Biocatalytic kinetics

As enantiomericaUy pure sulfoxides are excellent chiral auxUiaries for asymmetric synthesis, different approaches for biocatalytic asymmetric oxidations at the S-atom have been explored [30, 31]. Asymmetric peroxidaseorganic sulfides to sulfoxides in organic solvents opens up attractive opportunities by increased substrate solubility and diminished side reactions [32]. Plant peroxidases located in the cell wall are capable of oxidizing a broad range of structurally different substrates to products with antioxidant, antibacterial, antifungal, antiviral, and antitumor activities [33]. Hydroperoxides and their alcohols have been obtained in excellent e.e. in the biocatalytic kinetic resolution of secondary hydroperoxides with horseradish and Coprinus peroxidase [34]. 

Biocatalytic Dynamic Kinetic Resolution of (R,S)-1- 2,3-Dihydrobenzo[b]Furan-4-yl -Ethane-1,2-Diol. Most commonly used biocatalytic kinetic resolution of racemates often provide compounds with high e.e., although the maximum theoretical yield of product is only 50%. In many cases, the reaction mixture contains a roughly 50 50 mixture of reactant and product which have only slight differences in physical properties (e.g., a hydrophobic alcohol and its acetate), and thus separation may be very difficult. These issues with kinetic resolutions can be addressed by employing a Dynamic Kinetic Resolution process involving a biocatalyst or biocatalyst with metal-catalyzed in situ racem-ization (26,27). 

Scheme 89 Biocatalytic kinetic resolution of racemic hydroxymethylphosphinates 271...

Biocatalytic kinetic resolution of racemic hydroxymethylphosphinates 271 via their lipase-promoted acetylation in supercritical carbon dioxide as the reaction medium was investigated. The reaction was fastest when pressure was closer to the critical pressure at 11 MPa the reaction rate reached its maximum when the pressure was increased to 15 MPa. The optimal conditions were obtained at 13 MPa (yields 50%, 30% ee). The stereoselectivity of the reaction depended on solvent, substituents at phosphorus, and solubility of substrates in SCCO2. The best results were obtained with the Candida antarctica lipase (Novozym 435) (Scheme 89) [183, 184]. 

Scheme 6.60 Biocatalytic kinetic resolutions of 155 by hydrolysis and of 156 by acylation.

Faber K, Honig H, Kleewein A (1995) Recent developments determination of the selectivity of biocatalytic kinetic resolution of enantiomers - the enantiomeric ratio . In Roberts SM (ed) Preparative Biotransformations. Wiley, New York... 

The reduction of racemic 5 is a nice example of product-selective biocatalytic kinetic resolution. This means that the enantiomer that is reduced affords only one of two possible diastereomers. Enzyme reductase from the cells of Saccharomyces cerevisiae (baker s yeast) selectively reduces the carbonyl group in the S-enantiomer of 5 into alcohol with an S configuration on the new stereogenic center, hence producing (IS, 2S)-6. Due to the C-H acidify of the a-C atom in 5, it easily tautomerizes to enols 5a and 5b in equilibrium (Scheme 5.36). 

In 2010, Janssen and co-workers reported that the kinetic resolution of p-phenylalanine catalysed by a tandem biocatalytic system composed of phenylalanine aminomutase (PAM) and phenylalanine ammonia lyase (PAL) yielded the corresponding enantiopure (5)-p-phenylalanine in good yield (48%) and excellent enantiomeric excess of >99% ee (Scheme 4.13). The process was based upon the PAM-catalysed, reversible, enantioselective transformation of (I )-p-phenylalanine to (S)-a-phenylalanine. The latter one was transformed in a PAL-catalysed regioselective process into ( )-cinnamic acid, with liberation of ammonia. This constituted an example of a tandem biocatalytic, kinetic resolution in which one enzyme catalysed the equilibration between the substrate and reaction intermediate, while the other shifted this equilibrium between the substrate towards the final product... 

