Azido glycoside primer: a versatile building block for the biocombinatorial synthesis of glycosphingolipid analogues

A lactoside primer, 12-azidododecyl beta-lactoside, was synthesized via the Koenigs-Knorr method by glycosylation of 1,12-dodecyldiol with perbenzoylated lactosyl bromide. The presence of the 2-O-acyl substituent in the donor gave the beta-lactoside, and an excess of acceptor ensured monoglycosylation of the diol. Mesylation of the omega-hydroxyl group in the aglycon, followed by displacement of the mesylate with azide and subsequent O-debenzoylation gave the desired omega-azidododecyl beta-lactoside. The azido glycoside primer was examined in mouse B16 melanoma cells for its feasibility as a building block for oligosaccharide biosynthesis. Uptake of the azido glycoside primer by B16 cells resulted in the sialylation of the galactose residue of the primer to give a glycosylated product having the same glycan as in ganglioside GM3. After 24 h incubation of B16 cells with the primers, the amount of sialylated omega-azidododecyl beta-lactoside primer was 75% of the amount of sialylated n-dodecyl beta-lactoside. However, after 48 h incubation, both primers gave equal amounts of the sialylated products. Interestingly, the remaining azido glycoside primer after 48 h incubation was 5.6-fold greater than that of the alkyl primer, indicating degradation of the alkyl primer to a larger extent than the omega-azido glycoside primer. The facile chemical synthesis and the efficient uptake in cells make the azido glycoside primer a versatile building block for the biocombinatorial synthesis of glycolipid oligosaccharides.     

Study on systematizing the synthesis of the a-series ganglioside glycans GT1a, GD1a, and GM1 using the newly developed N-Troc-protected GM3 and GalN intermediates

A first systematic synthesis of the glycan parts of the a-series gangliosides (GT1a, GD1a, and GM1) utilizing the newly developed N-Troc-protected GM3 and galactosaminyl building blocks is described. The key processes, including the assembly of the GM2 sequence and its conversion into the 3-hydroxy acceptor, were facilitated mainly by the high degree of participation and chemoselective cleavability of the Troc group in the galactosaminyl unit. Furthermore, the novel GM2 acceptor served as a good coupling partner during glycosylation with galactosyl, sialyl galactosyl, and disialyl galactosyl donors, successfully producing the GM1, GD1a, and GT1a glycans.     

Synthesis and selectin-binding activity of N-deacetylsialyl Lewis X ganglioside

A novel analogue of sialyl Lewis X ganglioside, N-deacetylsialyl Lewis X ganglioside, was synthesized. Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-alpha-D-galacto-2-nonulopyranosylonate-(2 --> 3)-2,4,6-tri-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(trimethylsilyl)ethyl [2-acetamido-6-O-benzyl-2-deoxy-3-O-(4-methoxybenzyl)-beta-D-glucopyranosyl]-(1 --> 3)-[2,4,6-tri-O-benzyl-beta-D-galactopyranosyl]-(1 --> 4)-2,3,6-tri-O-benzyl-beta-D-galactopyranoside to give the desired pentasaccharide in high yield. The glycosylation of the pentasaccharide acceptor, which was derived from its precursor by removal of the 3-methoxybenzyl group, with the phenyl 1-thioglycoside derivative of L-fucose using N-iodosuccinimide-trifluoromethanesulfonic acid as promoter, produced the hexasaccharide. Proper manipulation of the protecting groups of the hexasaccharide afforded the corresponding glycosyl imidate, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol. Selective reduction of the azido group, N-acylation with octadecanoic acid, and the complete removal of the protecting groups gave the desired N-deacetylsialyl Lewis X ganglioside. L-Selectin bound more strongly to N-deacetylsialyl Lewis X ganglioside than to the sialyl Lewis X ganglioside, whereas E- and P-selectins bound equally well to the two gangliosides.     

