Chemoenzymatic synthesis of the sialyl Lewis X glycan and its derivatives

A combination of recombinant FKP and α-(1→3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5′-diphosphate-β-l-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.     

Synthesis of dimeric lactose and dimeric (sialyl) Lewis(X) glycolipids

To investigate structural requirements for the homophilic interaction between carbohydrates on planar model membranes, divalent derivatives with enforced proximity between the two carbohydrate epitopes (lactose, Lewis(X), and sialyl Lewis(X)) were synthesized by use of a dimeric membrane anchor as scaffold.     

High level expression of monomeric and dimeric human alpha1,3-fucosyltransferase V

alpha3/4-Fucosyltransferases play a crucial role in inflammatory processes and tumor metastasis. While several human fucosyltransferases (FucTs) with different acceptor substrate specificities have been identified, the design of specific inhibitors for therapeutic approaches is hampered by the lack of structural information. In this study, we evaluated the expression of different constructs of human fucosyltransferase V to generate the large amounts required for structural studies. The truncated constructs lacking the transmembrane region and the cytosolic N-terminus, were expressed in baculovirus-infected Trichoplusia ni (Tn) insect cells and in two non-lytic expression systems, stably transfected human HEK 293 and T. ni cells. Since secretion of some glycosyltransferases is controlled by formation of dimeric molecules via disulfide bonds, one of the fucosyltransferase V constructs contained the N-terminal cysteine residue 64 for dimerization, whereas this residue was replaced in the other construct by serine. In both human and insect cells dimerization did not prove to be essential for efficient expression and secretion. On the basis of enzymatic activity, the yield of secreted fucosyltransferase V was approximately 10-fold higher in stably transfected insect cells than in HEK 293 cells. In particular the monomeric form of the enzyme provides a valuable tool for structural analyses to elucidate the fine specifity of fucosyltransferase V-mediated fucosylation of Lewis type glycans.     

A solid-phase synthesis for beta-turn mimetics of sialyl Lewis X

A solid-phase synthesis of heterocyclic beta-turn mimetics of sialyl Lewis X, which is a natural carbohydrate ligand of selectins, was established. This synthetic method could be very useful for drug discovery of selectin antagonists using combinatorial chemistry techniques.     

New derivatives of reducing oligosaccharides and their use in enzymatic reactions: efficient synthesis of sialyl Lewis a and sialyl dimeric Lewis x glycoconjugates

The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyaldi tol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out.     

The alpha (1,3)-fucosyltransferase Fuc-TIV,but not Fuc-TVII,generates sialyl Lewis X-like epitopes preferentially on glycolipids

Fuc-TIV and Fuc-TVII are the two alpha(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLe(x)), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLe(x)-like epitopes in transfected Chinese hamster ovary (CHO)-Pro(-)5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLe(x). Surprisingly however, Fuc-TIV-generated sLe(x) was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80-90% of Fuc-TVII-generated sLe(x) was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLe(x) on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivo specificity for acceptor substrates with Fuc-TVII generating sLe(x) preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLe(x) is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates.     

Core saccharide dependence of sialyl Lewis X biosynthesis

The sialyl-Lewis X (SLe^x) determinant is important in leukocyte extravasation, metastasis and bacterial adhesion. The role of the protein, N-glycan and O-glycan core structures for the biosynthesis of SLe^x in vivo by fucosyltransferases (FucTs) is not known. Immunoglobulin G (IgG) Fc fusion proteins of α₁-acid glycoprotein (AGP), P-selectin glycoprotein ligand-1 (PSGL-1) or CD43 were used to probe the specificity of FucT-III-VII expressed alone in 293T and COS cells or together with O-glycan core enzymes in Chinese hamster ovary (CHO)-K1 cells. Western blotting with the monoclonal antibodies CSLEX and KM93 showed that FucT-III and V-VII produced SLe^x on core 2 in CHO cells. Only FucT-V, -VI and, with low activity, -VII worked on core 3 on CD43/IgG, but no SLe^x was detected with CSLEX on PSGL-1/IgG with core 3. KM93 stained SLe^x on core 2, but was not reactive with SLe^x on core 3. FucT-III, V-VII made SLe^x on N-glycans of AGP/IgG in CHO, but not in COS and 293T cells, even though the same FucTs could make SLe^x on CD43/IgG and PSGL-1/IgG in these cells. Our results define the specificities of FucT-III-VII in SLe^x biosynthesis on O-glycans with different core structures and the fine specificity of the widely used anti-SLe^x monoclonal antibody, KM93.     

