Simple and convenient synthesis of a fluorinated GM4 analogue

Article abstract of DOI:10.1016/j.jfluchem.2007.02.013

A series of fluorinated galactosides, dodecyl 2-deoxy-2-fluoro-β-d-galactopyranoside (2F Gal), dodecyl 4-deoxy-4-fluoro-β-d-galactopyranoside (4F Gal) and dodecyl 6-deoxy-6-fluoro-β-d-galactopyranoside (6F Gal), was chemically synthesized and introduced to B16 cells to serve as scaffolds for cellular enzyme glycosylation. Results showed that the presence of fluorine exercised significant effects on cell viability. Among the fluorinated galactosides used, 2F Gal was glycosylated to afford a GM4 analogue.

Full text of DOI:10.1016/j.jfluchem.2007.02.013

Journal of Fluorine Chemistry 128 (2007) 562–565
www.elsevier.com/locate/fluor
Short communication
Simple and convenient synthesis of a fluorinated GM4 analogue
*
Maria Carmelita Kasuya, Ayaka Ito, Kenichi Hatanaka
Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
Received 30 January 2007; received in revised form 16 February 2007; accepted 16 February 2007
Available online 25 February 2007
Abstract
A series of fluorinated galactosides, dodecyl 2-deoxy-2-fluoro-b-D-galactopyranoside (2F Gal), dodecyl 4-deoxy-4-fluoro-b-D-galactopyrano-
side (4F Gal) and dodecyl 6-deoxy-6-fluoro-b-D-galactopyranoside (6F Gal), was chemically synthesized and introduced to B16 cells to serve as
scaffolds for cellular enzyme glycosylation. Results showed that the presence of fluorine exercised significant effects on cell viability. Among the
fluorinated galactosides used, 2F Gal was glycosylated to afford a GM4 analogue.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Sialylation; Galactoside primer; Fluorinated saccharide; GM4 analogue
1. Introduction
saccharide primers. In this research, the replacement of a
hydroxyl group by fluorine atom in the galactose residue was
pursued to establish the effect in cellular uptake and
glycosylation. Fluorinated galactosides with dodecyl aglycon
(2F Gal, 4F Gal and 6F Gal) were chemically synthesized and
applied to prime oligosaccharide synthesis via cellular
enzymatic glycosylation.
Structurally, GM4 (NeuAca2 ! 3Galb1 ! Cer) is the
simplest ganglioside. Kuhn and Weigandt first revealed GM4
as a minor ganglioside of human brain [1]. The occurrence of
this ganglioside has also been detected in chicken cerebellum,
mouse erythrocytes, rat kidney and frog liver. GM4 exhibits
interesting biological activities—marked immunosuppressive
activity in vitro and ability to prevent experimental encepha-
lomyelitis in guinea pigs [2], among others. The biological
significance but very low availability of GM4 from natural
sources propels the design and synthesis of GM4 and its
derivatives. One of the most challenging synthetic considera-
tions involves the regio- and stereoselective sialic acid
incorporation.
2. Results and discussion
The chemical synthesis of the fluorinated galactosides
required multi-step sequences. As shown in Scheme 1,
modification of the starting material (1) gave a protected
galactal [6–7] derivative (2) that was fluorinated and transformed
to a trichloroacetimidate derivative (3). Subsequent glycosyla-
tion using dodecyl alcohol with t-butyldimethylsilyl triflate in
dichloromethane as solvent, followed by deacylation afforded
dodecyl 2-deoxy-2-fluoro-b-D-galactopyranoside, 2F Gal (4).
On the other hand, a 4,6-benzylidene derivative [8–10] proved to
be a useful intermediate for the synthesis of 4F Gal and 6F Gal
primers. Glycosylation [5] of dodecyl alcohol with peracetyl
glucose (5) in the presence of a Lewis acid followed by
benzilidenationandbenzylationafforded(6) asshowninScheme
2. Reduction of the benzylidene group of (6) followed by
fluorination gave compound (7) which was debenzylated.
However, difficulty of purification prompted further acetylation
and finally, deprotected to afford dodecyl 4-deoxy-4-fluoro-b-D-
galactopyranoside, 4F Gal (8). Similarly, 6F Gal was prepared
Facile synthesis of ganglioside analogues could be achieved
by biocombinatorial method [3]. Amphiphilic saccharide
primers that resemble intermediates in the biosynthetic
pathway could be recognized by cellular enzymes and used
as substrates for glycosylation [4]. As we described recently,
incorporation of dodecyl b-D-galactoside primer to B16 cells
resulted to monosialylation of the galactoside primer [5]. This
finding initiated experiments to determine the effect of
substituting the hydroxyl group of glycoside units and to
establish the requirements for efficient cellular glycosylation of
* Corresponding author. Fax: +81 3 5452 6355.
E-mail address: hatanaka@iis.u-tokyo.ac.jp (K. Hatanaka).
0022-1139/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2007.02.013

