Enzymatic kinetic method

Deleted. Three experiments that use mercury have been deleted. The paper chromatography experiment is deleted because thin-layer chromatography is predominantly used in its place today. For space reasons, the presentation of catalytic methods and the corresponding experirnent are deleted from the text in favor of enzymatic kinetic methods. Also, the anion chromatography separation of cobalt and nickel is omitted. 

Despite its widespread application [31,32], the kinetic resolution has two major drawbacks (i) the maximum theoretical yield is 50% owing to the consumption of only one enantiomer, (ii) the separation of the product and the remaining starting material may be laborious. The separation is usually carried out by chromatography, which is inefficient on a large scale, and several alternative methods have been developed (Figure 6.2). For example, when a cyclic anhydride is the acyl donor in an esterification reaction, the water-soluble monoester monoacid is separable by extraction with an aqueous alkaline solution [33,34]. Also, fiuorous phase separation techniques have been combined with enzymatic kinetic resolutions [35]. To overcome the 50% yield limitation, one of the enantiomers may, in some cases, be racemized and resubmitted to the resolution procedure. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

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. ... 

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. 

A variety of further methods for the enzymatic kinetic resolution of racemic hydroperoxides (equation 14) has been published. For comparison they are summarized in Table 5. 

This problem was solved by Adam and coworkers in 1994-1998. They presented a high-yielding and diastereoselective method for the preparation of epoxydiols starting from enantiomerically pure allyhc alcohols 39 (Scheme 69). Photooxygenation of the latter produces unsaturated a-hydroxyhydroperoxides 146 via Schenck ene reaction. In this reaction the (Z)-allylic alcohols afford the (5, 5 )-hydroperoxy alcohols 146 as the main diastereomer in a high threo selectivity (dr >92 8) as racemic mixmre. The ( )-allylic alcohols react totally unselectively threolerythro 1/1). Subsequent enzymatic kinetic resolution of rac-146 threolerythro mixture) with horseradish peroxidase (HRP) led to optically active hydroperoxy alcohols S,S) and (//,5 )-146 ee >99%) and the... 

Alternative synthetic approaches include enantioselective addition of the organometallic reagent to quinoline in the first step of the synthesis [16], the resolution of the racemic amines resulting from simple protonation of anions 1 (Scheme 2.1.5.1, Method C) by diastereomeric salts formation [17] or by enzymatic kinetic resolution [18], and the iridium-catalyzed enantioselective hydrogenation of 2-substituted quinolines [19]. All these methodologies would avoid the need for diastereomer separation later on, and give direct access to enantio-enriched QUINAPHOS derivatives bearing achiral or tropoisomeric diols. Current work in our laboratories is directed to the evaluation of these methods. 

We have introduced kinetics as the primary method for studying the steps in an enzymatic reaction, and we have also outlined the limitations of the most common kinetic parameters in providing such information. The two most important experimental parameters obtained from steady-state kinetics are kcat and kcat/Km. Variation in kcat and kcat/Km with changes in pH or temperature can provide additional information about steps in a reaction pathway. In the case of bisubstrate reactions, steady-state kinetics can help determine whether a ternary complex is formed during the reaction (Fig. 6-14). A more complete picture generally requires more sophisticated kinetic methods that go beyond the scope of an introductory text. Here, we briefly introduce one of the most important kinetic approaches for studying reaction mechanisms, pre-steady state kinetics. 

The fastest steps in an enzymatic process cannot be observed by conventional steady-state kinetic methods because the latter cannot be applied to reactions with half-times of less than about 10 s. Consequently, a variety of methods have been developed18 56-593 to measure rates in the range of 1 to 1013 s... 

Hydroperoxo-ferric intermediate, termed also Compound 0, is the immediate precursor of the main catalytic intermediate Compound I in peroxidase enzymatic cycle. Attempts to study this intermediate directly in reactions of hydrogen peroxide with HRP using fast kinetic methods have been inconclusive, possibly because it is not accumulated in sufficient concentrations.90,91 However, Compound 0 could be prepared and studied by EPR and optical absorption spectroscopy via cryoreduction of... 

Enzymatic Kinetic Resolution of N-Boc-Amino Add-Thioesters Coupled with Base-catalyzed Racemization Recently, a new method leading to the preparation of a number of aryl-glycines of the L-configuration has been published. The method is based on the hydrolysis of N-Boc-amino acid thioesters 15 catalyzed by an industrial preparation of the protease subtilisin (Scheme 13.16) [43]. 

