DHAP-aldolases

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,... 

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).

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,... 

D-fructose 1,6-bisphosphate 2 (FruA E.C. 4.1.2.13), D-tagatose 1,6-bisphosphate 4 (TagA E.C. 4.1.2.40), L-fuculose 1-phosphate 5 (FucA, E.C. 4.1.2.17), and L-rhamnulose 1-phosphate 4 (RhuA, E.C. 4.1.2.19). From previous studies, we have DHAP aldolases with all four possible specificities readily available, we have demonstrated their broad substrate tolerance for variously substituted aldehydes, and we have investigated their stereoselectivity profile with non-natural substrates [3-6]. 

While DHAP aldolases produce ketose derivatives, access to biologically important and structurally more diverse aldose isomers is achievable by use of enzymatic ketol isomerase interconversions. For this purpose, we had previously shown that L-rhamnose isomerase (Rhal E.C. 5.3.1.14) and L-fucose isomerase (Fuel E.C. 5.3.1.3) from E. coli display a relaxed substrate tolerance. Both enzymes convert sugars and their derivatives with distinct stereopreference at C2 and common (3R)-OH configuration, but tolerate alterations in configuration or substitution pattern at subsequent positions of the chain (Scheme 2.2.5.5) [11, 12]. 

Several DHAP aldolases having different stereospecificities were tested for their acceptance of this phosphonomethyl substrate mimic as the aldol donor, individual enzymes belonging to both Glass 1 and 11 types were found to catalyze the stereoselective addition of 14 to various aldehydes, providing bio-isosteric non-hydrolyzable analogues of sugar 1-phosphates in high yields (for example, 16/17 Scheme 2.2.5.7) [25, 26]. 

The Schiff-base-forming types (class I) are known only for the two former aldolases (FruA, TagA), which are found usually in mammalian or (as an exception) in specific microbial organisms, whereas the Zn2+-dependent type (class II) comprises all four DHAP aldolases which are commonly found in bacteria [43], Typically, type I FruA enzymes are tetrameric proteins composed of subunits of 40 kDa [191,192], while the type II FruA are dimers of 39 kDa subunits [193]. RhuA and FucA enzymes are homotetrameric with a subunit molecular weight of 25 kDa and 30 kDa respectively [194,195],... 

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... 

For any one of the DHAP aldolases, the absolute configuration at the newly created stereocenter at C-3 is invariably conserved upon reaction with any electrophile, apparently for mechanistic reasons [199] no exceptions are known so far. For the stereogenic center at C-4, the relative positioning of the aldehyde carbonyl in the transition state, and thus the relative configuration in the product, usually follows closely that of the natural substrates. Depending on the nature of the enzyme used and on the pattern of substitution present in the aldehydic component, a distinct fraction of the 4-epimeric diastereomers may also be observed which is presumably the result of incorrect binding of the respective aldehyde (cf. Sect. 3.1). 

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... 

Scheme 14. In-situ formation of dihydroxyacetone arsenate and vanadate esters as donor substrates for DHAP aldolases...

Like the DHAP aldolases, the class II 3-hexulose 6-phosphate aldolase from a unique formaldehyde-fixing system of the methylotrophic bacterium Methyl-omonas Ml5 utilizes a ketose phosphate, i.e. D-ribulose 5-phosphate (128), as the aldol donor component for which it has a stringent requirement [374], On the... 

The transaldolase (EC 2.2.1.2) is an ubiquitous enzyme that is involved in the pentose phosphate pathway of carbohydrate metabolism. The class I lyase, which has been cloned from human [382] and microbial sources [383], transfers a dihydroxyacetone unit between several phosphorylated metabolites. Although yeast transaldolase is commercially available and several unphosphorylated aldehydes have been shown to be able to replace the acceptor component, preparative utilization has mostly been limited to microscale studies [384,385] because of the high enzyme costs and because of the fact that the equilibria usually are close to unity. Also, the stereochemistry of transaldolase products (e.g. 38, 40) [386] matches that of the products from the FruA-type DHAP aldolase which are more effortlessly obtained. 

