Aldol aldolases

Enzymatic synthesis relying on the use of aldolases offers several advantages. As opposed to chemical aldolization, aldolases usually catalyze a stereoselective aldol reaction under mild conditions there is no need for protection of functional groups and no cofactors are required. Moreover, whereas high specificity is reported for the donor substrate, broad flexibility toward the acceptor is generally observed. Finally, aldolases herein discussed do not use phosphorylated substrates, contrary to phosphoenolpyruvate-dependent aldolases involved in vivo in the biosynthetic pathway, such as KDO synthetase or DAHP synthetase [18,19]. 

Although a variety of secondary aldols can be prepared by aldolase antibody 38C2-catalyzed cross-aldol reactions, tertiary aldols are typically not accessible via intermolecular cross-aldol reactions. For preparation of enan-tiomerically enriched tertiary aldols, aldolase antibody 38C2-catalyzed retro-aldol reactions can be used (Section 6.3.2). 

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... 

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). 

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. 

Due to mechanistic requirements, most of these enzymes are quite specific for the nucleophilic component, which most often is dihydroxyacetone phosphate (DHAP, 3-hydroxy-2-ox-opropyl phosphate) or pyruvate (2-oxopropanoate), while they allow a reasonable variation of the electrophile, which usually is an aldehyde. Activation of the donor substrate by stereospecific deprotonation is either achieved via imine/enamine formation (type 1 aldolases) or via transition metal ion induced enolization (type 2 aldolases mostly Zn2 )2. The approach of the aldol acceptor occurs stereospecifically following an overall retention mechanism, while facial differentiation of the aldehyde is responsible for the relative stereoselectivity. 

The charged group introduced into products by the aldol donors (phosphate, carboxylate) facilitates product isolation and purification by salt precipitation and ion exchange techniques. Although many aldehydic substrates of interest for organic synthesis have low water solubility, at present only limited data is available on the stability of aldolases in organic cosolvents, thus in individual cases the optimal conditions must be chosen carefully. 

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 ... 

Similar to DHAP aldolases, the 3-hexulose 6-phosphate aldolase found in Methylomonas Ml 5 is highly specific for the aldol donor component D-ribulose 5-phosphate, but accepts a wide variety of aldehydes as replacement for formaldehyde as the acceptor. With propanal,... 

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... 

Like many other antibodies, the activity of antibody 14D9 is sufficient for preparative application, yet it remains modest when compared to that of enzymes. The protein is relatively difficult to produce, although a recombinant format as a fusion vdth the NusA protein was found to provide the antibody in soluble form with good activity [20]. It should be mentioned that aldolase catalytic antibodies operating by an enamine mechanism, obtained by the principle of reactive immunization mentioned above [15], represent another example of enantioselective antibodies, which have proven to be preparatively useful in organic synthesis [21]. One such aldolase antibody, antibody 38C2, is commercially available and provides a useful alternative to natural aldolases to prepare a variety of enantiomerically pure aldol products, which are otherwise difficult to prepare, allovdng applications in natural product synthesis [22]. 

Figure 10.11 Aldol reactions catalyzed in vivo by the 2-keto-3-deoxy-6-phospho-o-gluconate and 2-keto-3-deoxy-6-phospho-o-galactonate aldolases.

Figure 10.19 Oxidative enzymatic generation of dihydroxyacetone phosphate in situ for stereoselective aldol reactions using DHAP aldolases (a), and extension by pH-controlled, integrated precursor preparation and product liberation (b).

Figure 10.21 Aldolase-catalyzed asymmetric synthesis of uncommon L-configured sugars (a), and selected examples of carbohydrate-related product structures that are accessible by enzymatic aldolization (b).

Such aldolase-catalyzed bidirectional chain elongation ( tandem aldolization) of simple, readily available dialdehydes has been developed into an efficient method for the generation of higher carbon sugars (e.g. (87)/(89)) by simple one-pot operations (Figure 10.32) [126,156]. The choice offuranoid (87) or pyranoid (89) nature of the products can be determined by a suitable hydroxyl substitution pattern in a corresponding cycloolefinic precursor (86) versus (88)). The overall specific substitution... 

Application of an aldolase to the synthesis of the tricyclic microbial elicitor (-)-syringolide (Figure 10.34) is another excellent example that enzyme-catalyzed aldolizations can be used to generate sufficient quantities of enantiopure material in multistep syntheses of complex natural and unnatural products [159]. Remarkably, the aldolase reaction established absolute and relative configuration of the only chiral centers that needed to be externally induced in the adduct (95) from achiral precursor (94) during the subsequent cyclization events, all others seemed to follow by kinetic preference. 

The metabolism of P-hydroxy-a-amino adds involves pyridoxal phosphate-dependent enzymes, dassified as serine hydroxymethyltransferase (SHMT) (EC 2.1.2.1) or threonine aldolases (ThrA L-threonine selective = EC 4.1.2.5, L-aHo-threonine selective = EC 4.1.2.6). Both enzymes catalyze reversible aldol-type deavage reactions yielding glycine (120) and an aldehyde (Eigure 10.45) [192]. 

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