M.C. Kasuya et al. / Journal of Fluorine Chemistry 128 (2007) 562–565
563
Scheme 1. Chemical synthesis of 2F Gal primer. (i) HBr–AcOH, CH2Cl2; (ii) CuSO4 Zn dust, AcOH (71%, 2 steps); (iii) SelectfluorTM, MeCN, H2O (32%); (iv)
CCl3CN, K2CO3, CH2Cl2; (v) C12H25OH, TBSOTf, CH2Cl2 (36%, 2 steps); (vi) MeONa, MeOH (89%).
Scheme 2. Chemical synthesis of 4F and 6F Gal primers. (a) C12H25OH, ClCH2CH2Cl, BF3ꢀEt2O (51%); (b) NaOMe, MeOH (100%); (c) PhCH(OMe)2, DMF, CSA;
(d) BnBr, NaH, DMF (76%, 2 steps); (e) TFA, Et3SiH, CH2Cl2 (87%); (f) Tf2O, CH2Cl2, pyridine; (g) TBAF, THF (35% 2 steps); (h) Pd/C, H2 then Ac2O, pyridine
(47%, 2 steps); (i) NaOMe, MeOH (93%); (j) 80% AcOH (aq) (87%); (k) DAST, CH2Cl2 (30%); (l) Pd/C, H2 (16%).
from peracetyl galactose (9) as shown in Scheme 2. Treatment of
the benzylidene derivative (10) with acetic acid followed by
fluorination gave compound 11 which was debenzylated to
afford dodecyl 6-deoxy-6-fluoro-b-D-galactopyranoside, 6F Gal
(12). Fluorine was introduced at the 2, 4 and 6 positions of the
galactose unit by treatment of corresponding galactosyl
derivative with SelectfluorTM, tetrabutylammonium fluoride
(TBAF) and N,N-diethylamino-sulfur trifluoride (DAST),
respectively [11–15]. The chemical synthetic methods for the
synthesis of all the primers were carried out according to the
references cited therein. The structures of the synthesized
compounds were confirmed from the 1H and 13C NMR results.
The 13C NMR spectra of the fluorinated galactoside primers in
comparisonwiththedodecylb-D-galactosideare showninFig. 1.
The primers were administered to mouse melanoma B16
cells to verify their potential as scaffolds for oligosaccharide
synthesis using cells. Cell culture, incubation of cells with
primer and lipid extraction from cell and medium fractions
were carried out as described previously [3,5]. After the
incubation of B16 cells with 50 mM of the primers for 48 h,
lipids from the culture media and from the cell homogenate
were collected and were extracted. Among the three kinds of
primers, 2F Gal did not affect cell morphology and viability
(Fig. 2). In contrast, 4F Gal primer exhibited slight toxicity to
cells and 6F Gal primer was cytotoxic. The HPTLC results of
the lipids confirmed that the primers were taken-up by the cells
as shown in Fig. 3. However, only the 2F Gal was glycosylated
and the elongated product released to the culture medium. The
structure of the glycosylated product was determined. The
MALDI TOF mass spectral results obtained suggested a
sialylated galactoside primer. Treatment of the elongated 2F
Gal with 2,3-sialidase (cloned from S. typhimurium LT2 and
expressed in E. coli) confirmed the structure of the glycosyla-
tion product, a GM4 analogue. From the results, we could infer
that 2F Gal diffused through the cell membrane to the Golgi
where the primer functioned as acceptor substrate for the sialyl
transferase.
In a related work, we reported that a fluorous-tagged
lactoside primer (6-(perfluorohexyl)hexyl 4-O-(b-D-galacto-
pyranosyl)-b-D-glucopyranoside) could be taken in by B16
melanoma cells, the saccharide chain elongated by cellular
enzymes to afford a GM3-type oligosaccharide, and the
Fig. 1. 13C NMR spectra of dodecyl b-D-galactopyranoside and fluorinated galactoside primers in MeOH-d4.

