key: cord-0039518-xjfvzdjg authors: Schauer, Roland; Kamerling, Johannis P. title: Chemistry, biochemistry and biology of sialic acids() date: 2008-05-29 journal: nan DOI: 10.1016/s0167-7306(08)60624-9 sha: b9197522c6f6c8b536e91e67389e0a1b7c8d59be doc_id: 39518 cord_uid: xjfvzdjg nan Since the discovery of N-acetylneuraminic acid, the most universal sialic acid, at the end of the 1930s [1,2] as well as the structural and stereochemical elucidation of its free and bound forms at the end of the 1960s (reviewed in ref. [3] ), there has been a continual increase in the number of sialic acid types (1 994: more than 40) recognized to occur in a variety of living organisms. It is now generally accepted that naturally occurring sialic acids are monosaccharides which influence many important biological and pathological phenomena. In previous articles [3-51, the former literature has been extensively reviewed with respect to a number of chemical, biological, metabolic, functional as well as historical aspects. Since 1982 [4, 5] , the proliferation of the literature on the chemistry, biochemistry and (molecular) biology of sialic acids has accelerated dramatically, and several short reviews dealing with specific aspects of sialic acids have appeared [6-121. The aim of this chapter is to collate a mixture of, what is in our opinion, relevant information published before 1982 and new data appeared since then. Because of the profusion of biochemical and biological data published in the last few years, it was necessary to select those reports which, we believe, reflect potential trends in the future development of sialobiologyr In Table 1 a survey of the 43 naturally occurring members of the sialic acid family [ 13-5 13 , together with their abbreviations and typical biological sources, is presented. The general name "sialic acid" is derived from the Greek "sialos", meaning saliva. Taking into account the Rules for Carbohydrate Nomenclature, as recommended by the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (1999, the mother-molecule neuraminic acid, which does not occur in free form in nature, due to its immediate cyclization to form an internal Schiff base, is systematically named 5-amino-3,5-dideoxy-~-gbcero-~galacto-non-2-ulosonic acid (Fig. 1 ) , and abbreviated as Neu, whereby the D-notation is implied in the trivial name. Chemically, this nine-carbon-containing monosaccharide is a 2-keto-carboxylic acid, a deoxysugar, and an aminosugar. The amino group is generally N-acetylated (5-acetam~do-3,5-d~deoxy-~-g~cero-~-galacto-non-2-ulopyranoson~c acid; fish glycoconjugates [511 a If possible, typical biological sources have been indicated. In the case of a sialic acid structure being proven by mass spectrometry andor NMR spectroscopy, reference numbers refer in general to such studies or to review articles including this sialic acid. N-acetylneuraminic acid; Neu5Ac) ( Fig. 1 ) or N-glycolylated (5-hydroxyacetamido-3,5-d~deoxy-~-glycero-~-galacto-non-2-ulopyranoson~c acid; N-glycolylneuraminic acid; Neu5Gc). As is evident from Table 1 , the hydroxyl groups may be free, esterified (acetylated, lactylated, sulfated, phosphorylated) or etherified (methylated). In the case where a sialic acid bears a hydroxy group instead of an amino group at C5, so-called deaminated neuraminic acid, it is systematically named 3 -deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Fig. l) , and is abbreviated as Kdn (2-keto-3-deoxynononic acid; also in this case the D-notation is part of the trivial name). Sialic acids are relatively strong acids, e.g. Neu5Ac has a pK, value found in the range of 2.2-3.0 in various studies with an average of 2.6. This strong acidity is responsible for processes such as autohydrolysis of sialic-acid-containing carbohydrate chains. Conformationally, sialic acids adopt the 2Cs chair conformation, having the glycerol side chain in an equatorial orientation [52] . Due to hydrogen bonding of HO7 and H08, leading to a trans-orientation of these groups, the glycerol side chain is not as flexible as one might expect. This has consequences for the course of the mild periodate oxidative degradation of this moiety in the case of substitution at C9 [53] . Free sialic acids have mainly the b-anomeric ring structure (>93%), whereas glycoconjugate-bound sialic acids occur specifically in the a-anomeric form [54] . In nucleotide-bound sialic acids, i.e. CMP-Neu5Ac (Fig. 2) , a P-configuration for the glycosidic bond is present [55] . Crystalline Neu5Ac occurs specifically in the 0-anomeric form [56] . With respect to 0-acetylation patterns in sialic acids, it is worthwhile to mention that spontaneous migration of 0-acetyl groups can occur between C7, C8 and C9. At pH values at which no significant de-0-acetylation is observed (e.g. physiological pH values), Neu5,7Ac2 can readily transform into Neu5,9Ac2, whereas Neu5,7,9Ac3 yields an equilibrium of Neu5,7,9Ac3 and Neu5,8,9Ac3 in a molar ratio of approximately 1:l; Neu4,5Ac2 does not give rise to 0-acetyl migrations [23] . Also starting from a-Neu5,8,9Ac3 4-aminophenylthio-glycoside a 1 : 1 equilibrium between the 8,9-and 7,9-di-O-acetylated derivatives is established [57] . 9-0-Acetylated N-acylneuraminic acids have been found in both glycoconjugates and oligolpolysaccharides of different biological origin ( Table 1) . The same is true for 7-0-acetylated N-acylneuraminic acids. It should be emphasized that Neu5,9Ac2 and other side-chain-0-acylated sialic acids occur in man[%] (see also sections 8.4.2 and 8.4.3), e.g. in human colon. Ungulates form the major source for 4-0-acetylated N-acylneuraminic acids [ 131, whereas minor sources are monotremes [19] , guinea pigs [21] and humans [59] . In an evaluation of the naturally occurring 0-acylation patterns, it is evident that 0-acyl groups are most frequently found at C9 of both NeuSAc and NeuSGc. 0-Methylated and 0-sulfated N-acylneuraminic acids have only been found in lower animals, e.g. in echinoderms [13, 31, 42] . 5-N-Acyl-2-deoxy-2,3-didehydro-neuraminic acids like Neu2enSAc (S-acetamido-2,6anhydro-3,5-d~deoxy-~-g~cero-~-galacto-non-2-enon~c acid, Fig. 3 ) occur in free form in nature. Moreover, they can be generated from corresponding CMP-N-acylneuraminic acids in a non-enzymatic elimination reaction, occurring under physiological and, much faster, under alkaline conditions [34, 35] . Small amounts of Neu2enSAc are formed by a water elimination side reaction from NeuSAc during influenza-B-virus-sialidase-catalyzed desialylations of sialoglycoconjugates [60] . S-N-Acety1-2,7-anhydro-neuraminic acid (Neu2,7anSAc, Fig. 3 ), which occurs in free form in nature [36, 37] , can be generated from NeuSAc(a2-3)Gal(fi 1-containing glycoconjugates using a sialidase isolated from the leech Macrobdella decara [38,6 13. Members of the sialic acid family occur mainly in bound form in higher animals, from the echinoderms onwards in evolution, but also in some viruses, various bacteria, protozoa, and pathogenic fungi [8, 13, 69] . Generally, they are constituents of glycoproteins [20, 21, 25, 40, [46] [47] [48] 5 1,70-I071 (Table 2) , glycolipids [13,3 1,44 ,108-1241 (Table 3) , and oligosaccharides [ 16, 19, 25, 76, (Table 4 ), usually occurring as terminal monosaccharide units, and of homo-and heteropolysaccharides [24, 26, 27, 49 ,156-I851 (Table 5 ). However, glycosylphosphatidylinositol membrane anchors have also been shown to contain sialic acid [ 1861. Neu5Ac-and Neu5Gc-containing glycans frequently occur together, whereby ratio differences reflect species specificity, tissue specificity or physiological fluid specificity. Also Kdn has been found together with NeuSAc/SGc in specific glycoconjugates (Table 2 ). It should be noted that the presence of Neu5Gc in normal human tissue and soluble glycoproteins has not been established conclusively [ 13,187,1881. As is evident from Table 2 , NeuSAc/SGc-and Kdn-containing elements in Nand O-glycoprotein glycans do occur in many different microenvironments, however, with a restricted glycosidic linkage specificity. In general, N-acylneuraminic acids are a-2,3-or a-2,6-linked with D-galactose (Gal), a-2,3-or a-2,6-linked with N-acetyl-Dgalactosamine (GalNAc), a-2,6-linked with N-acetyl-D-glucosamine (GlcNAc), or a-2,8linked with other N-acylneuraminic acids. These types of glycosidic linkages have been firmly established by different analytical methods, including NMR spectroscopy [76, 77] . The sialic-acid-containing N-linked carbohydrate chains form part of the complex (Nacetyllactosamine and N,N'-diacetyllactosediamine subtypes; mono-, di-, tri-, tri'-, and tetraantennae) or the hybrid type of structures [ 1891. For the mucin-type 0-glycans, the N-acylneuraminic-acid-containing sequences are extensions of most of the wellestablished core types [ 1901. In addition, terminal NeuSAc(a24)Gal [75] , terminal NeuSAc(a24)GlcNAc [96] , and internal Fuc( 14)NeuSGc [97] sequences have been reported, and only one example of a terminal NeuSAc(a2-9)NeuSAc(a2-dimer has Table 2 Sialic-acid-containing elements in N-and 0-glycoproteins a Partial structure N 0 Ref(s). [751 + [70, 72, 73] + [70, 73] + + [76, 77] + + [77, 78] + [79, 80] + + [77, 81] [25, 84] + [25, 84] + + [76, 77] + [821 + ~3 1 + [791 + + [771 {NeuSAc(a2-8)},-with 4Ac, 7Ac, 9Ac or 9Lt + [loo] {Neu5Ac/SGc(a2-8)},-with 4Ac, 7Ac or 9Ac + [loo] { NeuSGc(a2-05)},,- In the case of a specific fragment being established by ' H NMR spectroscopy, the reference refers to such a study or to a review that includes the fragment. S means sulfate. been described [98] . In a number of structures not only sialic acid is responsible for the acidic character of the carbohydrate chain, but also sulfate. Here, an unusual example is the Neu5Ac(a2-3)[6S]GaI@ 14)GlcNAc element [82] . The list of sialic-acid-containing sequences, in which N-acylneuraminic acid is replaced by Kdn is continuously growing ( Table 2) . Interestingly, also structural elements occur which have not been found so far for N-acylneuraminic acids, e.g. General structural information with respect to glycoprotein glycans is presented in volume 29a of the New Comprehensive Biochemistry series, and detailed information with respect to poly-N-acylneuraminyl-containing glycoproteins and Kdn-containing glycoproteins in the present volume 29b. Several of the sialic-acid-containing sequences present in Nand 0-glycoprotein glycans, also occur in glycolipids, milk and urinary oligosaccharides, and in (1ipo)polysaccharides of different biological origin. An impression of this overlap in structures can be obtained from an inspection of the structural data in Tables 3-5 , summarizing sialic-acid-containing elements of glycolipids, structures of milk and urinary sialo-oligosaccharides, and structures or elements of sialic-acid-containing (lipo)polysaccharides, respectively. It is interesting to note that for both glycoprotein and glycolipid glycans a sequence has been found comprising the oligomerization of Neu5Gc residues through their anomeric centers and N-glycolyl moieties, Neu5Gc(a2-05)Neu5Gc(a2-05)Neu5Gc(a2- [3 1,lO 11 ; in the case of the glycolipid material the NeuSGc residues are also 8-U-methylated [3 I] . In microbial polysaccharides, besides internal 8and 9-substituted NeuSAc residues, also internal 4-and 7-substituted Neu5Ac units have been frequently found (Table 5 ). It should be noted that some of the sialic-acid-containing glycan fragments and polysialic acids are specifically found in lipopolysaccharides and capsular polysaccharides of pathogenic bacteria, leading to severe problems in the development of suitable vaccines. A typical source for sialo-oligosaccharides generated from N-and O-linked glycans is the urine of sialidosis and I-cell disease patients [191-1991, though this is not discussed in detail here (see section 10.5, and volume 30 of the New Comprehensive Biochemistry series). Patients with other inborn errors of metabolism, like aspartylglycosaminuria [76, 152] or P-mannosidosis [ 1531 excrete small amounts of structurally unusual sialooligosaccharides, of which the formulae have been included in the footnotes of Table 4 . Finally, sequence information of the already mentioned glycosylphosphatidylinositol anchor is available, showing that the glycan core consists of Mana-Mana-Man-[NeuSAc- The phenomenon of intramolecular lactone formation, often reported for polysialic acid [200-2021 and for gangliosides [203-2061, has not been detected so far in glycoprotein sialoglycans. In the case of a NeuSAc(a2-8)NeuSAc sequence, lactonization affords a NeuSAc(a2-8,1-9)Neu5Ac element (Fig. 4) , whereby the COOH group of one residue reacts with H 0 9 of an adjacent residue, to give a six-membered ring. Similarly NeuSAc(a2-9)NeuSAc can be converted into Neu5Ac(a2-9,1-8)NeuSAc, and NeuSAc(a2-3)Gal into Neu5Ac(a2-3,1-2)Gal or Neu5Ac(a2-3,1-4)Gal. In a polysialic acid chain of a-2,8-linked NeuSAc residues the a-2,8/ 1 ,9-lactonization can be effected under relatively mild conditions, like mild acid treatment [200] , yielding a water-insoluble polymer. The NeuSAc(a2-9,1-8)Neu5Ac formation in a polysialic acid chain can only be realized by carbodiimide treatment [20 11 , illustrating a more difficult condensation with the secondary H 0 8 group. The difference in reactivity between the primary and the secondary OH group has been nicely illustrated for the alternating a-2,8/a-2,9polysialic acid of E. coli K92 (Table 5 ) [202] . For gangliosides, it has been stated that ganglioside lactones occur also as such in nature [206] , and that in this way the negative charge of a ganglioside under physiological conditions can be modulated. Lactonization makes the oligosaccharide chain also more rigid, which may have important biological implications. Treatment with carbodiimide or glacial acetic acid can even convert a Neu5Ac(a2-8)NeuSAc(a2-3)Gal(~ 1-into a NeuSAc(a2-8,1-9)NeuSAc(a2-3,1-2)Gal(Pl-sequence [204, 205] . Over the years NeuSAc has been prepared by a variety