Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn).

Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid), a unique deaminated member of the sialic acid family, has emerged as a new building block of glycoconjugates from a wide variety of organisms, ranging from bacteria to mammals. In particular, the presence of Kdn has been demonstrated in different rat organs and tissues, but not in liver. Here we report on the detection and quantitation of Kdn in rat liver and on its variations with postnatal development and aging. We have previously established the optimal conditions for derivatization of Kdn with 1,2-diamino-4, 5-methylene-dioxybenzene (DMB), and detection by reverse-phase HPLC. Analysis of whole liver homogenates and different subcellular fractions reveals that Kdn is fundamentally present in the cytosolic fraction as nucleotide precursor. The expression of Kdn, Neu5Gc, and Neu5Ac changes unevenly with age. While the content of Neu5Ac, the major species, and Neu5Gc decreases to a different extent from newborn to old animals, Kdn content decreases from newborn to trace amounts in adult rats and increases again with aging. Thus, expression of Kdn, Neu5Gc, and Neu5Ac appears to be independently regulated.     

Rapid access to uronic acid-based mimetics of Kdn2en from D-glucurono-6,3-lactone

A concise route to novel mimetics of Kdn2en, based on delta4-uronic acids, from D-glucurono-6,3-lactone is presented. Uronic acid-based mimetics in which an aliphatic ether (O-glycoside), a thioether (S-glycoside), or acetamide takes the place of the natural C-6 glycerol sidechain of the sialic acid were synthesized from the key intermediate, methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate bromide.     

A new polymer of 8,9-di-O-glucosylated 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid from a cell wall of Brevibacterium casei AEI Ac-2114T

A polysaccharide containing the residues of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) was found in the cell wall of the Brevibacterium casei strain AEI Ac-2114T . The polymer structure was elucidated by analyzing one-dimensional spectra of 1H and 13C NMR and bidimentional experiments 1H/13C-COSY, TOCSY, 1H/13C-gHSQC, and 1H/13C-gHMBC. The polymer is built up of the 2--> 4-linked Kdn residues substituted by beta-D-Glcp residues at 8- and 9-hydroxyls; such a polymer with disubstituted Kdn residues was found for the first time. A glycosylated teichoic acid of the 1,3-poly(glycerophosphate) type was also identified among other anionic polymers of cell wall.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Identification of Streptomyces coelicolor M145 genomic region involved in biosynthesis of teichulosonic acid–cell wall glycopolymer

The cell wall of the model actinomycete Streptomyces coelicolor M145 has recently been shown to contain the novel glycopolymer teichulosonic acid. The major building block of this polymer is 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn), suggesting initial clues about the genetic control of biosynthesis of this cell wall component. Here, through genome mining and gene knockouts, we demonstrate that the sco4879–sco4882 genomic region of S. coelicolor M145 is necessary for biosynthesis of teichulosonic acid. Specifically, mutants carrying individual knockouts of sco4879, sco4880 and sco4881 genes do not produce Kdn-containing glycopolymer and instead accumulate the minor cell wall component poly(diglycosyl 1-phosphate). Our studies provide evidence that this region is at least partly responsible for biosynthesis of Kdn, whereas flanking genes might control the other steps of teichulosonic acid formation.     

A new polymer of 8,9-Di-O-Glucosylated 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid from a cell wall of Brevibacterium casei ACM Ac-2114T

A polysaccharide containing the residues of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) was found in the cell wall of the Brevibacterium casei strain ACM Ac-2114^T. The polymer structure was elucidated by analyzing one-dimensional spectra of ¹H and ¹³C NMR and bidimentional experiments ¹H/¹H-COSY, TOCSY, ¹H/¹³C-gHSQC, and ¹H/¹³C-gHMBC. The polymer is built up of the 2 → 4-linked Kdn residues substituted by β-D-Glcp residues at 8- and 9-hydroxyls; such a polymer with disubstituted Kdn residues was found for the first time. A glycosylated teichoic acid of the 1,3-poly(glycerolphosphate) type was also identified among other anionic polymers of cell wall.     

