Dependent aldolases synthesis

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... 

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. 

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... 

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]. 

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]. 

The use of this enzyme in multi-step synthesis is relatively recent. Clapes et al. have reported the first example of FSA-mediated synthesis of iminocyclitols [53]. The synthetic strategy is similar to the one previously described for DHAP-dependent aldolases without the need for the dephosphorylation step. AldoUc reaction of DHA with N-Cbz-3-aminopropanal catalyzed by FSA followed by selective catalytic reductive aminahon furnishes the naturally occurring imino-sugar D-fagomine (Scheme 4.22). 

FSA is an interesting novel tool in chemoenzymatic synthesis. It use greatly simplifies the enzymatic procedures based on the use of DffAP-dependent aldolases since it is employs non-phosphorylated donors and, as a consequence, the reaction products do not require a subsequent dephosphorylation step. Unfortu-natly, only one stereoconfiguration of the aldol adduct is accessible. 

Enzymatic synthesis relying on the use of aldolases offers several advantages. As opposed to chemical aldolization, aldolases usually catalyze a stereoselective aldol reaction under mild conditions there is no need for protection of functional groups and no cofactors are required. Moreover, whereas high specificity is reported for the donor substrate, broad flexibility toward the acceptor is generally observed. Finally, aldolases herein discussed do not use phosphorylated substrates, contrary to phosphoenolpyruvate-dependent aldolases involved in vivo in the biosynthetic pathway, such as KDO synthetase or DAHP synthetase [18,19]. 

While the lyases that transfer a pyruvate unit form only a single stereogenic center, the group of dihydroxyacetone-phosphate-(DHAP, 41)-dependent aldolases create two new asymmetric centers, one each at the termini of the new C-C bond. A particular advantage for synthetic endeavors is the fact that Nature has evolved a full set of four stereochemically-complementary aldolases [189] (Scheme 6) for the retro-aldol cleavage of diastereoisomeric ketose 1-phosphates— D-fructose 1,6-bisphosphate (42 FruA), D-tagatose 1,6-bisphosphate (43 TagA), L-fuculose 1-phosphate (44 FucA), and L-rhamnulose 1-phosphate (45) aldolase (RhuA). In the direction of synthesis this formally allows the... 

To date, 2-deoxy-D-ribose 5-phosphate aldolase (DERA) is the only acetaldehyde-dependent aldolase being applied in organic synthesis. Thus the stereoselectivity of DERA is significant, all known enzymes from different organisms showing the same preferences, limiting the field of application to syntheses in which specifically the DERA-catalyzed enantiomer is needed. 

Serine hydroxymethyltransferase is a PLP-dependent aldolase. It catalyzes interconversion between glycine and various P-hydroxy-a-amino acids, such as serine and threonine, via formation of a quinoid intermediate derived from PLP with the amino acid substrate (Scheme 2.9). This aldolase-type reaction is of interest as an asymmetric synthesis of a-amino acids via C-C bond formation. 

Pyruvate-dependent aldolases catalyze the breaking of a carbon-carbon bond in nature. This reaction can, however, be reversed if an excess of pyruvate is used, establishing one new stereocenter in the course of it. The natural function of phosphoenolpyruvate (PEP)-dependent aldolases on the other hand is to catalyze the synthesis of a-keto acids. Since PEP is a very reactive, unstable and difficult to prepare substrate, they are not commonly used in synthesis. 

In the chemical synthesis of complex natural products, the concept of bidirectional chain synthesis has also been shown effective for the bi-directional backbone extension, as implemented by DHAP-dependent aldolases.37 As such, highly complex carbohydrate mimics may be obtained in a single pot... 

Pyruvate-dependent aldolases have catabolic activity in vivo, whereas their counterparts utilizing phosphoenolpyruvate as the donor substrate are involved in the biosynthesis of keto acids. Both classes of enzymes have been used in synthesis to prepare similar a-keto acids. The enzymes catalzye this type of reaction in vivo and their stereoselectivity are presented together in this section (Schemes 5.27, 5.28). 

