Aldolases donor substrate

Due to mechanistic requirements, most of these enzymes are quite specific for the nucleophilic component, which most often is dihydroxyacetone phosphate (DHAP, 3-hydroxy-2-ox-opropyl phosphate) or pyruvate (2-oxopropanoate), while they allow a reasonable variation of the electrophile, which usually is an aldehyde. Activation of the donor substrate by stereospecific deprotonation is either achieved via imine/enamine formation (type 1 aldolases) or via transition metal ion induced enolization (type 2 aldolases mostly Zn2 )2. The approach of the aldol acceptor occurs stereospecifically following an overall retention mechanism, while facial differentiation of the aldehyde is responsible for the relative stereoselectivity. 

Figure 10.2 Nucleophilic donor substrates of preparatively useful aldolases.

Figure 6.10 Broad donor substrate tolerance of sialic acid aldolase in synthesis of fluorinated sialic acid analogues...

Aldolases are part of a large group of enzymes called lyases and are present in all organisms. They usually catalyze the reversible stereo-specific aldol addition of a donor ketone to an acceptor aldehyde. Mechanistically, two classes of aldolases can be recognized [4] (i) type I aldolases form a Schiff-base intermediate between the donor substrate and a highly conserved lysine residue in the active site of the enzyme, and (ii) type II aldolases are dependent of a metal cation as cofactor, mainly Zn, which acts as a Lewis acid in the activation of the donor substrate (Scheme 4.1). 

The stereochemistry of the aldol reaction is highly predictable since it is generally controlled by the enzyme and does not depend on the structure or stereochemistry of the substrates. Aldolases generally show a very strict specificity for the donor substrate (the ketone), but tolerate a broad range of acceptor substrates (the aldehyde). Thus, they can be functionally classified on the base of the donor substrate accepted by the enzyme. 

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... 

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]. 

Owing to the narrow specificity of the DHAP aldolases for the donor substrate DHAP (41), direct access to this essential compound is vital to the development of synthetic applications. Commercial offers of the compound, however, are prohibitively expensive for preparative-scale applications. A further problem is that 41 is relatively unstable in solution and, particularly at elevated pH values, readily decomposes according to an Elcb pathway via an enediol intermediate... 

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

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... 

For both types of enzymes, with few exceptions, highly predictable product stereochemistry is observed, as the stereoselectivity is determined by the enzyme and does not depend on the structure or configuration of the substrate. Aldolases are fairly specific for the donor component but exhibit a more relaxed specificity for the acceptor. The aldolases that have been investigated for use in synthesis are broken down into four main groups, based on the structure of the donor substrates (Scheme 5.2). 

Scheme 5.2. The four main groups of aldolase reactions classified by their donor substrate (1) Dihydroxyacetone phosphate (DHAP)- dependent aldolases, (2) phosphoenol pyruvate (PEP)-and pyruvate-dependent aldolases, (3) 2-deoxyribose-5-phosphate aldolase (DERA), a member of the acetaldehyde-dependent aldolases, and (4) glycine-dependent aldolases (GDA).

Despite the broader substrate tolerance for aldehyde acceptors by DHAP-dependent aldolases, the donor substrate specificity is narrow. With the exception of DHAP, only a few donors have shown to be acceptable as weak substrates (Scheme 5.6).20a 21a 26... 

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). 

The dephosphorylation of 5-chloro and 5-bromo-D-xylulose-l-phosphate was carried out by the addition of acid phosphatase. After purification, 5-chloro-D-xylu-lose and 5-bromo-D-xylulose were recovered as pure compounds in 47 and 12% yields, respectively, from DHAP. In this study, we have shown that DHAP generated from glycidol 7 can be used in situ as a donor substrate of FruA in the presence of 2-halo-acetaldehydes 20 as acceptor substrates for the synthesis of 5-halo-D-xylulose 19. Given that DHAP aldolases display a broad specificity towards acceptor substrates, this strategy can be applied generally to the synthesis of various analogs of monosaccharides. 

Here we account on our research about a novel chemo-enzymatic approach to the synthesis of iminosugars from N-protected-aminoaldehydes as donor substrates and aldolases as biocatalysts for the key aldol addihon reaction. 

Our group has recently cloned a truncated Pd2,6ST containing 17-497 amino acid residues as N-hexohistine tagged protein and explored its application in the one-pot three-enzyme system for preparative synthesis of functionalized o2,6-sialosides (25). The tolerance of donor substrate modification by the purified Pd2,6ST was tested using the one-pot three-enzyme system, in which CMP-sialic acid derivatives were generated in situ from sialic acid precursors by the aldolase and NmCSS. An extremely relaxed donor substrate specificity was observed for Pd2,6ST. The preparative-sacle reactions were then carried out at... 

One normally expects antibodies to have a low tolerance to substrate modifications, however an ongoing feature of these aldolase antibodies is their wide scope. They accept a remarkable range of aldol donors and acceptors and perform crossed-, intramolecular- and retro-variants of this reaction, with high yields, rates, and stereospecificities [81,82,83]. Substrate modification experiments have revealed that when acetone is the aldol donor in a ketone-aldehyde crossed aldol reaction, stereoinduction is linked to attack of the sz-face of a prochiral aldehyde with typically >95% ee and when hydroxyacetone is the donor substrate, attack occurs preferentially at the re-face of the aldehyde leading to a diastereomeric a,P-dihydroxy ketones with the two stereogenic centers having an a-syn configuration. This reaction leads to stereospecificities of typically 70 to >99% ee. 

There have been more than 20 aldolases isolated, eight of which have been explored for organic synthesis (6). Aldolases possess two interesting common features the enzymes are specific for the donor substrate but flexible for the acceptor component, and the stereochemistry of aldol reaction is controlled by the enzyme not by the substrates. In our previous study, we have described the use of lipases, hexokinases, glycosyl transferases and rabbit muscle aldolase for the synthesis of certain fluorosugars (7). This review describes our recent development in aldolase-catalyzed reactions for the synthesis of fluorosugars. 

Fuc 1-P aldolase, Rha 1-P aldolase, and TDP aldolase also use DHAP as the donor substrate in aldol condensation. Fuc 1-P aldolase catalyzes the reversible condensa-... 

Extensive substrate specificity studies have indicated that only pyruvate is acceptable as the NeuAc aldolase donor substrate11161. However, this enzyme has broad acceptor specificity, and over sixty aldoses have been characterized as substrates. Substitutions at C2, 4 and 6 of ManNAc are allowed, with only a slight preference for absolute stereochemistry at C4, 5 and 61118-120, 125-13°1, Some pentoses and their analogs are also substrates, although 2 and 3 carbon molecules are not accepted. 

Figure 14.1-26. Aldol addition reaction catalyzed in v/Vo by KHG aldolase and the donor substrate specificity of this enzyme.

Although both types of aldolases are highly specific for the donor substrate they show some flexibility as far as the structure of the acceptor is concerned. Some of them accept variety of unnatural compounds. The stereoselectivity of the addition is in general controlled by the enzyme and does not depend on the structure of substrate. This rule is in force to almost all know aldolases with only few exceptions. 

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. 

Aldolases are a group of C—C bond forming enzymes with widespread applications. The stereoselective aldol addition reaction catalyzed by aldolases represents an attractive alternative to conventional chiral organic chemistry methods for chemical and pharmaceutical industries. Aldolases are classified according to both their proposed catalytic mechanism and the structure of the donor substrate, their sources and microbial production processes being presented in this chapter. To design appropriate bioreactors for aldol synthesis, the characteristics of aldolase biocatalysts obtained after purification procedures in free and immobilized form are discussed. 

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