Aldolase addition

A salient shortcoming of the catalyst is the relatively low conversion rate obtained with any of the substrate analogs tested ( < 1 %) which in practice must be compensated by utilization of large amounts of enzyme and long reaction times. Thus, in reactions leading to thermodynamically unfavorable products it has been observed under equilibrating conditions that—in common with results discussed above for other aldolases — additions do not proceed fully stereospecifically at the reaction center [369],... 

Fig. 5. Peptide bonds in rabbit muscle and liver aldolases hydrolyzed by cathepsin M. The COOH-terminal sequences of both enzymes are shown. The primary sites of cleavage are shown by the large arrows and the secondary sites by the small arrows. With muscie aldolase, additional peptides derived from the last 20 amino acid residues, but not including proline 342, are recovered (unpublished observation).

This cleavage is a retro aldol reaction It is the reverse of the process by which d fruc tose 1 6 diphosphate would be formed by aldol addition of the enolate of dihydroxy acetone phosphate to d glyceraldehyde 3 phosphate The enzyme aldolase catalyzes both the aldol addition of the two components and m glycolysis the retro aldol cleavage of D fructose 1 6 diphosphate... 

TKsubstrate pNZYTffiS IN ORGANIC SYNTHESIS] (Vol 9) D-Glyceraldehyde-3-phosphate[591-57-l]aldolase-cataly zed additions... 

There are two distinct groups of aldolases. Type I aldolases, found in higher plants and animals, require no metal cofactor and catalyze aldol addition via Schiff base formation between the lysiae S-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily ia microorganisms and utilize a divalent ziac to activate the electrophilic component of the reaction. The most studied aldolases are fmctose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn " -containing aldolase from E. coli. In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetone phosphate [57-04-5] (DHAP). 

Fig. 6. FDP-aldolase-catalyzed addition of electrophiles (94) with DHAP (139—146). Representative R groups ia (94) are given as (a—j) (a) methyl, CH (b)...

KDPG is a member of a yet unexplored group of aldolases that utilize pymvate or phosphoenol pymvate as the nucleophile in the aldol addition. They are quite tolerant of different electrophilic components and accept a large number of uimatural aldehydes (148). The reaction itself, however, is quite specific, generating a new stereogenic center at the C-4 position. 

Aldol reactions occur in many biological pathways, but are particularly important in carbohydrate metabolism, where enzymes called aldolases catalyze the addition of a ketone enolate ion to an aldehvde. Aldolases occur in all organisms and are of two types. Type 1 aldolases occur primarily in animals and higher plants type II aldolases occur primarily in fungi and bacteria. Both types catalyze the same kind of reaction, but type 1 aldolases operate place through an enamine, while type II aldolases require a metal ion (usually 7n2+) as Lewis acid and operate through an enolate ion. 

Table t. Products from Complementary Aldolase Catalyzed Additions of Dihydroxyacetone Phosphate to Simple Aldehydes... 

Tabic 2. Fructose 1,6-Bisphosphate Aldolase Catalyzed Additions of Dihy-droxyacelone Phosphate to Sugar Phosphates... 

Table 3. Selection of Products Derived from Fructose 1,6-Bisphosphatc Aldolase Catalyzed Additions of Dihydroxyacetonc Phosphate to Respective Aldehydes...

Commercial A -acetylneuraminic acid aldolase from Clostridium perfringens (NeuAcA EC 4.1.3.3) catalyzes the addition of pyruvate to A-acetyl-D-mannosamine. A number of sialic acid related carbohydrates are obtained with the natural substrate22"24 or via replacement by aldose derivatives containing modifications at positions C-2, -4, or -6 (Table 4)22,23,25 26. Generally, a high level of asymmetric induction is retained, with the exception of D-arabinose (epimeric at C-3) where stereorandom product formation occurs 25 2t The unfavorable equilibrium constant requires that the reaction must be driven forward by using an excess of one of the components in order to achieve satisfactory conversion (preferably 7-10 equivalents of pyruvate, for economic reasons). 

