Catalysis covalent

Covalent catalysis is a general term applied when a catalyst forms full covalent bonds with the substrate, not just intermolecular non-covalent interactions. The nucleophilic catalysis by an amine given in Eq. 9.9 is a specific example where a nucleophile acts as a covalent catalyst. 

As another example, consider the decarboxylation of acetoacetate. This compound will decarboxylate under acidic conditions with heating. However, the addition of aniline catalyzes the reaction, so it occurs at less acidic pH and ambient temperature (Eq. 9.10). Formation of the imine between aniline and the p-keto acid leads to a species that is protonated and can act as a good electron sink during the decarboxylation. Hydrolysis of the enamine product gives the ketone and regenerates the catalyst, thus leading to turnover. 

One especially successful example of organocatalysis is the chiral amine catalyst shown developed by MacMillan and co-workers. The key feature of such systems is that forming the iminium ion creates a much lower-lying LUMO relative to that found in the starting enone. This makes the structure much more susceptible to nucleo- 

Austin, J. E, and MacMillan, D. W. C. Enantioselective Organocataly tic Indole Alkylations. Design of a New and Highly Efeclive Chiral Amine for Iminium Catalysis. /. Am. Chem. Soc., 124,11 (2002). 

The term covalent catalysis was chosen by Bender and Komiyama (1978) 

Reactive immunization is a fundamentally different approach to selecting antibody pockets that contain functional groups. This method employs mechanism-based inhibitors as haptens these molecules react covalently with appropriately functionalized antibodies, allowing direct selection of active clones from large pools of inactive variants. When a suitable substrate is used in place of the inhibitor, reactive residues in the selected antibodies can often mediate its conversion into product. 

This strategy has yielded catalysts for several reactions. For example, phosphonylat-ing agents have been used to generate antibodies that promote ester hydrolysis by a 

Aldolase antibodies obtained by reactive immunization are notable for high activity, broad substrate specificity, and high selectivities [53]. Rate accelerations are typically in the range 105 to 107-fold over background. Although the k /K values are 102 to 104 lower than those of aldolase enzymes, these are among the most efficient antibody catalysts described to date. Their efficacy is all the more notable in light of the inherently complex, multistep process they catalyze. 

Despite the absence of stereochemical information in the reactive immunogen, the aldolase antibodies promote carbon-carbon bond formation with surprisingly high selectivity. For instance, the enamine formed from acetone adds to the si face of various aldehydes with ee s in excess of 95% [53], In other examples, Robinson annula-tions have been carried out with high enantioselectivity [55], tertiary aldols and other compounds have been successfully resolved [56], and enantiopure intermediates have been prepared for the synthesis of various natural products [57, 58]. 

Studies aimed at identifying the active site of an enzyme (cf. 2.4.1.1) have shown that, during catalysis, a number of enzymes bind the substrate by covalent hnkages. Such covalent linked enzyme-substrate complexes form the corresponding products much faster than compared to the reaction rate in a non-catalyzed reaction. Examples of enzyme functional groups which are involved in covalent bonding and are responsible for the transient intermediates of an enzyme-substrate complex are compiled in Table 2.8. Nucleophilic catalysis is dominant (examples 1-6, Table 2.8), since amino acid residues are present in the active site of these enzymes, which 

The endiol formed from dihydroxyacetone-3-phosphate in the presence of enzyme isomerizes into glyceraldehyde-3-phosphate. 

These two examples show clearly the significant differences to chemical reactions in solutions. The enzyme driven acid-base catalysis takes place selectively at a certain locus of the active site. The local concentration of the amino acid residue acting as acid or base is fairly high due to the perfect position relative to the substrate. On the other hand, in chemical reactions in solutions all reactive groups of the substrate are nonspecifically attacked by the acid or base. 

A number of peptidase and esterase enzymes react covalently in substitution reactions by a two-step nucleophilic mechanism. In the first step, the enzyme is acylated in the second step, it is dea-cylated. Chymotrypsin will be discussed as an example of this reaction mechanism. Its activity is dependent on His and Ser, which are positioned in close proximity within the active site of the enzyme because of folding of the peptide chain (Fig. 2.16). 

An exceptionally reactive serine residue has been identified in a great number of hydrolase enzymes, e. g., trypsin, subtilisin, elastase, acetylcholine esterase and some lipases. These enzymes appear to hydrolyze their substrates by a mechanism analogous to that of chymotrypsin. Hydrolases such as papain, ficin and bromelain, which are distributed in plants, have a cysteine residue instead of an active serine residue in their active sites. Thus, the transient intermediates are thioesters. 

In this process, the presence of p-nitrobenzoic acid (PNBA) as an additive was necessary to improve the yield of the product obtained as a single anh -diastereoisomer in good to excellent enantioselectivity. The presence of PNBA likely helped the formation of the iminium ion species. The final functionalised products have been further elaborated exploiting aldol and reduction reactions. 

The intermolecular aldol reaction of ketones with aldehydes has been intensively investigated since the seminal discovery of the i-proline catalysed process.Most of the modified organocatalysts, successively reported for this fundamental reaction, were amide derivatives of i-proline, able to work at lower loading in more environmentally friendly media and displaying better stereocontrol (Chapter 5). 

