Sialic aldolase

Commercial A -acetylneuraminic acid aldolase from Clostridium perfringens (NeuAcA EC 4.1.3.3) catalyzes the addition of pyruvate to A-acetyl-D-mannosamine. A number of sialic acid related carbohydrates are obtained with the natural substrate22"24 or via replacement by aldose derivatives containing modifications at positions C-2, -4, or -6 (Table 4)22,23,25 26. Generally, a high level of asymmetric induction is retained, with the exception of D-arabinose (epimeric at C-3) where stereorandom product formation occurs 25 2t The unfavorable equilibrium constant requires that the reaction must be driven forward by using an excess of one of the components in order to achieve satisfactory conversion (preferably 7-10 equivalents of pyruvate, for economic reasons). 

N-Acetylneuraminic acid aldolase (or sialic acid aldolase, NeuA EC 4.1.3.3) catalyzes the reversible addition of pyruvate (2) to N-acetyl-D-mannosamine (ManNAc (1)) in the degradation of the parent sialic acid (3) (Figure 10.4). The NeuA lyases found in both bacteria and animals are type I enzymes that form a Schiff base/enamine intermediate with pyruvate and promote a si-face attack to the aldehyde carbonyl group with formation of a (4S) configured stereocenter. The enzyme is commercially available and it has a broad pH optimum around 7.5 and useful stability in solution at ambient temperature [36]. 

Figure 10.10 Natural substrates of the 2-keto-3-deoxy-monno-octosonic acid aldolase, and nonnatural sialic acids obtained by KdoA catalysis.

The power of directed evolution has been demonstrated by the conversion of an aldolase into a new kind of aldolase. Wong and coworkers evolved a pyruvate-dependent sialic acid aldolase... 

Figure 6.1 Alteration of substrate specificity of sialic acid aldolase by directed evolution...

Figure 6.3 Synthesis of modified sugars with sialic acid aldolase mutants...

Figure 6.9 Broad acceptor substrate tolerance of sialic acid aldolase in synthesis of nonnatural disaccharides...

Hsu, C.C., Hong, Z.Y., Wada, M. etal. (2005) Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (l-KDO) aldolase. Proceedings of the National Academy of Sciences of the United States of... 

Woodhall, T., Williams, G., Berry, A. and Nelson, A. (2005) Creation of a tailored aldolase for the parallel synthesis of sialic acid mimetics. Angewandte Chemie-International Edition, 44, 2109-2112. 

Huang, S.S., Yu, H. and Chen, X. (2007) Disaccharides as sialic acid aldolase substrates synthesis of disaccharides containing a sialic acid at the reducing end. Angewandte Chemie-International Edition, 46, 2249-2253. 

Of the known classes of aldolase, DERA (statin side chain) and pyruvate aldolases (sialic acids) have been shown to be of particular value in API production as they use readily accessible substrates. Glycine-dependent aldolases are another valuable class that allow access to p-hydroxy amino acid derivatives. In contrast, dihydroxy acetone phosphate (DHAP) aldolases, which also access two stereogenic centres simultaneously,... 

Flitsch and Turner reported the generation of a sialic acid DCL using an aldolase enzyme [36,37], The DCL design is shown in Scheme 2.7, and is based on the cleavage of sialic acid (58a) to A-acetylmaimosamine (56a) and sodium pyruvate (57), catalyzed by an aldolase enzyme. 

Scheme 2.7 Aldol reaction of ManNAc analogues and sodium pyruvate to produce sialic acid, catalyzed by A-acetylneuraminic acid (NANA) aldolase.

Several enzymatic procedures have been developed for the synthesis of carbohydrates from acyclic precursors. Aldolases appear to be useful catalysts for the construction of sugars through asymmeteric C-C bond formation. 2-deoxy-KDO, 2-deoxy-2-fluoro-KDO, 9-0-acetyl sialic acid and several unusual sugars were prepared by a combined chemical and enzymatic approach. Alcohol dehydrogenases and lipases have been used in the preparation of chiral furans, hydroxyaldehydes, and glycerol acetonide which are useful as building blocks in carbohydrate synthesis. 

