Khushi L. Matta

Use of 1,2-dichloro 4,5-dicyanoquinone (DDQ) for cleavage of the 2-naphthylmethyl (NAP) group

Abstract The 2-naphthylmethyl (NAP) group is a versatile group for protection of hydroxyl functions. It is stable to 4% TFA in CHCl 3 , hot 80% HOAc–H 2 O, SnCl 2 –AgOTf and HCl–EtOH, but it can readily be removed with DDQ in CH 2 Cl 2 .

Fucosylation of Disaccharide Precursors of Sialyl LewisX Inhibit Selectin-mediated Cell Adhesion

We showed previously that HL-60 and F9 mouse embryonal carcinoma cells will take up and deblock peracetylated Galβ1–4GlcNAcβ-O-naphthalenemethanol (Galβ1–4GlcNAc-NM) and use the disaccharide as a primer of oligosaccharide chains (Sarkar, A. K., Fritz, T. A., Taylor, W. H., and Esko, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3323–3327). We now report that another disaccharide, acetylated GlcNAcβ1–3Gal-naphthalenemethanol (GlcNAcβ1–3Gal-NM), has even greater potency and that both compounds will inhibit sialyl LewisX(sLex)-dependent cell adhesion. When fed to U937 cells, acetylated forms of Galβ1–4GlcNAc-NM and GlcNAcβ1–3Gal-NM primed oligosaccharides in a dose-dependent manner. Analysis of compounds assembled on Galβ1–4GlcNAc-NM showed only one product, namely Galβ1–4(Fucα1–3)GlcNAc-NM. In contrast, GlcNAcβ1–3Gal-NM generated Galβ1–4GlcNAcβ1–3Gal-NM, Galβ1–4(Fucα1–3)GlcNAcβ1–3Gal-NM, NeuAcα2–3Galβ1–4GlcNAcβ1–3Gal-NM, and NeuAcα2–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Gal-NM. Both compounds decreased the incorporation of [3H]fucose into cellular glycoconjugates, without affecting the incorporation of [3H]mannosamine, a precursor of sialic acid residues. Moreover, the overall extent of sialylation was not affected based on the reactivity of cells to fluorescein isothiocyanate-conjugatedMaackia amurensis lectin. Priming inhibited expression of sLex on cell surface glycoconjugates, which reduced E-selectin-dependent cell adhesion to tumor necrosis factor-α-activated human umbilical vein endothelial cells. GlcNAcβ1–3Gal-NM and Galβ1–4GlcNAc-NM represent starting points for making enzyme-specific, site-directed inhibitors of glycosyltransferases that could act in living cells.

Synthesis of p-nitrobenzyl and p-nitrophenyl 1-trioglycopyranosides☆☆☆

Reaction of 2,3,4-trio-O-acetyl-alpha-L-fucopyranosyl bromide (1) with thiourea (2), followed by reductive cleavage of the product, gave 2,3,4-tri-O-acetyl-1-thio-beta-L-fucopyranose (4). Reaction of 4 with p-nitrobenzyl bromide followed by O-deacylation yielded p-nitrobenzyl 1-thio-beta-L-fucopyranoside (6). Similar reaction conditions were used for the synthesis of p-nitrobenzyl 1-thio-beta-D-fucopyranoside (11) and 1-thio-alpha-D-mannopyranoside (16). A facile preparation of O-acylated p-nitrophenyl 1-thioglycopyranosides was achieved by condensing the appropriate glycosyl halide with sodium p-nitrobenzenethioxide in N,N-dimethylformamide.

The selective acetylation of primary alcohols in the presence of secondary alcohols in carbohydrates

Abstract Treatment of primary-secondary sugar diols with ethyl acetate in the presence of Woelm neutral alumina produced selectively the corresponding primary monoacetates in good yield. No di-acetate was formed in a detectable amount.

A Comparison of the Binding Specificities of Lectins from Ulex Europaeus and Lotus Tetragonolobus

The L-fucose-binding lectins present in the seeds of Ulex europaeus and Lotus tetragonolobus have been isolated by affinity chromatography. The binding specificities of the lectins were compared by hemagglutination inhibition analysis. The lectins behaved similarly with respect to a variety of synthetic saccharides. D-Fucose derivatives were inactive as inhibitors. In general, the alpha -L-fucopyranosyl derivatives were better inhibitors than the beta-L-fucopyranosyl derivatives. Aromatic aglycones increased the inhibitory activity of the saccharides with p-nitrophenyl-alpha-L-fucopyranoside being one of the best inhibitors. The major difference between the two lectins was the observation that the Ulex lectin required higher concentrations of saccharides to bring about complete inhibition of hemagglutination than did the Lotus lectin.