Derivatives of daunorubicin containing an inosine fragment

Condensation of daunorubicin or its (13 R, S)-dihydro derivative with inosine dialdehyde in the presence of NaBH3CN yielded novel derivatives of anthracycline antibiotics with incorporated inosine residue: 3'-deamino-3'-[(2" R)-(hypoxanthyl-9)-(6" S)-hydroxymethylmorpholino-N4"]- daunorubicin and (13 R,S)-dihydro-3'-deamino-3'-[(2" R)-(hypoxanthyl-9)-(6" S)- hydroxymethylmorpholino-N4"]-daunorubicin. The compounds did not inhibit growth of Bacillus mycoides and were less cytotoxic in vitro and less toxic in vivo than the parent antibiotics.     

Synthesis of selected aminodeoxy analogues of galabiose and globotriose

Six aminodeoxy 2-(trimethylsilyl)ethyl (Me3SiEt) glycoside analogues of galabiose (4'- and 6'-aminodeoxy) and globotriose (6"-, 4"-, 2"-, and 6'-aminodeoxy) were synthesized by glycosylation of protected Me3SiEt galactoside and lactoside acceptors with azido-substituted monosaccharide donors, followed by reduction of the azido groups and removal of the protecting groups.     

Synthesis of a hexasaccharide,the repeating unit of O-deacetylated GXM of C. neoformans serotype A

beta-D-GlcpA-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp-(1-->3)[-beta-D-Xylp-(1-->2)]-alpha-D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A, was synthesized as its allyl glycoside. Thus, 3-O-selective acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside afforded 2, and subsequent glycosylation of 2 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate furnished the beta-(1-->2)-linked disaccharide 4. Debenzylidenation followed by benzoylation gave allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-3-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (5), and selective 3-O-deacetylation gave the disaccharide acceptor 6. Coupling of 6 with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate yielded the trisaccharide 8, and subsequent deallylation and trichloroacetimidation gave 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-[2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)]-4,6-di-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9). Condensation of the trisaccharide donor 9 with the disaccharide acceptor 6 gave the pentasaccharide 10 whose 2-O-deacetylation gave the acceptor 11. Glycosylation of 11 with methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate and subsequent deprotection gave the target hexasaccharide.     

Synthesis of a spacer-linked derivative of heptopyranosyl(α1-3)heptopyranose expressed in lipooligosaccharide and lipopolysaccharide

Glycosylation of penta-O-acetyl heptopyranosyl trichloroacetimidate with the 3-OH acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside, gave the desired α1-3-linked disaccharide in a 94% yield. The oct-enopyranoside moiety of the disaccharide was converted to the heptoside by oxidative cleavage with osmium tetroxide/NaIO(4) and subsequent reduction with NaBH(4). The resulting α1-3-linked heptose disaccharide was converted to a tricholoroacetaimidate derivative containing a benzoyl group at C-2. This donor was glycosylated with 2-(carbobenzoxyamino)-1-ethanol to give an α spacer-linked disaccharide derivative in a 90% yield. Zemplén deacylation of the derivative and subsequent hydrogenolysis gave a 2-aminoethyl glycoside of heptopyranosyl(α1-3)heptopyranose.     

肝癌组织中GD3合成酶的纯化

ＧＤ３合成酶是膜嵌合蛋白,定位于高尔基体,经过制作微粒体,Ｔｒｉｔｏｎ增溶、ＡＨ－Ｓｅｐｈａｒｏｓｅ及ＧＭ３－Ｇｌａｓｓｂｅａｄｓｅ及ＧＭ３－Ｇｌａｓｓｂｅａｄｓ亲和层析等步骤,二乙基亚硝胺诱发大鼠肝癌组织中的ＳＴ２被纯化了３１５９７倍,得率为０．３５％。ＳＤＳ－聚丙烯酰胺凝胶电泳后银染色呈１条蛋白着色带,分子量为５５ｋｄ。     

