DHAP Synthesis from Dihydroxyacetone

However, wider prachcal applicahons of aldolases require cheap and ready access to DHAP. Expensive or toxic reagents, multistep purification procedures, and functional group protechon complicate the chemical strategies. A promising alternative would combine a short and inexpensive DHAP synthesis followed by an in situ aldolization reaction catalyzed by aldolase. By this approach, DHAP was recently obtained by DHA phosphorylation catalyzed by DHA kinase with ATP 

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Fig. 4. Ether phospholipid synthesis from dihydroxyacetone-phosphate. (A) Dihydroxyacetone-P acyl transferase (DHAPAT). The first step of ether phospholipid synthesis is catalyzed by peroxisomal DHAPAT. This enzyme is a required component of complex ether lipid biosynthesis and its role cannot be assumed by a cytosolic enzyme that also forms acyldihydroxyacetone-P. (B) Ether bond formation by alkyl-DHAP synthase. The reaction that forms the 0-alkyl bond is catalyzed by alkyl-DHAP synthase and is thought to proceed via a ping-pong mechanism. Upon binding of acyl-DHAP to the enzyme alkyl-DHAP synthase, the pro-f hydrogen at carbon atom 1 is exchanged by enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP complex. The carbon atom at the 1-position of DHAP in the enzyme complex is thought to carry a positive charge that may be stabilized by an essential sulfhydryl group of the enzyme thus, the incoming alkox-ide ion reacts with carbon atom 1 to form the ether bond of alkyl-DHAP. It has been proposed that a nucleophilic cofactor at the active site covalently binds the DHAP portion of the substrate.

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... 

Figure 26.1 PATHWAY INTEGRATION Sources of intermediates in the synthesis of triacylglycerols and phospholipids. Phosphatidate, synthesized from dihydroxyacetone phosphate (DHAP) produced In glycolysis and fatty acids, can be further processed to produce triacylglycerol or phospholipids. Phospholipids and other membrane lipids are continuously produced in all cells.

The aldol reaction is extensively used in Nature as in the laboratory to make C-C bonds and some aldolases have been used in asymmetric synthesis. One of the most popular has been the fructose-6-phosphate aldolase44 from rabbit muscle, familiarly known as RAMA. The enzymatic reaction combines the enol from dihydroxyacetone phosphate (DHAP) 142 with glyceraldehyde-3-phosphate 143 in a diastereo- and enantioselective aldol reaction. PO in these diagrams means phosphate. 

Figure 19.2 schematically depicts the primary pathways of prokaryotic and eukaryotic glycerophospholipid biosynthesis. Note that the center pathway shown in purple occurs in both prokaryotic and eukaryotic cells. Phosphatidic acid, the branch point between the synthesis of fats and other glycerophospholipids, can be made via three different pathways in eukaryotes-from glycerol-3-phosphate (Figure 19.3), from diacylglycerol (Figure 19.2), and from dihydroxyacetone phosphate (DHAP) (see here). 

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... 

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]. 

FIGURE 21-21 Glyceroneogenesis. The pathway is essentially an abbreviated version of gluconeogenesis, from pyruvate to dihydroxyacetone phosphate (DHAP), followed by conversion of DHAP to glycerol 3-phosphate, which is used for the synthesis of triacylglycerol. 

Fructose 1,6-biphosphate aldolase from rabbit muscle in nature reversibly catalyzes the addition of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde 3-phosphate. The tolerance of this DHAP-dependent enzyme towards various aldehyde acceptors made it a versatile tool in the synthesis of monosaccharides and sugar analogs [188], but also of alkaloids [189] and other natural products. For example, the enzyme-mediated aldol reaction of DHAP and an aldehyde is a key step in the total synthesis of the microbial elicitor (—)-syringolide 2 (Fig. 35a) [190]. 

Figure 6-6. Synthesis of fatty acids and triacylglycerols from glucose. G-6-P = glucose 6-phosphate F-6-P = fructose 6-phosphate F-1.6-P = fructose 1,6-bisphosphate DHAP = dihydroxyacetone phosphate AcCoA = acetyl CoA VLDL = very-low-density lipoprotein.

Figure 17-29. Synthesis of [3, 4 -13C2]-thymidine from [2, 3 -13C2]-dihydroxyacetone phosphate with triosephosphate isomerase (TPI) and D-2-deoxyribose-5-phosphate (DHAP). Asterisks indicate the positions selectively labeled with 13C. Other positions that can be isotopically substituted are marked with , A, and V. Reprinted from Ouwerkerk et al.12511.

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... 

PDOs are useful for synthesis of polymers, aircraft de-icing, and as food additives [84]. The synthesis of 1,3-PDO was reported at a titer of up to 130 gl reported in 2003 [14] as the result of an effort by DuPont and Genencor. 1,3-PDO was produced in E. coli through the glycolytic triose dihydroxyacetone phosphate (DHAP), which is reduced and dephosphorylated to glycerol, followed by dehydration and reduction to 1,3-PDO. The strategy involved the use of ATP-related glucose transport rather than the native PEP-related mechanism. Additionally, the previously uncharacterized native reductase YqhD was used for the final reduction from 3-hydroxypropanal to 1,3-PDO. The process has become industrially successful, supported by patents for the production [85, 86] and purification [87] of 1,3-PDO. 

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). 

Here we recount the latest research on chemoenzymatic multistep and cascade strategies for the synthesis of iminocyclitols, carbohydrates, and deoxysugars from N-protected ami noaldehydes, hydroxyaldehydes, and simple alkylaldehydes, respectively. The key step in all of them is the stereoselective aldol addition reaction of dihydroxyacetone phosphate (DHAP) and its unphosphorylated analogs to the acceptor aldehydes using DH AP-dependent and dihydroxyacetone- (DH A)-utilizing aldolases, respectively, as biocatalysts. 

Typical applications of the DHAP aldolases indude the synthesis of monosaccharides and derivatives of sugars from suitable functionalized aldehyde precursors. High conversion rates and yields are usually achieved with 2- or 3-hydroxyaldehydes, because for these compounds reaction equilibria benefit from the cyclization of the products in aqueous solution to give more stable fiiranose or pyranose isomers (Figure 5.32). For example, enantiomers of glyceraldehyde are good substrates, and stereoselective addition of dihydroxyacetone phosphate produces enantiomerically pure ketohexose... 

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