of methods. Several biological sources have been explored to isolate this sialic acid in high amounts. Among them are edible birds nest substance [207] , urine of sialuria patients [65] , colominic acid {Neu5Ac(a2-8)}, produced by E. coli strains [208] , and hen's egg chalaza, egg-yolk membranes and delipidated egg yolk [209, 210] . The large scale organic synthesis of Neu5Ac is still complicated (see section 6.1). However, recent biotechnological routes, using sialate-pyruvate lyase (aldolase; see section 9.5), readily yield large amounts of Table 5 Survey of structures or elements of sialic-acid-containing microbial polysaccharides Neu5Ac(a2-3/6)Gal(@ l4)GlcNAc(@l-3)Gal(fi1-4)Hep- [ 1741 this sialic acid (see section 6.1). Efficient procedures to prepare Neu5Gc from colominic acid via de-N-acetylation, N-acryloylation and reductive ozonolysis, followed by acid or enzymatic hydrolysis of the formed (NeuSGc(a2-X)}, has appeared in refs. [211, 212] . Also porcine submandibular gland is a good source for the large scale preparation of NeuSGc, whereas Neu5,9Ac2 can be prepared from bovine submandibular gland [2 13) . Campylobacter jejuni 0 1 [I751 Besides the occurrence of sialic acids [(0-acetylated) Neu5Ac and Kdn] in microbial polysaccharides (Table 5) , some lipopolysaccharides have shown to contain sialic-acidlike monosaccharides. They differ from sialic acids in the presence of an additional amino function at C7, a deoxy function at C9, and in the configuration at the chiral centers: 5,7-diamino-3,5,7,9-tetradeoxy-~-glycero-~-manno-non-2-ulosonic acid and 5,7-diamino-3,5,7,9-tetradeoxy-~-g~cero-~-ga~acto-non-2-u~osonic acid [2 14,2 14al. So far, the two amino groups have been found to be substituted in different combinations, yielding acetamido, formamido, (R)-3-hydroxybutyramido, 4-hydroxybutyramido, or acetamidino functions. Also 0-acetylation can occur, whereas the (R)-3-hydroxybutyryl group at C7 can be used to link monosaccharides in a polysaccharide chain. Typical species are Pseudomonas aeruginosa strains [214] , Shigella boydii type 7 [214] , Vibrio salmonicida [214a] , Vibrio cholerae 0 2 [215] , Vibrio alginolyticus strain 945-80 [216] , Salmonella arizonae 0 6 1 [2 171, Yersinia ruckeri 0 1 [2 181, Legionella pneumophila strain 1 [219, 220] , and PseudomonasJluorescens ATCC 49271 [220a] . For a review, see ref. [220] , but it should be noted that the absolute configuration of 5,7-diamino-3,5,7,9tetradeoxy-~-glycero-~-ga~acto-non-2-u~osonic acid was earlier assigned as D-glycero-Lgalacto-[2 14,2 14a,2 16-2 19,220al. For the staining of sialic acids in tissues and cells, a great variety of techniques is available. Classical histochemistry of sialic-acid-containing glycoconjugates makes use of either binding of cationized dyes (e.g. Alcian blue, cationized ferritin, ruthenium red) or selective periodate oxidation (derivatization of generated aldehyde groups of sialic acids with e.g. p-dimethylaminobenzaldehyde, dansylhydrazine, rhodamine, biotidferritin-conjugated avidin). The second approach is strongly dependent on sidechain modifications [53] . Comprehensive reviews on this subject have appeared [6,22 11 . Nowadays, specific lectins are frequently used to detect the presence of glycosidically bound sialic acids in complex carbohydrates. However, the specificity of lectins is generally broad, and positive information has always to be checked in control experiments using e.g. sialidases in the presence and absence of inhibitors. For the histochemical analysis, lectins may be conjugated e.g. with gold particles, peroxidase, rhodamine or fluorescein isothiocyanate [6] . A large series of lectins which recognize sialic acid have been demonstrated to occur in nature, and most of their biological sources have been summarized in refs. [6, 11, 222] ; see also refs. [223-2271. For updated reviews focusing on lectins, see the present volume 29b of the New Comprehensive Biochemistry series. Generally, lectins have been isolated from lower animals such as prawns, snails, crabs, spiders, scorpions, lobsters, slugs, oysters, but also from plants, rat brain and B cells. While some of these lectins bind to both NeuSAc and NeuSGc, others are specific for NeuSAc. 0-Acetylation may also influence the binding strength of the lectin, both in a positive and in a negative manner (see section 10.3). For introductory glycoprotein analysis, in answering questions like "what monosaccharides are in the glycoprotein glycans?", two sialic-acid-specific plant lectins, having also a glycosidic linkage specificity, have been included in commercially available kits. These lectins are the agglutinins from Muuckia umurensis and Sumbucus nigra, being diagnostic for NeuSAc(a2-3)Gal and NeuSAc(a2-6)Gal/GalNAc elements, respectively. For screening purposes, the lectins are labelled with digoxigenin-succinyl-E-amidocaproic acid hydrazide (DIG, a spacer-linked steroid hapten digoxigenin). After SDS/PAGE of the (g1yco)protein mixture and Western blotting, sialoglycoprotein bands with a-2,3and/or a-2,6-linked NeuSAc can be labelled by one or both of these DIG-labelled lectins, whereby the detection is carried out in an enzyme immuno-assay using a digoxigeninspecific antibody conjugated to alkaline phosphatase, followed by color development with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride [228, 229] . Viruses and antibodies can also be used for the detection of sialic acid in complex carbohydrate systems. The sialic-acid-binding properties of a number of viruses have been established, and it has been shown that influenza C virus, bovine corona virus and encephalomyelitis virus recognize 9-0-acetylated sialic acids (see also sections 8.4.2, 9.1 and 10.3). Microtiter-plate and nitrocellulose-membrane assays have been developed that use the hemagglutinin (receptor) and the receptor destroying activities (sialate 9-0acetylesterase) of the influenza C virus to specifically detect bound 9-0-acetylated sialic acids in glycoproteins [230,23 I] . Although the recognition site for Neu5,9Ac2 and the esterase activity are located on the same viral glycoprotein, these activities can be separated using different temperatures for the binding (4°C) and the enzyme reaction (4-methylumbelliferyl acetate or a-naphtyl acetate as substrates; 20-37°C). Other approaches are based on binding of the virus to immobilized ligands, and detection of the virus with monoclonal antibodies, whereby the esterase was selectively inactivated by the use of diisopropylfluorophosphate (DFP) [232] . Virus particles have also been labelled with radioactive isotopes [232] or biotin [233] . The application of the assay with virus particles for the staining of 0-acetylated sialic acids in tissue sections will be described in section 8.4.2. Recently, a new technique using a soluble chimera of the hemagglutininesterase portion of the hemagglutinin-esterase-fusion-glycoprotein from influenza C virus and the Fc portion of human IgG, has been reported [234, 235] . Such a chimera retains both its recognition and enzymatic functions, and also has the binding properties of the Fc portion of IgG. The probing can be carried out on blots and TLC plates taking into account precautions for the recognition and esterase activities (this has to be inhibited by DFP in the test) as discussed above. An interesting electrochemical method for the determination of bound sialic acid has been developed, making use of a potentiometric four-channel thick-film sensor [236] . The sialidase sensor consists of a bilayer of a membrane containing Clostridium perfringens sialidase immobilized in a poly(viny1 acetate)-polyethylene copolymer, which is placed on top of an H+-selective poly(viny1 chloride)-poly(viny1 acetate) indicator membrane. The enzyme-induced release of bound sialic acid leads to a concomitant decrease in pK, of the carboxyl function of sialic acid. This decrease affords a local pH change inside the sialidase-containing sensor membrane, which is monitored by the H+-selective indicator membrane. The pH optimum of the sialidase sensor was pH 4 for sialyllactose, mucin and colominic acid. Finally, TLC analysis may also be of great help in the screening of biological materials for the presence of sialic acids. This item will be discussed in section 5.3.1. The characterization of the type of sialic acid in sialoglycoconjugates is frequently carried out after release and (partial) isolation. The cleavage of sialic acid from sialic-acidcontaining material is mainly performed by two methods, namely, acid hydrolysis and enzymatic hydrolysis. Both approaches have advantages and disadvantages. In the case of acid hydrolysis, problems arise with respect to de-0-esterification, which complicates quantitative analysis procedures. With respect to the enzymatic hydrolysis with sialidases, linkage specificity as well as a reduced or complete lack of susceptibility have to be taken into account. Moreover, in most cases much lower amounts of sialic acids are released by sialidases than by acid hydrolysis, which may be due to the different accessibility of the sialic acids in the biomolecules to be analyzed. Additionally, in work-up procedures and analyses, pH values below 4 and over 6 should be avoided to prevent migration of the 0-acetyl group at C7/C8 and hydrolysis of 0-acetyl groups as much as possible [23, 57] . Several approaches have been reported for the effective acid hydrolysis of the labile a-2,3-, a-2,6-and a-2,8-linkages. All these procedures suffer from being not optimal in giving the real spectrum of sialic acids originally present in the sialoglycoconjugate under study, especially in the case of a mixture of (0-acylated) N-acylneuraminic acids. Terminal sialic acids are released in high yield (and low destruction) using a two-step acid hydrolysis procedure comprising treatment with formic acid (pH 2, 1 h, 70°C), followed by HCl (pH 1, 1 h, SOOC). After each step, the liberated sialic acids must be recovered by centrifugation, ultrafiltration or dialysis [I 1,12,213,237] . It should be noted that in the case of a spectrum of (0-acylated) N-acylneuraminic acids, the supernatant, ultrafiltrate or diffusate of the formic acid hydrolysis contains the majority of the 0-acylated N-acylneuraminic acid, whereas that of the HCl hydrolysis contains High-molecular-mass isoenzyme (P. Roggentin, personal communication) (see also section 9.2). ' Sialyllactose, pH 5.0-5.1 (acetate buffer) and pH 5.8-6.0 (phosphate buffer). J Depending on the buffer system used. mostly Neu5Ac and Neu5Gc. In the case of low-molecular-mass substances, isolations can be carried out by gel-permeation chromatography. Although these conditions do not lead to significant de-N-acylation, de-0-acylation has been shown to occur to an extent of about 30-50%. One has to consider that milder acidic conditions result in incomplete release of sialic acids. Acid hydrolysis with acetic acid (2M, 3 h, SOOC) as suggested in ref. [238] did not improve the yield of 0-acylated N-acylneuraminic acids [ l 11. When focusing on sialic acid analysis, the use of H2S04 (0.05 M) is not recommended because of work-up problems. In connection with the HPLC analysis of Neu5Ac and NeuSGc, microwave hydrolysis in 2 M acetic acid has shown to be an interesting alternative [239] . In the methanolysis procedure, as used for the standard quantitative monosaccharide GLC analysis of glycoconjugates [240, 241] , 1 M methanolic HCl (24 h, 85OC) is applied. However, under these conditions released sialic acids are completely de-N, 0-acylated, which makes this approach unsuitable for the characterization of different types of sialic acid. It is, however, a reliable approach for the determination of the total amount of a mixture of (0-acetylated) N-acylneuraminic acids. When using a milder methanolysis procedure (0.05 M methanolic HCl, 1 h, 80°C) the de-N-acylation but not the de-0-acylation is strongly reduced [242] . In the quantitative determination of N-acylneuraminic acids in poly-N-acylneuraminicacid-containing glycoproteins, the release of free sialic acid was shown to be optimal using a combined mild acid hydrolysis (0.1 M TFA, 3 h, 8O0C)/subsequent mild methanolysis (0.05 M methanolic HCl, 1 h, SOOC) [243] . This method is also advised for the analysis of the Kdn content in Kdn-containing glycoproteins. In contrast, optimal release of Kdn from poly-Kdn-containing glycoproteins is obtained by mild methanolysis with a longer incubation time [243] . In the latter case the standard conditions of methanolysis also give good results. The enzymatic release of N-acylneuraminic acids can be carried out under such mild conditions (low temperature, pH 5 4 ) , that destruction, migration, and de-0-acylation is kept at a minimum. In Table 6 the substrate specificity of commercially available sialidases from Arthrobacter ureafaciens, Yibrio cholerae, Clostridium perfringens, Salmonella typhimurium, and Newcastle disease virus, using simple N-acylneuraminyllactose substrates is compared (for comprehensive reviews on sialidases, see refs. [5, 33, . It is evident that the sialidases show different ratios of cleavage rates for the a-2,3-, a-2,6-and a-2,8-linkages. The finding of a strong preference of the Newcastle disease virus sialidase for a-2,3-linkages holds also for other viral sialidases, such as those from fowl plague virus and influenza A2 virus. The latter enzyme also has a low specificity for a-2,g-linkages [5] . Among the bacterial sialidases, A. ureafaciens sialidase has a certain preference for a-2,6linkages. The S. typhimurium sialidase is the only bacterial sialidase with a viral sialidase-like pronounced preference for a-2,3-linkages [247] . Recently, two sialidases from Bacteroides fragilis having a higher preference for the cleavage of a-2,8linkages, when compared with a-2,3-and a-2,6-linkages, were isolated [248] . The sialidase, recently isolated from Macrobdella leech, cleaves only NeuSAc(a2-3)Gal linkages [61] . In general, Neu5Ac residues are released faster than NeuSGc residues. In a study using different N-and 0-glycoproteins with a-2,3-and/or a-2,6-linked N-acylneuraminic acids as substrates [antifreeze glycoprotein, ovine submandibular gland glycoprotein, a I -acid glycoprotein; Neu5Ac(a2-6)GalNAc(a l-O)Thr/Ser, NeuSAc/SGc(a2- and sialidases from A. ureafaciens, V cholerae, C. perfringens, Newcastle disease virus, fowl plague virus, and influenza A2 virus, roughly similar patterns of substrate specificity as for sialyllactoses were found. However, it was demonstrated that the core oligosaccharide andor the protein structure may also influence the rate of release for different glycosidic linkages [249] . In the case of S. typhimurium sialidase, also N-glycoprotein a-2,3-sialoglycans were susceptible to efficient cleavage, but not mucin 0-glycoprotein sialoglycans [247] . The most recently discovered sialic acid, NeuSGcAc, could not be released with V cholerae sialidaseE431. A comparison of the different commercially available sialidases shows that the C. perfringens sialidase iso-enzyme with a molecular mass of about 63 kDa has the broadest specificity. It should be noted that C. perfringens in fact produces two sialidases, the larger of which (63kDa) is commercially available (P. Roggentin, personal communication) (see also section 9.2). As described for the acid hydrolysis procedure, the work-up of enzymatically released sialic acids can be achieved employing various methods, depending on the starting sialoglycoconjugate material. 