Enzyme-catalyzed aldol addition aldolase

Recent advances in enzyme-catalyzed aldol addition reactions for biocatalytic carbon-carbon bond formation by means of aldolases are described. They offer an exceptionally stereoselective and green tool for asymmetric framework construction and preparation of innovative molecules with engineered enz5unes. 

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). 

As pointed out before, the mechanism of aldolase-catalyzed aldol addition is different for class I and class II enzymes. 

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... 

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. 

Aldolases are part of a large group of enzymes called lyases and are present in all organisms. They usually catalyze the reversible stereo-specific aldol addition of a donor ketone to an acceptor aldehyde. Mechanistically, two classes of aldolases can be recognized [4] (i) type I aldolases form a Schiff-base intermediate between the donor substrate and a highly conserved lysine residue in the active site of the enzyme, and (ii) type II aldolases are dependent of a metal cation as cofactor, mainly Zn, which acts as a Lewis acid in the activation of the donor substrate (Scheme 4.1). 

Here, we will focus on the enzymatic routes since enzymatic preparation of DHAP is usually coupled with the aldol addition catalyzed by the aldolase representing genuine multi-enzyme systems. 

Asymmetric C-C bond formation is the most important and most challenging problem in synthetic organic chemistry. In Nature, such reactions are facilitated by lyases, which catalyze the addition of carbonucleophiles to C=0 double bonds in a manner that is classified mechanistically as an aldol addition [1]. Most enzymes that have been investigated lately for synthetic applications include aldolases from carbohydrate, amino acid, or sialic acid metabolism [1, 2]. Because enzymes are active on unprotected substrates under very mild conditions and with high chemo-, regio-, and stereoselectivity, aldolases and related enzymes hold particularly high potential for the synthesis of polyfunctionalized products that are otherwise difficult to prepare and to handle by conventional chemical methods. 

Another promising route was reported in patent and open hterature by both DSM and Diversa [13, 14]. This route employs a 2-deoxy-D-ribose 5-phosphate aldolase (DERA) that catalyzes a tandem aldol addition in which two equivalents of acetaldehyde (AA) are added in sequence to chloroacetaldehyde (CIAA) to produce a lactol derivative that is similar to the 3,5-dihydoxy side chain of synthetic statins (Figure 6.2e). Diversa screened environmental libraries for novel wild-type DERAs and identified an enzyme that was both tolerant to increased substrate concentrations and more active than DERA from E. coli in the target reaction [13]. 

One of the reactions in the metabolism of carbohydrates by the glycolytic pathway is a type of aldol addition. In this reaction D-fructose (as the 1,6-diphosphate ester) is formed from D-glyceraldehyde and 1,3-dihydroxypro-panone (both as monophosphate esters). The process is readily reversible and is catalyzed by an enzyme known as aldolase ... 

An asymmetric C-C coupling, one of the most important and challenging problems in synthetic organic chemistry, seems to be most appropriate for the creation of a complete set of diastereomers because of the applicability of a convergent, combinatorial strategy [38-40]. In Nature, such reactions are facilitated by lyases which catalyze the (usually reversible) addition of carbo-nucleophiles to C=0 double bonds, in a manner mechanistically most often categorized as aldol and Claisen additions or acyloin reactions [41], The most frequent reaction type is the aldol reaction, and some 30 lyases of the aldol type ( aldolases ) have been identified so far [42], of which the majority are involved in carbohydrate, amino acid, or hydroxy acid metabolism. This review will focus on the current state of development of this type of enzyme and will outline the scope and limitations for their preparative application in asymmetric synthesis. 

In addition to the larger families of preparatively useful aldolases, some less common aldolases have been evaluated lately for preparative use. A range of mechanistically distinct enzymes, which are actually categorized as transferases but which also catalyze aldol-related additions through the aid of cofactors such as pyridoxal 5-phosphate (PLP), thiamine pyrophosphate (TPP), tetrahydro-folate (THF), or coenzyme A (CoA), are becoming more frequently applied in organic synthesis. Because of their emerging importance and/or commercial availability, a selection of these enzymes and examples of their synthetic utility will be included in further separate chapters. 

