Neu5Ac aldolase

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... 

N-acetyl-D-neuraminic acid GlcNAc+pyravate Neu5Ac-aldolase... 

Neu5Ac aldolase has also been used for the synthesis of 3-deoxy-D-mfl7j jo-octulosonic acid (KDO, 17) [16,17,20]. Condensation of o-arabinose (18) with pyruvate gave a mixture of KDO and 4-epi-KDO (19). Wong and coworkers have since reported the isolation of a KDO aldolase which produces KDO with complete stereospecificity at C-4 and also accepts a wide variety of carbohydrate substrates [51]. N-Acetylneuraminate synthase, found in Neisseria meningitidis, has been used to catalyse the condensation of 6-azido-6-deoxy-N-acetylmannosamine with phosphoenolpyruvate to give 9-azido-9-deoxy-Neu5Ac [52]. 

Table 1. A sample of marmo-configurated sugars tested as substrates for Neu5Ac aldolase ...

Free sialic acids are used by the enzymes of sialic acid metabolism, CMP-sialate synthase and Neu5Ac aldolase. However the majority of sialic acid-metabolising and -recognising proteins interact with glycosidically bound sialic acid. There-... 

Figure 7-5. Deactivation of Neu5Ac-Aldolase depending on the reactor material1471.

Figure 7-7. Synthesis of N-acetyl-neuraminic acid with Neu5Ac-aldolase equilibrium conversion as a function of the concentration of both substrates1471.

In contrast, Neu5Ac aldolase turned out to be very useful for synthetic purpose because it tolerate wide range of unnatural substrates [21,23]. The enzyme also named sialic acid aldolase (EC 4.1.3.3.) catalyze reversible reaction of A/-acetyl-D-mannosamine (15) and pyruvate (Scheme 3) [33-35],... 

Neu5Ac aldolase has been isolated from many sources including bacteria and mammals. Significant interest toward sialic acid analogs and commercial availability of the aldolase, have led to the intensive studies on this enzyme, which resulted in the knowledge that despite its highly specificity for pyruvate [36] it accepts a number of different acceptor... 

Table(2). Neu5Ac analogs prepared using Neu5Ac aldolase.

Neu5CBz (entry 9) and 5-azido-5-deacetamido-Neu (entry 12) have been prepared by treatment of the corresponding 2-modified mannose derivatives with pyruvate in the presence of Neu5Ac aldolase. The C-5 azido group may be easily converted to the free amine. Synthesis of 7-amino-substituted Neu5Ac derivative has been presented [50] via azido derivative as an alternative route to the particularly difficult chemical... 

Scheme (4). Neu5Ac aldolase catalyzed reaction of A/-acetyl-D- and L-mannosamine...

Unlike the most known aldolases the stereochemical outcome of reactions catalyzed by Neu5Ac aldolase depends on the structure of the substrate. In substrates with the natural S configuration at C-3... 

Some observations indicate that the stereochemical outcome of the reaction seems to be under thermodynamic control, and stereochemistry at C-4 can be reversed. For example, in the Neu5Ac aldolase catalyzed synthesis of KDO, a mixture of (5)-C-4 and (7 )-C-4 were isolated when D-arabinose was the substrate (Scheme 5) [47,48]. 

A sequential aldol reaction leading to sialic acid derivatives has been also presented [59], Combination of acetaldehyde dependent aldolase (DERA) and Neu5Ac aldolase catalyzed reactions led to (R)-C-4 keto acids. In this case, however, one-pot reaction sequence was not possible due to the incompatibility of the reaction conditions for two enzymes. 

As Neu5Ac aldolase accepts a range of substrates (more than 60 are known) in place of ManNAc, this enzyme can also used to synthesize Neu5Ac derivatives [149]. For example, if ManNAc is replaced by D-mannose, Neu5Ac aldolase can be used for the preparative-scale synthesis of KDN (3-deoxy-(5-D-g/ycer0-D-g /acto-2-nonulosonic acid Fig. 7) [150],... 

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