4-0-Acetylated neuraminic acids in any glycoconjugate are resistant to most sialidases tested so far; only viral sialidases show a low but significant activity. With the exception of Streptococcus sanguis sialidase [250] , the presence of 0-acetyl substituents at C7-C9 leads to a reduced rate of cleavage by all sialidases, so that prolonged incubations are necessary for an efficient release [251] . More information has been collected in a detailed study with bacterial and viral sialidases and 4-methylumbelliferyl (MU) a-glycosides of 4-, 7-, and 9-0-acetylated N-acetylneuraminic acids as substrates [252] . In contrast to the other sialidases tested, the fowl plague virus sialidase catalyzes a slow release of Neu4,5Ac2 from a-Neu4,5Ac2-MU. The recent finding of {Neu5Gc(a2-05)}, elements in a glycoprotein [ l o l l initiated a kinetic study of the enzymatic and non-enzymatic hydrolysis of Neu5Gc(a2-05)Neu5Gc and Neu5Gc(a2-8)NeuSGc [253] . It turned out that at pH < 3.8 the rate of acid hydrolysis of the unusual a-2,05-linkage was greater than that of the normal a-2,8-linkage. However, at pH > 3.8 reverse results were obtained; NeuSGc(a2-8)NeuSGc released a small but detectable amount of Neu5Gc even at pH 6. The a-2,05-linkage was only partially cleaved by C. perfringens and l ! cholerae sialidases, and was essentially resistant to A . ureafaciens sialidase. The detection of sialidases that can release Kdn is so far highly limited. The liver of the loach Misgurnus fossilis was found to contain a sialidase capable of releasing both Neu5Ac and Kdn from sialoglycoconjugates [39] . While the sialidases investigated so far in detail require an NH-acyl group at C5 for full activity, the loach enzyme can handle both NH-acyl and OH functions at C5. The rainbow trout also turned out to be a useful source for the isolation of a sialidase, active in releasing both Neu5Ac and Kdn [254] . Recently, a sialidase was isolated from Sphingobacterium multiuorum that specifically released a-2,3-, a-2,6-and a-2,8-linked Kdn; Neu5Ac and Neu5Gc were not liberated [255] . Before fractionation, pools of free sialic acids can be freed from contaminants by several methods, including ion-exchange chromatography, cellulose chromatography, reversed-phase chromatography, preparative TLC [6,11 , 121. Generally, one of the purification procedures for the pool of sialic acids comprises Dowex ion-exchange chromatography at low temperature. After passage through a cation-exchange resin (Dowex SOW-XS, H+-form), the pool of sialic acids is adsorbed to an anion-exchange resin (Dowex 2-X8 or 1-X8, HCOO--form). Elution from the anion-exchange resin is generally carried out with &2 M formic acid. The ion-exchange chromatography should be carried out rapidly, as prolonged contact of acylneuraminic acids with the resin or the solvent systems used may result in degradation, 0-acetyl migration and/or de-0-acetylation. After rotary evaporation or lyophilization, the sialic acid pools are stored at -20°C. In general, mixtures of sialo-oligosaccharides from {Neu5Ac(a2-8)},, (NeuSAc(a2-9)},, {-8)Neu5Ac(a2-9)Neu5Ac(a2-),, {Neu5Gc(a2-8)},, and (Kdn(a2-8)},, are generated by limited acid hydrolysis [211, 256, 257] . Depending on the polysialic acid, different conditions have been applied. Also attention has been paid to the intramolecular self-cleavage of polysialic acids such as {Neu5Ac(a2-8)}, [258] . Adjacent COOH groups with a high pK, (3.9-5.5) act as proton donors for general acid catalysis. The lability is seen under mild acidic conditions, that can be encountered in various physiological situations. {Neu5Ac(a2-8)},3 is substantially more unstable than {Neu5Ac(a2-8)}2-3. A highly useful enzyme for the depolymerization of polysialyl carbohydrate chains, yielding oligosialyl compounds, is endo-sialidase (endo-N) produced by infection of E. coli K1 with a lytic bacteriophage [9, 259, 260] . The enzyme is specific in cleaving a-2,8-linkages, and requires at least five Neu5Ac or Neu5Gc residues for activity. A limited digestion of {NeuSAc(a2-8)},-R affords mainly {Neu5Ac(a2-8)}4, with some {Neu5Ac(a2-8)} 1-3. Alternating a-2,8/a-2,9-polysialyl chains, as present in some bacterial polysaccharides, are also cleaved, but a-2,9-linked polysialyl chains are resistant. Poly-Kdn and {Neu5Gc(a2-05)}, are not substrates for endo-N. A similar endo-sialidase associated with phage particles, namely, E. coli bacteriophage $92, has been reviewed in ref. [261] . One of the oldest methods used to detect and to quantitate sialic acids is colorimetry [3, 6, 12, 237, 262] . When carried out on non-purified samples, the influence of contaminants interfering with the assays has to be taken into account. Greatest problems are encountered when using cells or tissue extracts, as the level of contamination is inevitably high. Moreover, factors such as non-identical reactions of different sialic acids in the same assay and the non-specificity of the reactions for the sialic acids are important. Although several colorimetric methods have been developed in the past, only two main procedures are currently routinely applied, namely the orcinol/Fe3+/HC1 assay, known as the "Bial" reaction, and the periodic acidhhiobarbituric acid assay. For microadaptations of these two different tests, see ref. [l 11. In the first assay, the sample is mixed with orcinol, FeC13 and concentrated HCl and heated at 96°C. The formed purple to red-violet chromophore is extracted with isoamyl alcohol and its absorbance measured at 572 nm. Because of the use of HCl, the method can be used to quantitate the total amount of both free and glycosidically bound sialic acids. Due to the strongly acidic conditions, ester groups are released. As the assay does not discriminate between bound and free sialic acids, it is widely used to monitor the presence of sialic acids in either form during fractionation of biological material. It should be noted that other monosaccharides, especially pentoses, hexoses and uronic acids interfere with the assay, which is of importance when small amounts of sialic acid are present. In the second assay, only suitable to quantitate free sialic acids, sialic acid is oxidized by periodate at 37°C under strongly acidic conditions. The oxidation leads to the formation of a prechromogen, a six carbon aldehyde, which then yields the chromogen fi-formyl pyruvic acid by aldol cleavage between C4 and C5. The chromogen reacts with thiobarbituric acid to give a red chromophore, the absorbance of which is measured at 549 and 532nm. In principle two approaches can be followed, called the Warren method and the Aminoff method. Differences between the methods lie in the acidity of the initial periodate oxidation and in the solvent used for the extraction of the pigment (cyclohexanone, Warren; acidic 1-butanol, Aminoff). It should be noted that substituents in the glycerol side chain severely influence the periodate oxidation. Therefore, in the case of ester substituents, a prior saponification is necessary (0.1 M NaOH, 37°C). Types of free sialic acid which do not yield the chromogen are negative in this test. Several compounds have been shown to interfere with the periodate/thiobarbituric acid assay, most especially 2-deoxyribose, 2-keto-3-deoxyaldonic acids other than Kdn, disaccharides such as lactose and maltose, and unsaturated fatty acids. The greatest errors arise in the quantitation of sialic acids from cellular extracts or homogenates containing membrane and nucleic acid material. Therefore, prior ion-exchange column chromatography and removal of lipids by ether extraction are of advantage. Special attention to the periodic acidthiobarbituric acid assay of Kdn has been paid in ref. [243] . When both tests are used in combination, a differentiation between total and free sialic acid is possible, allowing the calculation of the amount of glycosidically bound sialic acid. In a new approach for the direct determination of free and bound sialic acid, an acidic ninhydrin assay has been proposed [263] . Heating of solutions of sialic-acid-containing material with ninhydridacetic acid/37% HCl at 100°C yields a stable chromophore, the absorbance of which can be measured at 470nm. In view of the comments made in section 5.1 with respect to the release of sialic acid by sialidases, a quantification procedure for bound sialic acid based on the enzymatic analysis of pyruvate, formed after sialidase/aldolase treatment [6, 264] , should be handled with care. Of the various fluorimetric assays available for sialic acid analysis, the method which allows the discrimination between sialic acids with or without 0-acyl groups at C8 and/or C9 may be of interest [6, 265] . After mild periodate oxidation, formaldehyde, derived from C9 in the case of non-substituted H 0 9 and H 0 8 is derivatized with acetylacetone in the presence of ammonium acetate, leading to the fluorigen 3,Sdiacetyl-1,4-dihydr0-2,6-dimethylpyridine (4 10 nm excitation, 5 10 nm emission). It is evident that all contaminants producing formaldehyde under the influence of periodate will interfere with this sialic acid analysis. Finally, for the quantitative estimation of the 0-acyl content of sialic-acid-containing samples (Hestrin assay), also a colorimetric assay is routinely used. The method is based on the reaction with hydroxylamine in alkaline medium yielding hydroxamates, which form with Fe(C104)3 red chromophores, the absorbance of which is measured at 520 nm [262] . From the beginning of free sialic acid analysis, TLC has played a major role in screening and tentative assignment procedures, and over the years several solvent systems for both cellulose and silicagel plates have been reported [6, 237, 262] . One of the most popular TLC methods for the analysis of (substituted) N-acylneuraminic acids comprises the use of plastic HPTLC plates precoated with cellulose and 1-propanol/l-butanol/O. 1 M HCl (2:1:1, v/v/v) as solvent system. It shows the best and most reproducible separation of different sialic acids, and is less sensitive to interfering substances when compared with other systems. Generally, the visualization of the different sialic acids is carried out by spraying with the orcinol/Fe3+/HC1 reagent [237] , yielding typical purple bands. For quantitative purposes densitometry is also used. It should be noted that due to differences in cellulose quality (impurities), even after pre-washing of the plates, the reproducibility of Rf values is relatively low. Therefore, analyses should be carried out in the presence of an appropriate reference sialic acid mixture on a separate lane. To give an impression of the separation capacity of cellulose plates, Table 7 summarizes the Rf values of a series Table 7 Thin-layer chromatographic migration rates (Rf) of sialic acids on 0.1 mm cellulose plates using 1-propanol/lbutanol/O.l M HCI (2: 1 : 1, v/v/v) [ of N, 0-acyl-neuraminic acids from one experiment [6] . In the case of radio-labelled sialic acids, the bands can also be traced by radio-TLC-scanning. Application of the TLC method in a two-dimensional procedure with intermediate ammonia treatment gives information about the type of N-acylneuraminic acid of the constituting NO-acylneuraminic acids. In this way a differentiation is possible for example between co-migrating Neu9AcSGc and Neu5,7Ac2 [6] . In principle, de-0-acetylations can also be carried out by sialate 0-acetylesterases [266] . For the analysis of oligomers of a-2,g-linked NeuSAc, NeuSGc or Kdn, a TLC procedure on silicagel with the solvent system 1 -propanol/25% ammonia/water (1 2:2:5, v/v/v) has shown good results [267] . In this way, mixtures of {Neu5Ac(a2-8)}2 to {Ne~SAc(a2-8)}~4, {Ne~5Gc(a2-8)}~ to {Neu5G~(a2-8)}~~, or (Kdn(a2-8)}2 to {Kdn(a2-8)}7 are well separated. It should be noted that (NeuSAc(a2-X)}, and {Neu5Gc(a2-8)}, can be visualized also with a resorcinol spray reagent, whereas for { Kdn(a2-8)}, the orcinol spray reagent is needed. Initially, the separation of sialic acids was mainly carried out by cellulose chromatography at low temperature [6, 237] . However, nowadays HPLC fractionations using different column materials, elution protocols and detection techniques have replaced this approach [6, 11, 268] . The application of HPLC has also introduced a rapid method for tentative assignments of sialic acids in complex mixtures, based on elution times of known standards, being more reliable when more than one HPLC procedure is