The heterogenised catalysts could be conveniently recovered together after an acidic treatment, reused for up to ten runs maintaining the level of enantioselectiviiy, although with a slight decrease in diol peld. 

The iminium strategy, elegantly illustrated by MacMillan, using chiral imidazolidinones as organocatalysts in a Diels-Alder reaction was then 

4 Reduction of Carbon-Oxygen, Carbon-Nitrogen and Carbon-Carbon Double Bonds 

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If the enzyme-catalyzed reaction is to be faster than the uncatalyzed case, the acceptor group on the enzyme must be a better attacking group than Y and a better leaving group than X. Note that most enzymes that carry out covalent catalysis have ping-pong kinetic mechanisms. 

As shown in Figure 16.10, this reaction mechanism involves nucleophilic attack by —SH on the substrate glyceraldehyde-3-P to form a covalent acylcysteine (or hemithioaeetal) intermediate. Hydride transfer to NAD generates a thioester intermediate. Nucleophilic attack by phosphate yields the desired mixed carboxylic-phosphoric anhydride product, 1,3-bisphosphoglycerate. Several examples of covalent catalysis will be discussed in detail in later chapters. 

CHYMOTRYPSIN FRUCTOSE-2,6-BISPHOSPHATASE ILLUSTRATE COVALENT CATALYSIS... 

Catalytic mechanisms employed by enzymes include the introduction of strain, approximation of reactants, acid-base catalysis, and covalent catalysis. 

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

The structural and chemical mechanisms used by enzymes to achieve transition state stabilization have been reviewed in detail elsewhere (e.g., see Jencks, 1969, Warshel, 1998, Cannon and Benkovic, 1998, Copeland, 2000, Copeland and Anderson, 2002 and Kraut et al., 2003). Four of the most common strategies used by enzymes for transition state stabilization—approximation, covalent catalysis, acid/base catalysis, and conformational distortion—are discussed below. 

Enzymes are often considered to function by general acid-base catalysis or by covalent catalysis, but these considerations alone cannot account for the high efficiency of enzymes. Proximity and orientation effects may be partially responsible for the discrepancy, but even the inclusion of these effects does not resolve the disparity between observed and theoretically predicted rates. These and other aspects of the theories of enzyme catalysis are treated in the monographs by Jencks (33) and Bender (34). 

In contrast to the reactions of the cycloamyloses with esters of carboxylic acids and organophosphorus compounds, the rate of an organic reaction may, in some cases, be modified simply by inclusion of the reactant within the cycloamylose cavity. Noncovalent catalysis may be attributed to either (1) a microsolvent effect derived from the relatively apolar properties of the microscopic cycloamylose cavity or (2) a conformational effect derived from the geometrical requirements of the inclusion process. Kinetically, noncovalent catalysis may be characterized in the same way as covalent catalysis that is, /c2 once again represents the rate of all productive processes that occur within the inclusion complex, and Kd represents the equilibrium constant for dissociation of the complex. 

Finally, in the sense that the imposition of conformational restrictions or specific solvent effects on an organic molecule are forms of strain, non-covalent catalysis by the cycloamyloses may provide a simple model for the investigation of strain and distortion effects in enzymatic reactions. 

Another alternative is for the enzyme to actually form a covalent bond between the enzyme and the substrate. This direct, covalent participation of the enzyme in the chemical reaction is termed covalent catalysis. The enzyme uses one of its functional groups to react with the substrate. This enzyme-substrate bond must form fast, and the intermediates must be reasonably reactive if this kind of catalysis is going to give a rate acceleration. 

A simple example of non-covalent catalysis is the intramolecular acyl transfer [3] to [4] which is catalysed by a-CD but retarded by /3-CD (Griffiths and Bender, 1973). As seen by the constants in Table 1, the... 

Table 1 Non-covalent catalysis of intramolecular acyl transfer [3]—> [4].°...

Many of the basic elements of enzyme catalysis have been illustrated here, including binding of substrate, multifunctional catalysis, microenvironmental effects, covalent catalysis, and strain effects. The most remarkable rate enhancements reported to date are those brought about by apolar derivatives of PEI, a polycation. These rate enhancements are very en-... 

Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product. Many of the enzymes that involve covalent catalysis are hydrolytic enzymes these include proteases, lipases, phosphatases and also acetylcholinesterase. Some of these enzymes possess a serine residue in the active site, which reacts with the substrate to form an acylenzyme intermediate that is attacked by water to complete the hydrolysis (Fignre 3.3). 

Figure 3.3 (a) Covalent catalysis the catalytic mechanism of a serine protease. The enzyme acetylcholinesterase is chosen to illustrate the mechanism because it is an important enzyme in the nervous system. Catalysis occurs in three stages (i) binding of acetyl choline (ii) release of choline (iii) hydrolysis of acetyl group from the enzyme to produce acetate, (b) Mechanism of inhibition of serine proteases by diisopropylfluorophosphonate. See text for details. 

Proton transfers are particularly common. This acid-base catalysis by enzymes is much more effective than the exchange of protons between acids and bases in solution. In many cases, chemical groups are temporarily bound covalently to the amino acid residues of the enzyme or to coenzymes during the catalytic cycle. This effect is referred to as covalent catalysis (see the transaminases, for example p. 178). The principles of enzyme catalysis sketched out here are discussed in greater detail on p. 100 using the example of lactate dehydrogenase. 

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