Comparative study of carbohydrate chains released from the oviducal mucins of the two very closely related amphibian species Bombina bombina and Bombina variegata

The eggs of amphibians are surrounded by an extracellular matrix, termed jelly coat, which is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by the oviduct and play an important role in the fertilization process. From a structural and chemical point of view, these oviducal mucins are very different from one species to another and they could be involved in the species-specificity of gamete interactions or could influence the parasite tropism. Bombina bombina and Bombina variegata are the two most closely related species within the genus, which hybridize readily in nature. Divergence occurred during geographic isolation estimated at 2-7 million years ago. The oviducal mucins of these species have been studied at the carbohydrate level, and the primary structures of 28 compounds have been established by NMR spectroscopy. The carbohydrate chains released from the oviducal mucins of the two species were similar and characterized by the common sequences GlcNAc(beta 1-3)[Fuc(alpha 1-4)]GlcNAc(beta 1-6) and GlcNAc(alpha 1-4)Gal(beta 1-4)Gal(beta 1-3) attached to GalNAc-ol (core 2). Nevertheless, some differences confirmed the strict species-specificity of amphibian oviducal carbohydrate chains observed previously. On the one hand, the presence of beta Gal 1,4-linked to beta GlcNAc in B. bombina, but not in B. variegata, can indicate that beta 4GalT: beta GlcNAc and beta 4GalT: beta Gal are two distinct glycosyltransferases. On the other hand, deaminoneuraminic acid (Kdn) is present in B. bombina, and N -glycolylneuraminic acid (NeuGc) in B. variegata. Although the enzymes involved in the biosynthesis of Kdn are not as well characterized, it can be suggested that at least one step of the biosynthetic pathway of NeuAc has been disrupted, leading the B. bombina oviducal NeuAc-9-synthase to use Man-6-P as a substrate, instead of ManNAc-6-P.     

Cell wall anionic polymers of Streptomyces sp. MB-8, the causative agent of potato scab

The cell wall of Streptomyces sp. MB-8 contains a major teichoic acid, viz., 1,3-poly(glycerol phosphate) substituted with N-acetyl-alpha-D-glucosamine (the degree of substitution is 60%), a minor teichoic acid, viz., non-substituted poly(glycerol phosphate), and a family of Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid)-containing oligomers of the following general structure: [carbohydrate structure: see text]. The composition of the oligomers was established using MALDI-TOF mass spectroscopy. The present study provides the second example of the identification of Kdn as a component of cell wall polymers of streptomycetes, which are the causative agents of potato scab.     

Enzymatic synthesis of Kdn oligosaccharides by a bacterial alpha-(2-->6)-sialyltransferase.

Synthesis of CMP-deaminoneuraminic acid (CMP-beta-D-Kdn) and its enzymatic transfer reaction using bacterial alpha-(2-->6)-sialyltransferase were examined. CMP-beta-D-Kdn was prepared from methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-beta-D-galacto-2- nonulopyranosonate (2) in 24% overall yield. Enzymatic synthesis of Kdn oligosaccharide with CMP-beta-D-Kdn (10.2 mumol), methyl beta-D-lactosaminide (7, 8.1 mumol) and purified sialyltransferase (80 munits) afforded Kdn-alpha-(2-->6)-Gal-beta-(1-->4)-GlcNAc-beta-1-OMe in 77% yield.     

Chemoenzymatic synthesis of C8-modified sialic acids and related α2-3- and α2-6-linked sialosides

Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and 8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized from corresponding 5-O-modified six-carbon monosaccharides and pyruvate using a sialic acid aldolase cloned from Pasteurella multocida strain P-1059 (PmNanA). In addition, α2-3- and α2-6-linked sialyltrisaccharides containing Neu5Ac8Me and Kdn8Deoxy were also synthesized using a one-pot multienzyme approach. The strategy reported here provides an efficient approach to produce glycans containing various C8-modified sialic acids for biological evaluations.     

