Acetylneuraminic aldolase and

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-... 

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which N-acetylneuraminic acid (NeuAc) is generated in situ from IV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

Brunetti, P., Jourdian, G. W., and Roseman, S., 1%2, The sialic acids III. Distribution and properties of animal N-acetylneuraminic aldolase, J. Biol. Chem. 237 2447-2453. Burnet, F. M., 1948, Mucins and mucoids in relation to influenza virus action. III. Inhibition of virus haemagglutination by glandular mucins, Aust. J. Exp. Biol. Med. Sci. 26 371-329. 

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]. 

Mahmoudian, M., Noble, D., Drake, C.S. etal. (1997) An efficient process for production of N-acetylneuraminic acid using V-acetyl neuraminic acid aldolase. Enzyme and Microbial Technology, 20 (5), 393—400. 

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. 

Figure 6. Synthesis of 9-0-acetyl-N-acetylneuraminic acid. The aldol acceptor was prepared from N-acetylmannosamine and isopropenyl acetate in DMF catalyzed by protease N obtained from Amano. The aldol condensation was carried out by using N-acetylneuraminic acid aldolase as catalyst.

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. 

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... 

A-Acetyl neuraminic acid aldolase [from Clostridium perfringens, A-acetylneuraminic acid pyruvate lyase] [9027-60-5] [EC 4.1.3.3]. Purified by extraction with H20, protamine pptn, (NH4)2S04 pptn, Me2CO pptn, acid treatment at pH 5.7 and pptn at pH 4.5. The equilibrium constant for pyruvate + n-acetyl-D-mannosamine ++ /V-acetylneuraminidate at 37° is 0.64. The Km for A-acetylneuraminic acid is 3.9mM in phosphate at pH 7.2 and 37°. [Comb and Roseman Methods in Enzymology 5 391 1962). The enzyme from Hogg kidney (cortex) has been purified 1700 fold by extraction with H20, protamine sulphate pptn, (NH4)2S04 pptn, heat treatment between 60-80°, a second (NH4)2S04 pptn and starch gel electrophoresis. The Km for A-acetylneuraminic acid is 1.5mM. [Brunetti et al. JBC 237 2447 1962). 

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],... 

M.-J. Kim, W. J. Hennen, H. M. Sweers, and C.-H. Wong, Enzymes in carbohydrate synthesis 79-Acetylneuraminic acid aldolase catalyzed reactions and preparation of 7V-aceiyl-2-deoxy-D-neuraminic acid derivatives, J. Am. Chem. Soc. 770 6481 (1988). 

W. Fitz and C.-H. Wong, Combined use of subtilisin and Al-acetylneuraminic acid aldolase for the synthesis of a fluorescent sialic acid, J. Org. Chem. 59 8279 (1994). 

Immobilized sialyl aldolase (50 mL of gel, 68 U) was added to a mixture of 88% pure A-acetylmannosamine (20 mmol), sodium pyruvate (180 mmol), 1,4-dithiothreitol (0.2 mmol), and sodium azide (20 mg) in 0.05 M potassium phosphate buffer, pH 7 (150 mL). The suspension was gently stirred under nitrogen for 4 d at 37°, the reaction being monitored by t.l.c. in 7 3 propanol - water. The gel was removed by filtration, washed with the buffer, and A-acetylneuraminic acid (2) was isolated by chromatography on Dowex 1 X8 (HCOj-) resin, using a gradient of formic acid as the eluant, in 66% yield. The gel was used in four successive runs. Starting from 17 g of 88% pure A-acetylmannosamine, the procedure allowed the synthesis of 14 g of A-acetylneuraminic acid (2). In the end, the recovered gel retained 80% of its enzymic activity. 

The preparation of trisaccharide 63 illustrates the activation and enzymic coupling of the 9-acetate of vV-acetylneuraminic acid, This involves the utilization of enzymes in a cascade of reactions which probably do not occur in cells (a) synthesis of Neu5,9Ac2 from the 6-acetate of vV-acetylmannosa-mine with the catabolic sialyl aldolase, (b) activation with CMPNeu5Ac synthetase, and (c) coupling. Acetylation in cells seems posterior to coupling. Terminal nonreducing vV-acetyl-9-O-acetylneuraminic acid residues appear... 

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... 

N-Acetylneuraminic acid aldolase (NeuAc aldolase) is commercially available and has been the subject of much attention [49]. NeuAc aldolase catalyzes the aldol reaction between pyruvate and mannose or mannose derivatives. The enzyme activates the donor as its enamine, similar to the Type I aldolase described above (Scheme 5.21). The enzyme has been used for the synthesis of aza sugars and var-... 

In a related protocol, the acetaldehyde trimer 54 from the generic RibA oligomerization was found to be a substrate for the N-acetylneuraminic acid aldolase (NeuA EC 4.1.3.3) which catalyzed the addition of pyruvate. By this means, a tetradeoxy-L-arahi o-2-nonulosonic acid 56 was obtained in 55% yield [116]. A one-pot, tandem operation was complicated by the fact that temperature requirements for optimum activity and stability of the two catalysts were not compatible. 

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]. 

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