Effect of fluorous tags on glycosylation of saccharide primers in animal cells

Article abstract of DOI:10.1246/bcsj.82.1409

A series of fluorous-tagged saccharide primers with different contents of fluorous atoms was synthesized and introduced into mouse melanoma B16 cells. The primers did not affect cell morphology and viability at a concentration of 50 μM. The numerous fluorine atoms did not pose a steric barrier to primer assimilation into cells and did not affect cellular-enzyme-catalyzed glycosylation. The lactoside primers were sialylated to afford GM3-type oligosaccharide. On the other hand, the GlcNAc primers were galactosylated to afford a lactosamine derivative that was further sialylated by cellular enzymes to afford a sialylated lactosamine.

Full text of DOI:10.1246/bcsj.82.1409

©
2009 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 82, No. 11, 1409–1415 (2009) 1409
Effect of Fluorous Tags on Glycosylation of
Saccharide Primers in Animal Cells
1
2
1
Maria Carmelita Z. Kasuya, Mami Tojino, Shinya Nakano,
2
1
Mamoru Mizuno, and Kenichi Hatanaka*
1Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505
2Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0004
Received May 28, 2009; E-mail: hatanaka@iis.u-tokyo.ac.jp
A series of fluorous-tagged saccharide primers with different contents of fluorous atoms was synthesized and
introduced into mouse melanoma B16 cells. The primers did not affect cell morphology and viability at a concentration of
50 ¯M. The numerous fluorine atoms did not pose a steric barrier to primer assimilation into cells and did not affect
cellular-enzyme-catalyzed glycosylation. The lactoside primers were sialylated to afford GM3-type oligosaccharide. On
the other hand, the GlcNAc primers were galactosylated to afford a lactosamine derivative that was further sialylated by
cellular enzymes to afford a sialylated lactosamine.
Glycolipids are ubiquitous in membranes of biological
tain a fluorine atom at different positions of the galactose
residue was chemically synthesized and introduced to B16
cells. Although the primers were taken up by cells, only 2F Gal
primer was sialylated by B16 cells to give a ganglioside GM4
analog. Saccharide primers with numerous fluorine atoms in the
aglycon unit were likewise used. Cellular uptake of a fluorous-
tagged lactoside primer, 6-(perfluorohexyl)hexyl 4-O-(¢-D-
systems and play essential roles in physiological processes.
However, their ready availability is hampered by synthetic
means that remain to be daunting. Recently, the saccharide
1
primer method has been reported as a promising approach
for the production of glycolipids. The strategy is easy and
convenient. Significantly, the use of saccharide primers and
cells simplifies the rather tedious conventional method that
requires several chemical synthetic steps. The cells used
already provide the glycosyl donor and enzyme required in
this strategy. Moreover, glycosylation is regio- and stereo-
specific. Among the requirements of the saccharide primer
method include (a) amphiphilic saccharide primer that is
synthetically accessible in only a few steps; (b) assimilation
6
galactopyranosyl)-¢-D-glucopyranoside resulted to sialylation
of the terminal galactose residue to afford a GM3-type
oligosaccharide. Significantly, the presence of many fluorine
atoms in the aglycon unit did not have any adverse effect to the
cells nor inhibit cellular enzyme-catalyzed glycosylation. The
fluorous tag was employed to facilitate the separation of the
products from the culture medium by simple extraction with a
2
7
of the primer without damage to cells; and (c) recognition
fluorous solvent.
3
of saccharides by cellular enzymes in the Golgi, the site of
In this research, several saccharide primers (lactose and
GlcNAc derivatives) with different lengths of fluorous tags
were chemically synthesized and introduced to mouse mela-
noma B16 cells to establish the effect of numerous fluorine
atoms in the aglycon unit on the production of glycolipids via
saccharide primer strategy. The lactoside primers and GlcNAc
primers were prepared by conventional chemical synthetic
methods and introduced to mouse melanoma B16 cells.
glycosylation. Since glycosylation of the primer is parallel
to glycosylation of natural precursors in the biosynthetic
pathway, the kind of cell used determines the products. The
4
length of the aglycon unit is crucial to ensure that the primers
interact with the lipid bilayer and reach the Golgi where
elongation takes place. The hydrophobic (aglycon moiety) and
hydrophilic (saccharide moiety) balance also plays an impor-
tant role not only in cellular uptake but more importantly, in the
subsequent release of the glycosylation products to the culture
medium.
Results and Discussion
The lactoside primers [Lac H8F4 (each number indicates the
number of methylene and difluoromethylene, respectively), Lac
H6F6, Lac H4F8, Lac H3F8, Lac H2F8, and Lac H2F10] and
GlcNAc primers [GN H8F4, GN H6F6, GN H4F8, GN H3F8,
and GN H2F8] (Chart 1) were synthetically accessible in a
few steps as shown in Scheme 1. The saccharide primers were
prepared with conventional methods of glycosylation of the
partially fluorinated alcohol with either the lactose or GlcNAc
Saccharide primers with fluorine atoms in the saccharide or
aglycon moieties have been reported as building blocks for
the synthesis of gangliosides. When administered to mouse
melanoma B16 cells, the primers were sialylated by cellular
enzymes and the elongated products were released to the
5
culture medium. A series of fluorinated galactosides, dodecyl
2
-deoxy-2-fluoro-¢-D-galactopyranoside (2F Gal), dodecyl 4-
deoxy-4-fluoro-¢-D-galactopyranoside (4F Gal), and dodecyl
-deoxy-6-fluoro-¢-D-galactopyranoside (6F Gal), which con-
8
derivative. After the glycosylation, deacylation was accom-
6
plished under Zemplen conditions.
Published on the web November 10, 2009; doi:10.1246/bcsj.82.1409