Cancer-associated elevation of α(1→3)-L-fucosyltransferase activity in human serum

GDP‐fucose:N‐acetylglucosaminide α(1 → 3)‐L‐fucosyltransferase activity was measured in sera of patients with various cancers using a synthetic substrate, N‐acetyl‐2′‐O‐methyllactosamine, as an acceptor. One hundred twenty‐four of the 169 patients showed significantly high levels of the enzyme activity when compared to healthy controls, irrespective of the location of their tumor. However, enzyme levels were in the normal range in patients with non‐neoplastic diseases, such as infectious disease, liver disease, and other inflammatory problems as well as in leukemic patients. The chromatofocusing profile of the enzyme using PBE‐94 gel over a pH grandient from pH 6.0 to 4.0 demonstrated that the level of the enzyme eluted at pH 5.4 was markedly elevated in the sera of stomach and ovarian cancer patients. A correlation was established between α(1 → 3)‐L‐fucosyltransferase activity and the presence of malignancy which may be used to evaluate the utility of the enzyme as a tumor marker.

Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation.

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

ProcessingO-glycan core 1, Galβ1-3GalNAcα-R. Specificities of core 2, UDP-GlcNAc: Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase and CMP-sialic acid:Galβ1-3GalNAc-R α3-sialyltransferase

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 β6-GlcNAc-T) and CMP-sialic acid: Galβ1-3GalNAc-R α3-sialyltransferase (EC 2.4.99.4; α3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 β6-GlcNAc-T activity; core 2 β6-GlcNAc-T from mucin secreting tissue (named core 2 β6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAcβ1-6(GlcNAcβ1-3)GalNAc-R] and blood group I [GlcNAcβ1-6(GlcNAcβ1-3)Galβ-R] branches; core 2 β6-GlcNAc-T in leukemic cells (named core 2 β-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 β6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Galβ1-3GalNAcα-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. α3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Galβ1-3GalNAcα-Bn. Galβ1-3(6-deoxy)GalNAcα-Bn and 3-deoxy-Galβ1-3GalNAcα-Bn competitively inhibited core 2 β6-GlcNAc-T and α3-sialyltransferase activities, respectively.

Synthesis of oligosaccharides containing the X-antigenic trisaccharide (α-l-Fucp-(1→3)-[β-d-Galp-(1→4)]-β-d-GlcpNAc) at their nonreducing ends

Abstract The “armed” methyl 2,3,4-tri- O -benzyl-1-thio-β- l -fucopyranoside was reacted with “disarmed” phenyl O -(tetra- O -acetyl-β- d -galactopyranosyl)-(1→4)-6- O -benzyl-2-deoxy-2-phthalimido-1-thio-β- d -glucopyranoside in the presence of CuBr 2 -Bu 4 NBr complex to give phenyl O -(2,3,4,6-tetra- O -acetyl-β- d -galactopyranosyl)-(1→4)- O -[(2,3,4-tri - O -benzyl-α- l -fucopyranosyl)-(1→3])-6- O -benzyl-2-deoxy-2-phthalimido-1-thio-β- d -glucopyranoside ( 6 ) as a novel glycosyl donor. The glycosylating capability of 6 was further examined using N -iodosuccinimide-triflic acid as a reagent. This led to the synthesis of a tetrasaccharide and a pentasaccharide incorporating the X-antigenic structure represented by 6 .

Inhibition of human HT-29 colon carcinoma cell adhesion by a 4-fluoro-glucosamine analogue

Cell surface glycoconjugates play an important role in cellular recognition and adhesion. Modification of these structures in tumour cells could affect tumour cell growth and behaviour, including metastasis. 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-α-D-glycopyranose (4-F-GlcNAc) was synthesized as a potential inhibitor and/or modifier of tumour cell glycoconjugates. The effect of this sugar analogue on the adhesive properties of human colon carcinoma HT-29 cells was evaluated. Treatment of HT-29 cells with 4-F-GlcNAc led to reduced cell surface expression of terminal lactosamine, sialyl-Lex and sialyl-Lea, as determined by Western blotting and flow cytometry. The aberrant expression of these oligosaccharide structures on the HT-29 cell surface resulted in: (1) decreased E-selectin mediated adhesion of human colon cells to human umbilical cord endothelial cells (HUVEC); (2) impaired adhesion of HT-29 cells to β-galactoside binding lectin, galectin-1; and (3) reduced ability to form homotypic aggregates. After exposure to 4-F-GlcNAc, lysosomal associated membrane proteins (lamp) 1 and 2, and carcinoembryonic antigen (CEA) detected in HT-29 cells were of lower molecular weight, probably due to impaired glycosylation. These results strongly suggest that modification of tumour cell surface molecules can alter tumour cell adhesion and that tumour cell surface oligosaccharides may be suitable targets for therapeutic exploitation.