Enzymes Catalysed aldol reactions

More effort was therefore invested in the application of synthetic methodologies for these alkaloids and some straightforward chemo-enzymatic approaches were recently developed [150]. An enzyme-catalysed aldol reaction was again a crucial step in that synthetic route and is strongly reminiscent of Wong s research strategy relating to the chemo-enzymatic synthesis of pyrrolizidines mentioned earlier. 

Synthesis of syringolide using an enzyme-catalysed aldol reaction... 

The solution to the chemoselectivity problem is minimal protection ap-mcthoxybcnzyl group for the hydroxyaldehyde and an acetal 158 for the diol unit in the product 157 of the enzyme-catalysed aldol reaction. The solution to the alkene geometry problem is going to be intramolecular trapping by the one remaining free OH group. 

A Wadsworth-Emmons reaction of the phosphonate (250) with the aldehyde (251) has been used as a key step in a total synthesis of analogues (252) and (253) of topostin B-1, an inhibitor of mammalian DNA topoisomerase The enzyme-catalysed aldol condensation between the phosphonate aldehyde (254) and dihydroxyacetone phosphate is followed by spontaneous intramolecular olefination of the product to give the cyclitol (256) in spite of the reaction being below pH 7 at all times.Attempts at a similar reaction of the homologue (255) were unsuccessful probably because (255) is a poor substrate for the aldolase. 

A comprehensive review (260 refs.) on the synthesis of carbohydrates from noncarbohydrate sources covers the use of benzene-derived diols and products of Sharpless asymmetric oxidation as starting materials, Dodoni s thiazole and Vogel s naked sugar approaches, as well as the application of enzyme-catalysed aldol condensations. The preparation of monosaccharides by enzyme-catalysed aldol condensations is also discussed in a review on recent advances in the chemoenzymic synthesis of carbohydrates and carbohydrate mimetics, in parts of reviews on the formation of carbon-carbon bonds by enzymic asymmetric synthesis and on carbohydrate-mediated biochemical recognition processes as potential targets for drug development, as well as in connection with the introduction of three Aldol Reaction Kits that provide dihydroxyacetone phosphate-dependent aldolases (27 refs.). A further review deals with the synthesis of carbohydrates by application of the nitrile oxide 1,3-dipolar cycloaddition (13 refs.). ... 

A similar aldol reaction is encountered in the Krebs cycle in the reaction of acetyl-CoA and oxaloacetic acid (see Section 15.3). This yields citric acid, and is catalysed by the enzyme citrate synthase. This intermediate provides the alternative terminology for the Krebs cycle, namely the citric acid cycle. The aldol reaction is easily rationalized, with acetyl-CoA providing an enolate anion nucleophile that adds to the carbonyl of oxaloacetic acid. We shall see later that esters and thioesters can also be converted into enolate anions (see Section 10.7). 

The reaction catalysed by citrate synthase in the Krebs cycle (see Section 15.3) is primarily an aldol reaction, but the subsequent step, hydrolysis of a thioester linkage, is also catalysed by the same enzyme. This is shown below. 

In several recent applications of enzyme catalysis, the snbstrates on which the enzymes act are not the kind of snbstrates that are natnral to the enzyme. However, enzyme catalysed synthesis of hexoses in the laboratory depends solely on enzymes acting on natural or near natnral snbstrates. The relevant enzymes are the aldolases (EC 4.1.2 aldehyde-lyases) since they catalyse an aldol type of C-C bond forming aldol addition reaction. The aldolases most commonly join two C-3 units, called donor and acceptor, and two new stereocentra are formed with great stereoselectivity. 

The shikimate pathway begins with a coupling of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate to give the seven-carbon 3-deoxy-D-araWno-heptulosonic acid 7-phosphate (DAHP) (Figure 4.1). This reaction, shown here as an aldol-type condensation, is known to be mechanistically more complex in the enzyme-catalysed version several of the other transformations in the pathway have also been found to be surprisingly... 