Table 4. IV-Acetylneuraminic Acid Aldolase Catalyzed Preparative Aldol Additions with Pyruvate...

Deoxy-D- /rce/ o-D- a/ac7i7-nonulosonie Acid (KDN) V-Acetylneuraminic Acid Aldolase Catalyzed Preparative Aldol Additions with Pyruvate Typical Procedure27 ... 

Mechanistically similar to the pyruvate lyases, 2-deoxy-D-ribose 5-phosphate aldolase (EC 4.1.2.4) catalyzes the addition of acetaldehyde to D-glyceraldehyde 3-phosphate. 

A number of lyases are known which, unlike the aldolases, require thiamine pyrophosphate as a cofactor in the transfer of acyl anion equivalents, but mechanistically act via enolate-type additions. The commercially available transketolase (EC 2.2.1.1) stems from the pentose phosphate pathway where it catalyzes the transfer of a hydroxyacetyl fragment from a ketose phosphate to an aldehyde phosphate. For synthetic purposes, the donor component can be replaced by hydroxypyruvate, which forms the reactive intermediate by an irreversible, spontaneous decarboxylation. 

Unfortunately, the phosphorylated form of the starting aldehyde is expensive, and dephosphorylation by a phosphatase requires an additional step. Therefore, the challenge was to obtain a mutant aldolase that not only accepts nonphos-phorylated substrates but also turns over the enantiomeric aldehyde (29) stereoselectively with formation of (30), which is a precursor of carbohydrate (31) (see Scheme 2.8) [74] ... 

In another intriguing direded evolution study, the stereochemical course of aldol additions was significantly altered in a different sense [78] rather than evolving aldolase mutants that seledively accept stereoisomers of substrates, the... 

N-Acetylneuraminic acid aldolase (or sialic acid aldolase, NeuA EC 4.1.3.3) catalyzes the reversible addition of pyruvate (2) to N-acetyl-D-mannosamine (ManNAc (1)) in the degradation of the parent sialic acid (3) (Figure 10.4). The NeuA lyases found in both bacteria and animals are type I enzymes that form a Schiff base/enamine intermediate with pyruvate and promote a si-face attack to the aldehyde carbonyl group with formation of a (4S) configured stereocenter. The enzyme is commercially available and it has a broad pH optimum around 7.5 and useful stability in solution at ambient temperature [36]. 

The D-fructose 1,6-bisphosphate aldolase (FruA EC 4.1.2.13) catalyzes in vivo the equilibrium addition of (25) to D-glyceraldehyde 3-phosphate (GA3P, (18)) to give D-fructose 1,6-bisphosphate (26) (Figure 10.14). The equilibrium constant for this reaction of 10 strongly favors synthesis [34]. The enzyme occurs ubiquitously and has been isolated from various prokaryotic and eukaryotic sources, both as class I and class II forms [30]. Typically, class I FruA enzymes are tetrameric, while the class II FruA are dimers. As a rule, the microbial class II aldolases are much more stable in solution (half-lives of several weeks to months) than their mammalian counterparts of class I (few days) [84-86]. 

Even an entirely different enzyme can be changed to the one that has enolase activity. One representative example is the changing of a lipase to an aldolase utilizing the basicity of the catalytic triad via a simple mutation. The resulting promiscuous lipase has been demonstrated to catalyze the aldol reaction and Michael addition as shown in Fig. 23. 

Aldol addition, 2 63-64 acetone, 1 164 Aldolases, 3 675 4 711 Aldol process, for higher alcohol manufacture, 2 27t, 41-43 Aldonic acid, 14 132 Aldoses, 4 696... 

NAM is produced by base-catalysed epimerization of N-acetyl-o-glucosamine (NAG), generating an unfavourable 1 4 mixture of NAM NAG. NAG, although not a substrate for the aldolase, inhibits the reaction. In addition, excess pyruvate is required to push the equilibrium in favour of product formation (Scheme 1.31). Although 90% yields can be obtained at laboratory scale using E. coli NANA aldolase using a NAG NAM mixture, the NANA product is difficult to separate from the excess pyruvate required to achieve this. 

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