1
410 Bull. Chem. Soc. Jpn. Vol. 82, No. 11 (2009)
Fluorous-Tagged Saccharide Primers
The primers were administered to the culture medium of
following pattern of results for both fluorous-tagged primers
with Lac (Figures 2A and 2B) and GlcNAc (Figures 2C and
2D): (1) HPTLC of the lipids from the cell fraction showed that
the primers were taken in by the cells, (2) the bands on the cell
fraction corresponding to primers with more fluorine atoms are
more intense, (3) except for Lac H4F8 and GN H4F8 having
the same kind of fluorous tag, HPTLC of the culture medium
fraction showed a new band corresponding to a glycosylated
primer, and (4) most of the products of glycosylation are found
in the culture medium. The products of glycosylation were
separated using fluorous solid phase extraction cartridges and
the mass spectra of the products of glycosylation were taken.
Results suggested sialylation of the fluorous-tagged lactoside
primers to afford the following GM3-type oligosaccharides and
the respective yield: sialylated (SA) Lac H8F4 (17.8%), SALac
H6F6 (14.1%), SALac H3F8 (1.8%), SALac H2F8 (3.7%),
and SALac H2F10 (1.2%). On the other hand, the fluorous-
tagged GlcNAc primers were initially galactosylated to give a
lactosamine derivative that was further sialylated. From the
HPTLC results of the culture medium fraction, the absence
of a band corresponding to the lactosamine product suggests
that after galactosylation of the GlcNAc primers, sialylation
follows almost instantaneously and the product subsequently
released to the culture medium. Incorporation of GlcNAc
primers to B16 cells afforded the sialylated lactosamines
and the respective yield: SAGalGN H8F4 (2.2%), SAGalGN
H6F6 (1.7%), SAGalGN H3F8 (0.8%), and SAGalGN H2F8
mouse melanoma B16 cells and the effect on cellular enzyme-
mediated glycosylation by the number of fluorine atoms in the
aglycon unit was determined. All the primers did not exhibit
any adverse effects on B16 cell morphology and viability as
shown in Figure 1. After 48-h incubation of cells (seeded cell
6
number: 2 © 10 cells per 10 cm dish; 7 mL DMEM-F12) in the
presence of the primers (50 ¯M), the cell and culture medium
fractions were collected and the lipids were extracted.
Separation of the lipids and analysis by HPTLC gave the
OH
OH
OH
OH
O
O
O
O
OR
HO
HO
OR
HO
HO
OH
OH
NHAc
Lac (Lactoside) primer
GN (GlcNAc) primer
F
F
F
F
F
F
R:
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F F F
H8F4
H2F8
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
H6F6
H3F8
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F F F
F
F
F
F F F
F F
F
F
F
(
12.8%).
H2F10
H8F4 = 9,9,10,10,11,11,12,12,12-nonafluorododecyl
H4F8
Sialylation of the primers was anticipated since B16 cells
produce GM3 as a major ganglioside. The sialyl transferases
of B16 cells recognized and elongated the primers to give a
sialylated dodecyl lactoside derivative (GM3-type oligosac-
charide) and a sialylated dodecyl lactosamine derivative. The
H6F6 = 7,7,8,8,9,9,10,10,11,11,12,12,12-tridecafluorododecyl
H4F8 = 5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,12- heptadecafluorododecyl
H2F10 = 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,12-henicosafluorododecyl
H2F8 = 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl
H3F8 = 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11- heptadecafluoroundecyl
6
monosialylated primers were confirmed to be ¡-(2¼3)-linked
Chart 1.
except for monosialylated GN H2F8 which may possibly be a
A
B
OAc
OAc
O
OAc
OAc
OAc
O
O
OAc
O
i
AcO
O
O
AcO
OAc AcO
OAc
ii
OAc AcO
OR
OAc
OAc
OH
HO
OH
O
OH
O
O
OH
HO
OR
OH
OAc
OAc
O
AcO
AcO
O
iii
iv
AcO
AcO
C H C4F
9
OAc
8
16
N
NHAc
O
C H12C6F13
6
C H C F
R:
4
8
8
17
17
17
C H C F
3
6 8
OAc
OH
C H C F
2 4 8
O
AcO
AcO
v
O
HO
HO
C2H4C10F21
OR
OR
NHAc
NHAc
Scheme 1. Synthesis of fluorous-tagged (A) lactoside primers: (i) ROH, BF3¢OEt2, CH2Cl2, rt; or HBr­AcOH, CH2Cl2, rt then ROH,
Ag2CO3, AgClO4, CH2Cl2, MS4A, rt; (ii) NaOMe, MeOH and (B) GlcNAc primers: (iii) TMSOTf, 1,2-dichloroethane; (iv) ROH,
CSA, 1,2-dichloroethane, 90 °C, 2 h; (v) NaOMe, MeOH.

