Ketones, reaction with enzymes

Enzymes are very sophisticated systems that apply sound chemical principles. The side-chains of various amino acids are used to supply the necessary bases and acids to help catalyse the reaction (see Section 13.4). Thus, the enzyme aldolase binds the dihydroxyacetone phosphate substrate by reacting the ketone group with an amine, part of a lysine amino acid residue. This forms an imine that becomes protonated under normal physiological conditions. 

Thiamine diphosphate (TPP, 3), in cooperation with enzymes, is able to activate aldehydes or ketones as hydroxyalkyl groups and then to pass them on to other molecules. This type of transfer is important in the transketo-lase reaction, for example (see p. 152). Hydroxyalkyl residues also arise in the decarboxylation of 0x0 acids. In this case, they are released as aldehydes or transferred to lipoamide residues of 2-oxoacid dehydrogenases (see p. 134). The functional component of TPP is the sulfur- and nitrogen-containing thiazole ring. 

Enzymes with oxyanion holes are now known to catalyze a wide range of reactions with substrates that have a carbonyl moiety. The examples discussed in this chapter include thioesters, oxygen esters, peptides, and ketones (Figure 4.1). Two classes of high-energy intermediates with oxyanions are generated in these reactions (Table 4.3), a tetrahedral intermediate and an enolate. These reactions are... 

This reaction is quite special in that it is an aldol-type addition in which a thioester is the donor (nucleophile) and a keto acid is the acceptor (electrophile). From the discussion in Section 18-8E, you will see that reactions of this kind involving an ester as the donor and an aldehyde or ketone as the acceptor can be achieved in the laboratory only under rather special conditions. For the thioester to function as a nucleophile at the a carbon under the restraints imposed by having the reaction occur at the physiological pH, the catalyzing enzyme almost certainly must promote formation of the enol form of the thioester. The enol then could add to the ketone carbonyl with the assistance of a basic group on the enzyme. This kind of catalysis by enzymes is discussed in Section 25-9C. 

The first step in the overall synthetic scheme (Scheme 6) is the condensation of an appropriate carboxylic acid with trifluoroacetaldehyde. The carboxylic acid is chosen to impart specificity for the target enzyme. In one example,[28 the dianion of cyclohexanepropanoic acid (29) was formed by the addition of LDA and then quickly condensed with trifluoroacetaldehyde to form the p-hydroxy acid 30 as a racemic mixture of erythro- and threo-isomers. The p-hydroxy acid 30 is then protected with TBDMSOTf forming 31. Diphenyl phosphorazidate, TEA, and benzyl alcohol were then utilized in a Curtius rearrangement of the protected alcohol 31, which proceeds through an isocyanate intermediate that yields the protected amino alcohol 32 upon reaction with benzyl alcohol. In order for this step to occur at an appreciable rate, a second equivalent of triethylamine had to be added. The amino alcohol 32 was then deprotected and coupled with Boc-Phe-Leu-OH to give the trifluoromethyl alcohol 33, which was oxidized to the corresponding trifluoromethyl ketone 34 as a 1 1.2 mixture of diastereomers using the Dess-Martin periodinane procedure. Thus far, the compound shown in Scheme 6 is the only compound that has been synthesized by this method, but it is reasonable to assume that many other similar fluoro ketones can be produced by this scheme. 

An experiment with an irreversible inhibitor should carry with it a control experiment involving the addition of a substrate if the location of the reaction with inhibitor is at the active site, then the addition of a substrate will slow down the rate of inhibition. For example, the reactivity of papain (5 pM) with a 1.71 pM solution of 4-toluenesulphonylamidomethyl chloromethyl ketone suffers a drop of 1.68-fold when the substrate (methyl hippurate) is changed from 12.7 to 21.1 mM. The inhibitor which reacts covalently with the enzyme should carry either a radioactive or spectroscopic tag which would enable the location of the altered amino acid to be determined in the sequence, and hence in the three-dimensional X-ray crystallographic map of the enzyme. An alternative approach is to design an inhibitor with groups (analogous to those attached to the substrate) which force it to bind at the active site (Scheme 11.18). 

Baeyer-ViUiger oxidation involves NADPH and flavin (FAD) as cofactors and was originally proposed by Walsh et al. based on data obtained from cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (Fig. 24) [156]. In a first step, enzyme-bound flavin is reduced, followed by the addition of oxygen yielding a hydroperoxide anion. Reaction with the ketone substrate gives a Criegee intermediate, which is then converted into the product under dissociation of water. The cofactor FAD is recovered via oxidation with NADP+. 

