DHAP dihydroxyacetone preparations

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).

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... 

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. 

DHAP has also been prepared by phosphorylation of dihydroxyacetone with glycerol kinase in the presence of ATP, with in situ regeneration of ATP, giving yields in excess of 80%. The use of chemical methods260 c for the preparation of DHAP generates a pure product, which results in a cleaner aldol reaction. In an improved and commonly used chemical preparation, the protected dimer of dihydroxyacetone was phosphorylated with diphenyl chlorophosphate, followed by hydrogenolysis and hydrolysis, to give clean DHAP in 61% yield (Scheme 5.8).28... 

We have developed preparative enzymatic syntheses of several unusual hexoketoses using fructose-1,6-diphosphate aldolase (FDP-aldolase, E.C.4.1.2.13) as catalyst and dihydroxyacetone phosphate (DHAP) and an aldehyde as substrates (15). The enzyme appears to be very specific for DHAP but will accept a variety of aldehydes as acceptors. The ketose-1-phosphates prepared are converted to the phosphate free ketoses after removal of the phosphate group by acid- or phosphatase-catalyzed hydrolysis. The ketoses can be isomerized stereospecifically to aldoses catalyzed by glucose isomerase (E.C.5.3.1.5.) from Flavobacteriuum arborescens. The equilibrium mixtures of aldoses and ketoses are then separated by chromatography on Dowex 50 (Ba ) or Dowex 1 (HSO "). Figure 1 illustrates the preparation of a mixture of 6-deoxy-6-fluoro-D-fructose... 

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... 

Synthesis of DHAP. Both RAMA and Fuc-l-P aldolase require DHAP as the nucleophilic component. Although DHAP is commercially available, it is too expensive for synthetic use, and must be synthesized for preparative-scale enzymatic reactions. DHAP has been prepared via three major routes enzymatically from FDP using a combination of RAMA and triose isomerase (TIM, E.C. 5.3.1.1) (25), chemically, by phosphorylation of dihydroxyacetone dimer (26), and enzymatically by phosphorylation of dihydroxy acetone catalyzed by glycerokinase (Scheme 8) (25). 

The preparation of DHAP by phosphorylation of dihydroxyacetone with glycerokinase is most effective for large-scale (mole) syntheses of... 

Since many of the aldolases use DHAP as the nucleophilic component of the reaction, attention has been given to devising efficient methods for its preparation starting from dihydroxyacetone. The synthesis of DHAP can be achieved in high overall yield according to the procedure shown... 

An ingenious alternative to the use of DHAP is to exploit the fact that organic arsenates can be spontaneously formed in situ from the corresponding alcohol and inorganic arsenate (HOAsOj ). Thus dihydroxyacetone arsenate (S) can be prepared in solution, and it has been shown... 

Attempts to use a mixture of dihydroxyacetone and a small amount of inorganic arsenate, which spontaneously forms dihydroxyacetone arsenate which is a mimic of DHAP and is accepted by FDP aldolase as a substrate, were impeded by the toxicity of arsemate [1411, 1412]. However, the use of borate ester mimics of DHAP offers a valuable nontoxic alternative for preparative-scale reactions [1413]. 

Valuable dihydroxyacetone phosphate (DHAP), a building block for the synthesis of monosaccharide analogues, was prepared by chemoenzymatic two-step procedure using inexpensive rac-glucidol (Scheme 2)/ ... 

Gholson et al. (1976) have studied quinolinic acid (QA) biosynthesis, in a cell-free system prepared from E. coli mutants. In this system QA is synthesized by the condensation of aspartate and a [3- C]dihydroxyacetone phosphate which is incorporated into the C-4 of QA. An FAD-requiring reaction is catalyzed by two partially purified proteins which they call quinolinate synthetase. Quinolinate synthetase is composed of protein A (MW about 35,(X)0) and protein B(MW about 85,000). Preincubation of A and B proteins leads to inactivation of at least the A protein and DHAP prevents this inactivation reaction. Neither the A nor the B protein binds aspartate- C but in the presence of both proteins aspartate is bound to an entity with an apparent MW greater than the B protein. 

While pyruvate aldolases form only a single stereogenic center, the aldolases specific for dihydroxyacetone phosphate (DHAP, 22) as a nucleophile create two new asymmetric centers at the termini of the new C—C bond. Particularly useful for synthetic applications is the fact that nature has evolved a full set of four stereochemically unique aldolases [27] for the retroaldol cleavage of ketose 1-phosphates 23-26 (Fig. 12). In the direction of synthesis this formally allows the deliberate preparation of any one of the possible four diastereomeric aldol adducts in a building block fashion [15,22,27] by simply choosing the complementary enzyme and starting materials for full control over constitution and absolute configuration of the desired product. 

Apparently, all DHAP aldolases are highly specific for the donor component 22 for mechanistic reasons [29]. For synthetic applications, two equivalents of 22 are conveniently generated in situ from commercial fructose 1,6-bisphosphate 23 by the combined action of FruA and triose phosphate isomerase (EC 5.3.1.1) [93,101]. The reverse, synthetic reaction can be utilized to prepare ketose bisphosphates, as has been demonstrated by an expeditious multienzymatic synthesis of the (3S,4S) all-cis-configurated D-tagatose 1,6-bisphosphate 24 (Fig. 13) from dihydroxyacetone 27, including a cofactor-dependent phosphorylation, by employing the purified TagA from E. coli (Fig. 13) [95,96]. 

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