Enzyme Aldol condensation

From the many enzymes that are known to make and break C-C bonds, we first chose the two transferases, transketolase (TKT) and transaldolase (TAL), both from the Gram-negative bacterium Escherichia coli. While project B21 evolved, we learned that this microorganism holds other and so far unknown enzymes which are of interest for asymmetric syntheses. One transketolase-like enzyme, 1-deoxy-D-xylulose 5-phosphate synthase (DXS), turned out to be the first enzyme of a novel biosynthetic pathway leading to isoprenoids in bacteria, algae, and plants. The other, fructose 6-phosphate aldolase (ESA) - while similar to transaldolase - allows the direct use of the inexpensive dihydroxyacetone in aldol condensations. 

The medicinal chemistry of Alzheimers is complicated by the fact that the etiology of this disease is still far from clear. Evidence points to an association with decreased levels of acetyl choline in the brain. Many of the drugs that have been introduced to date for treating this disease thus comprise agents intended to raise the deficient levels of that neurotransmitter by inhibiting the loss of existing acetylcholine due to the action of cholinesterase. A compound based on an indene that, perhaps surprisingly, inhibits that enzyme has been proposed for the treatment of Alzheimer s. Aldol condensation of piperidine aldehyde (4-2) with the indanone (4-1) from cyclization of 3,4-dimethoxycinnamic acid leads to the olefin (4-3). Catalytic reduction removes the double bond to afford donepezil (4-4) [3]. 

Erom the results presented in Figure 6.4 it is clear that the loss of enzyme activity over time is dramatic when either ClAA or AA concentrations exceed 100 mM, resulting in half-life times in the range of 5-7 h at industrially relevant concentrahons. The first aldol condensation product also rapidly inactivates DERA at concentrahons above 100 mM. Although the final product 1 seems to have a less pronounced effect on the stability of the enzymes. Figure 6.4 indicates that the achvity in the presence of the compound is low. 

Cleavage of Fructose 1,6-Bisphosphate The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase, catalyzes a reversible aldol condensation (p. 485). Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, glyceraldehyde 3-phosphate, an aldose, and dihydroxyacetone phosphate, a lcetose ... 

C2 conformation, unlike Neu5Ac occurring in the 2CS conformation), because the conformation of the aldol condensation products is due to the stereochemistry of the substrate on C-3. But in addition to that, the 3-position on the substrate is critical for the enzyme stereoselectivity. Indeed we first observed that condensation of D-arabinose with pyruvate in tenfold excess led to two diastereomers, 4-epi-KDO and KDO in the 56 44 ratio (Scheme 4) [35],... 

From this we can conclude that two pKa values can be as much as eight units apart and AG will still be less than 50 kj / mol, low enough to permit rapid enzymatic reactions. However, for transfer of a proton from a C-H bond to a catalytic group, for example, to form an enolate ion for an aldol condensation (Chapter 13), the intrinsic barrier is known to be about 50 kj / mol.141 In this case, to allow rapid enzymatic reaction either the thermodynamic barrier must be very low, as a result of closely matching pKa values, or the enzyme must lower the intrinsic barrier. It may do both. 

When an ionic organic reaction (the kind catalyzed by most enzymes) occurs a nucleophilic center joins with an electrophilic center. We use arrows to show the movement of pairs of electrons. Tire movement is always away from the nucleophile which can be thought of as "attacking" an electrophilic center. Notice the first step in the second example at right. The unsaturated ketone is polarized initially. However, this is not shown as a separate step. Rather, the flow of electrons from the double bond, between the a- and (1-carbons into the electron-accepting C=0 groups, is coordinated with the attack by the nucleophile. Dotted lines are often used to indicate bonds that will be formed in a reaction step, e.g., in an aldol condensation (right). Dashed or dotted lines are often used to indicate partially formed and partially broken bonds in a transition state, e.g., for the aldol condensation (with prior protonation of the aldehyde). However, do not put arrows on transition state structures. 

Polycarboxylic acid synthases. Several enzymes, including citrate synthase, the key enzyme which catalyzes the first step of the citric acid cycle, promote condensations of acetyl-CoA with ketones (Eq. 13-38). An a-oxo acid is most often the second substrate, and a thioester intermediate (Eq. 13-38) undergoes hydrolysis to release coenzyme A.199 Because the substrate acetyl-CoA is a thioester, the reaction is often described as a Claisen condensation. The same enzyme that catalyzes the condensation of acetyl-CoA with a ketone also catalyzes the second step, the hydrolysis of the CoA thioester. These polycarboxylic acid synthases are important in biosynthesis. They carry out the initial steps in a general chain elongation process (Fig. 17-18). While one function of the thioester group in acetyl-CoA is to activate the methyl hydrogens toward the aldol condensation, the subsequent hydrolysis of the thioester linkage provides for overall irreversibility and "drives" the synthetic reaction. 