A total synthesis of the antibiotic (K)-fridamycin E was accomplished using as a key chiral building block, the (S)-diol product obtained through EH-catalyzed kinetic resolution of 2-methyl-2-(oct-2-yn-l-yl)oxirane, followed by stereoinversion of the remaining (K)-epoxide by subsequent chemical hydrolysis (Figure 8.51). The biocatalytic kinetic resolution (E-value = 66) was performed with lyophilized cells of Methylobacterium sp. FCC 031. Chemical hydrolysis of the remaining... 

Dynamic kinetic resolution (DKR) of racemic substrate proceeds through asymmetric reduction when the substrate does racemize and the product does not under the applied experimental conditions. For example, by dynamic kinetic resolution of a-alkyl S-keto ester, one isomer, out of the four possible products for the unselective reduction (Scheme 33.18), can be selectively synthesized. Various examples of biocatalytic kinetic resolution through reduction are summarized in Scheme 33.19. A recent development in dynamic kinetic resolution has been reviewed. 

Stereoinversion Stereoinversion can be achieved either using a chemoenzymatic approach or a purely biocatalytic method. As an example of the former case, deracemization of secondary alcohols via enzymatic hydrolysis of their acetates may be mentioned. Thus, after the first step, kinetic resolution of a racemate, the enantiomeric alcohol resulting from hydrolysis of the fast reacting enantiomer of the substrate is chemically transformed into an activated ester, for example, by mesylation. The mixture of both esters is then subjected to basic hydrolysis. Each hydrolysis proceeds with different stereochemistry - the acetate is hydrolyzed with retention of configuration due to the attack of the hydroxy anion on the carbonyl carbon, and the mesylate - with inversion as a result of the attack of the hydroxy anion on the stereogenic carbon atom. As a result, a single enantiomer of the secondary alcohol is obtained (Scheme 5.12) [8, 50a]. 

Comparison of whole cell biocatalytic reaction kinetics for recombinant Escherichia coli with periplasmic-secreting or cytoplasmic-expressing organophosphorus hydrolase... 

In the present work, for detail kinetic studies, we compared biocatalytic reaction kinetics for four types of whole cell biocatalyst systems whole cells with periplasmic-secreting OPH under trc or T7 promoters and whole cells with cytoplasmic-expressing OPH imder trc or T7 promoters. 

Table 1. Kinetic parametral for Paraoxon-biocatalytic reaction using the OPH-expressing whole cells. All data wrae based on unit cell concentration (1 mg-dry cell weight ml" ).

May O., Verseck, S., Bommarius, A. and Drauz, K. (2002) Development of dynamic kinetic resolution processes for biocatalytic production of natural and nonnatural L-amino acids. Organic Process Research Development, 6 (4), 452-457. 

Rhaman and coworkers [112,113] studied the adsorption of lipase on [MgAl] LDH and its biocatalytic activity for butyl oleate synthesis. They demonstrated that up to 277 and 531 mgg-1 of lipase were adsorbed on [MgAl-N03] and [MgAl-Dodecylsulfate] LDH, respectively, showing the highest adsorption capacity of the anionic clays compared to smectite or inorganic phosphate. Recently, we reported the adsorption isotherms of urease on [ZnRAl] LDH under various experimental conditions (pH, buffer) [117]. The kinetic study showed the fast adsorption process (less than 60 min) (Figure 15.3). 

Since the first report on the ferrocene mediated oxidation of glucose by GOx [69], extensive solution-phase studies have been undertaken in an attempt to elucidate the factors controlling the mediator-enzyme interaction. Although the use of solution-phase mediators is not compatible with a membraneless biocatalytic fuel cell, such studies can help elucidate the relationship between enzyme structure, mediator size, structure and mobility, and mediation thermodynamics and kinetics. For example, comprehensive studies on ferrocene and its derivatives [70] and polypy-ridyl complexes of ruthenium and osmium [71, 72] as mediators of GOx have been undertaken. Ferrocenes have come to the fore as mediators to GOx, surpassing many others, because of factors such as their mediation efficiency, stability in the reduced form, pH independent redox potentials, ease of synthesis, and substitutional versatility. Ferrocenes are also of sufficiently small size to diffuse easily to the active site of GOx. However, solution phase mediation can only be used if the future biocatalytic fuel cell... 

