is racemized and the resolution repeated. An elegant alternative to this tedious, terative procedure is the dynamic, kinetic racemate resolution (Figure 12.6), which combines a fast racemization equilibrium (5)-A (,R)-A with an enantioselective transformation in order to accomplish the dera-cemization process. In addition to the chemical methods (such as thermal-, acid/base-, deprotonation/protonation processes) enzyme-catalyzed methods also find application for this racemization process. The use of racemases (EC. 5.1) has recently been reviewed . 

The approach is ideally suited to the study of IVR on fast timescales, which is the most important primary process in imimolecular reactions. The application of high-resolution rovibrational overtone spectroscopy to this problem has been extensively demonstrated. Effective Hamiltonian analyses alone are insufficient, as has been demonstrated by explicit quantum dynamical models based on ab initio theory [95]. The fast IVR characteristic of the CH cliromophore in various molecular environments is probably the most comprehensively studied example of the kind [96] (see chapter A3.13). The importance of this question to chemical kinetics can perhaps best be illustrated with the following examples. The atom recombination reaction... 

Another variation is the mode-locked dye laser, often referred to as an ultrafast laser. Such lasers offer pulses having durations as short as a few hundred femtoseconds (10 s). These have been used to study the dynamics of chemical reactions with very high temporal resolution (see Kinetic LffiASURELffiNTS). 

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. 

Fig. 2.6. Dependence of enanhomeric excess on relative rate of reaction and extent of conversion with a chiral reagent in kinetic resolution. [Reproduced from J. Am. Chem. Soc. 103 6237 (1981) by permission of the American Chemical Society.]...

Now. comparing this to Eq. (4-131). one sees that they appear different. If one di ides through the second equation by k4/k2, the algebraic forms can be made to be identical. The lesson is this The kinetics alone offers no distinction separately identifying the substrate that interacts with the catalyst and incorporating the chemical sense of the process will, one hopes, provide the resolution. 

The route from kinetic data to reaction mechanism entails several steps. The first step is to convert the concentration-time measurements to a differential rate equation that gives the rate as a function of one or more concentrations. Chapters 2 through 4 have dealt with this aspect of the problem. Once the concentration dependences are defined, one interprets the rate law to reveal the family of reactions that constitute the reaction scheme. This is the subject of this chapter. Finally, one seeks a chemical interpretation of the steps in the scheme, to understand each contributing step in as much detail as possible. The effects of the solvent and other constituents (Chapter 9) the effects of substituents, isotopic substitution, and others (Chapter 10) and the effects of pressure and temperature (Chapter 7) all aid in the resolution. 

Revisions of the continuous-flow method have been made to allow observations along the length of the flow tube rather than at right angles.5 This method, fast continuous flow, eliminates the dead time during which the reaction cannot be observed. Kinetic data can be extracted to a time resolution of nearly 10 p,s, but the mathematics is more complicated in this limit, because the mixing and chemical reaction occur on the same time scale. Rate constants nearly as large as the diffusion-controlled value have been determined in favorable cases.6... 

This type of asymmetric conjugate addition of allylic sulfinyl carbanions to cyclopen-tenones has been applied successfully to total synthesis of some natural products. For example, enantiomerically pure (+ )-hirsutene (29) is prepared (via 28) using as a key step conjugate addition of an allylic sulfinyl carbanion to 2-methyl-2-cyclopentenone (equation 28)65, and (+ )-pentalene (31) is prepared using as a key step kinetically controlled conjugate addition of racemic crotyl sulfinyl carbanion to enantiomerically pure cyclopentenone 30 (equation 29) this kinetic resolution of the crotyl sulfoxide is followed by several chemical transformations leading to (+ )-pentalene (31)68. 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

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