Primary amine catalysis (usually involving a lysine residue) has been recognised to play an important role in various enzyme-catalysed reactions. Examples are the conversion of acetoacetate to acetone catalysed by acetoacetate decarboxylase, the condensation of two molecules of S-aminolevulinic acid catalysed by -aminolevulinic deshydratase during the biosynthesis of porphyrins, and the reversible aldol condensation of dihydroxy-acetone phosphate with glyceraldehyde which in the presence of aldolase yields fructose-1-phosphate (64) (For reviews see, for example, Snell and Di Mari,... 

This NADPH reaction is typically stereo- and chemoselective, though the stereochemistry is rather wasted here as tile next step is a dehydration, typical of what is now an aldol product, and occurring by an enzyme-catalysed ElcB mechanism. 

Mammals produce sialic acid by aldolic condensation of phosphoenolpyruvate and Ai-acetylmannosamine 6-phosphate (reaction 12.1). A kinase enzyme catalyses the phosphorylation of A -acetylmannosamine and a phosphatase catalyses the hydrolysis of the phosphate of sialic acid. These phosphorylation and dephosphorylation steps are irreversible, such that the synthesis can be total even with low concentrations of the substrate. A variation of reaction (12.1), observed with the bacterium Neisseria meningitidis, uses non-phosphated /-acetylmannosamine. However, these were not the enzymes used in the preparative synthesis, which used instead a microbial aldolase which catalyses equilibrium (12.2). This enzyme probably plays a catabolic role in these organisms, but it functions in the synthetic sense in the presence of an excess of pyruvate. 

An enzymic counterpart of these complex base-catalysed rearrangements of sugars may be the reaction catalysed by 4-phospho-3,4-dihydroxy-2-butanone synthetase. The enzyme catalyses the formation of the eponymous intermediate in secondary metabolism from ribulose 5-phosphate. Labelling studies indicated that C1-C3 of the substrate became C1-C3 of the product, that H3 of the substrate derived from solvent and that C4 was lost as formate. X-ray crystal structures of the native enzyme and a partly active mutant in complex with the substrate are available. The active site of the enzyme from Met ha-nococcus jannaschii contains two metals, which can be any divalent cations of the approximate radius of Mg " or Mn ", the two usually observed. Their disposition is very reminiscent of those in the hydride transfer aldose-ketose isomerases, but also to ribulose-5-phosphate carboxylase, which works by an enolisation mechanism, so the enolisation route suggested by Steinbacher et al. is repeated in Figure 6.14, as is the Bilik-type alkyl group shift, for which an equivalent reverse aldol-aldol mechanism cannot be written. 

One successful approach is to use the enzyme DERA (chapter 29) to combine two molecules of MeCHO and one of C1CH2CH0 in a double aldol reaction. Each aldol creates a new chiral centre the first is controlled entirely by the enzyme but the second, using the chiral aldehyde 189 as the electrophile, could be affected by 1,3-control.31 As might be expected for an enzyme-catalysed reaction, the catalyst dominates and the same stereoselectivity is found 190. The product is isolated in the form of the lactol 191. 

Non-enzyme-catalysed bond-forming and -breaking reactions, such as the aldol and Claisen condensations, require strongly basic conditions to form the carbanion intermediates. In living systems, the carbanion formed in Claisen-type condensation is the a-anion of an acylthioester, usually acyl coenzyme A, rather than the usual acyl oxygen ester. 

We mentioned the citric acid cycle earlier but we have not so far discussed the chemistry involved. The citric acid cycle allows metabolism to shunt carbon atoms between small molecules, and the key step is the synthesis of citric add from oxaloacetate and acetyl CoA. The reaction is essentially an aldol reaction between the enol of an acetate ester and an electrophilic ketone, and the enzyme which catalyses the reaction is known as citrate synthase. 

The next step is reduction of the ketone group. This NADPH reaction is typically stereo-and chemoselective, although the stereochemistry is rather wasted here as the next step is a dehydration, typical of what is now an aldol product, and occurring by an enzyme-catalysed ElcB mechanism. The elimination is known to be a cis removal of H and OH, and the double bond is exclusively trans ( ). Finally in this cycle, the double bond is reduced using another molecule of NADPH to give the saturated side chain. 