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. 

Asymmetric routes to lamivudine have recently been reviewed. A number of these are biocatalytic, the most elegant of which is a highly enantioselective kinetic resolution process based on the use of cytidine deaminase from E. coli. The process is particularly impressive given that the reaction site is five atoms away from the nearest chiral centre (Scheme 1.38). 

A model of such structures has been proposed that captures transport phenomena of both substrates and redox cosubstrate species within a composite biocatalytic electrode.The model is based on macrohomo-geneous and thin-film theories for porous electrodes and accounts for Michaelis—Menton enzyme kinetics and one-dimensional diffusion of multiple species through a porous structure defined as a mesh of tubular fibers. In addition to the solid and aqueous phases, the model also allows for the presence of a gas phase (of uniformly contiguous morphology), as shown in Figure 11, allowing the treatment of high-rate gas-phase reactant transport into the electrode. 

We will first discuss some examples of biocatalytic polymerisation in biological systems, followed by a review of recent man-made systems and the design rules that are emerging. Unique features related to control of polymerisation (both in terms of kinetics and thermodynamics) will be discussed, followed by a review of (potential) applications in biomedicine and nanotechnology. 

The use of a monolithic stirred reactor for carrying out enzyme-catalyzed reactions is presented. Enzyme-loaded monoliths were employed as stirrer blades. The ceramic monoliths were functionalized with conventional carrier materials carbon, chitosan, and polyethylenimine (PEI). The different nature of the carriers with respect to porosity and surface chemistry allows tuning of the support for different enzymes and for use under specific conditions. The model reactions performed in this study demonstrate the benefits of tuning the carrier material to both enzyme and reaction conditions. This is a must to successfully intensify biocatalytic processes. The results show that the monolithic stirrer reactor can be effectively employed in both mass transfer limited and kinetically limited regimes. 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

Furthermore, it can be shown that, in the limiting cases of first-order kinetics [Equation (11.35) also holds for this case] and zero-order kinetics, the equal and optimal sizes are exactly the same. As shown, the optimal holding times can be calculated very simply by means of Equation (11.40) and the sum of these can thus be used as a good approximation for the total holding time of equal-sized CSTRs. This makes Equation (11.31) an even more valuable tool for design equations. The restrictions are imposed by the assumption that the biocatalytic activity is constant in the reactors. Especially in the case of soluble enzymes, for which ordinary Michaelis-Menten kinetics in particular apply, special measures have to be taken. Continuous supply of relatively stable enzyme to the first tank in the series is a possibility, though in general expensive. A more attractive alternative is the application of a series of membrane reactors. 

The enzyme-catalyzed regio- and enantioselective reduction of a- and/or y-alkyl-substituted p,5-diketo ester derivatives would enable the simultaneous introduction of up to four stereogenic centers into the molecule by two consecutive reduction steps through dynamic kinetic resolution with a theoretical maximum yield of 100%. Although the dynamic kinetic resolution of a-substituted P-keto esters by chemical [14] or biocatalytic [15] reduction has proven broad applicability in stereoselective synthesis, the corresponding dynamic kinetic resolution of 2-substituted 1,3-diketones is rarely found in the literature [16]. 

By what appears to be a convenient coincidence, it turns out that the barriers that contribute to ksp for a more realistic kinetic scheme, notably the bi bi ping pong scheme adopted by the majority of hydrolases that are currently employed in biocatalytic resolutions reactions, are equally simple to identify. Figure 2.4 shows the barriers that contribute. By straightforward manipulation of the kinetic equations one obtains Equation 16 ... 

This source of multiplicity is probably the most intrinsic one in catalytic and biocatalytic reactors, for it occurs due to the nonmonotonic dependence of the intrinsic rate of reaction upon the concentration of reactants and products. Although a decade ago, nonmonotonic kinetics of catalytic reactions were considered the exceptional case, today it is clear that nonmonotonic kinetics in catalytic reactions are much more widespread than previously thought. The reader can learn more about various examples from the long list of catalytic reactions exhibiting nonmonotonic kinetics [71-76]. 

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