Development of sensitive chemical and immunochemical methods for detecting sulfated sialic acids and their application to glycoconjugates from sea urchin sperm and eggs

Sulfated sialic acid (SiaS) is a unique sialic acid (Sia) derivative in which an additional anionic group is attached to a carboxylated monosaccharide. Very little is known about the occurrence and biologic function of SiaS, due to the limitations of analytical methods to detect it in minute amounts. In this study, to develop methods and probes for detecting and pursuing the functions of SiaS, we developed sensitive chemical and immunochemical detection methods. First, we synthesized as model compounds 4-methylumbelliferyl glycosides of 8-O- and 9-O-sulfated Sia consisting of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). Second, we applied fluorometric high performance liquid chromatography (HPLC) analysis to these synthetic glycosides. After acid hydrolysis of the samples, the liberated SiaS were labeled with a fluorescent reagent, 1,2-diamino-4,5-methylenedioxybenzene, and analyzed on fluorometric HPLC. We established an optimal elution condition for successful separation of 8-O- and 9-O-sulfated Neu5Ac, Neu5Gc, and Kdn on HPLC. Third, we generated a monoclonal antibody (mAb) 2C4 against SiaS using sea urchin egg components as the immunogen. mAb.2C4 recognizes both 8-O-sulfated Neu5Ac (Neu5Ac8S) and Neu5Gc8S, whereas the previously prepared mAb.3G9 only recognizes Neu5Ac8S. Finally, using the fluorometric HPLC and monoclonal antibodies, we demonstrated that glycoconjugates from sea urchin sperm exclusively contained Neu5Ac8S, whereas those from eggs contained Neu5Gc8S. Furthermore, we clarified the quantitative differences in the SiaS content in eggs and sperm from two different species of sea urchins. Immunostaining using mAb.2C4 showed that Neu5Gc8S is localized in the cortical granules in unfertilized eggs, whereas it is localized in the outer surface of the fertilization layer as well as in the inner surface of fertilized eggs. Thus, 8-O-sulfation is dependent on the species, gametic cell-type, site-localization of the eggs, and glycoconjugates.     

Anionic carbohydrate-containing cell wall polymers of Streptomyces melanosporofaciens and related species

The structures of cell wall anionic carbohydrate-containing polymers in Streptomyces melanosporofaciens VKM Ac-1864T and phylogenetically close organisms-S. hygroscopicus subsp. hygroscopicus VKM Ac-831T, S. violaceusniger VKM Ac-583T, S. endus VKM Ac-1331, S. endus VKM Ac-129, and S. rutgersensis subsp. castelarensis VKM Ac-832T--have been comparatively studied by chemical and NMR spectroscopic methods. The natural polymer of a new, previously unknown structure, Kdn (3-deoxy-D-glycero-Dgalacto-non-2-ulopyranosonic acid) with beta-galactose residues at C-9, has been found in the cell walls of all the strains under study. The cell walls of all the studied organisms contain three teichoic acids (TA): a predominant TA (1,3-poly(glycerol phosphate) with N-acetylated alpha-glucosaminyl substitutes by C-2 of glycerol, and minor TAs, 1,3- and 2,3-poly(glycerol phosphate) polymers without substitution. Their chains have O-acetyl and O-lysyl groups. Microorganisms of the above-mentioned species differ in the number of alpha-glucosaminyl substitutes and in the degree of their acetylation in the predominant teichoic acid.     

Effects of amine ligand bulk and hydrogen bonding on the rate of reaction of platinum(II) diamine complexes with key nucleotide and amino acid residues

NMR spectroscopy has been used to observe the effects of the amine ligand on the rate of reaction of platinum diamine and triamine complexes with DNA and protein residues. Whereas [Pt(dien)Cl]Cl and [Pt(dien)(D(2)O)](2+) have been known to react faster with thioether residues such as N-AcMet than with 5'-GMP, we found that [Pt(Me(4)en)(D(2)O)(2)](2+) appeared to react faster with 5'-GMP. To quantitatively assess the factors influencing the rates of reaction, rate constants at pH 4 were determined for the reactions of [Pt(en)(D(2)O)(2)](2+) [en = ethylenediamine] and [Pt(Me(4)en)(D(2)O)(2)](2+) with N-AcMet, N-AcHis, 5'-GMP, and Guo (guanosine). In each case the less bulky complex ([Pt(en)(D(2)O)(2)](2+)) reacts more quickly than does the bulkier [Pt(Me(4)en)(D(2)O)(2)](2+), as expected. Both complexes reacted faster with 5'-GMP; however, analysis of the rate constants suggests that the [Pt(en)(D(2)O)(2)](2+) complex favors reaction with 5'-GMP due to hydrogen bonding with the 5'-phosphate, whereas [Pt(Me(4)en)(D(2)O)(2)](2+) disfavors reaction with N-AcMet due to steric clashes. Bulk had relatively little effect on the rate constant with N-AcHis, suggesting that peptides or proteins that coordinate via His residues would not have their reactivity affected by bulky diamine ligands.     