The scope of this chapter does not allow nor attempt a comprehensive account of all developed processes to date. A detailed summary, in particular of aldol, Mannich, or ot-functionalisation reactions, can be found in excellent reviews written on the topic." Barbas and List reported an asymmetric, direct, intermolecular aldol reaction of acetones and aldehydes (Scheme 5.4), presumably via enamine formation of proline and acetone. As compared to its metal-catalysed alternatives, no preformation of the respective enolate is required, a mode of action that mimics metal-free aldolase enzymes. ... 

From a synthetic point of view, aldolases offer a number of advantages as catalysts for C—C bond formation. For example, they operate best on unprotected substrates, thus avoiding the problem of complex protection/ deprotection schemes for polyfunctional molecules (e.g. carbohydrates). They also catalyse C—C bond synthesis with high diastereoselectivity and enantioselectivity. Such simultaneous control is often difficult to achieve using non-enzymic aldol reactions. 

Fructose-b/fphosphate aldolase, aldolase (EC 4.1.2.13) a tetrameric lyase which reversibly cleaves fructose l,6-f>irphosphate into the two triose phosphates, dihydroxyacetone phosphate and o-glyceral-dehyde phosphate. The reaction is analogous to the aldol condensation (CH3CHO + CH3CHO -> CH3-CHOH-CH2-CHO), hence the name of the enzyme. The equilibrium concentrations are 89% fructose huphosphate and 11 % triose phosphate. The enzyme catalyses the condensation of a number of aldehydes with dihydroxyacetone phosphate, and can also cleave fructose 1-phosphate. Liver aldolase (aldolase B, M, 156,000, 4 subunits of A/, 39,000) cleaves fructose l,6-6isphosphate and fructose 1-phosphate at nearly the same rate. Muscle aldolase (aldolase A, M, 1, 000,4 subunits of M 41,000, pi 6.1), however, is more active with the hirphosphate. Aldolase from yeast is inhibited by cysteine, and reactivated by Fe, Zii and Co. Spinach leaf aldolase has a M, of only 120,000 (M, of subunits 30,000). 

The combination of organocatalysts and enzymes remains rare, and the first examples of asymmetric tandem reactions catalysed by this type of catalyst combination have been described only recently. For example in 2004, Cordova et al. worked out a one-pot procedure involving L-proline as catalyst in the first step of the reaction and the enzyme Amano I (lipase extracted from Pseudomonas cepacia) in the second step. As shown in Scheme 9.1, the aldol reaction between an aldehyde and acetone occurred to give the corresponding intermediate p-hydroxy ketone, which was subsequently submitted to kinetic resolution by treatment with the enzyme, affording the corresponding almost enantiopure acetate in moderate yields. 

This enzyme catalyses the decarboxylation of the ) -ketoacid oxaloacetate, with the same stoichiometry as acetoacetate decarboxylase. The former however, requires a Mn ion for activity and is insensitive to the action of sodium borohydride. This duality of mechanism is not unlike the one observed for enzymatic aldol condensation, where enzymes of Class 1 react by forming Schiff-base intermediates, whereas enzymes of Class II show metal ion requirements [47]. Oxaloacetate decarboxylase from cod also catalyses the reduction by borohydride of the enzymatic reaction product pyruvate. This is evidenced by the accumulation of D-lactate in presence of enzyme, reducing agent, and manganous ions. It has been proposed that both reduction and decarboxylation occur by way of an enzyme-metal ion-substrate complex in which the metal ion acts as an electron sink, thereby stabilizing the enolate ion formed in the decarboxylation reaction [48] ... 

By the reaction of D-glyceraldehyde and 1,3-dihydroxypropane (both as monophosphate ester), D-fructose as the 1,6-diphosphate ester is formed. The process is readily reversible and is catalysed by an enzyme known as aldolase. This is a type of aldol addition (known as biological aldol addition) and is one of the reaction in the metabolism of carbohydrates by the glycolic pathway. 

---