followed. Moreover, a rapid method for quantification of released sialic acids has become available. Due to the relatively short HPLC runs, also fast transitions between members of the sialic acid family due to migration of substituents, introduction of substituents, cleavage of substituents, or other (enzymatic) modifications can easily be monitored. First detailed reports on the separation of non-derivatized sialic acids deal with the application of Aminex A-28 or A-29 anion-exchange chromatography using 0.75 mM [269, 2701 or 0.5 mM [268] Na2S04 as eluting system and UV monitoring at 200 nm (nanomole range). In a different approach, fluorigenic derivatives of sialic acids, prepared by reaction with 1,2-diamin0-4,5-methylenedioxybenzene (DMB) in the presence of 2-mercaptoethanol and sodium hydrogensulfite, have been separated by C reversed-phase HPLC [27 1-2731, using acetonitrile/methanol/water (9:7:84, v/v/v) as solvent system and fluorescence monitoring at 373 nm excitation and 448 nm emission wavelengths. An appropriate cutoff filter may be used instead. The fluorescence labelling makes a relatively specific and highly sensitive (femto-to picomole range) detection possible. However, using radiolabelled sialic acids, it was found that the derivatization reaction is not quantitative [268] . For an adapted protocol, see ref. [l 11 . Another interesting approach is the conversion of NeuSAc/SGc into chemiluminescent quinoxalinone derivatives using 4,s-diaminophthalhydrazide dihydrochloride (a-keto acid derivatization) [274] . These derivatives are analyzed by reversed-phase HPLC (femtomole range), whereby the chemiluminescence detection follows the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) in alkaline solution. Other conversions of NeuSAc, useful for HPLC separations, include the derivatization with 4,4'-dicarboxy-2,2'-biquinoline [275] , 2-cyanoacetamide [276] , periodatethiobarbituric acid [277] , benzoic anhydride [278] , 4'-hydrazino-2-stilbazole [279] and 1,2-diamino-4,5-dimethoxybenzene [280] . Taking advantage of the separation capacity of the anion-exchange resin Car-boPac PAl, fractionation of non-derivatized sialic acids at neutral pH, using sodium acetate as eluent and pulsed amperometric detection (PAD) following postcolumn addition of alkali, has shown excellent results in terms of the wide array of sialic acids that can be separated, the sensitivity of the detection method (picomole range), and the relative ease of use for preparative work (without PAD detection) [268] . The general problem of quantification in PAD analyses, due to differences in detector response attributed to differences in the number of free hydroxyl groups of the various components separated, holds also for sialic acids. So far, only for a limited number of sialic acids relative detector response factors have been calculated (e.g. NeuSAc, 30 500; Neu5,9Ac2, 14 500; NeuSGc, 35 400) . It is important to note that pH values > 11, as usually applied in CarboPac-PAD analyses of oligosaccharides (for such a NeuSAc/NeuSGc separation, see ref. [28 l ]), will lead to rapid de-U-acylation of the 0-acylated sialic acids [282] . This phenomenon can even occur between the point of postcolumn alkali addition and the entry into the PAD detector [268] . In an evaluation of five different HPLC methods, it turned out that no single method is adequate to completely separate and quantitate complex mixtures of sialic acids [268] , and the use of multi-dimensional HPLC is advised. As a clear illustration of this statement, Table 8 is included. This evaluation also compares a series of essential features of the five HPLC methods, namely, sensitivity, specificity of detection, separation by number of hydroxyl groups or substituents, separation of isomers, preparative use, avoiding of ester migration during purification, and avoiding of ester loss during purification. In this comparison [268, 270, 271, 283, 284] the HPLC systems I (CarboPac PA-I) and 111 (TSK-ODS 120T, DMB derivatives) gave the highest averaged scores in terms of applicability. A major advantage of HPLC system V (Aminex A-29) is the short running time, only 5 4 min, which makes this approach highly attractive for studying enzymatic conversions. In order to obtain information about the structure of sialic acids, HPLC is a very useful technique to be applied in combination with mild chemical or enzymatic degradation methods. For instance, HPLC before and after alkaline treatment of a mixture of 0-acylated sialic acids can give information about the de-0-acylated sialic acids present, e.g. in terms of their N-acyl substituents. Also the linkage specificity of sialidases in releasing sialic acids from sialoglycoconjugates (see section 5 .1) can be monitored by HPLC. Furthermore, the specificity of enzymes involved in sialic acid metabolism can be studied in this way. In this respect, interesting results have been obtained with aldolase, cleaving sialic acids to N-acylmannosamine derivatives and pyruvate, and with sialate 9-O-acetylesterase, hydrolyzing 0-acetyl groups from C9 of sialic acids. The aldolase degrades Neu5Ac faster than Neu5Gc; a slow degradation has been observed for 0-acylated sialic acids, not affecting 4-0-acetylated sialic acids at all [5] . A typical example of the HPLC analysis of enzyme reactions, in which esterase and aldolase are involved, and including the non-enzymatic conversion of an 0-acetyl group from C7 to C9, is presented in Fig. 5 [6, 7, 36] . Other examples are the determination of sialidase activity (sialyllactose as substrate, Neu5Ac as product, and Neu2en5Ac as inhibitor), CMP-Neu5Ac synthase activity (disappearance of NeuSAc, appearance of CMP-NeuSAc), CMP-Neu5Ac phosphodiesterase activity (appearance of NeuSAc, disappearance of CMP-Neu5Ac) [36] . Recently, 5-N-acetyl-9-0-acetyl-2-(N-dansyl-4-aminophenylthio)-a-neuraminic acid has been proposed as a highly sensitive fluorescent substrate for the HPLC measurement of sialate 9-0-acetylesterase (334 nm excitation, 564 nm emission) [285] . As a thioglycoside, the compound is very stable in acidic aqueous solution and towards enzymatic hydrolysis by sialidases. In this context, it is also worthwhile mentioning that a sensitive HPLC assay has been developed for the tracing of sialyltransferase activity, making use of the synthetic fluorigenic acceptor lactose 2-[(2-pyridyl)amino]ethyl glycoside [286] . Details for a HPLC separation of CMP-NeuSAc, CMP-NeuSGc and CMP-Kdn on a DC-613 cation-exchange column are reported in ref. [ In addition to the fractionation procedures described for free sialic acids, several approaches have been reported for the separation of sialyl-oligomers. These compounds with a degree of polymerization up to 16 have been fractionated with varying results using conventional gel-filtration, TLC, DEAE-Sephadex A-25, and HPLC methods (see section 5.1 for preparation; see section 5.3.1 for TLC). A survey of literature has been included in ref. [287] . In general, mixtures of sialo-oligomers from (NeuSAc(a2-8)},, {NeuSAc(a2-9)},, {Neu5Gc(a2-8)},, or { Kdn(a2-8)}, can be isolated on a preparative scale via convential DEAE-Sephadex A-25 [256, 257] or DEAE-Toyopearl 650M [267] anion-exchange chromatography. HPLC procedures comprise anion-exchange and adsorption-partition chromatography. A mixture of { NeuSAc(a2-8)}2-16 has been efficiently separated on a Zorbax SAX anion-exchange column using 0.2-1 M NaCl in lOmM phosphate buffer pH 3.5 [287] . Also adsorptionpartition chromatography on polystyrene DC-6 13 using mixtures of 0.02-0.025 M sodium phosphate buffer pH 7.4 and acetonitrile as solvent system, has shown good results [267] . On Mono Q anion-exchange columns, excellent results were obtained in the separation of { N e u S A~( a 2 -8 ) )~-~~, {Ne~5Gc(a2-8)}~_lo, or {Kdn(a2-8)}~_~ after conversion into alditols with NaBH4 (or NaBT4), and using a NaCl gradient in Tris-HC1 buffer pH 8 as elution system [267, 288] . In this context, several studies have focused on the determination of the chain lengths of sialo-oligomers and -polymers (for a review of methods currently employed in the analysis of polysialic acids, see ref. [289] ), and recently a highly detailed adapted methodology for the analysis of a-2,8-linked sialooligomers and -polymers has appeared [290] . Using three variable assay procedures, providing overlapping information, details could be provided with respect to the degree of polymerization, the simultaneous identification of NeuSAc, Neu5Gc and Kdn when present in a single preparation, and the ability to distinguish qualitatively between reducing and non-reducing polymers. The developed approach may include mild periodate oxidation (degradation of non-reducing terminal unit) in combination with reduction (degraded glycerol side chain yielding C7-sialic acid; reducing unit if present affording the corresponding stereoisomeric alditols), whereas monomer analysis is carried out after sialidase or acid hydrolysis on CarboPac PA-1 with pulsed amperometric detection. In the structural analysis of glycoprotein-derived N-and 0-linked sialic-acid-containing carbohydrate chains, fractionation procedures based on HPLC play a major role. As this aspect is outside the scope of this chapter, no details are included. Typical examples, making use of anion-exchange chromatography (Mono Q, CarboPac), and normal (e.g. Lichrosorb-NH2) or reversed-phase chromatography, are found in refs. [25, 81, 84, 133, . In this context, it is also worth noting the recent use of highperformance capillary electrophoresis for the separation of glycoprotein-derived N-glycan chains [301] and 0-glycan chains [302] . Gas-liquid Chromatography combined with muss spectrometry As discussed in section 5.1, methanolysis of free and glycosidically bound sialic acids gives rise to the formation of methyl ester band a-methyl glycosides. Using the conditions of the standard quantitative monosaccharide analysis of glycoconjugates, de-N-acylatiodde-esterification takes place, which means that NO-acylneuraminic acid residues are converted into neuraminic acid methyl ester methyl glycoside (8, -96%; a, -4%). For the characterization by GLC the sialic acid methyl ester methyl glycoside is derivatized via N-acetylatiodtrimethylsilylation or pertrifluoroacetylation [241] . It should be noted that during the N-acetylation step H09 of NeuSAc, when not substituted, is partially 0-acetylated (-4%). Using milder methanolysis conditions [242] , an N-acetylation step is not necessary, yielding a method to determine both Neu5Ac and Neu5Gc by GLC. In principle, the latter approach is also suitable to determine sialic acids bearing only 0-alkyl groups. GLC analysis is generally carried out on SE-30 type column materials. Starting from free sialic acids (purified or as a pool), mainly present in their 8-anomeric forms, volatile sialic acid derivatives are generated using mild derivatization procedures such as esterification with diazomethane followed by trimethylsilylation [ 15,3031 or pertrimethylsilylation [ 1 1,3041. With respect to silylation cocktails, it should be noted that N-methyl-N-trimethylsilyl-2,2,2-trifluoroacetamide/pyridine leads to the formation of N-trimethylsilyl derivatives, yielding two different peaks for each sialic acid [36, 305] . Subsequent GLC analysis is generally carried out on SE-30 or OV-17 type column materials. Both types of derivatives are highly suitable for MS analysis, and GLC coupled with electron impact (EI) MS formed the basis for the development of a highly reliable mass spectrometric method for the identification of sialic acids. Originally set up for the GLC-EI MS analysis of mixtures of NO-acylneuraminic acids [303] , the method has also proved to be useful for the analysis of other naturally occurring sialic acids, of (partially) 0-methylated sialic acid methyl ester methyl glycosides as obtained in methylation analyses, and of synthetic sialic acid(s) (derivatives) [6, 11, 15] . In the following the principles of the EI MS identification procedure will be explained. Typical derivatives are trimethylsilylated methyl ester or pertrimethylsilylated derivatives of N, 0-acylneuraminic acids or of N-acyl-0-alkylneuraminic acids, acetylated N-acyl- 0-alkylneuraminic acid methyl ester methyl glycosides, trimethylsilylatedrnethylated N,N-acy1,methyl-neuraminic acid methyl ester methyl glycosides (methylation analysis), and acetylatedmethylated N,N-acy1,methyLneuraminic acid methyl ester methyl glycosides (methylation analysis). The determination of the type, number, and position of the 0-acyl or 0-alkyl groups as well as the type of the N-acyl group in neuraminic acids is facilitated by the highly specific EI mass spectra of the derivatized compounds. In Fig. 6 , a schematic survey is depicted showing the selected fragment ions A-H, which furnish the information (abundances and mlz values of the ions) necessary to deduce the complete structure of the sialic acids. Fragments A and B indicate the molecular mass of the sialic acid derivatives and thereby the type and the number of substituents. Fragments C-H can be used for the determination of the positions of the different substituents. Fragment A is formed from the molecular ion by the elimination of a methyl group originating from a trimethylsilyl substituent in trimethylsilylated (0-acylated0-alkylated) N-acylneuraminic acid derivatives. When RSI = CH3 (methylation analysis), the eliminated methyl group can also originate from the NN-acy1,methyl group. Fragment B is formed by elimination of the Cl part of the molecule. Eliminations of OCOCH3 in 0-acylated sialic acid derivatives and of NH2COCH3 in N-acetylneuraminic acid derivatives, which in principle give rise to the same mlz value as fragment B in the case of R1 =CH3, can be neglected. For 0-trimethylsilylated N 0-acylneuraminic acids (B-anomers) it holds that, when compared to their methyl esters, in their trimethylsilyl esters the intensity of fragment A decreases