Synthesis of a Spacer-Linked Derivative of Heptopyranosyl(α1-3)heptopyranose Expressed in Lipooligosaccharide and Lipopolysaccharide

Glycosylation of penta-O-acetyl heptopyranosyl trichloroacetimidate with the 3-OH acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside, gave the desired α1-3-linked disaccharide in a 94% yield. The oct-enopyranoside moiety of the disaccharide was converted to the heptoside by oxidative cleavage with osmium tetroxide/NaIO₄ and subsequent reduction with NaBH₄. The resulting α1-3-linked heptose disaccharide was converted to a tricholoroacetaimidate derivative containing a benzoyl group at C-2. This donor was glycosylated with 2-(carbobenzoxyamino)-1-ethanol to give an α spacer-linked disaccharide derivative in a 90% yield. Zemplén deacylation of the derivative and subsequent hydrogenolysis gave a 2-aminoethyl glycoside of heptopyranosyl(α1-3)heptopyranose.     

Expression of the Ganglioside GD3 in Human Meningiomas Is Associated with Monosomy of Chromosome 22

The ganglioside composition of 59 meningiomas has been compared with a molecular genetic analysis of chromosome 22 in the same specimens. Major gangliosides were GM3 (II3NeuAc-LacCer) and/or GD3 [II3(NeuAc)2-LacCer]. In specimens with no or partial deletions of chromosome 22, the GM3 ganglioside predominated, and the mean value for GM3, 61% of total sialic acid, was around four times higher than that for GD3. A loss of chromosome 22, found in 56% of the specimens, was shown to be associated with an increase in the proportion of ganglioside GD3, with the ratio between mean values of GM3 and GD3 being approximately 1:1. Logistic regression revealed that the probability of predicting monosomy of chromosome 22 by the GD3 proportion was 66%.     

Synthesis of Neu5Ac(alpha2-->6)[Gal(alpha1-->3)]GalNAcalpha and Neu5Ac(alpha2-->6)[Gal(beta1-->3)]GalNAcalpha trisaccharides as protected trifluoroacetamidopropyl glycosides

Protected sialo-containing trisaccharides, fragments of oligosaccharide chains of mucin glycoproteins, were synthesized. Regioselective sialylation of the primary hydroxyl group of (3-fluoroacetamidopropyl)-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-ben zyl)-alpha -D-galactopyranosyl)-alpha-D-galactopyranoside with methyl ester of peracetyl-beta-ethylthioglycoside of N-acetylneuraminic acid in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) yielded 39 and 25% of alpha- and beta-sialyl-(2-->6)biosides, respectively. Catalytic hydrogenolysis of the azide and benzyl groups of the alpha-anomer followed by N- and O-acetylation gave target trifluoroacetamidopropyl glycoside, Neu5Ac(alpha 2-->6)[Gal(alpha 1-->3)]GalNAc alpha-OSp, as a peracetate. An analogous coupling of the sialyl donor with (3-fluoroacetamidopropyl)-2-acetamido-2-deoxy-3-O- (2,3,4,6-tetra-O-acetyl)-beta-D-galactopyranosyl)-alpha-D-galactopyranos ide affords acetylated trifluoroacetamidopropyl glycoside Neu5Ac(alpha 2-->6)[Gal(beta 1-->3)]GalNAc alpha-OSp in a yield of 15% and the corresponding Neu5Ac(beta 2-->6)-anomer in a yield of 12%. After O-deacetylation and N-detrifluoroacetylation, these sialylbiosides can be used as ligands in preparing neoglycoconjugates.     