The aldol condensation and the reverse cleavage reaction catalyzed by these enzymes both involve a Schiff base. The cleavage reaction is similar to the acetoac-etate decarboxylase mechanism, with the protonated imine being expelled. The condensation reaction illustrates the other function of a Schiff base, the activation of carbon via an enamine (equation 2.40). 

Further developments are shown in Figure 4. On the basis that glucosamine reacted with pyruvic acid in the presence of alkali to yield pyrrole-2-carboxylic acid, in 1% yield, Gottschalk (21) proposed that sialic acid was formed by an aldol condensation reaction between N-ace-tylglucosamine and pyruvic acid. Kuhn and Brossmer (15) and Zilliken and Glick (22) showed that the reverse reaction also took place under alkaline conditions. Cornforth, Firth, and Gottschalk (23) synthesized crystalline N-acetylneuraminic acid (NANA) from N-acetylglucosamine and oxaloacetic acid (pH 11, 20°C). Under conditions less subject to misinterpretation, Heimer and Meyer (24) found that Vibrio cholerae enzymes cleaved NANA into an N-acetylhexosamine and pyruvic acid. 

Fructose-1,6-bisphosphate is cleaved by aldolase into two molecules of triose phosphate. This reaction represents the reversal of an aldol condensation (fig. 12.20). Most aldolases are highly specific for the upper end of the substrate molecule, requiring a phosphate group at C-l, a carbonyl at C-2, and specific steric configurations at C-3 and C-4. The nature of the remainder of the molecule is unimportant as far as the enzyme action is concerned. 

This reaction is an aldol condensation, in which a carbanion generated at C-2 of the acetyl group (by loss of a proton to water or to an acceptor group on the enzyme) adds to the carbonyl group of oxaloacetate (fig. 13.7). Coenzyme A is released from the product while it is still bound to the enzyme, so that the products of the reaction are free coenzyme A and citrate. 

The heat-labile enzyme which catalyzes this aldol condensation was purified eightfold from the extracts of E. coli. By combining this phos-phodesoxyriboaldolase with purified phosphoriboaldolase from yeast, Rackerl 7(b)-108 was able to demonstrate the long sought conversion of D-ribose into desoxy-D-ribose, e. g.,... 

On the basis of a catalytic system previously developed by the same group, Nicholls and collaborators [51] reported the preparation of an imprinted polymer for enantioselective formation of a C-C bond with properties of a metallo-enzyme aldolase type II. Polymers were imprinted using the two enantiomers of a 1,3-diketone, the (l.S, 35,45)-(75), and the corresponding (l/ ,3/ ,4/ )-(75), together with two 4-vinyl-pyridine held in place by a Co(II). The cross-aldol condensation... 

N-Acetyl-L-phenylalanylsarcosine amide, 348 O-Acetylserine, 148 N-0 Acyl migrations, 157 Acyl-enzyme, 342-350 Z-0-Acylisoamide, 295 1,4-Addition, 221-242 1,6-Addition, 231 L-Ala-L-Ala-pNA,350 f-Ala-T-Pro-pNA, 350 AicohoT dehydrogenase, 340-341 Aldehydes, 209-211 Aldol condensation, 304-306 2-Alkoxytetrahydrofuran, 86 2-Alkoxytetrahydropyran, 18, 85-90 Alkylation of enamine, 282 Alkylation of enolate, 280 C and 0-Alkylation, 240 O-Alkylbenzohydroximoyl chloride, 155... 

The only useful Cannizzaro reactions involving the use of aldehydes having one or two a-hydrogen atoms are those already described, in which the aldehyde first undergoes an aldol condensation. The direct dismutation of aldehydes of these types has been carried out successfully only by means of enzyme systems or catalytic metals (p. 95). Such reactions do not represent the true Cannizzaro reaction and as yet have found little practical use. The smooth and practically quantitative dismutation of straight-chain aliphatic aldehydes of four to seven carbon atoms under the influence of the enzymes of hog-liver mash 6 suggests that practical applications of this method may be found. 

Reymond and Chen88 have investigated the same set of antibodies for their ability to catalyze bimolecular aldol condensation reactions. The antibodies were assayed individually at pH 8.0 for the formation of aldol 111 from aldehyde 109 and acetone. None catalyzed the direct reaction, but in the presence of amine 110 three anti-52a and three anti-52b antibodies showed modest activity. In analogy with natural type I aldolase enzymes, the reaction is believed to occur by formation of an enamine from acetone and the amine, followed by rate-determining condensation of the enamine with the aldehyde. As in the previous example, the catalyst, which was characterized in detail, is not very efficient in absolute terms ( cat = 3 x 10-6 s 1 for the anti-52b antibody 72D4), but it is approximately 600 times more effective than amine alone. Moreover, the reactions with the antibody are stereoselective The enamine adds only to the si face of the aldehyde to give... 

---