relative to B. Fragment C is formed by elimination of C8-C9, with localization of the charge on position 7. In general, cleavage occurs between two alkoxylated carbon atoms, or between an acetoxylated and an alkoxylated carbon atom, rather than between two acetoxylated carbon atoms. In accordance with the fragmentation rules for partially methylated alditol acetates [306] , the charge is preferentially located on an ether oxygen instead of on an ester oxygen. Therefore, fragment C has only significant abundance if C7 bears an ether group. When an ester group is present at C7, this fragment ion is absent or hardly observable. Fragment D is formed from fragment C by consecutive eliminations of R20H and R40H. It is evident that the occurrence of this fragment ion is dependent on the presence of fragment C. Fragment E is formed by elimination of the whole side-chain C7-C8-C9 and the substituent at C5. This fragment ion is not observed if an 0-acyl group is attached to C4, illustrating that the transition state in the McLafferty rearrangement is more favored when the substituent at C4 is an ether group rather than an ester group. For 0-trimethylsilylated N, 0-acylneuraminic acids (B-anomers) it holds that, when compared to their methyl esters, in their trimethylsilyl esters the intensity of fragment E is much reduced but still present; instead, an additional fragment derived from fragment E by loss of Me3SiOH is clearly present. Fragment F contains C8-C9. Based on the same fragmentation rules as mentioned above for fragments C and D, this ion can only readily be formed if an ether group is attached to C8. Fragment G consists of the C4-CS part of the molecule. Fragment H, necessary to use for derivatives containing only 0-alkyl substituents, is formed by elimination of the C9 part, followed by elimination of R4OH and R70H. For instance, this fragment is useful to discriminate between an OSiMe3 group at C8 or C9 in trimethylsilylated partially methylated N-acylneuraminic acids. Finally, for quadrupole analyzers, in the high mass range the fragment ions A, B and C often are of low intensity, especially when only small amounts of material are available. In Table 9 a survey is presented of GLC retention times and of characteristic EI MS fragment ions for a series of naturally occurring sialic acids, analyzed as their trimethylsilylated methyl ester or as their pertrimethylsilylated derivatives [6,11,15,3 1,38,43,307] . Although the sialic acids predominantly occur in the B-anomeric form, the a-anomer could occasionally be detected separately from the B-anomer, in most cases as a small shoulder. As a typical example, in Figs. 7a,b the EI mass spectra of the trimethylsilylated methyl ester of B-NeuSAc and of the pertrimethylsilylated derivative of P-NeuSAc, respectively, are depicted. Additional spectra have been published in refs. [ 15, 43, 304, 307] . For a detailed survey of fragment ions of other derivatives mentioned, including mass spectra, and those obtained from periodate-oxidized sialic acids (C7-NeuSAc, C8-NeuSAc, C7-NeuSGc and C8-NeuSAc), see refs. [15, 308] ; for EI MS data of Neu4,8anSAc, see ref. [63] ; for EI MS data of permethylated Kdn, see ref. [46] . In additional studies, the suitability of chemical ionization (CI) for the GLC-MS analysis of pertrimethylsilylated N, 0-acylneuraminic acids has been investigated. Isobu- The RNeuSAc values of the PT derivatives on CP-Sil 5 (capillary column), using the program 5 midl40"C; 2Wmin up to 220°C; 15 mid220"C, are given relative to the PT derivative of P-NeuSAc. For the preparation of TM derivatives, see 1151; for the preparation of PT derivatives, see [304] . For an explanation of the minus signs, see text. tane [304] , as well as methane and ammonia[ll] were used as reactant gases. The CI mass spectra are characterized in the high mass range by [M+H]+ pseudomolecular ions, and typical major fragment ions derived from [M+H]+ by loss of R20H (fragment I), &OH (fragment I'), and RzOH+&OH (fragment 11). It was found that methane in particular gave CI spectra that also include several of the typical fragment ions observed in the EI spectra. In addition to GLC-MS, HPLC-CI MS with Aminex A-29 as column material and ammonium formate in water/acetonitrile as solvent system has been explored for the analysis of underivatized N, 0-acylneuraminic acids [6, 309] . Although the positive-ion mass spectra allow the discrimination between different N-acylneuraminic acids (NeuSAc, NeuSGc) and the determination of the degree of 0-acetylation (Neu5,9Ac2, Neu5,7,9Ac3, Neu5,7,8,9Ac4), the position of the 0-acetyl groups (Neu4,5Ac2, Neu5,7Ac2, Neu5,9Ac2) could not be established. For the latter assignment, the combination with specific elution positions of standards on the HPLC column is advised. In the interpretation of the various fragment ions, the open-chain structure of the sialic acids has been generally used. NeuSAc and NeuSGc have also been converted into phosphatidylethanolamine dipalmitoyl derivatives, and after separation by HPTLC and subsequent isolation, the sialic acid derivatives were analyzed by liquid secondary ion MS [281] . In both cases intense [M -HI-ions together with sodium attachment ions were detected. For the detection of Neu5Ac on human tumor mucin, after liberation with sialidase, electrospray MS has been used [310] . Free sialic acids, isolated after cleavage from glycoconjugate starting material, have been investigated, without derivatization, by FAB MS using 5% aqueous acetic acid solutions for loading into glycerol on the FAB target [4 I] . The positive and negative FAB mass spectra of each sialic acid showed clear [M+H]+ and [M-HI-pseudomolecular ions, respectively. Sialic acid mixture analysis (pg range) made the recognition of subgroups of sialic acids with the same molecular mass possible (e.g. NeuSAc(Ac)l, NeuSAc(Ac)z, NeuSAc(Ac)3, NeuSGc(Ac)3). However, a differentiation between positional isomers was not possible. Sialic acids were also studied after derivatization, which improves the sensitivity [4 I]. Direct peracylation failed to produce suitable derivatives, but reduction under acidic conditions followed by peracylation (perdeuteroacetylation or perpropionylation) gave good results. Generally, the sialic acids give rise to two major pseudomolecular ions, corresponding to the peracylated open chain form and an open-chain-derived lactone form, and a minor pseudomolecular ion corresponding to an open-chain-derived anhydrofonn (2,6 and/or 4,8). As the lactone peak is markedly reduced in the spectrum of Neu4,5Ac2, H 0 4 seems to be mainly involved in the lactonization. In the case of sialic acid mixtures, a fast sialic acid subgroup analysis based on molecular masses is possible; again, a differentiation between positional isomers cannot be achieved. Careful analysis of the negative FAB spectra of reduced and perpropionylated sialic acids in mixtures demonstrated that these spectra could also be used for quantitative purposes. As worked out for mixtures of NeuSAc, Neu5,9Ac2 and NeuSGc, an estimate of the relative amounts of these sialic acids can be given with an error of 1 &I 5%, when the sum of the intensities of the [M + HIf ions of the linear and the lactone forms of each component is compared, taking into account that the molar response of Neu5Gc is approximately 50% of that of Neu5Ac. In order to generate sialic-acid-derived compounds, which can be used to differentiate between positional isomers, use has been made of the rather difficult periodate oxidation under mild or more rigorous conditions [53, 308] . Under both conditions, the resulting aldehyde groups were derivatized with p-amino-benzoic acid ethyl ester, reductively introduced at acidic pH without loss of the native 0-acetyl functions [41] . Sialic acids treated in this way were additionally reduced and peracylated, and then analyzed by FAB MS. Mixtures of products with different ring sizes (original, lactonized, anhydro) and/or open chain forms, depending on the substitution pattern, are often obtained. Of the mono-0-acetylated N-acylneuraminic acids Neu4,5Ac2, Neu5,7Ac2, Neu5,9Ac2, Neu4Ac5Gc and Neu9Ac5Gc were investigated, however, no attention was paid to the behavior of Neu5,8Ac2. Neu5,7(8),9Ac3 and Neu7,9Ac25Gc were also included in these investigations. Although not discussed in this chapter, FAB MS is widely used in the characterization of glycoprotein-derived N-and 0-linked sialic-acid-containing carbohydrate chains. Typical information can be found in refs. [311,312]. Since the introduction of high-resolution ' H NMR spectroscopy for the structural analysis of glycoprotein-derived glycans, a huge amount of NMR data have been generated, and highly detailed reviews on N-linked [76] and 0-linked [77] carbohydrate chains have appeared. The continuous expansion in the amount of data has made it necessary to develop computerized search programs, and, in connection with the still growing Complex Carbohydrate Structural Database (CARBBANK), attention has been paid to the development of a NMR-spectroscopic data base of carbohydrate structures, called SUGABASE [3 131. Sialic-acid-containing oligosaccharides/glycopeptides constitute a considerable majority of the glycoprotein glycans. In addition to the two reviews mentioned above, a specific review focusing on the NMR spectroscopy of sialic acids has also been published [ 161. Free as well as glycosidically bound sialic acid give rise to highly characteristic 'H NMR parameters. The 'H NMR spectra are generally recorded in D20, and because of the pH dependency of the proton chemical shifts, the spectral data are standardized at pD 6-7. The choice of the pH is also of importance in view of the earlier discussed de-0-acylation, 0-acyl migration, and autohydrolysis. As a typical example of a free sialic acid, in Fig. 8 the 500MHz 'H NMR spectrum of Neu5Ac in D20 at pD 7 is depicted. The spectrum shows a minor and a major set of protons, reflecting the subspectra of the aand p-anomer of NeuSAc, respectively (a$ = 7:93), and especially the H3e,3a signals, resonating outside the bulk signal, can be used for the differentiation between both anomers. The effect of pH on the proton chemical shifts is clearly illustrated by respectively, and at pD 7.0 at 6 2.730 and 6 1.621, respectively. Over the years, a large number of (naturally occurring) sialic acids and related derivatives have been analyzed, and in [46] , and those of CMP-9amino-NeuSAc and CMP-9NAc-NeuSAc in ref. [316] . ' H NMR spectroscopy has shown to be an excellent method to monitor chemical and biochemical conversions of sialic acids, directly in the NMR-tube or by analysis of isolated reaction products. A typical example is the demonstration of the release of a-NeuSAc as the primary product of bacterial and viral sialidase action on Neu5Ac(a2-glycosides and oligosaccharides [3 17-3201. The initial formation of a-NeuSAc, as traced by 'H NMR spectroscopy, formed an excellent probe to investigate the kinetics of the mutarotation of NeuSAc by means of 'H NMR analysis in dependency of the pH [321] . At pD 5.4 the establishment of the equilibrium of mutarotation turned out to be rather slow, but at higher and lower pD values a more rapid establishment was observed, so that at pD 1.3 and pD 11.7 mutarotation was too fast to be measured. With the ability to generate a-NeuSAc in situ, the aldolase-catalyzed degradation of NeuSAc to pyruvate and N-acetylmannosamine (ManNAc) could also be investigated in more detail using ' H NMR spectroscopy [322] . Using sialidase (pH optimum 5.4) and aldolase (pH optimum 7.2) from C. perfringens and N-acetyl-2-azido-2-deoxy-a-neuraminic acid as substrate at pH 5.4, only released a-NeuSAc was found to be consumed by the aldolase, yielding specifically a-ManNAc followed by a fast mutarotation to a,P-ManNAc. These findings confirmed earlier work using Neu5Ac(a2-3)lactose as a-Neu5Ac-generating system and crystalline b-NeuSAc [319] . In the reversed reaction a-ManNAc is the substrate [322] . For more details about the aldolase-catalyzed degradation, see section 9.3. In connection with these studies, it has to be noted that under comparable conditions the activities of sialidase and aldolase in D20 are only about 50% of those in H20. With respect to 0-acetyl migrations, also the earlier mentioned (see section 2) spontaneous conversion at physiological pH of Neu5,7Ac2 into Neu5,9Ac2 and of Neu5,7,9Ac3 into Neu5,8,9Ac3 has been monitored by ' H NMR spectroscopy [23] . Furthermore, the NMR approach has shown its value in the determination of substrate specificities of various sialidases using substrates with differently linked sialic acid residues [323-3251. --- ' Sialic acid occurs in 5C2 conformation. forms; chemical shifts are relative to HOD at 6 4.750; H3a= H3 and Values are assigned relative to the HOD signal at 6 4.81 at 296 K. H8 and H8'. g H7 and H7'. Personal communication Y. Inoue; values are assigned relative to 2,2-dimethyl-2-silapentane-5-sulphonate in D 2 0 (set to 0 ppm). Note that in CMP-6-Kdn the H8 and H9 signals have been interchanged when compared with ref. [3 151 . On one line values may have to be interchanged. Sialic acid occurs in 'C4 conformation and is present in two tautomeric N m 4 Table 11 'H-Chemical shifts for the H3e and H3a signals of sialic acids as part of N-and 0-linked glycoprotein glycans. Chemical shifts are given in ppm relative to internal acetone in D 2 0 (6 2.225) at 300 K, or relative to internal 2,2-dimethyl-2-silapentane-5-sulphonate in D 2 0 set to 0 ppm (marked ') ~~~ ~ Another interesting pH phenomenon, for the first time demonstrated by 'H NMR spectroscopy, is the complete replacement of H3a by a D-atom, when free Neu5Ac is kept in an alkaline D20 solution at pD 9.0 [326] . In fact, H3a can be exchanged in the pH range 6.5-9.0, and the H-D exchange is reversible. In the 'H NMR spectrum the H3a signal disappears, and the coupling patterns of H3e and H4 alter. At pD 12.4, H3e