O-Acylated sialic acids 118

Mass Units of Characteristic Fragment-ions A-G Used for the Identification of Natural, N,O-Acylated Sialic Acids, and of the C7 and C8 Analogs of Neu5Ac and Neu5Gc as their Methyl Esters, Trimethylsilyl Ethers, by Mass Spectrometry"... 

Histochemical demonstration of most of the O-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation of this part of the molecule to an extent dependent on the number and position of the O-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanol91) may increase the staining reaction of a sialic acid. For example, the presence of O-acylated sialic acids has been demonstrated in colonic, epithelial mucin of man and various mammals (summarized in Ref. 91), in healthy and diseased, human small-intestine,188-190 in bovine submandibular gland,182 in mouse and rat erythrocyte-membranes,191 and in human lymphocytes.192... 

Muramic acid has been determined quantitatively by a procedure that involves its degradation to acetaldehyde via lactic acid. Acetaldehyde was measured spectrophotometrically, and other components present in hydrolysates of bacterial cell walls did not interfere with the determination. The analysis of natural and synthetic N- and O-acylated sialic acids by g.l.c. of the TMS ethers has been described. The 0-acyl groups were not lost when iV-trimethylsilyl-imidazole was used as the silylating reagent. A quantitative determination of neuraminic acid by g.l.c.-m.s. (using multiple ion-detection) has been reported. A modified periodate-resorcinol procedure, which avoids extraction of the... 

The preparation of acylneuraminic acids is desirable to provide characterized standards for analytical methods and as a basis for the development of synthetic and new analytical techniques. Several methods exist for this, involving hydrolysis of material containing glycosidically bound sialic acids by dilute aqueous or methanolic acids or by enzymic means. Problems exist with respect to O-acyl-sialic acid preparation due to their lability during acid hydrolysis and their reduced or non-susceptibility towards the action of sialidases. Standard techniques have been developed over the last 15 years in the authors laboratory and these are discussed in the light of alternative methods and improvements which have appeared in the literature. 

H. Ogura, K. Furuhata, S. Sato, and K. Anazawa, Synthesis of 9-O-acyl and 4-O-acetyl sialic acids, Carbohydr. Res. 167 11 (1987). 

The paper-chromatographic analysis of the nonulosaminic acids has been hampered by their lability toward acid and alkali. This holds particularly for the higher acylated sialic acids. The mobilities of iV-acetyl-O-acetyl-neuraminic acid, iV-acetylneuraminic acid and its methyl ester, and A -glycolyl- and methoxy-neuraminic acid are given in Table IV. 

Mild acid hydrolysis is less specific than sialidase digestion and also less expensive. Attention should be paid to the different conditions of hydrolysis required for various glycoconjugates and the slower release of 0-acylated sialic acids relative to Neu5Ac (Ledeen and Yu 1976) (see chapter C). To obtain optimal quantitation, a release curve for sialic acid should be prepared (Schauer et al 1975, O Kennedy 1979). In addition to this precaution, the destruction of sialic acid under the conditions of the hydrolysis should be assessed (e.g. Schauer et al 1975, O Kennedy 1979). The inclusion of a sialic acid standard under the conditions of hydrolysis will allow assessment of the level of destruction (Schauer et al 1975, Schauer 1978, Caimi et al 1979). 

Sialic acids generally occur in vertebrates, as is summarized in Refs. 19-21. Neu5Ac was isolated in our laboratory from the secreted mucus of the cyclostome Mi/xine glutinosa,20 a discovery in contrast to an earlier report by Wessler and Werner.3 The sialic acid content of the dried mucus is 0.5%. The greatest variety of N- and O-acyl derivatives of Neu was found in mammals, as will be reported later. 

The nature of the O-acyl substituents can be tentatively determined by t.l.c. as acylhydroxamates. However, the nature and position of the ester groups in sialic acids can only be unequivocally determined by g.l.c.-m.s. or by n.m.r. spectroscopy (see later). As O-acetyl groups have been found to migrate within the sialic acid side-chain90 (see... 

Although the different methods for the analysis of sialic acids thus far described give some insight into the structure of these compounds, mass spectrometry permits unambiguous determination of the chain length and the number, type, and position of N- or O-acyl substituents in Neu derivatives. Thus, a variety of new and rare sialic acids have been discovered in the past few years, and the presence of known, mainly O-acetylated, sialic ac ids has been established in a variety of biological materials.34-37,62 85,94,95-97-96-10 -107-147-149... 

Radioactive acetate is a cheaper, readily available precursor for experiments on the labelling of sialic acid in tissues or cells, and it effectively labels the N- and O-acyl groups of sialic acids.228 This method is of great value not only for the preparation of radioactive sialic acids having high specific radioactivity but also for metabolic studies of sialic acids. However, the sialic acids must be isolated before determination of the specific radioactivity, as other aeetylated hexosamines are also labelled. 

The sialic acid aldolase-catalyzed condensation of o-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. A-Substituted neuraminic acids could be prepared either directly from the corresponding A-substituted mannosamine, such as A-thioacyl derivatives [40], or after reduction and acylation of 5-azido-KDN [41]. Recently, A-carbobenzyloxy-o-mannosamine was converted, in a good yield, into the A-carbobenzyloxy-neuraminic acid, further used as a precursor of a derivative of castanospermine [42]. 

Sialic acid is the general name of a mily of nine-carbon sugars, which are N- and 0-acyl derivatives of neuraminic add, 5-amino-3,5-dideoxy-D-gi ero-D-ga/ac/o-2-nonulopyranosic add such as 5-N-acetylneuraminic acid (NeuSAc, 1) which is the most natural occurring sialic add [1]. 

---