Dihydroxyacetone arsenate

Figure 2. Mechanism of dihydroxyacetone/arsenate reaction with FDP aldolase. Both dihydroxyacetone and inorganic arsenate are not the inhibitor of the aldolase reactions. The rate constant for the arsenate ester formation is determined enzymatically (a plot of 1/v vs 1/E gives a non-zero intercept which is attributed to the rate at infinite enzyme concentration and that rate corresponds to the rate of nonenzymatic formation of the arsenate ester).

Figure 3. Selectivity of the FDP-aldolase reactions using DHAP vs. dihydroxyacetone/arsenate as a substrate. In the former case, the more stable sugar is obtained due to the reversible nature of the reaction. In the later case, both sugars were obtained in nearly equal amounts, because the reaction was found to be virtually irreversible and the formation of the arsenate ester was rate limiting.

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

A mixture of dihydroxyacetone and inorganic arsenate may replace DHAP and this mixture has been used in syntheses by Wong and coworkers. A dihydroxyacetone arsenate monoester probably forms in the rate-determining step, and is consumed in a fast, irreversible aldol reaction. The irreversibility imposed by this method may be useful with slowly reacting substrates, but the toxicity of arsenate limits its usefulness. Vanadate does not operate as a phosphate mimic in FDP aldolase catalyzed reacdons. ... 

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]. 

A mixture of dihydroxyacetone and inorganic arsenate can replace DHAP due to the transient formation of a monoarsenate ester which is recognized by the aldolase as a DHAP mimic21. This approach suffers from the high toxicity of arsenate, especially at the relatively high levels (>0.5 M) needed for efficient conversion, and from problems in product isolation. 

Figure 10.20 Substrate analogs of dihydroxyacetone phosphate accessible by the CPO oxidation method, and spontaneous, reversible formation of arsenate or vanadate analogs of dihydroxyacetone phosphate/n s/tu for enzymatic aldol additions.

Efforts to overcome the Dff AP dependence of aldolases have involved the in situ formation of arsenate [9] or borate [10] complexes with dihydroxyacetone (DHA), which could mimic a phosphate ester (Scheme 4.3). 

The same authors then introduced a second system, in which arsenate esters were used as phosphate ester mimics. Dihydroxyacetone readily reacts with arsenate and the resulting ester is accepted by FDP A as a substrate. Since the arsenate ester formation is an equilibrium reaction the desired product is released in situ and the arsenate is available again for the next catalytic cycle (Scheme 5.27) [47]. However, although only catalytic amounts of arsenate are necessary it remains a toxic metal. 

Figure 2. Use of dihydroxyacetone and arsenate as substrate in FDP-aldolase reactions. The rate constant was determined in Figure 3.

A linear plot of the reciprocal of the rate versus the reciprocal of the enzyme concentration shows a non-zero intercept. This intercept corresponds to the rate at infinite enzyme (Figure 3) concentration which is equal to the rate of forjpation of the dihydroxyacetone ester (2.4 X 10" s ). A previous study on glucose-e-phosphate dehydrogenase showed that glucose-6-arsenate was formed and the rate constant was 6.3 X 10 ° M s ... 

Figure 3. Reciprocal of the rate (M/min) of glycerophosphate dehydrogenase-catalyzed reduction of dihydroxyacetone in the presence of 5 mM arsenate vs the reciprocal of enzyme concentration (mg/mL). Reactions were carried out in Tris buffer (20 mM, pH 8.0) containing NADH (0.1 mM), dihydroxyacetone (50 mM), glycerophosphate dehydrogenase and sodium arsenate (5 mM). The decrease in absorbance at 340 nm was monitored versus time.

The FDP aldolase and two other aldolases require dihydroxyacetone phosphate as a substrate. Current methods for the preparation of dihydroxyacetone phosphate are from dihydroxyacetone by chemical or enzymatic synthesis, and from FDP by in situ enzymatic generation (6). Dihydroxyacetone phosphate can also be replaced with dihydroxyacetone and catalytic amounts of inorganic arsenate (18),... 

Spontaneous, reversible formation of arsenate and vanadate analogs of dihydroxyacetone phosphate in situ for enzymatic aldol additions. 

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