176 Sialic acid formation

The wide occurrence of sialic acids in nature is an indication of their great biological importance. As is described in other chapters in this book, the identification of the sialic acids was pioneered in several laboratories, while the biosynthetic mechanism of sialic acid formation was elucidated essentially by the work of Roseman s and Warren s groups in the United States (see Roseman 1962, Warren 1972, Schachter and Roden 1973, McGuire 1976, Schachter 1978). 

As outlined in sections II. 2-4, several enzymes are subject to negative feedback control during sialic acid formation. These are listed in Fig. 9. The central intermediates are UDP-GlcNAc and CMP-Neu5Ac. As has been described for other monosaccharide activation pathways, the negative feedback influences the first enzymic steps in the pathway (ScHACHTERand Roden 1973, Schachter 1978). 

The biosynthetic pathway for sialic acid formation in liver was well established nearly thirty years ago, by the elegant work from the groups of Roseman [9-11] and Warren [12] (Figure 1). The enzymatic conversion of UDP-A-acetyl glucosamine to A-acetylmannosamine is the critical step in the overall synthesis of sialic acid because the activity of this epimerase is controlled downstream in the pathway by feedback inhibition by CMP-A -acetylneuraminic acid [13]. Thus, production of ManNAc as a precursor for sialic acid biosynthesis is a closely regulated cellular process. 

UDP-GlcNAc-2-epimerase activity [24], has not been detected in many tissues other than liver suggesting that there may be alternate routes for sialic acid formation. Recently the gene for the epimerase has been used as a probe for Northern blot analysis of mRNA from various tissues. The lack of an epimerase mRNA signal from many of the tissues confirms the observations of earlier enzymatic studies [25, 26]. 

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]. 

Most nucleotide sugars are formed in the cytosol, generally from reactions involving the corresponding nucleoside triphosphate. GMP-sialic acids are formed in the nucleus. Formation of uridine diphosphate galactose (UDP-Gal) requires the following two reactions in mammahan tissues ... 

The sensors discussed so far are based on ligands covalently bound to the polymer backbone. Other methods of detection - often referred to as mix and detect methods - work by simple noncovalent incorporation of the polymer with the ligand of interest. Reichert et al. generated liposomes of polydiacetylene with sialic acid for the same purpose of detection as Charych s surface-bound polymers, but realized that covalent functionalization of the polymer was not necessary [17]. Through simple mixing of the lipid-bound sialic acid with the polymer before sonication and liposome formation, they were able to form a functional colorimetric recognition system (Fig. 8). 

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. 

In a similar manner, sialic acids are liable to lactone formation. In some cases the observed lactones have been suggested to possess enhanced biological... 

Sialic acid occurs naturally as both the N-acetylated and the N-glycoloylated form, the proportion in a given environment being different from one species to another.316 Oxidation of the acetyl group to the glycoloyl group occurs after the formation of 2-acetamido-2-deoxy-D-glucose.317... 

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. 

Synthesis of sialic acid in the nervous system also exhibits a unique, control mechanism that may be delineated from the observation of an increased formation of sialic acid (ganglioside) in rat brain during training.242... 

Nothing is yet known concerning the formation of lactvl groups found at 0-9 of sialic acids from different sources, reported in Section II. This is also true of the biosynthesis of methyl ether and sulfuric ester groups occurring in some natural sialic ac ids. 

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