Scheme 23. Stereodivergent synthesis of all eight possible hexokctoses from glyceraldehyde by DHAP aldolases...

The stereodivergent action of DHAP aldolases is also preparatively useful under conditions of thermodynamic control [202,230, 294]. Starting from racemic 3-hydroxybutanal 84 the enantiocomplementary nature of the FruA RhuA enzyme couple will extend to the selection of the dependent stereocenter at C-6 to yield the corresponding mirror imaged products 78 with high selectivity [189], Conversely, the FucA will contribute a different major diastereomer 173, albeit only at a low de because of more balanced energetic relations [362],... 

Two differently JV-acyl protected derivatives of (S)-serinal 197, readily available from D-glucosamine, have been subjected to aldolizations with DHAP aldolases to furnish the corresponding 5-aminoketoses 196/198 [220,235], The latter compounds are interesting in their own right or as further potential precursors for pyrrolidine-type azasugars. 

Fessner W-D (1992) A Building Block Strategy for Asymmetric Synthesis The DHAP Aldolases. In Servi S (ed) Microbial Reagents in Organic Synthesis. Kluwer Academic, Dordrecht, vol. 381, p 43... 

The use of aldolases and transketolase has opened the way to many highly multifunctional organic compounds [1]. In organic synthesis, the most widely used dihydroxyacetonephosphate (DHAP) aldolase is the commercially available fruc-tose-1,6-bisphosphate aldolase from rabbit muscle (FruA). This enzyme is a key enzyme of the glycolytic pathway, reversibly catalyzing the cleavage of fructose-... 

The dephosphorylation of 5-chloro and 5-bromo-D-xylulose-l-phosphate was carried out by the addition of acid phosphatase. After purification, 5-chloro-D-xylu-lose and 5-bromo-D-xylulose were recovered as pure compounds in 47 and 12% yields, respectively, from DHAP. In this study, we have shown that DHAP generated from glycidol 7 can be used in situ as a donor substrate of FruA in the presence of 2-halo-acetaldehydes 20 as acceptor substrates for the synthesis of 5-halo-D-xylulose 19. Given that DHAP aldolases display a broad specificity towards acceptor substrates, this strategy can be applied generally to the synthesis of various analogs of monosaccharides. 

DHAP-Aldolase-mediated Synthesis of tminosugars from N-Cbz-amino Aldehydes 301... 

Figure 19.3 DHAP-aldolase catalyzed aldol addition of DHAP to N-Cbz-aminoaldehydes. Reaction conversion to aldol adduct in gel emulsion (black bars) and dimethylformamide/ water 1 4 system (gray bars) for reactions catalyzed by (a) RAMA, (b) RhuA and (c) FucA.

The stereochemistry outcome of the aldol additions is an issue of paramount importance in the synthesis of iminosugars. Based on mechanistic considerations of the DHAP aldolases [29, 30] it can be assumed that the absolute configuration at C-3 (i.e. the stereocenter arising from DHAP) is independent of the acceptor used in the reaction. Analysis of the stereochemistry at C-4 (i.e. the one generated from the aldehyde) can be used to infer the kinetic stereoselectivity of the aldolases towards each of the N-protected amino aldehydes (Figure 19.4). For the selected... 

The chemoenzymatic strategies described heretofore are based on DHAP-depen-dent aldolases. One of the main drawbacks of DHAP aldolases is their strict specificity toward DHAP and a negligible activity with the unphosphorylated DHA analog (29) (Figure 19.8). The chemical synthesis of DHAP involves several steps in an approximately 70% overall yield. [37] Alternatively, multienzymatic methods coupled with the aldol reaction have also been described [38]. However, drawbacks such as the different and often incompatible optimal reaction conditions of... 

---