The capability of L-proline - as a simple amino acid from the chiral pool - to act like an enzyme has been shown by List, Lemer und Barbas III [4] for one of the most important organic asymmetric transformations, namely the catalytic aldol reaction [5]. In addition, all the above-mentioned requirements have been fulfilled. In the described experiments the conversion of acetone with an aldehyde resulted in the formation of the desired aldol products in satisfying to very good yields and with enantioselectivities of up to 96% ee (Scheme 1) [4], It is noteworthy that, in a similar manner to enzymatic conversions with aldolases of type I or II, a direct asymmetric aldol reaction was achieved when using L-proline as a catalyst. Accordingly the use of enol derivatives of the ketone component is not necessary, that is, ketones (acting as donors) can be used directly without previous modification [6]. So far, most of the asymmetric catalytic aldol reactions with synthetic catalysts require the utilization of enol derivatives [5]. The first direct catalytic asymmetric aldol reaction in the presence of a chiral heterobimetallic catalyst has recently been reported by the Shibasaki group [7]. 

Oxidations of carbon-heteroatom species often results in the destruction of a stereogenic center, as in the oxidation of a secondary alcohol to a ketone. In some instances, this reaction can be coupled with another to provide a chiral product (see Chapter 21). One example is the enzymatic acetylation of one enantiomer of a secondary alcohol, where a redox reaction with a transition metal catalyst equilibrates the unreactive isomer of the alcohol (Scheme 9.1).10 12 The redox reaction can also be performed by an enzyme.13... 

It is of great interest to compare this last value with the keto-enol equilibrium constant obtained similarly for acetone = 0.35 x 10-8). Indeed, in many enzyme-catalysed reactions, aldolisation for example, enamine formation is not rate-limiting, and the rate is usually controlled by subsequent electrophilic additions. Consequently, the rate depends on enamine reactivity and on the enamine concentration at equilibrium. Therefore, if one wants to compare the two processes, via enol and via enamine, in order to explain why the enamine route is usually preferred, the difference in equilibrium constants for enol and enamine formation must be taken into account. Data on ketone to enol and ketone to enamine equilibrium constants show that the enamine and enol concentrations are of similar magnitude even for relatively small concentrations of primary amine. Thereafter, since the enamine is much more reactive than the enol for reactions with electrophilic reagents (in a ratio of 4-6 powers of ten for proton addition), it can be easily understood why the amine-catalysed pathway is energetically more favourable. 

Reactions with these compounds suffer from very low substrate concentrations due to the low solubility of hydrophobic ketone substrates in aqueous media, which leads to unsatisfactory volumetric productivities. To achieve higher substrate concentrations, a biphasic reaction medium was introduced. The system water/ n-heptane (4 1) proved to be the most suitable system with regard to stability of the examined enzymes. The large-scale available (S)-specific ADH from R. erythropolis as well as FDH from C. boidinii are stable for long periods of time in this aqueous-organic solvent system. Preparative conversions with a variety of aromatic ketone substrates were carried out with this reaction medium. For example, p-chloroacetophenone was converted into the corresponding (S )-alcohol with >99% ee and 69% conversion. The obvious increase in volumetric productivity is due to the higher substrate concentrations. The reduction of p-chloroacetophenone... 

Figure 1.16 Left Schematic presentation of the 1,3-diaminopropanone core moiety as cysteine protease-directed and active site-spanning inhibitor principle (top). Upon reaction with the enzyme nucleophile, the ketone is reversibly converted to a hemithioketal (bottom). Right Peptidomimetic cysteine protease inhibitors of subsequent generations are depicted together with their inhibitory activity and primary targets.

Biological systems overcome the inherent unreactive character of 02 by means of metalloproteins (enzymes) that activate dioxygen for selective reaction with organic substrates. For example, the cytochrome P-450 proteins (thiolated protoporphyrin IX catalytic centers) facihtate the epoxidation of alkenes, the demethylation of Al-methylamines (via formation of formaldehyde), the oxidative cleavage of a-diols to aldehydes and ketones, and the monooxygenation of aliphatic and aromatic hydrocarbons (RH) (equation 104). The methane monooxygenase proteins (MMO, dinuclear nonheme iron centers) catalyze similar oxygenation of saturated hydrocarbons (equation 105). ... 

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