can also be replaced by a D-atom [327] . On the basis of this finding, the exchange experiments were also carried out in T20, yielding T-labelled NeuSAc, which was converted enzymatically into T-labelled CMP-Neu5Ac [326] . In glycosidically linked NeuSAc the H3 atoms are not exchangeable, rendering this specific labelling technique suitable for sialyltransferase experiments. In the H NMR structural-reporter-group concept, developed for the structural analysis of glycoprotein N-and 0-glycans, advantage is taken of the fact that a number of the H-atoms of the constituting monosaccharides, pccurring in special microenvironments, resonate outside the bulk-signal region [76, 77, 328] . In the case of glycosidically linked a-sialic acids the structural reporters are the H3a-and H3e-atoms, and the N-acetyl or N-glycolyl groups. The positions of the H3a,3e signals reflect not only structural information with respect to the type of sialic acid present, but also with respect to the coupled monosaccharide in terms of type of linkage and type of monosaccharide. Furthermore, 0-acyl substituents induce additional shifts for other H-atoms. In Table 11 a survey is presented of chemical shifts of H3e and H3a signals of sialic acid residues occurring in different linkage types as part of N-and 0-glycans. For a further fine-tuning of the chemical shifts within the presented ranges, influenced by the different microenvironments wherein the sialic acid residues occur, see refs. [76, 77] and the references cited in Table 2 . It should be noted that the presence of a certain sialic acid in a certain linkage also influences the structural-reportergroup signals of other monosaccharide residues [76, 77] . More detailed information with respect to the H3e,3a chemical shifts of sialic acids in sialo-oligomers and -polymers can be obtained from refs. [31, 47, 77, 329, 330] . A series of H3a,3e signals of sialic acids in milk-and glycolipid-derived oligosaccharides have been included in ref. [77] . More general NMR data of glycosidically linked sialic acids in glycolipids, milk and urinary oligosaccharides and (lipo)polysaccharides, if available, can be found in the references cited in Tables 3-5 (see also ref. [331] ). For a series of sialocarbohydrates, it has been shown that H6 of NeuSAc, easily traced from the TOCSY H3a,H6 correlation, also has potential value for discriminating between a-2,3-(6 3.63 & 0.01 1) and a-2,6-(6 3.70 * 0.01 7) linked NeuSAc in NeuSAc(a2-3/6)Gal(~l-O)R/NeuSAc(a2- [33 13 . However, for branched oligosaccharides, this rule is not valid if the monosaccharide in the branching position is in the alditol form [332] . The H3a,3e/NAc structural reporters of Neu5Ac have proved to be suitable in studying sialylation reactions in terms of positional specificity and branching (N-linked carbohydrate chains) specificity, using different sialyltransferases and CMP-Neu5Ac as donor [333-3351 (see also section 6.3). In more biophysical studies, several aspects of sialic acids have been investigated by NMR spectroscopy. Although so far mainly a-forms of bound sialic acid have been detected, good differentiation systems for aand p-forms are essential, and a number of empirical rules have been reported [336] . In a heteronuclear 2D-approach it could be demonstrated that the determination of the geminal C,H coupling constant 2J(C2,H3a) offers a unique criterion for the anomeric assignment in sialic acid glycosides (a, -8 Hz; p, -3 to -4Hz) [337] . Also the values of the vicinal C,H coupling constants 3J(C1,H3a) can be applied for this differentiation (a, -6 Hz; 0, -1 Hz) [55, 336, 338] . More details with respect to anomeric determinations have been reviewed in ref. [339] . 13C NMR data of sialic acids and glycoprotein-derived sialocarbohydrates have been reviewed in ref. [ 161. In several sialic-acid-related investigations, e.g. synthetic studies, I3C NMR spectroscopy forms part of the analysis techniques, and will not be reviewed in detail. For some additional data, more directly related with the glycoprotein glycan character of this chapter, see refs. [48,63,64, During the last ten years, activity in sialic acid chemistry has grown exponentially. Synthetic as well as biosynthetic routes for the preparation of sialic acids, sialic acid derivatives, analogues, glycosides and sialoglycoconjugates have been explored. The main reason for this considerable interest in preparing sialic acids and sialic-acidcontaining compounds lies in the fact that sialic acids were found to be among the most biologically important carbohydrate units in glycoconjugates. The progress in organic synthetic protocols and the availability of relevant enzymes in suitable amounts made it realistic to develop the sialic acid field from a preparative synthetic side. Initially, the main tools were to prepare suitable derivatives to study the properties of sialic acids, or to prepare substrates and inhibitors for sialidases, sialyltransferases or for sialic acid converting enzymes. Although these tools are still highly relevant, the preparation of sialo-oligosaccharides using strictly organic synthetic or enzymatic methods, or a mixture of both, are also receiving considerable attention. For relevant reviews on preparative (bio)synthetic aspects, see refs. [339,344-3531. 6 .1. Free sialic acids Several protocols have been followed for the organic chemical synthesis of NeuSAc. At first, the approaches were based on condensation reactions of (derivatives of) N-acetyl-D-mannosamine (ManNAc) or N-acetyl-D-glucosamine (GlcNAc) with (derivatives of) oxaloacetic acid (for a review, see ref. [3] ), however, the yields were very low. In this context, a procedure was worked out, that allowed modifications at Cl-C3 [354] . A total synthesis of NeuSAc from non-carbohydrate precursors has been reported in ref. [355] . Using a protocol for indium-mediated allylations of aldehydes, NeuSAc was prepared in good yields from a ManNAc precursor [356] . Furthermore, synthetic routes for NeuSAc have been proposed, based upon 1 -deoxy-1 -nitro-sugar chemistry, that should also allow the preparation of NeuSAc analogues, modified at several carbon atoms of the skeleton [357-3591. A separate route also yielded NeuSAc [360] . A synthesis starting with the aldol condensation of D-glucose (Glc) and oxaloacetic acid, followed by adaptation of the substituent at C5 has been described in ref. [361] . More recently, another approach for the organic synthesis of NeuSAc and NeuSAc derivatives, based on the cis-selective Wittig reaction of benzoyl 2,3-O-isopropylidene-a-~-lyxo-pentodialdo-1,4-furanoside with [(3S)-3,4-(isopropylidenedioxy)butyl]-triphenylphosphonium iodide as a first step, has been reported in ref. [362] . Comments on the acetylation of NeuSAc and its methyl ester have been published in ref. [363] . Starting from NeuSAc, in some derivatization reactions 1,4-as well as 1,7-lactone formation has been observed [349] . For the organic synthesis of Kdn, several routes were employed [364-3681, among them procedures starting from NeuSAc or from D-mannOSe (Man). NeuSAc-aldolase-catalyzed condensations of ManNAc and pyruvate [3, 33] , initially only investigated to understand sialic acid metabolism, have been optimized for preparative purposes. In principle, ManNAc can be generated from the cheaper GlcNAc in an alkaline epimerization process, yielding an epimeric mixture of which only the monosaccharide with the D-manno-configuration is recognized by the aldolase [369, 370] . However, ManNAc can also be generated from GlcNAc in a GlcNAc-epimerasecatalyzed isomerization [37 I] . A multigram-scale enzymatic synthesis based on the aldol condensation of ManNAc and pyruvate in the presence of phosphate, catalyzed by immobilized microbial NeuSAc-aldolase, has been reported [372] . A similar approach, using the aldolase enclosed in a dialysis membrane instead of being immobilized, has also been described [373] . Although in uiuo, the conversion of Neu5Ac into Neu5Gc occurs exclusively on the level of activated sialic acid (see section 8.4 .1), Neu5Gc can be prepared in uitro by incubating a mixture of N-glycolyl-D-mannosaminelN-glycolyl-D-glucosamine and pyruvate with immobilized aldolase [374] . Interestingly, a series of other sugars also turned out to be accepted by the aldolase, and Man and 2-deoxy-D-glucose in particular are excellent substrates [372,375-3771. In the case of Man as starting material, relatively moderate amounts of Kdn have been prepared [254, 376] . A series of free 0-acetylated sialic acids, i.e., Neu4,5Ac2, Neu5,9Ac2, Neu4,5,9Ac3, and Neu5,7,8,9Ac4, together with the benzyl ester a-glycosides of Neu5,7Ac2 and Neu5,7,9Ac3, have been synthesized by organic synthetic routes using protecting group techniques [378, 379] . Partially 0-acetylated sialic acid derivatives have also been prepared using more simple synthetic routes. @-Neu5,9Ac2 1,2Me2, fi-Neu4,5,9Ac3 1,2Me2, and @-Neu4,5,8,9Ac4 1,2Me2 were obtained from @-NeuSAcl,2Me2 by using N-acetylimidazole [53] . To realize 9-O-acetylations, also other acetylating reagents were applied, such as trimethyl orthoacetate [3 80, 38 I] , acetyl chloride [378] , and dimethylacetamide dimethyl acetal [382] . In a recent comprehensive study, in particularly the use of trimethyl orthoacetate and dimethylacetamide dimethyl acetal was explored using the 4-aminophenylthio, 4-nitrophenylthio, and 4-nitrophenyl glycosides of COOH-esterified a-Neu5Ac as acceptors, and depending on the acetylating reagent a range of partially 0-acetylated derivatives could be generated [57] . One of the naturally occurring 0-acetylated sialic acids, Neu5,9Ac~, has also been synthesized in an enzymatic way [383,384] on a gram-scale [372] . After the enzymatic acetylation of 0 6 of ManNAc, using isopropenyl acetate and protease N as a catalyst, 2-N-acetyl-6-O-acetyI-~-mannosamine was condensed with pyruvate as catalyzed by the aldolase. These two enzymatic steps turned out to be highly regio-and stereoselective. Following another route, Neu5,9Ac2 has been synthesized enzymatically by incubating Neu5Ac with trichloroethyl acetate in pyridine using porcine pancreas lipase as a catalyst [385] . An enzymatic synthesis of Neu5Ac9Lt has also been worked out [370, 384] . For the study of biochemical pathways, several isotopically labelled sialic acids and sialic acid derivatives have been prepared, both by enzyme-catalyzed synthesis and by organic synthesis. A survey of labelled sialic acids is presented in Table 12 [107, 344, 386] . In enzymatic procedures, use is generally made of the aldolase-catalyzed condensation of N-acybmannosamines and (phosphoenol)pyruvate, suitably labelled in one or both of the two synthons. For the preparation of N-[l-'4C]acetyl-and N -[ I-'4C]glycolylneuraminic acid, as well as O-['4C]acetylated sialic acids, surviving slices of submaxillary salivary glands incubated with [ 1 -I4C]acetate, followed by isolation of the glycoprotein fraction and mild acid hydrolysis, have been used. A number of these labelled sialic acids have been converted into their CMP-glycosides, and subsequently incorporated into glycoconjugates (see section 6.3). Of course, labelling of glycoconjugates can also be carried out by periodate oxidatiodtritiated borohydride reduction, thereby converting sialic acids, if chemically possible, into their radiolabelled C7 and C8 analogues. Using the latter approach, fluorescent probes (dansylhydrazine, dansylethylenediamine, fluoresceinamine) and EPR spin labels can also be incorporated (see references cited in ref. [344] ). The same holds for glycine [387] . Table 12 Survey of radiolabelled sialic acidsa Several interesting sialic acid variants and sialic acid derivatives have been synthesized, and a list is presented in Table . Both organic synthetic and aldolase-catalyzed routes have been followed. The major part of these compounds were prepared to study sialic acid metabolism (aldolase, CMP-NeuSAc synthase), sialic acid transfer (sialyltransferases), sialic acid release (sialidases), inhibition phenomena, or hemagglutinin-sialic acid interactions, and biological details are presented in sections 8-10. Compounds reported up to 1982 have been reviewed earlier [344] . Of special interest are the fluorescent and photoactivatable sialic acid derivatives [390,4 191 , which can be applied, after conversion into their corresponding CMP-glycosides, to detect enzyme activities or to follow biological processes (see sections 6.2 and 8.2). In the context of sialic acid variants, the following compounds are also of interest. In view of the similarity in acidity of a tetrazoie group and a carboxyl function, a variant of Neu5Ac has been prepared containing a CN4H instead of a COOH group [447] . Also the synthesis of a series of Neu5Ac derivatives with specifically introduced tert-butyldimethylsilyl groups have been reported [400, 448] . Furthermore, variants of 2d-2Ha-NeuSAc and 2d-2He,-NeuSAc, in which the carboxyl function has been replaced by a phosphono (PO3H2) group [449] , and a phosphonic acid analogue of Neu2en5Ac [450] , have been synthesized. In addition to 6-amino-2,6-dideoxy-sialic acids, as mentioned in Table 13 , the preparation of 2-C-hydroxymethyl derivatives [45 I] , and C6 and C7 analogues [452] have also been reported. Table 13 List of sialic acids, prepared along organic chemical or aldolase-catalyzed routes for use in biochemical studiesa 2d-2Ha,-Neu5Ac S-N-Acetyl-9-amino-9-deoxy-neuraminic acid S-N-Acetyl-9-azido-9-deoxy-neuraminic acid (methyl a-glycoside) S-N-Acetyl-9-(4-azidobenzamido)-9-deoxy-ne~uaminic acid S-N-Acetyl-9-(4-azidosalicylamido)-9-deoxy-neuraminic acid 5-N-Acetyl-9-benzamido-9-deoxy-neuraminic acid The organic synthesis of a long series of alkyl and aryl a-glycosides of N-acylneuraminic acids has been previously reported (for reviews, see refs. [339, 344] ). One of the famous condensation reactions (classical Koenigs-Knorr method) comprised the silver carbonate-promoted