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M.C. Kasuya et al. / Journal of Fluorine Chemistry 128 (2007) 562–565
Fig. 2. Effect on B16 cells after 48-h incubation in the (A) absence, or in the presence of primer (B) 2F Gal; (C) 4F Gal; (D) 6F Gal.
elongated product released by the cells to the culture medium
[16]. The fluorous tag did not exert a steric influence to cellular
uptake neither did the numerous fluorine at the aglycon exhibit
cytotoxicity. To take advantage of the positive effect of the
fluorine substituent to the cellular enzyme glycosylation of
primers, the hydroxyl group of the galactose residue was
replaced by fluorine. Relative to the hydroxyl group, the size of
the fluorine substituent does not dramatically influence to
create a steric hindrance to sialylation by the enzyme. Hence,
replacement of one hydroxyl unit by a fluorine atom is expected
to have only the hydrophobic effect on the conformation of the
galactose residue. Moreover, it was assumed that the presence
of only one fluorine atom in the saccharide unit would have no
adverse effects on the cells. However, results showed otherwise.
Interestingly, replacement of one hydroxyl unit by a fluorine
atom in different positions of the galactose residue elicited
different cellular responses. Modification at 2 position of the
galactose residue did not have adverse effects to the cell and 2F
Gal was glycosylated. Surprisingly, modification of 4 and 6
positions slightly, or greatly, affected cell viability. Conse-
quently, saccharide elongation of 4F and 6F Gal primers could
not possibly take place.
glycosylation, a terminal galactose residue [5]. However, the
type and position of substituent at the vicinity of the
glycosylation site (C3 position) and their positive effect on
cell viability are prerequisites for cellular enzyme glycosyla-
tion and substrate recognition by 2,3-sialyl transferase.
Although it may be too hasty to draw a conclusion and explain
the phenomenon based on present results, the position of the
substituent should also be a significant consideration for
oligosaccharide synthesis using primers and cells. Whether or
not other types of cells or substituents will give the same results
remain to be seen.
3. Concluding remarks
Fluoro-glycosyl compounds are of increasing importance in
biochemical studies [14]. For example, substitution of 2-OH by
fluorine in glycosides has been reported to alter biological
activities and improve antifungal, antiviral and anticancer
properties relative to parent compounds. In the light of
increased demand for fluorinated saccharides and gangliosides,
the simple and convenient method of the regio- and
stereoselective synthesis of the fluorine-containing GM4
analogue described in this study is a viable alternative to the
conventional yet tedious chemical synthetic method.
The differences in cellular response could be addressed
considering not only the conformation of primers but also
cellular enzyme specificity [13]. Even if it has been reported
that sialyl transferases are amenable to substrate modifications,
primers should be able to cross the plasma membrane, reach the
Golgi where the enzymes reside that is the site of glycosylation.
All three primers satisfy the requirement for cellular enzyme
4. Experimental
4.1. Chemical synthesis of primers
4.1.1. Chemical synthesis of 2F Gal primer
4.1.1.1. Preparation of 2,3,4,6-tetra-O-acetyl-a-bromo-D-ga-
lactopyranoside. 1,2,3,4,6-Penta-O-acetyl-D-galactopyrano-
side (1) (15.6 g, 40.0 mmol) was dissolved in dichloromethane
(280 ml) and to the solution was added 30% HBr in acetic acid
(26.0 ml). The solution was stirred for 1 h at 0 8C and for
another 6 h at room temperature. The solution was quenched
with ice water (600 ml), and the organic layer was extracted
with chloroform, neutralized with NaHCO3, washed with water
and with brine. The organic layer was dried over anhydrous
sodium sulfate, filtered and evaporated to dryness to afford the
desired product, 2,3,4,6-tetra-O-acetyl-a-bromo-D-galactopyr-
anoside, that was used for the next reaction without further
purification.
Fig. 3. B16 melanoma cells (1 ꢁ 106, 100 mm dish) were incubated for 48 h in
serum free 1:1 DMEM-F12 supplemented with transferrin and insulin (TI/DF)
in the absence or presence of 50 mM galactoside primer. Lipids were extracted
from the cell and culture medium fractions and separated by HPTLC. Lanes 1
and 6: Control (Me2SO only in TI-DF) for the cell and culture medium
fractions, respectively; lanes 2–5 and 7–10: Treated (50 mM galactoside primer)
for the cell and culture medium fractions, respectively; lanes 2 and 7, dodecyl
galactoside primer; lanes 3 and 8: 2F Gal primer; lanes 4 and 9: 4F Gal primer;
lanes 5 and 10: 6F Gal primer; lane 11: GM3 standard.
4.1.1.2. Preparation of 3,4,6-tri-O-acetyl-D-galactal
(2). 2,3,4,6-Tetra-O-acetyl-a-bromo-D-galactopyranoside
was dissolved in acetic acid (200 ml) and to the cooled solution
was slowly added zinc dust (30.0 g, 459 mmol) and CuSO4