Although TA from yeast is commercially available, it has rarely been used in organic synthesis applications, and no detailed study of substrate specificity has yet been performed. This is presumably due to high enzyme cost and also since the reaction equilibrium is near unity, resulting in the formation of a 50 50 mixture of products. In addition the stereochemistry accessible by TA catalysis matches that of FruA DHAP-dependent aldolase and the latter is a more convenient system to work with. In one application, TA was used in the synthesis D-fructose from starch.113 The aldol moiety was transferred from Fru 6-P to D-glyceraldehyde in the final step of this multi-enzyme synthesis of D-fructose (Scheme 5.60). This process was developed because the authors could not identify a phosphatase that was specific for fructose 6-phosphate and TA offered an elegant method to bypass the need for phosphatase treatment. 

In this sense, enzymatic catalysis is especially attractive for syntheses demanding highly regio- and stereoselectivity. Particularly dihydroxyacetone phosphate (DHAP) dependent aldolases are among the most suitable biocatalyst for imino-sugar synthesis due to their high stereoselectivity and chiral induction capacity [10, 12, 13]. The aldol addition of DHAP (1) to a synthetic equivalent of an... 

Serine Hydroxymethyltransferase Serinehydroxymethyltrans-ferase is a pyridoxed phosphate-dependent aldolase that catalyzes the cleavage of serine to glycine and methylene-tetrahydrofolate (as shown in Figure 10.5). Serine is the major source of one-carbon substituted folates for biosynthetic reactions. At times of increeised cell proliferation, the activities of serine hydroxymethyltransferase emd the enzymes of the serine biosynthetic pathway cue increased. The other product of the reaction, glycine, is also required in increased cimounts under these conditions (for de novo synthesis of purines). 

The aldolases which have been investigated for their synthetic utility can be classified on the basis of the donor substrate accepted by the enzyme. For the synthesis of 3-deoxy-2-ulosonic acids pyruvate- and phosphoenolpyruvate dependent aldolases are the most desirable enzymes as they are involved in the metabolism of sialic acids (or structurally related ones) in vivo. They use pyruvate or phosphoenolpyruvate as a donor to form 3-deoxy-2-keto acids (Table 1). Both of them add a three-carbons ketone fragment onto a carbonyl group of an aldehyde. The pyruvate dependent aldolases have a catabolic function in vivo, whereas the phosphoenolpyruvate dependent aldolases are involved in the biosynthesis of the keto acids. For synthetic purpose the equilibrium of the pyruvate dependent aldolases is shifted toward the condensation products through the use of an excess of pyruvate. 

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... 

A comprehensive review (260 refs.) on the synthesis of carbohydrates from noncarbohydrate sources covers the use of benzene-derived diols and products of Sharpless asymmetric oxidation as starting materials, Dodoni s thiazole and Vogel s naked sugar approaches, as well as the application of enzyme-catalysed aldol condensations. The preparation of monosaccharides by enzyme-catalysed aldol condensations is also discussed in a review on recent advances in the chemoenzymic synthesis of carbohydrates and carbohydrate mimetics, in parts of reviews on the formation of carbon-carbon bonds by enzymic asymmetric synthesis and on carbohydrate-mediated biochemical recognition processes as potential targets for drug development, as well as in connection with the introduction of three Aldol Reaction Kits that provide dihydroxyacetone phosphate-dependent aldolases (27 refs.). A further review deals with the synthesis of carbohydrates by application of the nitrile oxide 1,3-dipolar cycloaddition (13 refs.). ... 

Another labile phosphate species, which is needed as a cosubstrate for DHAP-dependent aldolase reactions, is dihydroxyacetone phosphate (Scheme 2.83, also see Sect. 2.4.1). Its chemical synthesis using phosphorus oxychloride is hampered by moderate yields. Enzymatic phosphorylation, however, gives significantly enhanced yields of a product which is sufficiently pure so that it can be used directly in solution without isolation [541, 542]. 

DHAP-dependent aldolases were also applied in the synthesis of new indoUzidine and quinoUzidine iminocyclitols with high configurational diversity [21]. To this end, we explore the use of R) and (S)-N-Cbz-piperidin-2-carbaldehyde ((S)-lOa and... 

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