A polymer with a backbone of 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, a teichuronic acid, and a beta-glucosylated ribitol teichoic acid in the cell wall of plant pathogenic Streptomyces sp. VKM Ac-2124.

Structures of cell wall anionic polymers of the strain Streptomyces sp. VKM Ac-2124, a causative agent of potato scab, which is phylogenetically the closest to plant pathogenic species S. setonii and S. caviscabies, were studied. The strain contains three anionic glycopolymers, viz., a teichuronic acid with a disaccharide repeating unit -->6)-alpha-d-Glcp-(1-->4)-beta-d-ManpNAc3NAcA-(1-->, a beta-glucosylated polymer of 3-deoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid (Kdn), and a beta-glucosylated 1,5-poly(ribitol phosphate). The strain studied is the second representative of plant pathogenic streptomycetes inducing potato scab disease, the cell wall anionic polymers of which were shown to contain a Kdn-polymer. Presumably, the presence of Kdn-containing structures in the surface regions of pathogens is essential for their efficient attachment to host plant cells.     

Photoaffinity labeling of a bacterial sialidase with an aryl azide derivative of sialic acid

A photoreactive radioiodinatable derivative of 2-deoxy-2,3-didehydro-5-N-acetylneuraminic acid (NeuAc2en), 5-N-acetyl-9-(4-azidosalicoylamido)-2-deoxy-2,3-didehydroneuram inic acid (ASA-NeuAc2-en) has been synthesized and used to label the active site of Clostridium perfringens sialidase. Like NeuAc2en, its aryl azide derivative is a strong competitive inhibitor of sialidase (Ki approximately 15 microM). The absorbance spectrum of ASA-NeuAc2en shows a characteristic aryl azide peak, which disappears upon photolysis with UV light. When its radioiodinated counterpart 5-N-acetyl-9-(4-iodoazidosalicoylamido)-2-deoxy-2,3-didehydrone uraminic acid ([125I]IASA-NeuAc2en) was photolyzed in the presence of C. perfringens sialidase a 72-kDa protein was labeled. Labeling occurred specifically in the active site since it was inhibited in the presence of NeuAc2en. Chemical cleavage of the photoaffinity-labeled 72-kDa protein demonstrates that specifically labeled peptides involved in the formation of the active site can easily be determined. ASA-NeuAc2en is a valuable new tool for the identification and structural/functional analysis of sialidases and other proteins, recognizing this sialic acid derivative.     

DNA-binding studies of mixed-ligand (ethylenediamine)ruthenium(II) complexes

A series of mixed-ligand ruthenium(II) complexes of the type [Ru(en)(2)bpy](2+) (bpy=2,2-bipyridine; 1), [Ru(en)(2)phen](2+) (phen=1,10-phenantroline; 2), [Ru(en)(2)IP](2+) (IP=imidazo[4,5-f][1,10]phenanthroline; 3), and [Ru(en)(2)PIP](2+) (PIP=2-phenylimidazo[4,5-f][1,10]phenanthroline; 4) have been isolated and characterized by UV/VIS, IR, and (1)H-NMR spectral methods. The binding of the complexes with calf thymus DNA has been investigated by absorption, emission spectroscopy, viscosity measurements, DNA melting, and DNA photo-cleavage. The spectroscopic studies together with viscosity measurements and DNA melting studies support that complexes 1 and 2 bind to CT DNA (=calf thymus DNA) by groove mode. Complex 2 binds more avidly to CT DNA than complex 1, complexes 3 and 4 bind to CT DNA by intercalation mode, 4 binds more avidly to CT DNA than 3. Noticeably, the four complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA.     

