Easy production of a glycolipid analogue using animal cells in culture

Article abstract of DOI:10.1002/cbdv.200900176

A glycolipid analogue, GM4-type ganglioside, was obtained by a combination of chemical synthesis and biosynthetic processes in animal cells with dodecyl β-d-galactoside (Gal C12) as primer. The primer was conveniently prepared in two steps: glycosylation, followed by deacetylation. The primer was introduced to mouse melanoma B16 cells to serve as substrate for cellular, enzyme-catalyzed glycosylation. Incubation of the cells in the presence of the primer resulted in sialylation of the galactose residue to afford a GM4 analogue that was released from the cells to the culture medium. The strategy of preparation of the GM4 analogue described in this study is a viable alternative to the existing methods. The saccharide-primer method is fast, convenient, not requiring expensive enzymes and glycosyl donors, and highly stereoselective.

Full text of DOI:10.1002/cbdv.200900176

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Easy Production of a Glycolipid Analogue Using Animal Cells in Culture
by Maria Carmelita Z. Kasuya and Kenichi Hatanaka*
Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
(phone: þ81-3-5452-6355; fax: þ81-3-5452-6356; e-mail: hatanaka@iis.u-tokyo.ac.jp)
A glycolipid analogue, GM4-type ganglioside, was obtained by a combination of chemical synthesis
and biosynthetic processes in animal cells with dodecyl b-d-galactoside (Gal C12) as primer. The primer
was conveniently prepared in two steps: glycosylation, followed by deacetylation. The primer was
introduced to mouse melanoma B16 cells to serve as substrate for cellular, enzyme-catalyzed
glycosylation. Incubation of the cells in the presence of the primer resulted in sialylation of the
galactose residue to afford a GM4 analogue that was released from the cells to the culture medium. The
strategy of preparation of the GM4 analogue described in this study is a viable alternative to the existing
methods. The saccharide-primer method is fast, convenient, not requiring expensive enzymes and
glycosyl donors, and highly stereoselective.
Introduction. – Gangliosides are cell-surface glycolipids with at least one sialic acid
residue [1]. Structurally, the simplest compound among the gangliosides is sialosylga-
lactosyl ceramide (aNeu5Ac2!3GalCer, GM4). GM4 is a minor ganglioside in
human brain, but found to be the major ganglioside in the liver of several species of
shark [2], in chicken embryonic liver [3], and egg yolk. Ganglioside GM4 has also been
detected in human myelin [4], human oligodendrocytes [5], mouse erythrocytes, rat
kidney, frog liver, and chicken cerebellum [6]. Except for its marked immunosuppres-
sive activity, the biological significance of ganglioside GM4 is not well-understood.
Because GM4 is scarce in natural sources, chemical [7–9] and enzymatic synthetic
methods remain to be the conventional strategy for GM4 production in quantitative
amounts. However, careful consideration of the synthetic design of GM4 is necessary to
overcome the challenges in the regio- and stereoselective incorporation of sialic acid.
Recently, we reported the preparation of gangliosides by the saccharide-primer
method, a novel strategy, where saccharide primers are used as scaffolds for glycan
synthesis in living animal cells [10][11]. The glycosylation of primers by cellular
glycosyltransferases is parallel with the glycosylation of natural precursors and
generates products with sequences depending on the biosynthetic pathway of the cell
used. Previously, we reported that mouse melanoma B16 cells take up and elongate the
dodecyl b-d-galactoside (Gal C12) primer [12]. We now report the convenient mass
production of a GM4 analogue in mouse melanoma B16 cells via the saccharide-primer
strategy as an alternative to the existing synthetic methods (Scheme 1).
Results and Discussion. – The saccharide primer Gal C12 was prepared in two steps
(Scheme 2). Glycosylation of dodecanol by tetraacetyl galactose in the presence of a
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Scheme 1. Synthesis of a GM4 Analogue in B16 Cells with Gal C12 as Primer
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Scheme 2. Chemical Synthesis of the Gal C12 Primer
1. Glycosylation: pentaacetylgalactose (10.0 g, 25.6 mmol), Me(CH2)11OH (10 g, 53.8 mmol, 2 equiv.),
BF3 ·OEt2 (4 equiv.), ClCH2CH2Cl, 4 h at 508. 2. Deacetylation: MeONa, MeOH, r.t., overnight.
Lewis acid, followed by deacylation with MeONa in MeOH afforded the Gal C12
primer.
Mouse melanoma B16 cells (5ꢀ106) were seeded into 15-cm dishes with DMEM/F-
12 medium and incubated at 378 for 48 or 72 h in a 5% CO2 atmosphere. Initially, cells
were treated with 50, 100, or 150 mm of Gal C12, to test the effect of the primer
concentration on cell growth and saccharide production. As evidenced by HPLC,
higher amounts of GM4 could be obtained with higher concentrations of primer.
However, 100 and 150 mm of Gal C12 exhibited slight to moderate cytotoxicity,
respectively (Fig. 1). Hence, 50 mm of primer was administered to the culture medium
to ensure continuous culture of the cells for several days. Repeated administration of
primer and harvest of culture medium was carried out for 12 d. As shown in Fig. 2, Gal
C12 did not exhibit adverse effects on the cell morphology and viability, although
formation of colonies became evident during the second week of incubation. Even if
the cells continued to glycosylate the primer, the amount of GM4 analogue produced
decreased with repeated primer administration and harvest for several days (Fig. 3).
After the cellular uptake of the primer, followed by glycosylation, the products were
released from the cells to the culture medium. The culture media from all the harvests
were pooled and the glycolipids were adsorbed on HP-20 resin and eluted with a
MeOH gradient (Fig. 4). The products were charged to SepPak columns for further
purification. Finally, purification by centrifugal partition chromatography (CPC) [13]
gave the GM4 analogue with high purity (Fig. 5). Analysis of the product was carried
out by ESI-MS. As shown in Fig. 6, the mass spectrum of the GM4 analogue showed a
peak at m/z 638.3, corresponding to sialylated dodecyl galactoside. Fragmentation
showed peaks at m/z 291.1 and 347.2, corresponding to sialic acid and dodecyl
galactoside, respectively. Treatment of the GM4 analogue with a-2,3 sialidase
confirmed the glycoside linkage. With this strategy, 4 mg of GM4 analogue was
isolated from 4.2 l of culture medium.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Fig. 1. Effect of the Gal C12 primer concentration on a) the cytotoxicity of B16 cells and b) the production
of the GM4 analogue after 48 h incubation with primer at different concentrations
Fig. 2. Effect of repeated Gal C12 primer administration and harvest on B16 cells

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Fig. 3. Profile of lipids obtained after repeated Gal C12 primer administration and harvest
Fig. 4. HPTLC of the lipid fractions obtained after separation from the culture media on HP-20 resin
eluted with a MeOH gradient