DHAP Syntheses

Apparently, all DHAP aldolases are highly specific for 41 as the donor component for mechanistic reasons [32, 50], which necessitates economical access to this compound for synthetic applications. Owing to the limited stability of 41 in solution, particularly at alkaline pH [172, 173], it is preferentially generated in situ to avoid high stationary concentrations. 

Multi-enzymatic artificial metabolism for in-situ generation of dihydroxyacetone phosphate from inexpensive sugars. 

Enzymatic in-situ generation of dihydroxyacetone phosphate for stereoselective aldol reactions using DHAP aldolases (box), and extension by pH-controlled, integrated precursor preparation and product liberation. 

Substrate analogs of dihydroxyacetone phosphate accessible by the GPO oxidation method. 

Because phytase, an inexpensive acid phosphatase, is only active at lotv pH but virtually inactive at pH 7.5 in tvhich aldolases have their catalytic optimum, this enables the independent staging of a one-pot synthetic cascade betvireen (1) transphosphorylation, (2) aldolization, and (3) product dephosphorylation simply by stvitching the pH [178]. 

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Regardless of the above, an efficient method of DHAP preparation is still essential. Several chemical routes of DHAP synthesis have been described in the literature. Of these routes, those starting from the DHA dimmer [12] or 1,3-dibromoacetone [13] are the most attractive since they provide a stable precursor of DHAP (Scheme 4.4). Their main drawback is the relatively low overall yield. 

Although many aldolases have been characterized for research purposes, the four aldolase enzymes described in Scheme 19.32 have not been used commercially to any significant extent. This is likely a result of their availability and the need for dihydroxyacetone phosphate (DHAP) (54), the expensive donor substrate required in these aldolase reactions (Scheme 19.32). A number of chemical and enzymatic routes have been described for DHAP synthesis, which could alleviate these concerns.9,258... 

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... 

When carbon rearrangements are balanced to account for net hexose synthesis, five of the glyceraldehyde-3-phosphate molecules are converted to dihy-droxyacetone phosphate (DHAP). Three of these DHAPs then condense with three glyceraldehyde-3-P via the aldolase reaction to yield 3 hexoses in the form... 

Figure 10.28 Complementary routes for the stereoselective synthesis of hydrolytically stable sugar phosphonates, either from the bioisosteric phosphonate analog of DHAP or from phosphonylated aldehydes.

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... 

In the case of L-rhamnulose-1-phosphate aldolase (RhaD), we found that the problem of phosphorylated substrate requirement (dihydroxyactone phosphate (DHAP)) could be overcome by a simple change in buffer. Thus, when using borate buffer, reversible borate ester formation created a viable substrate out of dihydroxyacetone, which is not otherwise accepted by the wild-type enzyme (Figure 6.6) [23]. The process was used in a one-step synthesis of... 

A practical, inexpensive one-step procedure was developed for the RhaD-catalyzed gram-scale synthesis of L-fructose. The requirement for DHAP as the donor substrate was circumvented by use of borate buffer, presumably by in situ formation of borate esters as a phosphate ester mimic. Racemic glyceraldehyde was also used, as the enzyme preferentially accepted the L-enantiomer as a substrate. The method can also be apphed to other products, including L-rhamnulose, and towards a two-step synthesis of L-iminocychtols. ... 

Dihydroxyacetone phosphate (DHAP) is used in liver and adipose tissue for triglyceride synthesis. 

An advantage of these enzymes is that they are stereocomplementary, in that they can synthesize the four possible diastereoisomers of vicinal diols from achiral aldehyde acceptors and DHAP (Scheme 4.2). Although this statement is generally used and accepted, it is not completely true since tagatose-l,6-bisphosphate aldolase (TBPA) from Escherichia coli-the only TBPA that has been investigated in terms of its use in synthesis-does not seems to control the stereochemistry of the aldol reaction when aldehydes different from the natural substrate were used as acceptors [7]. However, this situation could be modified soon since it has been demonstrated that the stereochemical course of TBPA-catalyzed C—C bond formation may be modified by enzyme-directed evolution [8]. 