Functionally and mechanistically reminiscent of the pyruvate lyases, the 2-deoxy-D-ribose 5-phosphate (121) aldolase (RibA EC 4.1.2.4) [363] is involved in the deoxynucleotide metabolism where it catalyzes the addition of acetaldehyde (122) to D-glyceraldehyde 3-phosphate (12) via the transient formation of a lysine Schiff base intermediate (class I). Hence, it is a unique aldolase in that it uses two aldehydic substrates both as the aldol donor and acceptor components. RibA enzymes from several microbial and animal sources have been purified [363-365], and those from Lactobacillus plantarum and E. coli could be induced to crystallization [365-367]. In addition, the E. coli RibA has been cloned [368] and overexpressed. It has a usefully high specific activity [369] of 58 Umg-1 and high affinity for acetaldehyde as the natural aldol donor component (Km = 1.7 mM) [370]. The equilibrium constant for the formation of 121 of 2 x 10M does not strongly favor synthesis. Interestingly, the enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates propional-dehyde 111, acetone 123, or fluoroacetone 124 can replace 122 as the donor [370,371], and a number of aldehydes up to a chain length of 4 non-hydrogen atoms are tolerated as the acceptor moiety (Table 6). 

Fructose 1,6-biphosphate aldolase from rabbit muscle in nature reversibly catalyzes the addition of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde 3-phosphate. The tolerance of this DHAP-dependent enzyme towards various aldehyde acceptors made it a versatile tool in the synthesis of monosaccharides and sugar analogs [188], but also of alkaloids [189] and other natural products. For example, the enzyme-mediated aldol reaction of DHAP and an aldehyde is a key step in the total synthesis of the microbial elicitor (—)-syringolide 2 (Fig. 35a) [190]. 

FDP aldolase catalyzes the reversible aldol addition reaction of DHAP and d-glyceraldehyde 3-phosphate (D-Gly 3-P) to form d-FDP (Fig. 14.1-1). The equilibrium constant for this reaction has a value of -104 m-1 in favor of FDP formation. The enzyme has been isolated from a variety of eukaryotic and prokaryotic sources, both in type I and type II forms[7 21). Generally, the type I FDP aldolases exist as tetramers (M.W. 160 KDa), while the type II enzymes are dimers (M. W. 80 KDa). For the... 

Figure 14.1-26. Aldol addition reaction catalyzed in v/Vo by KHG aldolase and the donor substrate specificity of this enzyme.

Because o-glucose has twice as many carbons as pymvate, it should not be surprising that one of the steps in the biosynthesis of o-glucose is an aldol addition. An enzyme called aldolase catalyzes an aldol addition between dihydroxyacetone phosphate and... 

The substrate for the first enzyme-catalyzed reaction of glycolysis is a six-carbon compound (o-glucose). The final product of glycolysis is two molecules of a three-carbon compound (pyruvate). Therefore, at some point in the series of enzyme-catalyzed reactions, a six-carbon compound must be cleaved into two three-carbon compounds. The enzyme aldolase catalyzes this cleavage (Figure 24.12). Aldolase converts o-fructose-l,6-diphosphate into o-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The enzyme is called aldolase because the reverse reaction is an aldol addition reaction (Section 19.13). The reaction proceeds as follows ... 

Aldolases are a group of C—C bond forming enzymes with widespread applications. The stereoselective aldol addition reaction catalyzed by aldolases represents an attractive alternative to conventional chiral organic chemistry methods for chemical and pharmaceutical industries. Aldolases are classified according to both their proposed catalytic mechanism and the structure of the donor substrate, their sources and microbial production processes being presented in this chapter. To design appropriate bioreactors for aldol synthesis, the characteristics of aldolase biocatalysts obtained after purification procedures in free and immobilized form are discussed. 

The FDP-aldolase catalyzed DHAP-addition to glyoxylic acid, examined in detail by Wandrey and Bossow-Berke under continuous flow reaction conditions [18], required the determination of the relative and absolute configuration. Employing the SAMP-/RAMP-hydrazone method, the aldol reaction of 1 with benzyl glyoxylate afforded a doubly protected adduct, which was deprotected with trifluoroacetic acid anhydride (TFAA) and subsequent debenzylation with H2/ Pd-C to give the final product, identical with the enzyme product. The independent chemical synthesis and the enzymatic route are compared in scheme 4. It... 

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