Enzymatic synthesis of a sialyl Lewis X dimer from egg yolk as an inhibitor of E-selectin

A dimeric sialyl Lewis X (SLe^x) glycopeptide was synthesized enzymatically in three steps from an N-linked oligosaccharide prepared from egg yolk. Treatment of delipidated hen egg yolk with the protease Orientase and neuraminidase gave a dimeric N-acetyllactosamine-containing oligosaccharide linked to asparagine. Addition of sialic acid and fucose catalyzed by -2,3-sialyltransferase and -1,3-fucosyltransferase provided the dimeric SLe^x, which was shown to be as active as monomeric SLe^x as an inhibitor of E-selectin with IC₅₀ 0.75 mM. The synthetic dimeric SLe^x of the mucin type (i.e. SLe^x linked to the 3- and 6-OH groups of Gal) is, however, about five times as active as the monomer. It is suggested that dimeric SLe^x glycopeptides of the mucin type would be effective ligands for E-selectin.     

Transfection of the c-erbB2/neu gene upregulates the expression of sialyl Lewis X, alpha1,3-fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell line.

The pCMV4 plasmid containing the cancer-promoting gene, c-erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the 'neo' selectable marker. Several clones showing stable expression of c-erbB2/neu were established and characterized by determination of c-erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and alpha1,3-fucosyltransferases and the biological behavior of 7721 cells after c-erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLe(x) expression on the surface of mock cells was high, whereas expression of SDLe(x), Lex and SLe(a) was absent or negligible. This is compatible with the abundant expression of alpha1,3-fucosyltransferase VII, very low expression of alphafucosyltransferase III/VI, and almost absent expression of alpha1,3-fucosyltransferase IV in the mock cells. After transfection of c-erbB2/neu, expression of SLe(x) and alpha1,3-fucosyltransferase VII were simultaneously elevated, but that of alphafucosyltransferase III/VI was not altered. The expression of both SLe(x) and alpha1,3-fucosyltransferase VII correlated positively with the expression of c-erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c-erbB2/neu-transfected cells. These increases also correlated positively with the expression intensities of c-erbB2/neu, SLe(x) and alpha1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLe(x) (KM93) and SDLe(x) (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLe(x) and SLe(a) did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of alpha1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c-erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of alpha1,3-fucosyltransferase VII and its specific product, SLe(x), and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the c-erbB2/neu gene is proposed to be a metastasis-promoting gene, and its effects are at least partially mediated by the increased expression of alpha1,3-fucosyltransferase VII and SLe(x).     

Chemoenzymatic synthesis of a MUC1 glycopeptide carrying non-natural sialyl TF-beta O-glycan

A MUC1 type of glycopeptide was synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol using benzyl and benzylidene-protected beta-D-Gal-(1-->3)-beta-D-GalNAc-Ser/Thr (TF-beta: a stereoisomer of the Thomsen-Friedenreich antigen). The synthetic glycopeptide was released from the resin with reagent K, and the resulting benzylated glycopeptide was deprotected under conditions of low-acidity trifluoromethanesulfonic acid (TfOH). The glycopeptide carrying duplicate non-natural O-glycans was dominant in the products, but was accompanied by a considerable amount of the glycopeptide missing one of the O-glycans. In contrast, the native alpha-glycoside was relatively stable under the acidic debenzylation conditions as shown by a parallel experiment with the glycopeptide involving alpha-D-GalNAc-Ser/Thr linkage. Enzymatic glycosylation with CMP-NeuAc was successful with both natural and non-natural O-glycans of the synthetic glycopeptide.     

