Crystal structures of sialyltransferase from Photobacterium damselae

Sialyltransferase structures fall into either GT-A or GT-B glycosyltransferase fold. Some sialyltransferases from the Photobacterium genus have been shown to contain an additional N-terminal immunoglobulin (Ig)-like domain. Photobacterium damselae α2–6-sialyltransferase has been used efficiently in enzymatic and chemoenzymatic synthesis of α2–6-linked sialosides. Here we report three crystal structures of this enzyme. Two structures with and without a donor substrate analog CMP-3F(a)Neu5Ac contain an immunoglobulin (Ig)-like domain and adopt the GT-B sialyltransferase fold. The binary structure reveals a non-productive pre-Michaelis complex, which are caused by crystal lattice contacts that prevent the large conformational changes. The third structure lacks the Ig-domain. Comparison of the three structures reveals small inherent flexibility between the two Rossmann-like domains of the GT-B fold.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.     

Lactones of disialyl lactose: characterisation by NMR and mass spectra

The lactonisation of alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose) was investigated. (1)H and (13)C NMR chemical shifts of disialyl lactose and alpha-Neup5Ac-(2-->8, 1-->9)-alpha-Neup5Ac-(2-->3, 1-->2)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose-dilactone) were assigned based on 1D and 2D NMR results, including edited HSQC, HSQC-TOSCY and HMBC. The time course of lactonisation was followed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with electrospray ionisation (ESI) mass spectrometry (MS) detection. The rate of lactonisation between alpha-(8)Neu5Ac and alpha-(3)Neu5Ac residues (lactonisation at the alpha-(2-->8) linkage) was faster than that of lactonisation between alpha-(3)Neu5Ac and Gal residues (lactonisation at the alpha-(2-->3) linkage). The mass spectra of disialyl lactose, its lactones, alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac (alpha-(2-->8) disialic acid) and alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc-lactone (3'-sialyllactose-lactone) showed that the alpha-(2-->8) linkage between Neu5Ac residues is difficult to cleave in the ESI-MS, compared with the alpha-(2-->3) linkage between Neu5Ac and Gal residues.     

Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae: incorporation of disialic acid

The lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterized biochemically to date in H. influenzae is Lic3A, an alpha-2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., M?nsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both alpha-2,3- and alpha-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance.     

Regulation of sialic acid O-acetylation in human colon mucosa

The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate-7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotide-sugar level [Shen et al., Biol. Chem. 383 (2002), 307-317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMP-Neu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-N-acetyl-9-O-acetyl-neuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2 could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMP-Neu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galbeta1,3(4)R- residues.     

Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.     

A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.     

Large-scale production of the carbohydrate portion of the sialyl-Tn epitope, alpha-Neup5Ac-(2-->6)-D-GalpNAc, through bacterial coupling

Alpha-Neup5Ac-(2-->6)-D-GalpNAc, the carbohydrate portion of sialyl-Tn epitope of the tumor-associated carbohydrate antigen, was prepared by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains overexpressed the CMP-Neup5Ac biosynthetic genes and the alpha-(2-->6)-sialyltransferase gene of Photobacterium damsela. C. ammoniagenes contributed to the production of UTP from orotic acid. Alpha-Neup5Ac-(2-->6)-D-GalpNAc was accumulated at 87 mM (45 g/L) after a 25-h reaction starting from orotic acid, N-acetylneuraminic acid, and 2-acetamide-2-deoxy-D-galactose.     

Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins.

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAc alpha-], BSA-[Gal(beta 1-3)GalNAc(alpha-], and BSA-[Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc(alpha-], were prepared. alpha GalNAc derivatives were galactosylated employing crude beta-galactosidase from bovine testes. The use of oversaturated donor solutions (pNP beta Gal) enhanced the yields up to 60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated products. Further treatment of the disaccharides with CMP-Neu5Ac and alpha 2-3 sialyltransferase from pork liver led to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide components. The results were monitored by HPLC and MALDI-TOF.     

Chemo-enzymatic synthesis of C-9 acetylated sialosides

A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-O-(beta-D-galactopyranosyl)-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)]-l-serine acetate (1) has been accomplished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically converted into CMP-Neu5,9Ac(2) (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43]. The resulting compound was then employed for the enzymatic glycosylation of the C-3' hydroxyl of chemically prepared glycosylated amino acid 10 using recombinant rat alpha-(2-->3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give, after deprotection of the N(alpha)-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The N(alpha)-CBz-protected intermediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.     

Glycan microarrays for screening sialyltransferase specificities

Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein-streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcbeta1-4GlcNAc)(n) including N-glycans, rat alpha2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1-4 and porcine alpha2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.     

Synthesis of Neu5Ac- and Neu5Gc-alpha-(2-->6')-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-alpha-(2-->6')-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-alpha-(2-->6')-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4',6'-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145

A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg(2+) for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.     

Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver alpha2,6-sialyltransferase

2-Keto-3-deoxy-D- glycero -D- galacto -nononic acid (KDN) was introduced into asialotransferrin and N -acetyllactosamine (LacNAc) from CMP-KDN by using rat liver Galbeta1-->4GlcNAc alpha2, 6- sialyltransferase to form KDN-transferrin and KDN-LacNAc. These structures contain terminal KDNalpha2-->6Gal-residues, a glycotope that has not yet been described in natural glycoconjugates. KDN was transferred to all four Gal residues in asialotransferrin by this enzyme. The incorporation efficiency of KDN from CMP-KDN into asialotransferrin was about half that of Neu5Ac from CMP-Neu5Ac, based on the V max/ K m values for these donor substrates, 0.0527 min-1and 0.119 min-1, respectively. The KDNalpha2-->6Gal linkage was resistant to exosialidase treatment, in contrast to the sensitivity of the Neu5Acalpha2-->6Gal linkage. Interestingly, Sambucus sieboldiana agglutinin (SSA) was shown to prefer KDN-transferrin to the corresponding Neu5Ac-transferrin, as estimated by slot-blot analysis. The use of an alpha2,6-sialyltransferase to synthesize neoglycoproteins containing KDN has not been previously reported. Their facile synthesis using CMP-KDN and sialyltransferases with different specificities offers new possibilities to study the function of neo-KDN- glycoconjugates, and to explore their use in glycotechnology.      

Identification of sialic acids on Leishmania donovani amastigotes

The presence of Neu5Ac on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, has been reported recently. Here we report the occurrence of Neu5Ac as a major component on amastigotes, as well as Neu5Gc, Neu5,9Ac2 and Neu9Ac5Gc as indicated by fluorimetric high performance liquid chromatography and gas liquid chromatography/electron impact mass spectrometry. Furthermore, binding studies with Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and various Siglecs, showed the presence of both (alpha2 --> 6)- and (alpha2 --> 3)-linked sialic acids; their binding was reduced after sialidase pretreatment. Western blotting of amastigote membrane glycoproteins with SNA demonstrated the presence of two sialoglycoconjugates of Mr values of 164000 and 150000. Similarly, binding of MAA demonstrated the presence of five distinct sialoglycans corresponding to molecular masses of 188, 162, 136, 137 and 124 kDa. Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid (alpha2 --> 6)-linked to GalNAc, demonstrated the occurrence of two 9-O-acetylated sialoglycans with Mr 158000 and 150000, and was corroborated by flow cytometry; this binding was abolished by recombinant 9-O-acetylesterase pretreatment. Our results indicate that Neu5Ac [(alpha2 --> 6)- and (alpha2 --> 3)-linked], as well as Neu5Gc and their 9-O-acetyl derivatives, constitute components of the amastigote cell surface of L. donovani.     

Synthesis of disaccharide Neu5Gcalpha(2-6)GalNAcalpha as a spacer glycoside

The first synthesis of the Neu5Gc analogue of SiaTn disaccharide, which can be detected in breast tumors by immunochemical methods, is reported. The regioselective sialylation of (3-trifluoroacetamidopropyl)-2-azido-2-deoxy-alpha-D-galactopyranoside with peracetate of the methyl ester of N-acetoxyacetylneuraminic acid beta-ethylthioglycoside in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) resulted in the derivatives of alpha- and beta-sialyl(2-->6)galactosaminide in 39 and 32% yields, respectively. The catalytic hydrogenolysis of the azido group and subsquent N- and O-acetylation of the alpha-anomer gave the trifluoroacetamidopropyl glycoside peracetate. Removal of the protective groups led to glycoside Neu5Gc alpha(2-->6)GalNAc alpha-O(CH2)3NH2. Using the Neu5Gc derivative with acetoxyacetyl groups at positions O9 and O4 as a donor increases the alpha-selectivity of sialylation to afford the alpha- and beta-anomers in 69 and 8% yields, respectively.     

Potential sialyltransferase inhibitors based on neuraminyl substitution by hetaryl rings

Replacement of the neuraminyl residue by a wide range of aryl rings in transition-state analogs of CMP-Neu5Ac led to readily accessible and potent inhibitors of alpha-(2-->6)- and alpha-(2-->3)-sialyltransferases. The synthesis of a series of potential sialyltransferase inhibitors in which the neuraminyl residue is replaced by hetaryl methylphosphonate residues (thiazole, benzothiazole, benzoxazole, benzothiophene and thiophene) is described in this paper.     

Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases

This paper presents kinetic properties of the transfer of several synthetic 9-substituted sialic acid analogues onto N- or O-linked glycoprotein glycans by four purified mammalian sialyltransferases: Gal beta 1,4GlcNac alpha 2,6sialyltransferase, Gal beta-1,4(3)GlcNAc alpha 2,3-sialyltransferase, Gal beta 1,3GalNAc alpha 2,3sialyltransferase, and GalNAc alpha 2,6sialyltransferase. The substituents at C-9 of the sialic acid analogues introduce special biochemical characteristics: 9-Amino-NeuAc represents, up to the present, the first derivative that is resistant toward bacterial, viral, and mammalian sialidases but is transferred by a sialyltransferase. 9-Acetamido-NeuAc, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc differ in size and hydrophobic character from each other and from parent NeuAc. 9-Azido-NeuAc may be used to introduce a photoreactive label. The kinetic properties of the four sialyltransferases with regard to the donor CMP-glycosides differed distinctly depending on the structure of the substituent at C-9. CMP-9-amino-NeuAc was only accepted as donor substrate by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase (rat liver), but the Km value was 14-fold higher than that of parent CMP-NeuAc. In contrast, 9-azido-NeuAc was readily transferred by each of these four enzymes. 9-Acetamido-NeuAc, which is a receptor analogue for influenza C virus, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc were also accepted by each sialyltransferase, but incorporation values differed significantly depending on the enzyme used. For the first time, the resialylation of asialo-alpha 1-acid glycoprotein with 9-substituted sialic acid analogues by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase is demonstrated.