M.C. Kasuya et al. / Journal of Fluorine Chemistry 128 (2007) 562–565
565
(3.00 g, 18.8 mmol) dissolved in water. The solution was stirred
for 30 min at 0 8C and for another 18 h at room temperature.
The solution was filtered through a bed of Celite. Chloroform
was added and the organic layer was neutralized with NaHCO3
and washed successively with water and with saturated sodium
chloride. The organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated in vacuo. Separation by silica
gel column chromatography (ethyl acetate:hexane, 1:2)
afforded the desired product (2) (2 steps: 7.76 g, 71%).
(268 mg, 0.56 mmol) was carried out by dissolving in 3 ml
methanol and to the solution was added sodium methoxide
(18 mg, 0.33 mmol). The mixture was stirred for 30 min at
room temperature and then neutralized with cation-exchange
resin (DIAION SK1B (H+ form), filtered and evaporated to
afford n-dodecyl 2-deoxy-2-fluoro-b-D-galactopyranoside
(174 mg, 89%).
Chemical synthesis of 4F and 6F Gal primers were carried
out according to literature cited
4.1.1.3. Preparation of 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D-
galactopyranoside. To a mixture of 3,4,6-tri-O-acetyl-D-
galactal (2) (949 mg, 3.49 mmol), Selectfluor (1.48 g,
4.19 mmol) was added acetonitrile (15 ml) and water (3 ml)
and the resulting solution was stirred for 16 h at room
temperature and refluxed for an additional 30 min. The solution
was evaporated to dryness and subsequent separation by silica
gel column chromatography (ethyl acetate:hexane, 1:2)
afforded the desired product (344 mg, 32%).
4.2. Cellular uptake of glycoside primers
Cell culture, incubation of cells with primer and identifica-
tion of glycosylated product were carried out according to the
literature cited in the text.
Acknowledgements
This work was supported partially by a grant for
‘‘Development of Novel Diagnostic and Medical Applications
through Elucidation of Sugar Chain Functions’’ from the New
Energy and Industrial Technology Development Organization
(NEDO).
4.1.1.4. Preparation of O-(3,4,6-tri-O-acetyl-2-deoxy-2-
(3). 3,4,6-
fluoro-D-galactopyranosyl)trichloroacetimidate
Tri-O-acetyl-2-deoxy-2-fluoro-D-galactopyranoside (1.09 g,
3.55 mmol) and K2CO3 (2.45 g, 17.8 mmol) were dissolved
in dichloromethane (50 ml) and the solution stirred for 1 h. To
the solution was added acetonitrile (1.08 ml, 10.7 mmol). The
solution was stirred for 16 h, then filtered through a bed of
Celite and evaporated in vacuo to afford the desired product (3)
that was used in the next reaction without further purification.
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