The main drawback of the DHAP-dependent aldolases is their strict specificity for the donor substrate. Apart from the scope limitation that this fact represents, DHAP is expensive to be used stoichiometrically in high-scale synthesis, and labile at neutral and basic pH, and therefore its effective concentration decreases over time in enzymatic reaction media, hindering the overall yield of the aldol reaction. In addition, due to the presence of a phosphate group in both DHAP and the... 

Scheme 4.3 (a) Use of arsenate ester of DHA as mimic of DHAP. (b) Two-step synthesis of iminocyclitols using borate ester of DHA as substrate of the Rha-1 PA. 

Fessner and Sinerius, in their seminal work [14], showed that the depicted strategy was adaptable to the synthesis of DHAP analogs modified at the phosphate group and from two of them they could identify the aldol adduct as mixtures with remaining starting material. 

DHAP-Dependent Aldolases in the Core of Aza Sugar Synthesis... 

When DHAP-dependent aldolases are used as catalyst of the aldol reaction, a phosphorylated azido or amino polyhydroxyketone is obtained. The phosphate may be cleaved enzymatically or reductively cleaved under the hydrogenation conditions of the next step in which the azide is reduced to the amine. Intramolecular imine formation occurs spontaneously when the azide is reduced. The intramolecular reductive amination is the second key step of the aldolase-mediated synthesis of iminocyclitols. In general, delivery of hydrogen onto five- and six-membered ring imines occurs from the face opposite to the C4 hydroxyl group. 

Lemaire et al. have developed a efficient fructose-1,6-bisphosphate aldolase (FBPA)-mediated synthesis of aminocyclitol analogs of valiolamine [34], This one-pot route involves the formation of two C—C bonds where four stereocenters are created. The first C—C bond formation reaction is catalyzed by the aldolase, coupling DHAP to nitrobutyraldehydes the other one is the result of a highly stereoselective intramolecular Henry reaction occurring on the intermediate nitroketone under acidic conditions during the aldolase-catalyzed reaction and phytase-catalyzed phosphate hydrolysis coupled step (Scheme 4.13). 

Rare or unnatural monosaccharides have many useful applications as nonnutritive sweeteners, glycosidase inhibitors and so on. For example, L-glucose and L-fructose are known to be low-calorie sweeteners. In addition, rare or unnatural monosaccharides are potentially useful as chiral building blocks for the synthesis of biologically active compounds. Therefore, these compounds have been important targets for the development of enzymatic synthesis based in the use of DHAP-dependent aldolases alone or in combination with isomerases. Fessner et al. showed that rare ketose-1-phosphates could be reached not only by aldol addition catalyzed by DHAP-dependent aldolases, but by enzymatic isomerization/ phosphorylation of aldoses [35]. Thus, for example, L-fructose can be prepared... 

Scheme 4.14 L-Fructose synthesis starting from glycerol and DHAP using a multi-enzyme system with galactose oxidase, Rhal PA, catalase and APase.

DHAP-dependent aldolases have also been used as key step in the synthesis of several complex natural products starting from achiral precursors. Thus, the sex pheromone (+)-exo-brevicomin can be synthesized in a multi-step route starting with the stereospecific aldol addition between DHAP and 5-oxohexanal or its 5-dithiane-protected analog catalyzed by FBPA from rabbit muscle ( RAMA ) as the key step by which the absolute configuration of the target is estabUshed (Scheme 4.16) [40]. 

Sphydrofuran is a structurally unique secondary metabolite produced by a variety of Streptomycetes strains. The first total synthesis of this compound was based in the use of RAMA to catalyze the aldol addition of chloroacetaldehyde with DHAP that provides two of the three chiral centers of the target molecule. The third quaternary center was introduced via a highly diastereoselective Grignard addition of allylmagnesium bromide (Scheme 4.17) [41]. 

Shimagaki et al. reported the synthesis of the C11-C16 fragment of the penta-mycin based in the stereoselechve C—C bond formation reaction catalyzed by FBPA [45]. Pentamycin is a polyene macrolide antibiotic, whose configurations at C15 and C14 would correspond to those of the C3 and C4 posihons of an aldol constructed from addihon of DHAP-derived from FBP by use of FBPA and TIM-to the corresponding aldehyde catalyzed by FBPA (Scheme 4.19). 

DHAP-dependent aldolases have also been used in the synthesis of the C3-C9 fragment of aspicilin [46] and of the C12-C20 fragment of amphotericin [47]. 

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