NANA aldolase

Carbon-carbon bond lyases, used in the reverse, synthetic direction have also enjoyed significant application in the pharmaceutical industry. For example 7/-acetyl-D-neuraminic acid (NANA), an intermediate in the chemoenzymatic synthesis of the influenza virus sialidase inhibitor zanamavir, may be synthesized using NANA aldolase. 

Catabolic enzyme NANA aldolase catalyses cleavage of NANA to form NAM and pyruvic acid, the latter being a more attractive material for a chemoenzymatic process. It has long been known that the reverse reaction may be used for NANA synthesis. However, this approach to a manufacturing process also has complications. 

NAM is produced by base-catalysed epimerization of N-acetyl-o-glucosamine (NAG), generating an unfavourable 1 4 mixture of NAM NAG. NAG, although not a substrate for the aldolase, inhibits the reaction. In addition, excess pyruvate is required to push the equilibrium in favour of product formation (Scheme 1.31). Although 90% yields can be obtained at laboratory scale using E. coli NANA aldolase using a NAG NAM mixture, the NANA product is difficult to separate from the excess pyruvate required to achieve this. 

As an alternative to chemical epimerization, NAG epimerase may be used to maintain a constant NAM NAG ratio in a one-pot reaction with pyruvate and NANA aldolase. The epimerase is itself inhibited by pymvate, which must, therefore, be added continuously or via aliquots to the reaction. In a refined version of this reaction at laboratory scale, Kragl et al produced NANA by a continuous process, using a membrane reactor to contain both enzymes in solution. 

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

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. 

NANA NANA aldolase/ Nac-epimerase aldol reaction 45 Kragl, 1995... 

In considering the application of enzyme catalysis to DCC, we were encouraged by the thermodynamic resolution of a dynamic mixture of aldol products by Whitesides and co-workers through the use of a broad-specificity aldolase to lead to reversible formation of carbon-carbon bonds under mild conditions.35 For the current investigation36 we chose a related enzyme, N-acetylneuraminic acid aldolase (NANA aldolase, EC 4.1.3.3), which catalyzes the cleavage of N-acetylneuraminic acid (sialic acid, 27a) to A-acetylmannosamine (ManNAc, 28a), and sodium pyruvate 29 in the presence of excess sodium pyruvate, aldol products 27a-c are generated from... 

Generation of the dynamic library proved straightforward a mixture containing equimolar amounts of the three substrates 28a-c and two equivalents of sodium pyruvate was incubated in the presence of NANA aldolase (Figures 29.2 and 29.3). Aliquots of the incubation mixture were withdrawn at intervals and analyzed by ion-exchange HPLC. 

It was evident that the aldol reaction had reached equilibrium after 16 hours incubation because the product distribution changed very little after this time. Evidence that the enzyme does indeed catalyze both aldol formation and cleavage on the timescale used was demonstrated by reequilibrating a mixture of sialic acid and D-mannose in the presence of the NANA aldolase. After incubation overnight the mixture contained, in addition to the initial components, KDN and sodium pyruvate, which could only arise through a retro-aldol cleavage of sialic acid followed by aldol formation of KDN. 

As determined by HPLC analysis of standard solutions of synthetic sialic acid. The control mixture contained D-mannose and ManNAc (0.5 pmol each), sialic acid (0.13 pmol), and NANA aldolase in buffer solution (0.2 U), total volume 40 pL. 

N-Acetylneuraminic Acid Aldolase Origin microorganisms Unitika N-Acetylneuraminic Acid Aldolase (Nana-Ald)... 

FIGURE 5. Reverse aldolization cleavage of NANA to N-acetylmannosamine (X) and pyruvate with aldolase enzyme, and to N-acetylmannosamine, N-acetylglucosEunine (XI)... 

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