condensation of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-(3-neuraminic acid or the corresponding methyl ester (Fig. 9 , structure A) with the appropriate alcohol, followed by removal of the protecting groups. In order to improve the yields, much attention has been paid to better catalysts. In these glycosidation reactions typical side reactions are the formation of unsaturated sialic acid derivatives (elimination of HCl) and of (3-glycosides (see section 6.3). For the preparation of simple (3-glycosides, N-acylneuraminic acids are often heated with the appropriate alcohol in the presence of an acid catalyst, followed by saponification of the formed ester. However, complex alcohols give rise to problems (for a review, see ref. [344] ). A mild and efficient Raney nickel-catalyzed deuteration procedure has been reported for Neu5Ac glycosides, with a rate of exchange at C8 > C9 > C7 >> C4 [453] . Attention has also been paid to the synthesis of N-, S-and Se-glycosides, which are sialidase stable [339, 454, 455] . Specific S-glycosides are used as sialic acid donors in sialoglycoconjugate organic synthesis (see section 6.3) . Early examples are the syntheses of the 4-nitrophenyl N-and S-glycosides of a-Neu5Ac [456] . Of special interest are the syntheses of 5-N-acetyl-2-azido-2-deoxy-aand p-neuraminic acids [322, 457, 458] . The azides can readily be converted into the corresponding 2-amino derivatives, and used in e.g. N-acylation reactions [459] . For a preparation of the 6-thioanalogue of 2azido-a-NeuSAc, see ref. [436] . In further investigations, a series of S-glycosides of a-Neu5Ac was synthesized (thiophenyl, 4-nitrothiophenyl, 4-aminothiophenyl, 2-mercaptopyridyl), starting from 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-~-neuraminic acid methyl ester and using triethylbenzylammonium chloride as a phase transfer catalyst 114541. These compounds turned out to be effective sialidase inhibitors. For the detection of sialidase activity both (naturally occurring) oligosaccharides and simple a-glycosides are used. In these assays, two approaches can be followed, namely, determination of the released (modified) sialic acid or identification of the released aglycon. In the case of a-glycosides, in which the released aglycon concentration is measured spectrophotometrically or detected on solid supports, substrates with synthetically introduced aglycons having specific chromogenic properties, are used. Among such substrates, released aglycons can be detected directly or after condensation with specific reagents. One of the oldest substrates is the 4-nitrophenyl glycoside of a-NeuSAc [456] , whereby released 4-nitrophenol is estimated by absorption at 400nm. In an adapted synthetic version, the compound has been prepared by coupling of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-(3-neuraminic acid methyl ester with sodium-nitrophenoxide in N, N-dimethylformamide, and subsequent deprotection [460] . Another suitable substrate is the 3-methoxyphenyl glycoside of a-NeuSAc, synthesized by coupling of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2deoxy-(3-neuraminic acid with 3-methoxyphenol in the presence of silver carbonate, followed by de-0-acetylation. Liberated 3-methoxyphenol is determined after coupling with the diazonium salt of 4-amino-2,5-dimethoxy-4'-nitroazobenzene (red colored product) [461] or with 4-aminoantipyrine in the presence of the oxidizing agent potassium ferricyanide (colored quinone) [462] . The most popular fluorigenic substrate is the 4-methylumbelliferyl glycoside of a-NeuSAc, which is prepared by different methods [344] . A convenient synthesis is the condensation of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-~-neuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in N,N-dimethylformamide, followed by deprotection [463] . Released 4-methylumbelliferone is measured at 360 nm (excitation)/440 nm (emission). Although the 4-methylumbelliferyl glycoside of a-Kdn has been synthesized starting from Neu5Ac [445] , also a direct route using the glycosyl chloride of peracetylated b-Kdn methyl ester and the sodium salt of 4-methylumbelliferone has been explored [254] . In addition, several 4-methylumbelliferyl a-glycosides of sialic acid variants and substituted sialic acids, including partial 0-acetylated ones, have been synthesized (e.g. refs. [252, 350, 399, 445, 464] . To develop a sensitive assay for the analysis of the linkage specificity of bacterial and viral sialidases, Neu5Ac(a2-3)-and NeuSAc(a2-6)Gal(P 1-O)C6H4N02 were synthesized enzymatically by using a-2,3-and a-2,6-sialyltransferase, respectively, CMP-NeuSAc (see section 6.3), and p-nitrophenyl-P-Galp [465] ; after cleavage of NeuSAc, p-nitrophenol can be released by additional treatment with b-galactosidase. For the localization of sialidase on electropherograms or for histochemistry, the chromogenic 5-bromo-indol-3-yl glycoside of a-NeuSAc has been synthesized by coupling of 5-N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2-deoxy-~-neuraminic acid methyl ester with 1 -acetyl-5-bromo-3-hydroxyindole, and subsequent deprotection [466] . The unstable intermediate 5-bromo-indoxyl, released by sialidase, is readily transformed into insoluble blue-green 5,Si-dibromo-indigo, which marks the sites of enzyme activity. To facilitate the screening of bacterial colonies or plaques for sialidase activity, the 5-bromo-4-chloro-indo1-3-yl glycoside variant has also been synthesized [467] . Using the same sialic acid synthon as starting product, the 4-azido-2-nitrophenyl S-glycoside of a-Neu5Ac has been prepared, which is a potential photoaffinity probe reagent for the screening of sialidases in tissues and the purification of sialic-acid-binding proteins [468] . The sialidase-resistant thioglycosyl linkage also makes the incorporation of 35 S possible. In order to detect sialate 9-0-acetylesterase activity, a highly sensitive fluorescent substrate, 5-N-acetyl-9-O-acetyl-2-[4-(dansylamino)phenylthio]-a-neuraminic acid, has been synthesized (see also sections 5.3.2 and 9.1) [285] . The regioselective acetylation at 0 9 of the dansylated S-glycoside was carried out with trimethyl orthoacetate. Other useful fluorescent substrates for sialate 0-acetylesterase assays comprise 5-N-acetyl-7, [469] , and the 4-[3-(fluoresceinyl)thioureido]phenyl S-glycoside of a-Neu5,9Ac2 [57] . In all cases the fluorescent groups have been coupled to the glycosidic 4-aminophenylthio group of 0-acetylated NeuSAc derivatives. In connection with the generation of a monoclonal antibody to free Neu5Ac for the purpose of establishing a simple and specific assay of NeuSAc in serum and urine, a broad series of sialic acid aand P-glycosides have been synthesized using substituted glycerol, substituted sphinganine and cholesterol as aglycons [470] . The synthesis of CMP-sialic acids is generally carried out enzymatically using CTP and CMP-sialic acid synthase as a catalyst [33,3 14,370,47 1,4721 . A multigram-scale one-pot synthesis of CMP-@-NeuSAc has been reported in ref. [473] . ManNAc, prepared by basecatalyzed epimerization of GlcNAc, was reacted with sodium pyruvate in the presence of NeuSAc-aldolase to yield NeuSAc (see section 6.1). For the formation of CMP-NeuSAc, CTP was generated in situ from CMP by using adenylate kinase, pyruvate kinase, and phosphoenolpyruvate, and reacted with NeuSAc in the presence of CMP-NeuSAc synthase (Fig. 10) . Instead of a one-pot synthesis, for practical reasons it is easier to generate and store crude solutions of NeuSAc and CTP. For the use of GlcNAc in combination with GlcNAc-epimerase, see ref. [474] . Experiments with cloned CMP-NeuSAc synthases from E. coli systems with NeuSAc and Kdn showed a high specificity for NeuSAc, thereby suggesting that in this case the 5-acetamido group is critical [384] . Chemical syntheses of CMP-NeuSAc, applying the phosphoramidite method [475] or using sialyl phosphites [476] , have also been described. Furthermore, a synthetic approach for the preparation of CMP-NeuSGc based on the phosphite method has appeared [477] . In addition to CMP-p-NeuSAc, CMP-@-Neu5,9Ac2, CMP-b-NeuSGc, and CMP-8-Kdn, a large series of artificial CMP-sialic acids have been prepared biochemically on microscale starting from the corresponding sialic acid (see references cited in Table 13) and CTP. Among them are CMP-9azido-NeuSAc, CMP-9amino-NeuSAc, CMP-9NAc-NeuSAc and other C9-modified CMP-sialic acids, CMP-NeuSAcNH2, CMP-NeuSAc4Me, and CMP-4d-NeuSAc [33,3 14,350,390,407,419,4781 . The CMP-sialic acids have found a broad application in enzymatic sialylations using different sialyltransferases (see sections 6.3 and 8.3). Several of the artificial CMP-sialic acids turned out to be suitable donors for asialo-a, -acid glycoprotein as acceptor with Gal(@lH)GlcNAc a-2,6-sialyltransferase from rat liver as a biocatalyst [3 16,402,4171 . The transfer of CMP-9amino-NeuSAc is of considerable interest, as a-linked 9amino-NeuSAc in sialoglycoconjugates is not a substrate for bacterial, viral or mammalian sialidases tested so far. CMP-9amino-NeuSAc and CMP-NeuSAcNHz have also been used as synthons to prepare fluorescent and photoactivatable analogues [419] . Because of the defined acceptor specificity, sialyltransferases in combination with fluorescent or photoactivatable donor CMP-sialic acids are excellent tools for selective introduction of a fluorescent or photoactivatable substituent to a distinct glycoconjugate. The latter reference [4 191 also includes kinetic data and information concerning the fluorimetric sialyltransferase assay. Typical fluorescent products comprise the CMP-sialic acids of S-N-acetyl-9-deoxy-9-(3-fluoresceinylthioureido)-neuraminic acid (CMP-9fluoresceinyl-Neu5Ac), 5-N-acetyl-9-(7-amino-4-methylcoumarinyl)acetamido-9-deoxy-neuraminic acid (CMP-9AMCA-NeuSAc), 5-N-acetyl-9-deoxy-9-(fluoresceinylaminomonochlorotriazinyl)amino-neuraminic acid (CMP-~MTAF-N~USAC), and N-(3-fluoresceinylthioureido-acetyl)neuraminic acid (CMP-NeuSfluoresceinyl). In the preparation of photoactivatable derivatives the NH29 group of CMP-9amino-NeuSAc has been substituted with a 4-azidobenzoyl, a 4-azidosalicyl, a 4-benzoylbenzoyl or a 4-azido[T]benzoyl group. In a similar way, CMP-NeuSAcNH2 has been labeled with a 4-azidobenzoyl group. Of special interest is the recently reported chemical synthesis of CMP-{NeuSAc(a2-8)NeuSAc} [479] ; an attempt to prepare this compound biosynthetically with NeuSAc(a2-8)NeuSAc and CMP-sialic acid synthase failed so far [472] . In addition to the preparation of regular CMP-sialic acids, synthetic approaches have been worked out for the organic synthesis of a S-(N-acetylneuraminy1)nucleoside analogue [480,48 11 and other CMP-sialic acid variants [482] . For the immobilization of sialic acids on Sepharose solid supports, which provides potentially useful affinity materials, see the references cited in ref. [344] . The preparation of an affinity adsorbent with immobilized sialic acid through a thioglycosidic linkage has been described in ref. [483] . Synthetic sialidase-stable a-Neu5,9Ac2 p-aminophenylthio glycoside has been immobilized directly or by a six-carbon long spacer group to agarose for lectin isolations [382] . The allyl glycoside of a-NeuSAc has been applied as a starting material for the synthesis of NeuSAc-neoglycoproteins and pseudopolysaccharides. These polymers containing multivalent sialic acid are in principle useful for various applications related with recognitionhindinglinhibition processes. Reductive ozonolysis of the allyl group (03, then MezS), followed by coupling of the formed aldehyde to protein carriers (E-aminogroup of lysine) by sodium cyanoborohydride-mediated reductive amination, yielded neoglycoproteins with varying amounts of NeuSAc [484, 485] . Copolymerization of the allyl glycoside with acrylamide generated a water-soluble pseudopolysaccharide [484] . In order to create a longer spacer arm for copolymerization with acrylamide, the allyl glycoside was converted into a 3-(2-aminoethylthio)propyl glycoside by reaction with cysteamine hydrochloride, after which the amino function was N-acryloylated [486] . The same principle of conjugation or copolymerization via N-acryloyl groups was also used for the preparation of sialo-oligosaccharide-neoglycoproteins and copolymers of sialo-oligosaccharides and acrylamide [487] . Using the strategy of reductive amination, p-formylphenyl glycoside of a-NeuSAc was also conjugated with proteins [488] , and starting from the p-nitrophenyl 0and S-glycosides, p-N-acryloylamino analogues were synthesized, which could be copolymerized with acrylamide, yielding water-soluble pseudopolysaccharides with Neu5Ac and acrylamide in different ratios [489] , or directly coupled with polylysine [490] . Using trimethyl orthoacetate, the NeuSAc residue of the S-glycoside-containing polymer was converted into Neu5,9Ac2 [489] . Finally, a series of interesting NeuSAc and Neu5,9Ac2-based dendrimers have been synthesized [49 11. The organic synthesis of oligosaccharides having terminal a-linked sialic acid has proved to be highly complex. The specific difficulties arise from three factors inherent in the sialic acid molecule. First, the carboxylic acid function at the anomeric center (C2) electronically disfavors oxonium ion formation. Secondly, from a steric point of view, the carboxyl function restricts the glycoside formation. Thirdly, the presence of a neighboring methylene group in the ring (C3), instead of a substituted carbon atom, eliminates the possible assisting andor directing effect of an adjacent substituent [346] . This means that side reactions can be relatively important, mainly the thermodynamically favored fi-glycoside and 2,3-dehydro-derivative formation, and low yields are quite often obtained. Initially, the synthesized glycosidic linkages comprised mainly NeuSAc(a2-6)Gal(fi 1-, NeuSAc(a24)GlcNAc((3 1-, NeuSAc(a2-6)Glc(fll-, NeuSAc(a2-3)Gal((3 1-, NeuSAc(a2-3)GlcNAc(fl1- [344] , and