Stereocontrolled and Convergent Entry to <i>CF</i><i>₂</i>-Sialosides: Synthesis of <i>CF</i><i>₂</i>-Linked Ganglioside GM4

Sialidase-resistant ganglioside analogues having biological activities similar to those of natural gangliosides are expected to be important probes for clarifying the biological functions of gangliosides. Focusing on difluoromethylene-linked (CF2-linked) α(2,3)sialylgalactose as a core structure of sialidase-resistant ganglioside mimics, we have developed novel, stereocontrolled, and efficient methodologies to synthesize CF2-sialosides based on Ireland−Claisen rearrangement. CF2-linked α(2,3)sialylgalactose and CF2-linked GM4 were synthesized

Stereocontrolled and Convergent Entry to <i>CF</i><i>₂</i>-Sialosides: Synthesis of <i>CF</i><i>₂</i>-Linked Ganglioside GM4

Sialidase-resistant ganglioside analogues having biological activities similar to those of natural gangliosides are expected to be important probes for clarifying the biological functions of gangliosides. Focusing on difluoromethylene-linked (CF2-linked) α(2,3)sialylgalactose as a core structure of sialidase-resistant ganglioside mimics, we have developed novel, stereocontrolled, and efficient methodologies to synthesize CF2-sialosides based on Ireland−Claisen rearrangement. CF2-linked α(2,3)sialylgalactose and CF2-linked GM4 were synthesized

Synthesis of <i>CH₂</i>-Linked α(2,3)Sialylgalactose Analogue: On the Stereoselectivity of the Key Ireland-Claisen Rearrangement

A CH2-linked α(2,3)sialylgalactose analogue was efficiently synthesized using an Ireland-Claisen rearrangement, which was developed recently by our group for constructing a CF2-sialoside. The reaction conditions of the rearrangement were optimized for α-stereoselective formation of the CH2-sialoside. On the basis of the observed temperature effects, the origin of the stereoselectivity of the Ireland-Claisen rearrangement is discussed. Moreover, reconstruction of the 2α-hydroxyl group on the galactose unit of the rearrangement product was achieved by means of stereoselective dihydroxylation and deoxygenation

Synthesis of All Possible Isomers Corresponding to the Proposed Structure of Montanacin E, and Their Antitumor Activity

Total synthesis of 4 and its three diastereomers is described. The key steps involve stereoselective formation of the tetrahydrofuran ring by a cascade cyclization of hydroxy tosylate 7 and an intermolecular cross metathesis between a tetrahydrofuran 5 and a γ-lactone 6. Spectroscopic data of 4 and biosynthetic hypothesis strongly suggest it to be montanacin E. Inhibitory activities of 4 and its isomers against six human solid tumor cell lines were also evaluated

Synthesis of <i>CH₂</i>-Linked α(2,3)Sialylgalactose Analogue: On the Stereoselectivity of the Key Ireland-Claisen Rearrangement

A CH2-linked α(2,3)sialylgalactose analogue was efficiently synthesized using an Ireland-Claisen rearrangement, which was developed recently by our group for constructing a CF2-sialoside. The reaction conditions of the rearrangement were optimized for α-stereoselective formation of the CH2-sialoside. On the basis of the observed temperature effects, the origin of the stereoselectivity of the Ireland-Claisen rearrangement is discussed. Moreover, reconstruction of the 2α-hydroxyl group on the galactose unit of the rearrangement product was achieved by means of stereoselective dihydroxylation and deoxygenation

Synthesis of <i>CH₂</i>-Linked α(2,3)Sialylgalactose Analogue: On the Stereoselectivity of the Key Ireland-Claisen Rearrangement

A CH2-linked α(2,3)sialylgalactose analogue was efficiently synthesized using an Ireland-Claisen rearrangement, which was developed recently by our group for constructing a CF2-sialoside. The reaction conditions of the rearrangement were optimized for α-stereoselective formation of the CH2-sialoside. On the basis of the observed temperature effects, the origin of the stereoselectivity of the Ireland-Claisen rearrangement is discussed. Moreover, reconstruction of the 2α-hydroxyl group on the galactose unit of the rearrangement product was achieved by means of stereoselective dihydroxylation and deoxygenation

Requirement of NEU3 sialidase activity for enhanced phosphorylation of EGFR and Src.

<p>(A) The sialidase activities of NEU3 wild type and the mutant (Y370 or N88D) in the EGFR-overexpressing clones were assayed with ganglioside as a substrate in independent experiments performed in triplicate (mean ±SD). (B) The phosphorylation levels of EGFR, Src and ERK1/2 in the NEU3 wild and mutant clones in EGFR-cells were measured by western blotting using the respective antibodies. Each value shown under the blot represents as a value relative to that in the vector controls.</p

NEU3-mediated potentiation of cell growth assessed by MTT assays and colony formation assays.

<p>(A) The cell growth curves of NEU3-transfected cells were compared with those of vector controls in the absence and presence of murine EGF (20 ng/ml). Three independent experiments were performed (mean ±SD). (B) The cell growth curves of EGFR- and EGFR/NEU3-transfected cells are shown with or without EGF in independent experiments performed in triplicate (mean ±SD). (C) Colony formation assays in the transfectants. The cells were plated at 1000 cells/well in six-well dishes with or without EGF, and the colonies were quantified after 7–14 days of culture. Representative images are shown. (D) Values represent means with standard deviations obtained from three independent experiments. In the right graph, the number of colonies over 3.0 mm in size was counted in independent experiments performed in triplicate (mean ±SD).</p

NEU3-mediated potentiation of anchorage- independent growth and in <i>vivo</i> growth in nude mice.

<p>(A) Anchorage-independent growth in soft agar. The cells (1 x 10⁵) were plated on soft agar with or without EGF, and two to three weeks later, the colony number and size were measured. Representative images are shown. (B) In the graph, the colony number on soft agar was calculated. The values are means with standard deviations obtained from three independent experiments. The graph in the right indicates the number of colonies over 200 μm in size. (C) <i>In vivo</i> growth in nude mice. Respective cells were subcutaneously transplanted into nude mice with injection of murine EGF (5 μg/ml) every two days, and the measured tumor weights are indicated. The results of experiments with the vector controls and NEU3-transfected cells (left graph) and those with EGFR- and EGFR/NEU3-transfected cells (right graph) are shown in the four groups (n = 3 or 4). (D) Tumor incidence in the mice transplanted with the cells. To confirm the results, other six groups (n = 4) of these cells were further examined with or without injection of EGF, as indicated in the lower table.</p

Alteration of glycolipids as a result of NEU3 catalytic reaction.

<p>Glycolipid changes due to NEU3 transfection were analyzed by thin- layer chromatography (A) in acidic (left panel) and neutral (right panel) fractions, and the relative changes in putative GM3 and Lac-cer were quantified in independent experiments performed in triplicate, as shown in the graphs below. (B, C) The effects of exogenously added Lac-cer (B) and GM3 (C) on EGFR phosphorylation in EGFR- and EGFR/NEU3-NIH-3T3 cells. Results are representative of two independent experiments. The values for phosphor-EGFR/EGFR relative to those for the vector controls with EGF are shown in the lower graphs.</p