1.3- Dihydroxyacetone phosphate, reactions with aldolase

The synthesis of iy -aldols from dihydroxyacetone (DHA, 55) is one of the best approaches for the stereoselective synthesis of carbohydrates. In nature, this process is facilitated by the dihydroxyacetone phosphate family of aldolases [2], and the organocatalyzed reaction of unprotected dihydroxyacetone mimics these processes. The synthesis of iyn-aidols requires the (Z)-enamine to react with an acceptor, and therefore only acyclic DHA derivatives can be considered as substrates. 

Stereospecifidly of Dihydroxyacetone Phosphate Reactions. In both the aldolase and triose phosphate isomerase reactions a carbon-hydrogen bond of the hydroxymethyl group is broken. Isotope exchange experiments with tritium have shown that both enzymes catalyze an equilibration between one hydrogen of the substrate and the hydrogen of water. ° The two enzymes do not attack the same hydrogen atom each is specific for only one position. In the projection shown (XII),... 

The reaction of dihydroxyacetone phosphate (DHAP) with racemic or (R)-3-azido-2-hydroxypropanol under the influence of rhamnulose-1-phosphate aldolase or fuculose-1-phosphate aldolase afforded azidoketose intermediates which were converted by way of palladium-mediated reductive amination into a number of novel compounds including 1,6-dideoxy-D-galactojirimycin (24), 1,6-dideoxy-L-altojirimycin (25), 1-deoxy-D-talojirimycin (26), 1-deoxy-L-mannojirimycin (27) and 1-deoxy-L-rhamnojirimycin (28). ... 

This reaction is followed by another phosphorylation with ATP catalyzed by the enzyme phosphofructoki-nase (phosphofructokinase-1), forming fructose 1,6-bisphosphate. The phosphofructokinase reaction may be considered to be functionally irreversible under physiologic conditions it is both inducible and subject to allosteric regulation and has a major role in regulating the rate of glycolysis. Fructose 1,6-bisphosphate is cleaved by aldolase (fructose 1,6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are inter-converted by the enzyme phosphotriose isomerase. 

A tandem enzymatic aldol-intramolecular Homer-Wadsworth-Emmons reaction has been used in the synthesis of a cyclitol.310 The key steps are illustrated in Scheme 8.33. The phosphonate aldehyde was condensed with dihydroxyacetone phosphate (DHAP) in water with FDP aldolase to give the aldol adduct, which cyclizes with an intramolecular Horner-Wadsworth-Emmons reaction to give the cyclo-pentene product. The one-pot reaction takes place in aqueous solution at slightly acidic (pH 6.1-6.8) conditions. The aqueous Wittig-type reaction has also been investigated in DNA-templated synthesis.311... 

L-Galactose is probably not produced from L-glycerose, since the latter inhibits glycolysis and, even so, reaction with dihydroxyacetone phosphate in the presence of aldolase yields L-sorbose 1-phosphate which, on stereochemical grounds, is also an unlikely precursor. A more plausible route is direct conversion from D-galactose (XXI) by complete reversal of stereo-... 

Enzymes are very sophisticated systems that apply sound chemical principles. The side-chains of various amino acids are used to supply the necessary bases and acids to help catalyse the reaction (see Section 13.4). Thus, the enzyme aldolase binds the dihydroxyacetone phosphate substrate by reacting the ketone group with an amine, part of a lysine amino acid residue. This forms an imine that becomes protonated under normal physiological conditions. 

Reaction of ribose 5-phosphate 116 with dihydroxyacetone phosphate, catalyzed by fructose 1,6-diphosphate aldolase from rabbit muscle (RAMA) affords the ketose diphosphate 117. Dihydroxyacetone phosphate was formed in situ from fructose 1,6-diphosphate by action of RAMA and triose phosphate isome-rase (TPI). The diphosphate 117 was dephosphorylated enzymatically using acid phosphatase, and the ketose 118 was reduced directly into the a-C-manno-side 119 by treatment with bistrimethylsilyltrifluoroacetamide, trimethylsilyl-triflate and triethylsilane (Scheme 28) [45]. 

Answer Problem 1 outlines the steps in glycolysis involving fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate. Keep in mind that the aldolase reaction is readily reversible and the triose phosphate isomerase reaction catalyzes extremely rapid interconversion of its substrates. Thus, the label at C-l of glyceraldehyde 3-phosphate would equilibrate with C-l of dihydroxyacetone phosphate (AG ° = 7.5 kJ/mol). Because the aldolase reaction has AG ° = -23.8 kJ/mol in the direction of hexose formation, fructose 1,6-bisphosphate would be readily formed, and labeled in C-3 and C-4 (see Fig. 14-6). 

Scheme 2 Proposed stabilizatioii of the enediolate form of dihydroxyacetone phosphate hy Zn(II) in the class II aldolase enz3une fructose l,6-bis(phosphate) aldolase, and its reaction with D-glyceraldehyde 3-phosphate to form n-fructose 1,6-bisphosphate.

The enzymatic aldol reaction represents a useful method for the synthesis of various sugars and sugar-like structures. More than 20 different aldolases have been isolated (see Table 13.1 for examples) and several of these have been cloned and overexpressed. They catalyze the stereospecific aldol condensation of an aldehyde with a ketone donor. Two types of aldolases are known. Type I aldolases, found primarily in animals and higher plants, do not require any cofactor. The x-ray structure of rabbit muscle aldolase (RAMA) indicates that Lys-229 is responsible for Schiff-base formation with dihydroxyacetone phosphate (DHAP) (Scheme 13.7a). Type II aldolases, found primarily in micro-organisms, use Zn as a cofactor, which acts as a Lewis acid enhancing the electrophilicity of the ketone (Scheme 13.7b). In both cases, the aldolases accept a variety of natural (Table 13.1) and non-natural acceptor substrates (Scheme 13.8). 

Actually, the reaction is a bit more complex than shown above because it does not take place on the free ketone. Instead, fructose 1,6-bisphosphate undergoes reaction with the side-chain -NH2 group of a lysine residue on the aldolase enzyme to yield an imine (Section 19.9), sometimes called a Schiff base. Protonation of the imine makes it more reactive a retro aldol-like reaction ensues, giving glyceraldehyde 3-phosphate and the imine of dihydroxyacetone phosphate and the imine is then hydrolyzed to yield dihy-droxyacetone phosphate. 

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... 

Fructose-1,6-diphosphate aldolase of rabbit muscle has been studied very extensively-and it is now commercially available. That of spinach leaves, obviously very accessible, was recently examined (Valentin and Bolte 1993). In the fundamental reaction of glycolysis (reaction 6.8), the donor is dihydroxyacetone phosphate. It can scarcely be varied, but there is more flexibility with the acceptor (David et al. 1991 Bednarski et al. 1989), and sometimes we can wander considerably from the subject of sugar chemistry. In any case, the vicinaZ-diol created at positions 3 and 4 (uloses numbering) has the D-threo configuration. Hence the condensation of the keto aldehyde 6.28 gives ketose 6.29 which, after isolation, is dephosphorylated enzymically in the presence of acid phosphatase. 

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. 

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