GD3 Prevalence in Adult Rat Retina Correlates with the Maintenance of a High GD3-/GM2-Synthase Activity Ratio Throughout Development

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a ganglioside of the lactosylceramide series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N-acetylneuraminic acid (NeuNAc)] to adult (30% of total gangliosidic NeuNAc). GD3-synthase specific activity increased about 1.5-fold from birth to day 7 and essentially plateaued thereafter. The GD3-/GM2-synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GD1a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other "b" pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3-/GM2-synthase activity ratio throughout development of the tissue.     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

GT3 Synthesis in the Proximal Golgi Occurs in a Compartment Different from Those for GD3 and GM3 Synthesis

Abstract: Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [³H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[³H]Gal. In (a), 1 µM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to ～50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of >90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited ～30 and 70%, respectively, in 7- and 10-day cells. In (b), >80% of the [³H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [³H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, <5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[³H]NeuAc incorporated ～20% of [³H]NeuAc into endogenous GT3, and this percentage was not affected by 1 µM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.     

Synthesis of alpha-series ganglioside GM1alpha containing C20-sphingosine

A synthesis of alpha-series ganglioside GM1alpha (III(6)Neu5AcGgOse4Cer) containing C20-sphingosine(d20:1) is described. Glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside with the glucosamine donor ethyl 3-O-acetyl-2-deoxy-4,6-O-[(4-methoxyphenyl)methylene]-2-phthalimido-1-thio-beta-D-glucopyranoside furnished a beta-(1-->4)-linked trisaccharide. Reductive cleavage of the p-methoxybenzylidene group followed by intramolecular inversion of its triflate afforded the desired trisaccharide, which was transformed into a trisaccharide acceptor via removal of the phthaloyl and O-acetyl groups followed by N-acetylation. A tetrasaccharide acceptor was obtained by glycosylation of the trisaccharide acceptor with dodecyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranoside, followed by removal of the p-methoxybenzyl group. Coupling of the tetrasaccharide acceptor with ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-thio-5-trichloroacetamido-D-glycero-D-galacto-2-nonulopyranosid)onate and subsequent radical reduction gave the desired GM1alpha saccharide derivative, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-eicosene-1,3-diol after conversion into the imidate.     

Synthesis of a GM3 ganglioside analogue carrying a phytoceramide moiety by intramolecular glycosylation as a key step

A novel analogue of ganglioside GM3, in which sphingosine was replaced with a phytosphingosine moiety, was synthesized by intramolecular glycosylation as a key step. Glucose, a reducing terminal of the saccharide, and phytoceramide were first tethered by succinic acid and the derivative used for the subsequent glycosidic bond formation. The obtained glycosyl phytoceramide was further glycosylated with the sialyl galactose residue to afford a fully protected GM3 derivative, which was converted into the desired, final compound by using conventional deprotection procedures.     

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.     

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.     

Synthesis of a hexasaccharide fragment of the O-deacetylated GXM of C. neoformans serotype B

beta-D-Xylp-(1-->4)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar B, was synthesized as its methyl glycoside. Thus, acetylation of allyl 3-O-benzoyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-acetyl-3,6-di-O-benzoyl-alpha-D-mannopyranoside (4). Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6. Deallylation followed by trichloroacetimidate formation gave the disaccharide donor 8, and subsequent coupling with allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-4,6-di-O-benzoyl-alpha-D-mannopyranoside (9), produced the tetrasaccharide 10. Reiteration of deallylation and trichloroacetimidate formation from 10 yielded the tetrasaccharide donor 12. The downstream disaccharide acceptor 18 was obtained by condensation of 5 with methyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenation, benzoylation, and selective 3-O-deacetylation. Coupling of 18 with 12 afforded the hexasaccharide 19, and subsequent deprotection gave the hexasaccharide glycoside 20. Selective 2"-O-deacetylation of 19 gave the hexasaccharide acceptor 21. Condensation of 21 with glucopyranosyluronate imidate 22 did not produce the expected heptasaccharide glycoside; instead, a transacetylation product 19 was obtained. Meanwhile, there was no reaction between 21 and the bromide donor 23.     

Mammalian sialyltransferase ST3Gal-II: its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated glycoproteins and specific gangliosides

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galβ1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin β subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.