DHAP-dependent aldolases

The main group of aldolases from the biocatalytic point of view is, arguably, the one that uses dihydroxyacetone phosphate (DHAP) as donor. Here, we will concentrate on that appHcations in which DHAP-dependent aldolase are part of a multi-enzyme system or, alternatively, on those in which the aldolase-catalyzed reaction is key in a multi-step synthetic pathway. 

DHAP-dependent aldolases produce 2-keto-3,4-dihydroxy adducts with high control of the configuration of the two newly formed stereogenic centers. However, while it can be assumed that the absolute configuration at C3 is independent on the acceptor used in the reaction, the configuration of the stereocenter generated from the addition to the aldehyde (C4 position) in some cases may depend on the structure and stereochemistry of the acceptor [6]. 

Scheme 4.2 Complementary stereochemistry of DHAP-dependent aldolases.

The main drawback of the DHAP-dependent aldolases is their strict specificity for the donor substrate. Apart from the scope limitation that this fact represents, DHAP is expensive to be used stoichiometrically in high-scale synthesis, and labile at neutral and basic pH, and therefore its effective concentration decreases over time in enzymatic reaction media, hindering the overall yield of the aldol reaction. In addition, due to the presence of a phosphate group in both DHAP and the... 

DHAP-Dependent Aldolases in the Core of Aza Sugar Synthesis... 

DHAP-dependent aldolases Pyruvate dependent aldolases Deoxyribose 5 -phosphate aldolase —h... 

When DHAP-dependent aldolases are used as catalyst of the aldol reaction, a phosphorylated azido or amino polyhydroxyketone is obtained. The phosphate may be cleaved enzymatically or reductively cleaved under the hydrogenation conditions of the next step in which the azide is reduced to the amine. Intramolecular imine formation occurs spontaneously when the azide is reduced. The intramolecular reductive amination is the second key step of the aldolase-mediated synthesis of iminocyclitols. In general, delivery of hydrogen onto five- and six-membered ring imines occurs from the face opposite to the C4 hydroxyl group. 

Rare or unnatural monosaccharides have many useful applications as nonnutritive sweeteners, glycosidase inhibitors and so on. For example, L-glucose and L-fructose are known to be low-calorie sweeteners. In addition, rare or unnatural monosaccharides are potentially useful as chiral building blocks for the synthesis of biologically active compounds. Therefore, these compounds have been important targets for the development of enzymatic synthesis based in the use of DHAP-dependent aldolases alone or in combination with isomerases. Fessner et al. showed that rare ketose-1-phosphates could be reached not only by aldol addition catalyzed by DHAP-dependent aldolases, but by enzymatic isomerization/ phosphorylation of aldoses [35]. Thus, for example, L-fructose can be prepared... 

DHAP-dependent aldolases have also been used as key step in the synthesis of several complex natural products starting from achiral precursors. Thus, the sex pheromone (+)-exo-brevicomin can be synthesized in a multi-step route starting with the stereospecific aldol addition between DHAP and 5-oxohexanal or its 5-dithiane-protected analog catalyzed by FBPA from rabbit muscle ( RAMA ) as the key step by which the absolute configuration of the target is estabUshed (Scheme 4.16) [40]. 

DHAP-dependent aldolases have also been used in the synthesis of the C3-C9 fragment of aspicilin [46] and of the C12-C20 fragment of amphotericin [47]. 

Fructose-6-Phosphate Aldolase An Alternative to DHAP-Dependent Aldolases ... 

Although fructose-6-phosphate aldolase (FSA) does not belong to the DHAP-dependent aldolases group, it deserves to be mentioned in this chapter as it can be considered as an alternative to those enzymes, or at least, an alternative to FBPA. FSA was described for the first time by Schiirmann and Sprenger in E. coli K-12 strain MG1655 [50]. The enzyme is a class I aldolase with a homodecameric... 

The use of this enzyme in multi-step synthesis is relatively recent. Clapes et al. have reported the first example of FSA-mediated synthesis of iminocyclitols [53]. The synthetic strategy is similar to the one previously described for DHAP-dependent aldolases without the need for the dephosphorylation step. AldoUc reaction of DHA with N-Cbz-3-aminopropanal catalyzed by FSA followed by selective catalytic reductive aminahon furnishes the naturally occurring imino-sugar D-fagomine (Scheme 4.22). 

Scheme 6.27 Transketolase versus DHAP-dependent aldolase.

In contrast to transketolase and the DHAP-dependent aldolases, deoxyribose aldolase (DERA) catalyzes the aldol reaction with the simple aldehyde, acetaldehyde. In vivo it catalyzes the formation of 2-deoxyribose-5-phosphate, the building block of DNA, from acetaldehyde and D-glyceraldehyde-3-phosphate, but in vitro it can catalyze the aldol reaction of acetaldehyde with other non-phosphorylated aldehydes. The example shown in Scheme 6.28 involves a tandem aldol reaction... 

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]. 

Dihydroxyacetone phosphate (DHAP) is the donor ketone that is utilized by the DHAP-dependent aldolases. These aldolases come under the class of lyases, just like the hydroxynitrile lyases (see Section 5.2.1.1). As for the HNLs, no cofactor... 

Two new stereocenters are established in the DHAP-dependent aldolases-cata-lyzed carbon-carbon bond formation. Consequently four different stereoisomers can be formed (Scheme 5.23). Enantioselective aldolases that catalyze the formation of just one of each of the stereoisomers are available fructose 1,6-diphosphate aldolase (FDP A), rhamnulose 1-phosphate aldolase (Rha 1-PA), L-fucu-lose 1-phosphate aldolase (Fuc 1-PA) and tagatose 1,6-diphosphate aldolase (TDP A). In particular the FDP A, that catalyzes the formation of the D-threo stereochemistry, has been employed in many syntheses. One such FDP A that... 

Scheme 5.23 The four different stereoisomers that can be formed are synthesized selectively by four different DHAP-dependent aldolases.

A significant drawback of the DHAP-dependent aldolases is that DHAP cannot be replaced by dihydroxyacetone. DHAP is expensive and it is labile at neutral and basic pH values. It can be synthesized chemically, however the procedures are not very atom efficient. Therefore alternative enzyme-catalyzed approaches have been developed. A variety of them can be performed in the... 

Another problem of the DHAP-dependent aldolases is that the product is phos-phorylated. The aldol reaction has therefore to be followed by a dephosphorylation step. In the sequences in Schemes 5.26-5.28 this deprotection is performed in situ. Otherwise several enzymes, such as phytase, are available for this purpose and the reaction normally proceeds under mild conditions [40, 43, 46]. 

The first group is the dihydroxyacetone phosphate (DHAP)-dependent aldolases, which use DHAP as the donor to produce 2-keto-l, 3, 4-trihydroxy motifs. The second group, the pyruvate- or phosphoenol pyruvate (PEP)-dependent aldolases, uses pyruvate to form 4-hydroxy-2-ketoacids. The third... 

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