Aldolase synthetic applicability

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. 

The KDO aldolase (KdoA, EC 4.1.2.23) is involved in the catabolism of the eight-carbon sugar d-KDO, which is reversibly degraded to D-arabinose (15) and pyruvate (Figure 10.10). The enzyme has been partially purified from bacterial sources and studied for synthetic applications [71,74]. It seems that the KdoA, similar to... 

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. 

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. 

U. Kragl, A. Godde, C. Wandrey, N. Lubin, and C. Auge, New synthetic applications of sialic acid aldolase, a useful catalyst for KDO synthesis. Relation between substrate conformation and enzyme stereoselectivity, J. Chem. Soc. Perkin Trans. 7 119 (1994). 

Representatives of all kinds have been explored for synthetic applications while mechanistic investigations were mainly focussed on the distinct FruA enzymes isolated from rabbit muscle [196] and yeast [197,198]. For mechanistic reasons, all DHAP aldolases appear to be highly specific for the donor component DHAP [199], and only a few isosteric replacements of the ester oxygen for sulfur (46), nitrogen (47), or methylene carbon (48) were found to be tolerable in preparative experiments (Fig. 7) [200,201], Earlier assay results [202] that had indicated activity also for a racemic methyl-branched DHAP analog 53 are now considered to be artefactual [203]. Dihydroxyacetone sulfate 50 has been shown to be covalently bound via Schiff base formation, but apparently no a-deprotonation occurred as neither H/D-exchange nor C-C... 

Owing to the narrow specificity of the DHAP aldolases for the donor substrate DHAP (41), direct access to this essential compound is vital to the development of synthetic applications. Commercial offers of the compound, however, are prohibitively expensive for preparative-scale applications. A further problem is that 41 is relatively unstable in solution and, particularly at elevated pH values, readily decomposes according to an Elcb pathway via an enediol intermediate... 

Fig. 35 Synthetic applications of (a) DHAP-, (b) pyruvate-, and (c) glycine-dependent aldolases...

The preparation of DHAP for synthetic applications has been accomplished both enzymatically and chemically (Scheme 5.7).27 DHAP can be generated enzymatically in situ from fructose 1,6-diphosphate, using FDP aldolase acting in its catabolic mode, and triosephosphate isomerase (TPI). Glyceraldehyde 3-phosphate (G3P) and DHAP are produced in this reaction, with the G3P rapidly undergoing isomerization to DHAP. 

Azido aldehydes were also utilized for the synthesis of 6-substituted D-fructopyranoside derivatives actives against Trypanosoma brucei parasite (Azema et al. 2000). Other interesting synthetic applications of DHAP aldolases are listed below ... 

Synthetic applications of threonine aldolase have been hampered due to the poor capacity for erythro/threo discrimination. Erythro-sdective threonine aldolase from Candida humicola has been used for the preparation of a key chiral building block in the synthesis of the immunodepressive lipid mycestericin D (Fig. 6.5.16). The conversion was purposely low to ensure a kinetic control and therefore maximizing the yield of the erythro product. 

Guisan JM, Fernandez-Lafuente R, Rodriguez V et al. (1993) Enzyme stabilization by multipoint covalent attachment to activated preexisting supports. In van den Tweel WJJ, Harder A, Buite-laar RM (eds). Stability and stabilization of enzymes, vol. 47. Elsevier, Amsterdam, pp 55-62 Henderson I, Garcia-Junceda E, Liu KK et al. (1994) Qoning, overexpression and isolation of the type II FDP aldolase firom E. coli for specificity study and synthetic application. Bioorg Med Chem 2 837-843... 

One of the drawbacks of DHAP aldolases is their strict specificity toward the donor substrate DHAP. DHAP is chemically unstable, particularly under alkaline conditions, and decomposes into inorganic phosphate and methyl glyoxal, both of which may inhibit the aldolase [4cj. Although the preparation [22] and synthetic applications of DHAP have reached a high degree of sophistication and efficiency [4h, 6e,i, 23], the preferred choice is by far the inexpensive unphosphorylated DHA nucleophile, which reduces costs and improves the atom economy of the process, especially when the phosphate group of the product must be removed in a separate reaction. In this connection, we focused our efforts on RhuA and FSA from E. coli [24]. 

Comparable with the situation for the sialic acid and KDO lyases (vide supra), a class I lyase complementary to the KDPGIc aldolase is known that has a stereopreference for the (4S) configuration (Figure 5.19). The aldolase, which acts on 2-keto-3-deoxy-6-phospho-D-galactonate (36) (KDPGal aldolase EC 4.1.2.21) and is less abundant [145, 146], has recently been studied for synthetic applications [147]. 

The class I FruA isolated from rabbit muscle ( RAMA ) is the aldolase used for preparative synthesis in the videst sense, because of its commercial availability and useful specific activity of 20 U mg. Its operating stability in solution is limiting, but recently more robust homologous enzymes have been cloned, e.g. from Staphylococcus camosus [152] or from the extremophilic Thcrmus aquaticus [153], vhich promise to be unusually stable in synthetic applications [154]. Attempts at catalyst immobilization have been performed vith rabbit muscle FruA, vhich has been covalently attached to microcarrier beads [58], cross-linked in enzyme crystals (CLEG) [59], or enclosed in a dialysis membrane [73]. It vas recently sho vn that... 

An exception is the dihydroxyacetone (DHA) utilizing aldolases such as the D-fructose-6-phosphate aldolase that also accepts hydroxyacetone, hydroxy-butanone, and glycoaldehyde. Such broad donor tolerance is almost unique among aldolases [8,82]. Aldolases can accept a broad structural variety of aldol acceptors, and this is what makes them highly important for synthetic applications. 

Aldol addition of DHAP to aldehydes is catalyzed by DHAP-dependent aldolases. Two stereogenic centers are formed and therefore four possible stereoisomers can be obtained. Although nature has evolved a set of four distinct stereocomplementary types (Scheme 10.3), so far, only three of the known DHAP-dependent aldolases, namely the D-fructose-l,6-bisphosphate aldolase (FruA), L-rhamnulose-1-phosphate aldolase (RhuA), and L-fuculose-1-phosphate aldolase (FucA), have found broad synthetic applicability due to their high stereoselectivity and broad acceptor tolerance [5,77]. DHAP-dependent aldolases are highly selective for the nucleophilic substrate DHAP, tolerating only few isosteric modifications [84-88]. 

The 2-keto-3-deoxyoctosonate (KDO) aldolase (KdoA EC 4.1.2.23) is an inducible enzyme in gram-negative microorganisms [78] where d-KDO 8 is a core constituent of the outer membrane lipopolysaccharide. The aldolases from Aerobacter cloacae and an Aureobacterium barkerei strain have been studied for synthetic applications [72,79]. Similar to the NeuA, the KdoA enzyme has a broad substrate specificity for aldoses in place for the natural acceptor D-arabinose (Table 2) while pyruvate was found to be irreplaceable. Preparative applications, e.g., that for the synthesis of KDO analogs 13/14 (Fig. 9), suffer... 

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