Aldolase DHAP-aldolases

Dihydroxyacetone phosphate-dependent aldolases (DHAP-aldolases) have been used widely for preparative synthesis of monosaccharides and sugar analogs (Fessner and Walter 1997 Wymer and Toone 2000 Silvestri et al. 2003). Among them, RAMA RhuA and FucA from E. coli are the most available aldolases, especially the former which was one of the first to be commercialized (Fessner and Walter 1997 Takayama et al. 1997). In many of the chemo-enzymatic strategies they are involved, the biocatalytic aldol addition to the configuration of the newly stereogenic centers is fixed by the enzyme. However, pertinent examples have been reported in which... 

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)...

When carbon rearrangements are balanced to account for net hexose synthesis, five of the glyceraldehyde-3-phosphate molecules are converted to dihy-droxyacetone phosphate (DHAP). Three of these DHAPs then condense with three glyceraldehyde-3-P via the aldolase reaction to yield 3 hexoses in the form... 

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. 

Four DHAP converting aldolases are known, these can synthesize different diastereomers with complementary configurations D-fructose (FruA EC 4.1.2.13) and D-tagatose 1,6-bisphos-phate (TagA, F.C 4.1.2.-), L-fuculose (FucA EC 4.1.2.17) and L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19)3. The synthetic application of the first (class 1 or 2) and the latter two types (class 2) has been examined. 

A mixture of dihydroxyacetone and inorganic arsenate can replace DHAP due to the transient formation of a monoarsenate ester which is recognized by the aldolase as a DHAP mimic21. This approach suffers from the high toxicity of arsenate, especially at the relatively high levels (>0.5 M) needed for efficient conversion, and from problems in product isolation. 

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,... 

Functionally related to FruA is the novel class I fructose 6-phosphate aldolase (FSA) from E. coli, which catalyzes the reversible cleavage of fructose 6-phosphate (30) to give dihydroxyacetone (31) and d-(18) [90]. It is the only known enzyme that does not require the expensive phosphorylated nucleophile DHAP for synthetic purpose. 

Figure 10.17 Kinetic enantiopreference of class II DHAP aldolases useful for racemic resolution of a-hydroxyaldehydes.

Apparently, all DHAP aldolases are highly specific for (25) as the donor component for mechanistic reason [30-33], a fact which requires an economical access to this compound for synthetic applications. Owing to the limited stability of (25) in solution, particularly at alkaline pH, it is preferentially generated in situ to avoid high stationary concentrations. 

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).

A tandem enzymatic aldol-intramolecular Homer-Wadsworth-Emmons reaction has been used in the synthesis of a cyclitol.310 The key steps are illustrated in Scheme 8.33. The phosphonate aldehyde was condensed with dihydroxyacetone phosphate (DHAP) in water with FDP aldolase to give the aldol adduct, which cyclizes with an intramolecular Horner-Wadsworth-Emmons reaction to give the cyclo-pentene product. The one-pot reaction takes place in aqueous solution at slightly acidic (pH 6.1-6.8) conditions. The aqueous Wittig-type reaction has also been investigated in DNA-templated synthesis.311... 

Aldolases catalyze asymmetric aldol reactions via either Schiff base formation (type I aldolase) or activation by Zn2+ (type II aldolase) (Figure 1.16). The most common natural donors of aldoalses are dihydroxyacetone phosphate (DHAP), pyruvate/phosphoenolpyruvate (PEP), acetaldehyde and glycine (Figure 1.17) [71], When acetaldehyde is used as the donor, 2-deoxyribose-5-phosphate aldolases (DERAs) are able to catalyze a sequential aldol reaction to form 2,4-didexoyhexoses [72,73]. Aldolases have been used to synthesize a variety of carbohydrates and derivatives, such as azasugars, cyclitols and densely functionalized chiral linear or cyclic molecules [74,75]. 

In the case of L-rhamnulose-1-phosphate aldolase (RhaD), we found that the problem of phosphorylated substrate requirement (dihydroxyactone phosphate (DHAP)) could be overcome by a simple change in buffer. Thus, when using borate buffer, reversible borate ester formation created a viable substrate out of dihydroxyacetone, which is not otherwise accepted by the wild-type enzyme (Figure 6.6) [23]. The process was used in a one-step synthesis of... 

The guilty party is the triose phosphate isomerase (TIM) reaction that interconverts DHAP and G3P. To be converted to pyruvate, the DHAP first has to be converted to G3P. TIM just moves the carbonyl group between the two carbons that don t have phosphate attached. TIM doesn t touch the phosphate. So, if the DHAP is labeled at the carbon that has the phosphate attached, the G3P that comes from DHAP will be labeled at the carbon with the phosphate attached. The carbon with the phosphate attached in the G3P that was produced directly by the aldolase reaction came from C-6 of glucose, but the carbon with the phosphate attached in the G3P that was produced from DHAP came from C-l of glucose. After TIM does it stuff, the carbon of G3P that has the phosphate will be... 

Recently, a chemoenzimatic catalized Henry reaction has been reported by El Blidi et al.53 Nitroaldol cyclization between the masked 3-hydroxy-4-nitrobutyraldehyde 72 and dihydroxyacetone phosphate (DHAP) 73, catalyzed by fructose-1,6-biphosphate aldolase (RAMA), afforded the nitro-cyclohexane 74 (Scheme 24). 

Of the known classes of aldolase, DERA (statin side chain) and pyruvate aldolases (sialic acids) have been shown to be of particular value in API production as they use readily accessible substrates. Glycine-dependent aldolases are another valuable class that allow access to p-hydroxy amino acid derivatives. In contrast, dihydroxy acetone phosphate (DHAP) aldolases, which also access two stereogenic centres simultaneously,... 

To this solution was added DHAP (1.61 mmol) followed by 30 mL water, and the pH was adjusted to 7.5 with 1 m NaOH. The mixture was bubbled with Ar and previously centrifuged aldolase" (60 U) was added. 

Figure 3. Selectivity of the FDP-aldolase reactions using DHAP vs. dihydroxyacetone/arsenate as a substrate. In the former case, the more stable sugar is obtained due to the reversible nature of the reaction. In the later case, both sugars were obtained in nearly equal amounts, because the reaction was found to be virtually irreversible and the formation of the arsenate ester was rate limiting.

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]. 

An advantage of these enzymes is that they are stereocomplementary, in that they can synthesize the four possible diastereoisomers of vicinal diols from achiral aldehyde acceptors and DHAP (Scheme 4.2). Although this statement is generally used and accepted, it is not completely true since tagatose-l,6-bisphosphate aldolase (TBPA) from Escherichia coli-the only TBPA that has been investigated in terms of its use in synthesis-does not seems to control the stereochemistry of the aldol reaction when aldehydes different from the natural substrate were used as acceptors [7]. However, this situation could be modified soon since it has been demonstrated that the stereochemical course of TBPA-catalyzed C—C bond formation may be modified by enzyme-directed evolution [8]. 

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... 

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