N-Acetvlneuraminic Acid Aldolase. A new procedure has also been developed for the synthesis of 9-0-acetyl-N-acetylneuraminic acid using the aldolase catalyzed reaction methodology. This compound is an unusual sialic acid found in a number of tumor cells and influenza virus C glycoproteins (4 ). The aldol acceptor, 6-0-acetyl-D-mannosamine was prepared in 70% isolated yield from isopropenyl acetate and N-acetyl-D-mannosamine catalyzed by protease N from Bacillus subtilis (from Amano). The 6-0-acetyl hexose was previously prepared by a complicated chemical procedure (42.) The target molecule was obtained in 90% yield via the condensation of the 6-0-acetyl sugar and pyruvate catalyzed by NANA aldolase (Figure 6). With similar procedures applied to KDO, 2-deoxy-NANA and 2-deoxy-2-fluoro-NANA were prepared from NANA. 

This enzyme [EC 4.1.3.3], also known as A-acetylneu-raminate aldolase, will convert A-acetylneuraminate to A-acetylmannosamine and pyruvate. The enzyme will also act on A-glycoloylneuraminate and on O-acetylated sialic acids, other than O -acetylated derivatives. 

In an interesting extension of this work, the Neu5Ac aldolase from E. coli was subjected to directed evolution to expand its catalytic activity for enantiomeric forms of the usual substrates to include A -acetyl-L-mannosamine and L-arabinose with formation of the synthetically important products L-sialic add and L-3-deoxy-L-manno-oct-2-ulosonic add (l-KDO) (163). The evolved Neu5Ac aldolases were characterized by sequence analysis, kinetics, stereoselectivity, and in one case even by an X-ray structure analysis. Again, remote mutations were identified. It is significant... 

Asymmetric C-C bond formation is the most important and most challenging problem in synthetic organic chemistry. In Nature, such reactions are facilitated by lyases, which catalyze the addition of carbonucleophiles to C=0 double bonds in a manner that is classified mechanistically as an aldol addition [1]. Most enzymes that have been investigated lately for synthetic applications include aldolases from carbohydrate, amino acid, or sialic acid metabolism [1, 2]. Because enzymes are active on unprotected substrates under very mild conditions and with high chemo-, regio-, and stereoselectivity, aldolases and related enzymes hold particularly high potential for the synthesis of polyfunctionalized products that are otherwise difficult to prepare and to handle by conventional chemical methods. 

Synthetic studies for sialic acid and its modifications have extensively used the catabolic enzyme N-acetylneuraminic acid aldolase (NeuA E.C. 4.1.3.3), which catalyzes the reversible addition of pyruvate (70) to N-acetyl-D-mannosamine (ManNAc, 11) to form the parent sialic acid N-acetylneuraminic acid (NeuSNAc, 12 Scheme 2.2.5.23) [1, 2, 45]. In contrast, the N-acetylneuraminic acid synthase (NeuS E.C. 4.1.3.19) has practically been ignored, although it holds considerable synthetic potential in that the enzyme utilizes phosphoenolpyruvate (PEP, 71) as a preformed enol nucleophile from which release of inorganic phosphate during... 

Scheme 2.2.5.23 Alternative pathways for sialic acid synthesis using the catabolic aldolase (NeuA) or the anabolic synthase (NeuS) enzymes.

The availability of both the cataboHc aldolase and the uniquely synthetic anabolic synthase made it possible to assemble a novel continuous assay for the determination of the metabolite N-acetylneuraminic acid [46]. A combination of both enzymes, in the presence of an excess of PEP, will start a cycle in which the determinant sialic acid will undergo a steady conversion of cleavage and re-syn-thesis as a futile cycle (Scheme 2.2.5.24). With each progression, however, 1 equiv of pyruvate is liberated simultaneously, which causes time-dependent signal amplification. Pyruvate is quantified spectrophotometrically by a corresponding NADH consumption when the system is coupled to the standard pyruvate dehy-... 