Synthesis of precursors for the dimeric 3-O-SO3Na Lewis X and Lewis A structures

Stereoselective syntheses of 3-O-SO₃Na-β-Gal-(1 → 4)-β-GlcNAc-(1 → 3)-β-Gal-(1 → 4)-GlcNAc-β-OBn (15) and 3-O-SO₃Na-β-Gal-(1 → 3)-β-GlcNAc-(1 → 3)-β-Gal-(1 → 3)-β-GlcNAc-(1 → 3)-β-Gal-(1 → 4)-Glc-β-OBn (25) were accomplished through the use of two novel glycosyl donors, namely, ethyl

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(8) and ethyl

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(18).     

A concise synthesis of the 6-O- and 6'-O-sulfated analogues of the sialyl Lewis X tetrasaccharide

The octyl glycoside of the sialyl Lewis X tetrasaccharide and its 6-O-sulfated and 6'-O-sulfated analogues were chemically synthesized in a concise manner starting from readily accessible monosaccharide intermediates. The synthesis involved formation of an orthogonally protected tetrasaccharide intermediate from which all three materials were prepared. A selective catalytic hydrogenolysis of four O-benzyl ethers in presence of a 4,6-O-benzylidene group was the key step in the synthetic scheme.     

Total synthesis of glycononaosyl ceramide with a sialyl dimeric Lex sequence

The first total synthesis of glycononaosyl ceramide with a sialyl dimeric Le^x sequence 1 is described. Regio- and stereo-selective glycosylations of sialyl donors 6,7,8 with the suitably protected Le^x trisaccharide acceptors 9,10 were performed to give the expected tetrasaccharides 15 and 21, which were converted into the corresponding donors 20 and 22. Boron trifluoride etherate-promoted glycosylation of 20 with pentasaccharide acceptor 11 afforded regioselectively the expected nonasaccharide 23. After replacing benzyl groups of 23 by acetyl groups, the anomeric acetate was transformed into the -trichloroacetimidate 27. The crucial coupling between 27 and (2S, 3R, 4E-3-O-benzoyl-2-N-tetracosanoylsphingenine 3 was executed to afford completely protected -glycoside 28. Finally, selective cleavage of the methyl ester and N,O-deprotection of 28 gave the target ganglioside 1.Dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.     

Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a' peptide

Starting from a tumor-associated synthetic MUC1-derived peptide MUC1a' and using a completely enzymatic approach for the synthesis of the core-2 sialyl Lewis X glycopart, the following glycopeptide was synthesized: AHGV[Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-O)]TSAPDTR. First, polypeptide N-acetylgalactosaminyltransferase 3 was used to site-specifically glycosylate MUC1a' to give MUC1a'-GalNAc. Then, in a one-pot reaction employing beta-galactosidase and core-2 beta6-N-acetylglucosaminyltransferase the core-2 O-glycan structure was prepared. The core-2 structure was then sequentially galactosylated, sialylated, and fucosylated by making use of beta4-galactosyltransferase 1, alpha3-sialyltransferase 3, and alpha3-fucosyltransferase 3, respectively, resulting in the sialyl Lewis X glycopeptide. The overall yield of the final compound was 23% (3.2 mg, 1.4 micromol). During the synthesis three intermediate glycopeptides containing O-linked GalNAc, Gal(beta1-4)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc, and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc, respectively, were isolated in mg quantities. All products were characterized by mass spectrometry and NMR spectroscopy.     

Diboronic acids as fluorescent probes for cells expressing sialyl Lewis X

A series of fluorescent diboronic acids was synthesized in nine steps as potential sensors for sialyl Lewis X (sLex). The fluorescent binding studies of these sensors with sLex were carried out in a mixed aqueous solution. Compound 7e was found to show the strongest fluorescence enhancement upon binding with sLex. Using cell cultures, 7e was shown to label sLex-expressing HEPG2 cells at 1 microM, while non-sLex-expressing cells were not labeled.     

Design, synthesis and biological evaluation of aryl-substituted sialyl Lewis X mimetics prepared via cross-metathesis of C-fucopeptides.

Several aryl substituted C-fucopeptides have been developed as sialyl Lewis X mimetics. Although the compounds have a much simpler structure compared to SLe(x), up to 3-times higher binding affinity toward E-selectin and > 1000 times toward P-selectin was observed. Furthermore, a convenient strategy for generating a number of analogues from a SLe(x) mimetic template at a very late stage of the synthesis was introduced, using a ruthenium catalyzed cross olefin metathesis under benchtop conditions.