Cytotoxic activity of platinum (II) complexes with tri-n-butylphosphine. Crystal structure of the dinuclear hydrazine-bridged complex, cis,cis-[PtCl(PBu3n)2(mu-N2H4)PtCl(PBu3n) 2] (ClO4)2.2CHCl3.

A series of platinum(II) tri-n-butylphosphine complexes having the formulas cis-[PtCl2L2], NEt4[PtCl3L], [PtCl(en)L]Cl, [Pt(en)L2](ClO4)2, sym-trans-[Pt2Cl4L2], [Pt2Cl2L4](ClO4)2, trans,trans-[PtCl2L(mu-N2H4)PtCl2L] trans,trans-[PtCl2L(mu-en)PtCl2L], and cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2 (L = tri-n-butylphosphine; en = ethylenediamine) have been synthesized and their cytotoxic activity in vitro and in vivo has been studied. The solution behavior of the novel dinuclear diamine-bridged platinum(II) complexes has been investigated by means of UV and 31P NMR spectroscopy. For the ionic hydrazine compound cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2, an x-ray structure determination is reported. Crystal data: space group P2(1)/a, a = 17.803(1), b = 18.888(3), c = 12.506(3) A, beta = 107.97(2) degrees, Z = 2, R = 0.052, RW = 0.058. The platinum coordination is approximately square-planar, with the bond lengths Pt-Cl = 2.358(5), Pt-N = 2.15(1), Pt-P(trans to Cl) = 2.260(5), and Pt-P(trans to N) = 2.262(6) A. All investigated compounds were cytotoxic in vitro against L1210 cells and showed no cross-resistance to cisplatin. On the other hand, no antitumor activity was observed vs L1210 leucemia in DBA2 mice.     

Crystal structures and spectroscopic studies of ternary compounds of Ni(II) with ethylenediamine and 5'GMP and 5'IMP

Two metal complexes [Ni(en)5'GMPH)2(H2O)2] (en).6.5H2O and [Ni(en)(5'IMPH)2(H2O)2].13H2O have been synthesized in the form of suitable crystals for x-ray crystallography (en = ethylenediamine, 5'GMP = guanosine 5'-monophosphate, 5'IMP = inosine 5'-monophosphate). The 5'GMP complex crystallizes in a monoclinic space group P21 (Z = 4) with a = 12.317(2), b = 28.417(4), c = 12.290(2)A, beta (deg) = 89.59(2). The 5'IMP complex is tetragonal, space group P4122 (Z = 4), with a = 12.119(3), b = 12.119(3), c = 28.560(4)A, beta (deg) = 90.0. The crystal structures of both complexes were refined from diffractometer data to conventional R values of 0.073 for the 5'GMP compound (5,284 observed reflections, 1,322 variables) and 0.030 for the 5'-IMP compound (1,529 observed reflections, 296 variables). In both structures, the Ni(II) is surrounded by two water molecules, one chelate ethylenediamine, and two nucleotide molecules. The synthesis was carried out from Ni(en)2Cl2.0.5H2O and the nucleotide in water medium. The dimer structure of the initial complex is broken, and one ethylenediamine is substituted by two molecules of the nucleotide with the N(7) of the purine ring in cis-position. Differences between both structures are largely due to retention in the structure or loss of the en molecule substituted and to the intermolecular hydrogen bonds of the en molecule coordinated. A third complex of composition [Ni(en)(5'IMPH)2(H2O)2] (en).6H2O similar to the 5'GMP complex has been obtained in the form of blue crystals, but unfortunately its crystal structure failed to be refined. This complex is isostructural with the monoclinic one.     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.     

Synthesis of delta4-beta-D-glucopyranosiduronic acids as mimetics of 2,3-unsaturated sialic acids for sialidase inhibition.

Mimetics of Neu5Ac2en and KDN2en, based on delta4-beta-delta-glucopyranosiduronic acids, have been synthesised. The Neu5Ac2en mimetic 5 showed inhibition of both bacterial and viral sialidases, with inhibition of the viral sialidase being comparable to that of Neu5Ac2en itself.