In the catalysis of the lyase from C. perfringens, the participation of lysine residues forming intennediary Schiff bases between enzyme and substrate molecules, and of histidine residues, has been demonstrated with the aid of photooxidation, reagents for histidine modification, and borohydride reduction in the presence of substrate.408-418 Thus, according to Frazi and coworkers,414 the lyase belongs to the class I lyases (aldolases). The catalytic mechanism proposed is outlined in Scheme 3. Evidence has been educed for the existence of a similar mechanism of cleavage of sialic acid by the lyase enriched from pig kidney.411... 

The difference between then and now can be attributed in major part to the advent of enzymes in organic synthesis. Aldolases are nature s catalysts for the poor man s chemical aldol reaction. These remarkable enzymes can produce rare and important deoxyulosonic acids such as sialic acid and KDO on a small or large scale. 

Sialic acid aldolase (SA EC 4.1.3.3), also named A-acetylneuraminate pyruvate lyase, has been extensively used by our group in its immobilized form, first for the synthesis of large amounts of A-acety lneuraminic acid [20] and then for many natural and unnatural sialic acids [21], SA catalyzes the reversible aldol reaction of A-acetylmannosamine and pyruvate to give A-acety lneuraminic acid with an optimum pH for activity of 7.5 and an equilibrium constant of 12.7 A/-1 in the synthetic direction (Scheme 3) [10],... 

Therefore, to achieve high conversion of the substrate a tenfold excess of pyruvate is usually needed. The enzymes from Clostridium perfringens and Escherichia coli are commercially available from Toyobo the E. coli enzyme has been cloned and overexpressed, which has considerably reduced its cost [22,23], Sodium borohydride inactivates the enzyme in the presence of either sialic acid or pyruvate, indicating that the enzyme belongs to the Schiff-base-forming class 1 aldolase. This aldolase was supposed to be a... 

Scheme 3 Reversible aldol addition reaction catalyzed by sialic acid aldolase.

The sialic acid aldolase-catalyzed condensation of D-mannose 8 and pyruvate led, in an excellent yield, to the synthesis of KDN 9 [33], a natural deaminated neuraminic acid first isolated from rainbow trout eggs [34] and then discovered in other species. The discovery that sialic acid aldolase accepts as substrates D-mannose substituted on the 2-position, even by bulky substituents such as phenyl, azido, or bromine, opened the route to novel unnatural sialic acid derivatives [35-39]. Pentoses also are substrates. N-Substituted neuraminic acids could be prepared either directly from the corresponding Af-substituted mannosamine, such as N-thioacyl derivatives [40], or after reduction and acylation of 5-azido-KDN [41]. Recently, AT-carbobenzyloxy-D-mannosamine was converted, in a good yield, into the N-carbobenzyloxy-neurarninic acid, further used as a precursor of a derivative of castanospermine [42]. 

Table 2 lists 37 sialic acid derivatives and analogues that have been synthesized with sialic acid aldolase by our group and others. This is a nice illustration of the great synthetic potential of the enzyme. In all of these examples, sialic acid derivatives or analogues with equatorial hydroxyl on C-4 are formed, corresponding to a new 45 chiral center and attack of the pyruvate from the si face of the aldehyde. In summary sialic acid aldolase accepts modifications on the 2, 4, 5, and 6 positions of the substrate without any change of its stereoselectivity. 

Table 2 Natural Sialic Acids and Analogues Synthesized with Sialic Acid Aldolase...

Scheme 4 Lack of steroselectivity observed in the sialic acid aldolase-catalyzed addition of pyruvate with D-arabinose.

Moreover a complete inversion of stereoselectivity has been reported with L-man-nose and L-rhamnose [49]. Table 3 lists KDO analogues with nine carbons that could be prepared as pure compounds using sialic acid aldolase. They are derived from L-hexoses belonging to l series with the R configuration at the 3- and 2-positions. In each case, KDO-type derivatives with equatorial hydroxyl on C-4 are formed, corresponding to a 4R chiral center and attack of the pyruvate from the re face of the aldehyde. Thns l-KDN... 

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