Not all organic chemists can be Involved in such exciting projects as the launching of a new anti-AIDS drug. But the chemistry used in this project was invented by chemists in other institutions who had no idea that it would eventually be used to make Crixlvan. The Sharpless asymmetric epoxlda-tion, the catalytic asymmetric reduction, the stereoselective enolate alkylation, and the various methods tried out for the enantiomerically pure amino indanol (resolution, enzymatic kinetic resolution) were developed by organic chemists in research laboratories. Some of these famous chemists like Sharpless invented new methods, some made new compounds, some studied new types of molecules, but all built on the work of other chemists. 

Enzymatic assay methods are classified as fixed-time assays, fixed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time pseudo-first-order conditions generally apply up to 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is > 10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0.1 Km), the initial rate will be directly proportional to initial substrate concentration (cf. Chapter 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear... 

Methods in which some property related to substrate concentration (such as absorbance, fluorescence, chemiluminescence, etc.) is measured at two fixed times during the course of the reaction are known as two-point kinetic methods. They are theoreticahy the most accurate for the enzymatic determination of substrates. However, these methods are technically more demanding than equifibrium methods and all the factors that affect reaction rate, such as pH, temperature, and amount of enzyme, must be kept constant from one assay to the next, as must the timing of the two measurements. These conditions can readily be achieved in automatic analyzers. A reference solution of the analyte (substrate) must be used for calibration. To ensure first-order reaction conditions, the substrate concentration must be low compared to the K, (i.e., in the order of less than 0.2 X K, . Enzymes with high K , values are therefore preferred for kinetic analysis to give a wider usable range of substrate concentration. 

Enzymatic Fluorometric Method I. There are several factors, such as enzyme concentration, substrate concentration, pH of buffer, and temperature, which can affect the kinetics of the enzyme catalyzed reaction. These factors should be optimized and carefully controlled In order to obtain the most sensitive and reproducible results. The results of the optimization studies are summarized In Table I. 

We describe the process development for the preparation of (R)-XU305, a key intermediate of roxifiban. This process evolved from a small-scale enzymatic kinetic resolution into a pilot plant-scale preparation. The combination of an unusual method of racemization of (S)-XU305 as a thioester permitted a dynamic enzymatic resolution to proceed, removing the need to recycle the less-reactive isomer in order to obtain acceptable overall yields. 

Now it is worth making enantiomerically enriched 90. One method already in the literature14 involved reduction of racemic 90 with horse liver alcohol dehydrogenase. This is an enzymatic kinetic resolution (chapters 28 and 29) and at 50% reduction the products are 31% unreacted ketone 90 in good ee, 33% of one enantiomer of the anti-alcohol 93 in perfect (100%) ee, and a trace of the vvn-alcohol 93. 

Syntheses of relatively simple chiral drugs on an industrial scale are the domain of catalytic or enzymatic methods. In the case of the calcium antagonist diltiazem [113] Sharpless asymmetric dihydroxylation (AD-reaction) is employed which works particularly efficiently for cinnamic acid derivatives such as 48-1. In fact diol 48-2 is obtained with good optical enrichment and is then converted into the target compound via 6 routine steps. Alternatively diltiazem is prepared via classical optical resolution or via enzymatic kinetic resolution of suitable intermediates [113]. 

Pre-steady-state (or transient-state) kinetic approaches, allowing the dissection of individual steps and intermediates in an enzymatic reaction, are superior to classical steady-state approaches. Pre-steady-state kinetic methods were first applied to DNA polymerases in late 1980s to early 1990s in classical studies of E. coli Pol I (Klenow fragment, and bacteriophage T7 DNA polymerase. These studies have served as a... 

Although computational methods are available for numerical treatment of enzymatic kinetics and estimation of parameters, still plotting methods are rather widesperad in enzymatic catalysis, while there are rather seldom used by researchers in heterogeneous catalytic kinetics. It is believed, that via plotting methods it is possible to recognized unexpected behavior and to better design experiments. 

Thus, plotting the reciprocal value of the rate vs 1/c, the values of the constants can be obtained (see 6.1). Several other methods are often used in enzymatic kinetics, like the Eadie-Hoftsee and Hanes-Woolf methods. Similarly, in the case of heterogeneous catalytic reactions, if the rate is given for instance by eq. (10.15) after rearrangement... 

C. B. Elliott, Application of Flow-Injection Analysis to Enzymatic Fluorescence Kinetic Methods. Diss. Ahstr. Int. B, 43 (1983) 3966. 

The concept of employing reaction-rate parameters to determine the initial analytical concentration of reactants dates back over 50 to 60 years to the early literature in biochemistry, radiochemistry, and gas-phase diffusion furthermore, among all the analyses performed in all the laboratories around this country, the number carried out by kinetic-based methods probably exceeds that carried out by thermodynamic methods and direct instrumental measurement combined. This comes as a surprise at first, until one considers the large numbers of enzymatic and other determinations done on multi-channel autoanalyzers used in clinical laboratories. Most of these rapid automated instruments use kinetic methods. 

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