over the years these glycosidic linkages, together with NeuSAc(a24)GalNAc(al-, still receive most of the attention. In view of the desire to prepare biologically relevant carbohydrate chains, this is understandable. In Fig. 9 a series of typical NeuSAc donors, introduced by different research groups with the aim of increasing both the glycosidation yield and the a-stereoselectivity, is depicted [339,34&348] . For each class of donors, some further information is presented in the following paragraphs. The oldest approach of synthesizing sialo-oligosaccharides is the one starting from 2-deoxy-2-halo-fi-NeuSAc derivatives (Fig. 9 , structure A). Methyl S-acetamido-4,7,8,9tetra-O-acetyl-2-ch~oro-2,3,5-t~deoxy-~-g~cero-fi-~-ga~acto-non-2-u~opyranosonate, X = C1, Y = Me, turned out to be a particularly useful donor [492] , and typical promoters are silver and mercury salts. Due to poor stereoselectivity, a$-glycoside mixtures are generally obtained, and HC1-elimination from the donor is a major side reaction. The reaction with secondary hydroxy groups in particular gave rise to problems. In the case of the aim to prepare NeuSGc-containing oligosaccharides, also the N-glycolyl group in the donor analogue is 0-acetylated [493] . For the synthesis of NeuSAc(a2-9)NeuSAq see ref. [494] . In another approach, a series of 3-substituted NeuSAc donors was prepared, starting from peracetylated [495] or perbenzylated [496, 497] Neu2enSAc methyl ester, thereby making use of the highly reactive 2,3-double bond to form adducts (Fig. 9 , structures B-F) (see also ref. [498] ). In the case of structure B as donor with silver triflate as a promoter, only fi-glycosidic linkages were created, and among several products, the NeuSAc(P2-8)NeuSAc linkage was synthesized [495] . Structure C yielded mainly (3-glycosidic linkages. From structure D, only the bromo variant is effective, although a$-glycoside mixtures are still formed. The bromo variant of structure D with silver triflate as a promoter has been applied in the synthesis of NeuSAc(a2-8)NeuSAc and NeuSAc(a2-9)NeuSAc linkages [499, 500] . Structures E and F form another series of donors, and E with X = Br and Y = SPh (mercury salts as promoter) has been shown to give particularly high glycosidation yields and a-stereoselectivity. A third type of donor involves the use of S-methyl or S-phenyl a-glysosides (Fig. 9 , structure G) [498, . Initially developed to synthesize S-glycosides, making use of sodium salts of the peracetylated Neu5Ac methyl ester aor 8-thioglycosides and suitable protected bromides [504-5061 (see also refs. [507, 508] ), this type of donors has shown to be highly attractive in 0-glycosidation reactions. In these couplings, frequently used promoters are dimethyl(methy1thio)sulfonium triflate or N-iodosuccinimideltriflic acid [347] . The choice of the solvent system is very important, as it greatly influences the stereoselectivity; e.g. acetonitrile gives mainly a-glycosidation. In addition to reports dealing with the synthesis of many monosialo-oligosaccharides, including those with a NeuSAc(a2-2)Glc, a NeuSAc(a2-3)GlcNAc, and a NeuSAc(a2-3)GalNAc sequence [509] , typical examples are the creation of NeuSAc(2-9)NeuSAc [5 10,5 1 11 and NeuSAc(a2-8)NeuSAc [512] linkages, as well as sialyl LeX sequences (native and variants) (ref. [513] and references cited therein). In this context, it is also interesting to note that several syntheses of sialo-oligosaccharides include the use of a separately prepared disaccharide donor with a terminal a-linked sialic acid [514] . Although in general the donors contain a N-acetyl group at C5, other examples have been reported with the phthaloyl (benzeneselenenyl triflate as a promoter [5 151) or the tert-butoxycarbonyl function as N-protecting group, e.g. in the case of the synthesis of Neu-containing glycoconjugates [5 16,5 171 . Following similar routes, Kdn-containing oligosaccharides have also been synthesized [5 181 . As a variation on this theme, the application of S-sialyl xanthates (SCSOEt) as donors in sialo-oligosaccharide synthesis has led to interesting results, including a high a-stereoselectivity [5 19-5221 . Here, also the use of 0-benzoyl protection instead of 0-acetyl protection has been proposed [523, 524] . Furthermore, the preparation of sialic acid S-glycosyl donors employing S,S'-bis( 1-phenyl-1H-tetrazol-5-y1)dithiocarbonate should be mentioned [525] . Another efficient donor combines parts of the structures F and G, yielding an a-thioglycoside with a SPh substituent at C3 (Fig. 9 , structure H) [526] . The high stereoselective a-sialylation was obtained using either methyl sulfenyl bromide/silver triflate or N-iodosuccinimide/triflic acid as promoters. Sialyl phosphites with trimethylsilyl triflate as a promoter have additionally been shown to be of practical use (Fig. 9 , structure I), affording good yields and a-stereoselectivity , and examples include the synthesis of sialyl LeX sequences [528] . For a detailed study on the evaluation of different sialyl phosphites, see ref. [530] . For the organic synthesis of sialo-oligosaccharides with di-or trimeric Neu5Ac elements, specific glycosyl donors have been prepared directly from NeuSAc(a2-8)NeuSAc or Neu5Ac(a2-8)Neu5Ac(a2-8)Neu5Ac [53 1-5331. Treatment of the free oligosaccharides with H+-resin in methanol, followed by 0-acetylation and subsequent replacement of the anomeric acetoxy group by a phenylthio function yielded the corresponding peracetylated methyl ester phenyl 2-thioglycosides, in which Neu5Ac residues are linked via a (a2-8,l-9) lactone ring (Fig. 9 , structure J for a disialosyl donor). For additional data with respect to the preparation of dimeric donors with structure A at the reducing site, see ref. [534] . In terms of preparative chemistry, the use of CMP (a 1-0) sequence on a microscale has been described in ref. [536] . A second example (Fig. 11) is the chemo-enzymatic synthesis on a preparative scale of NeuSAc(a2-3)Gal(p 14)[Fuc(ctl- 3)]GlcNAc(P l-O)CH2CH=CH2 (and analogues) using P-1,4-galactosyltransferase and recombinant a-2,3-sialyltransferase and a-1,3-fucosyltransferase with in situ regeneration of UDP-Gal, CMP-Neu5Ac and GDP-Fuc [541] . A third example is the one-pot enzymatic synthesis of Neu5Ac(a24)Gal(~l-4)GlcNAc (and analogues) based on a P-galactosidase-catalyzed galactosylation, using lactose as a donor and GlcNAc as an acceptor, and a pig liver a-2,6-sialyltransferase-catalyzed sialylation with in situ regeneration of CMP-NeuSAc [542] . A fourth example is the enzymatic synthesis of NeuSAc(a24)Gal(fi 14)GlcNAc(fi l-O)pent-4-ene, a precursor for the organic chemical synthesis of higher oligosaccharides [472] . The trisaccharide was synthesized starting from GlcNAc(fi l-O)pent-4-ene, UDP-Gal (in situ generated from UDP-Glc catalyzed by UDP-Gal 4-epimerase), and NeuSAc in a one-pot reaction employing fi-1,4-galactosyltransferase and a-2,6-sialyltransferase in a complete cofactor regeneration system. The availability of specific sialyltransferases will certainly contribute to a further expansion of this area. In this context, the recent finding of a novel sialyltransferase which catalyzes the transfer of Kdn from CMP-Kdn to the non-reducing termini of oligo/polysialyl chains, thereby capping a further elongation of a (NeuSGc(a2-8)}, chain, is of interest [545] . In the framework of the finding that two NeuSAc(a2-6)Gal(fil-4)GlcNAc units are the receptor determinants for the influenza virus hemagglutinin, these elements have been systematically anchored on a Gal residue in order to design structures capable of bimodal viral binding, and along chemo-enzymatic routes heptasaccharides with the general formula NeuSAc(a2-6)Gal(fi 14)GlcNAc(fi l-x)[NeuSAc(a2-6)Gal(fi 1- , where x and y are 2 and 3, 2 and 4, 2 and 6, 3 and 6, and 4 and 6, respectively, have been synthesized [546] . The concept of preparing compounds with a multivalent presentation of NeuSAc(a2-6)Gal(fi 1-4)GlcNAc((31-fragments on a linear or branched (via lysine) peptide backbone has been nicely worked out in ref. [547] . After the organic synthesis of a large series of peptide backbones in which GlcNAc((31-N)Asn units were incorporated, the oligosaccharide extensions were performed enzymatically by using fi-1,4-galactosyltransferase and a-2,6-sialyltransferase. Neu5,9Ac2-containing oligosaccharides have been prepared along both organic chemical and enzymatic routes. In a reaction with trimethyl orthoacetate, NeuSAc(a2-6)-Gal(fi14)Glc could be readily converted into Neu5,9Ac2(a2-6)Gal(filL4)Glc [380] . Employing CMP-Neu5,9Ac2 and immobilized porcine liver Gal((3 lL4)GlcNAc a-2,6-sialyltransferase, Neu5,9Ac2(a2-6)Gal(fi lL4)GlcNAc has been synthesized [478] . The Neu-SAc4Me-, Neu5Ac9Me-, and 8epi-NeuSAc-thioglycoside donors have been used to synthesize the corresponding sialoglycoconjugates [548] , whereas also thioglycoside donors of 4d-, 7d-, 8d-, and 9d-NeuSAc have been prepared [549] . In other enzymatic approaches, use has been made of a fi-D-gaiactoside a-2,3-transsialidase from Trypanosoma cruzi as biocatalyst (see also section 9. sequences, the GlcNAc unit should not be substituted with a Fuc residue, as in LeX or Lea determinants [551] . The trans-sialidase reaction has been used in the chemo-enzymatic preparation of a water-soluble polyacrylamide, bearing multivalent NeuSAc(a2-3)Gal(filL4)GlcNAc elements [553] . To solve the problem of the poor enzymatic a-2,3-sialylation of Gal 2-(trimethylsily1)ethyl (3-glycoside using all known a-2,3-sialyltransferases and CMP-NeuSAc, attention has been paid to the development of a sequence of enzymatic reactions, including cloned a-2,3-sialyltransferase and CMP-NeuSAc synthase, yielding an alternative active sialyl-donor-substrate in situ (e.g. NeuSAc(a2-3)1acto-N-tetraose), which can be used by trans-sialidase [554] . In this way it was possible to convert Gal 2-(trimethylsi1yl)ethyl P-glycoside into NeuSAc(a2-3)Gal 2-(trimethylsily1)ethyl 13-glycoside, a sialodisaccharide that can readily be transformed into a disaccharide donor, of interest for additional organic syntheses. In another study 4-MU-NeuSAc was tested as a donor with lactose as acceptor [555] . Starting from periodate treatedreductive aminated 4-MU-NeuSAc derivatives, interesting possibilities for the inclusion of fluorescent or photolyzable groups were demonstrated. Bacterial sialidases have also been explored in synthetic approaches. In a reverseenzyme reaction with A. ureafaciens sialidase, incubation of a concentrated solution of NeuSAc and lactose yielded NeuSAc(a24)Gal(P 1 4 ) G l c and Gal@ 14)[NeuSAc(a2-6)IGlc [556] . Similar experiments were carried out with immobilized K cholerae sialidase, using NeuSAc p-nitrophenyl a-glycoside as a donor. In this way NeuSAc(a2-x)Gal and NeuSAc(a2-x)Glc linkages could be produced, in which the a-2,6-linkage dominated over the a-2,3-linkage [557]. Transglycosylation of a NeuSAc unit using NeuSAc(a2-8)NeuSAc as a donor to Gal(P14)GlcNAc and Gal(b14)Glc was performed using sialidases of various origin [558] . Although the yields were low, a high regioselectivity was observed. The C. perfringens, A . ureufaciens and c! cholerae sialidases generated a-2,6-linkages, and the Newcastle disease virus sialidase a-2,3-linkages with the terminal Gal residue. For detailed information with respect to the synthesis of C-glycosides of sialic acids, see refs. [559-5631. As an example, the synthesis of a multivalent material that consists of the C-glycoside of NeuSAc, which is resistant to viral sialidase hydrolysis, should be mentioned [562] . Earlier studies have appeared on the X-ray crystallography of both crystalline P-NeuSAc. H20 and B-NeuSAclMe*lHzO [56, 564] . In an additional study, the crystal and molecular structure of a-NeuSAc1,2Me2 was also analyzed [565] . The C=O bond of the COOH function is approximately coplanar with the ring C-0 bond in a-NeuSAcl,2Me2, whereas in both P-NeuSAc and P-NeuSAclMe the C=O bond is found to be nearly eclipsed with the anomeric C-0 bond. In all three derivatives, the N-acetyl group is essentially planar, adopting the Z-conformation of a peptide bond. For a-NeuSAc 1,2Me2 and P-NeuSAclMe a hydrogen bond between the H-atom of H 0 7 and the carbonyl 0-atom of AcNHS was observed. The overall conformation of the glycerol side chain is the same for all three derivatives, as far as non-H atoms are concerned. In a-NeuSAc1,2Me2 a hydrogen bond between the H-atom of H 0 8 and the carbonyl 0-atom of the COOMe group is detectable. One of the oldest NMR studies on the conformation of NeuSAc is that focused on the spatial structure of aNeuSAc2Me in D20 [566, 567] . On the basis of 'H-'H coupling constants in combination with 13C spinlattice relaxation times (TI), a model could be constructed in which the amide H-atom of AcNHS is hydrogen-bonded to 0 7 , and the H-atom of H 0 8 is hydrogen-bonded to the ring-oxygen. A third hydrogen bond between the carbonyl 0-atom of AcNHS and the H-atom of H 0 4 was suggested on the basis of molecular model building. In this model, apparently, the anomeric center is not involved in any hydrogen bonding, leading to the same conformation for aand p-anomers. Independent of the models discussed above, the results fit the observation made by 'H NMR spectroscopy that H 0 7 and H 0 8 usually occur in a tuuns-orientation [53] . An NMR (H2O-suppressed 1 D TOCSY, ROESY, NOESY) study, carried out on NeuSAc(a2-3)Gal(~14)GlcNAc and NeuSAc(a2-6)Gal(B 14)GlcNAc in 85% H20/15% (CD3)2CO, and aimed to detect hydroxyl and amido protons, indicated that in both compounds, thus irrespective of the type of linkage, the H-atom of H 0 8 of NeuSAc is involved in a strong intramolecular hydrogen bond [568] . In view of the fact that 7epi-NeuSAc and 7Jepi2-NeuSAc are substrates for CMP-sialic acid synthase (see section 6.1), but not 8epi-Neu5Ac [411], a conformational study on the side chain conformation of these sialic acids and NeuSAc itself (all