FruA reactions

The natural acceptor aldehyde can be considerably varied among phosphorylated as well as unphosphorylated hydroxyaldehydes, which are both converted at comparable rates (Table 5)13-44 47. Although the catalytic reaction creates only a single stereocenter, the enzymes from yeast or spinach efficiently distinguish between adjacent configurations with preference for (3SAR)-i>yn isomeric ketose products44 47, which nicely parallel those derived from FruA reactions (Section 1.3.4.6.1). 

The only commercially available enzyme, FruA from rabbit muscle (class 1), is the most widely investigated, however, it is also the most sensitive under commonly used reaction conditions4. The other types, which can be isolated from overexpressing bacterial sources5-8, typically... 

The D-fructose 1,6-bisphosphate aldolase (FruA EC 4.1.2.13) catalyzes in vivo the equilibrium addition of (25) to D-glyceraldehyde 3-phosphate (GA3P, (18)) to give D-fructose 1,6-bisphosphate (26) (Figure 10.14). The equilibrium constant for this reaction of 10 strongly favors synthesis [34]. The enzyme occurs ubiquitously and has been isolated from various prokaryotic and eukaryotic sources, both as class I and class II forms [30]. Typically, class I FruA enzymes are tetrameric, while the class II FruA are dimers. As a rule, the microbial class II aldolases are much more stable in solution (half-lives of several weeks to months) than their mammalian counterparts of class I (few days) [84-86]. 

Based on the stereospecific transketolase-catalyzed ketol transfer from hydroxy-pyruvate (20) to D-glyceraldehyde 3-phosphate (18), we have thus developed a practical and efficient one-pot procedure for the preparation of the valuable keto-sugar 19 on a gram scale in 82% overall yield [29]. Retro-aldolization of D-fructose 1,6-bisphosphate (2) in the presence of FruA with enzymatic equilibration of the C3 fragments is used as a convenient in-situ source of the triose phosphate 18 (Scheme 2.2.5.8). Spontaneous release of CO2 from the ketol donor 20 renders the overall synthetic reaction irreversible [29]. 

In vivo, the D-fructose 1,6-bisphosphate aldolase (FruA EC 4.1.2.13) catalyzes the pivotal reaction of the glycolysis pathway the equilibrium addition of 41 to D-glyceraldehyde 3-phosphate (GA3P, 12) to give D-fructose 1,6-bisphosphate (42) [43]. The equilibrium constant of 104 M 1 strongly favors synthesis [229]. 

The scheme of thermodynamic equilibration, particularly for 3-hydroxyaI-dehydes 75, as described above for FruA catalysis (Scheme 16) can likewise be applied to reactions with RhuA, the enantiomeric enzyme [189,220]. As is to be expected from the complementarity of the stereoselectivities of these enzymes, the corresponding enantiomeric compounds ent-78 are enriched upon equilibration. Owing to the lower diastereoselectivity of the RhuA versus the FruA enzyme, however, the corresponding FucA configurated isomer 85 accumulates more rapidly at the expense of the thermodynamically less favored diastereomer ent-19 (cf. Scheme 18, Sect. 5.2). 

An interesting problem in stereoisomerism is found in the aldol reactions of the achiral aldehydes which are obtained by ozonolysis of the homoallylic alcohols 174. After stereospecific conversion by the FruA [230], the products can be readily induced to form an intramolecular glycoside 175 by acidic (R=OH) or alkaline treatment (R=C1), under which conditions the two equatorial ring hydroxyl groups completely direct the stereogenic acetal formation [234]. The corresponding RhuA catalyzed reactions deliver the enantiomeric... 

This procedure has also been used to prepare a series of hydrolytically stable dipyranoid disaccharide mimetics from homologous dihydroxy-a,oo-dialdehydes37 (Scheme 5.16). While the FruA and FucA reactions are selective for the (W)-aldchydc, the RhuA reactions are selective for the (S)-aldehydes, all giving thermodynamically more stable product with retention of the natural stereochemistry for the two newly formed carbon centers. 

Although TA from yeast is commercially available, it has rarely been used in organic synthesis applications, and no detailed study of substrate specificity has yet been performed. This is presumably due to high enzyme cost and also since the reaction equilibrium is near unity, resulting in the formation of a 50 50 mixture of products. In addition the stereochemistry accessible by TA catalysis matches that of FruA DHAP-dependent aldolase and the latter is a more convenient system to work with. In one application, TA was used in the synthesis D-fructose from starch.113 The aldol moiety was transferred from Fru 6-P to D-glyceraldehyde in the final step of this multi-enzyme synthesis of D-fructose (Scheme 5.60). This process was developed because the authors could not identify a phosphatase that was specific for fructose 6-phosphate and TA offered an elegant method to bypass the need for phosphatase treatment. 

Halo-sugars are very useful building blocks for designing further reactions by substitution of halogen atoms. From these compounds, we have developed a che-moenzymatic strategy giving 5-thio-D-xylose in four steps (the key step being catalyzed by FruA). 

FruA was added to the reaction mixture when the oxidation of L-glycerol-3-phos-phate catalyzed by L-GPO was complete. FruA was used for catalyzing the aldol addition of DHAP onto 2-chloro- or 2-bromoacetaldehydes 20 to give the aldol adduct 5-chloro or 5-bromo-D-xylulose-l-phosphate. The yield was quantitative. 

Scheme 7. Formation of a series of hydrolytically stable dipyranoid disaccharide mimetics from homologous dihydroxy- ,ta-dienes by tandem enzymatic aldolizations. Abbreviations for reaction conditions FruA /RhuA /FucA = 1.) O3, then (CHjljS 2.) aldolase, DHAP 3.) phosphatase. This notation is used throughout subsequent diagrams (except when indicated otherwise)...

Under carefully chosen reaction conditions by using a combination of RibA and another aldolase of different specificity, the intermediate 52 from the first RibA reaction can formally be intercepted by the second aldolase through the addition of a different nucleophile. In the presence of DHAP and FruA indeed predominant formation of the dideoxyketose 55 (17% yield) could be induced [116]. Due to the reversible nature of a thermodynamically unfavorable product, and because of the competition of both enzymes for the same substrate and the long reaction times required, a number of different side products and several stereoisomers have to be accounted for. 

A particularly elegant example is shown in Scheme 11 5-deoxy-5-ethyl-D-xy-lulose (19) was prepared from glycerol by a four step, one-pot chemoenzymatic reaction sequence. The key step was the aldol reaction catalyzed by fructose-1,6-bisphosphate aldolase (FruA) from Staphylococcus carnosus [56]. 

Although many aldolases have been characterized for research purposes, these enzymes have not been developed commercially to any significant extent. This is likely due to the availability of the various biocatalysts and the need for dihydroxyacetone phosphate (DHAP) (44), the expensive donor substrate required in nearly all aldolase reactions. A number of chemical and enzymatic routes have been described for DHAP synthesis, which could alleviate these concerns [12], In terms of the enzyme supply issue, this may change with the introduction of products from Boehringer Mannheim and their Chirazyme Aldol reaction kit. They have three kits, each containing a different aldolase fructose-1,6-diphosphate FruA) (EC 4.1.2.13), L-rhamnulose-1-phosphate RhuA (EC 4.1.2.19), and L-fuculose-1-phosphate (FucA) (EC 4.1.2.17). As more screening... 

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