M. C. Z. Kasuya et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 11 (2009) 1411
Control
Lac H8F4
Lac H6F6
Lac H2F8
LacH3F8
Lac H4F8
Lac H2F10
Control
GN H2F8
GN H8F4
GN H6F6
GN H3F8
GNH4F8
6
Figure 1. Effect of fluorous-tagged primers on B16 cells after incubation for 48 h. B16 melanoma cells (2 © 10 , 100 mm dish) were
incubated for 48 h in serum free 1:1 DMEM­F12 (7 mL) supplemented with transferrin and insulin (TI/DF) in the absence or
presence of 50 ¯M primer.
mixture of ¡-(2¼3) and ¡-(2¼6)-linked as evidenced by the
presence of two bands as shown in the HPTLC results in
Figure 2D.
primer is either in trace amounts only or is totally absent from
the HPTLC of the culture medium fraction. One of the
requirements to be satisfied for cellular glycosylation is that
primers should reach the Golgi, the site of glycosylation. The
presence of these primers in the cell fraction based on HPTLC
results could be inferred by the following possibilities: (1) both
Lac H4F8 and GN H4F8 primers did not reach the Golgi
because the cells preferentially remain at the membrane due to
strong interaction between the fluorous tag and the membrane
components and (2) the Lac H4F8 and GN H4F8 primers
reached the glycosylation site but were not recognized by the
glycosyl transferases. The presence of a larger number of
fluorine atoms could not readily account for the absence of
products because Lac H2F10 that has more fluorine atoms than
Lac H4F8 was sialylated. Whether this phenomenon is specific
only for B16 cells or not, Lac H4F8 primer was also introduced
to African green monkey Vero cells. As shown in Figure 4, Lac
H4F8 primer likewise did not give any glycosylation product.
Interestingly, the GlcNAc primers were galactosylated
initially to afford a lactosamine that was further elongated by
sialyl transferases. It has been reported that a terminal ¢-
9
galactoside is a prerequisite for cellular mediated sialylation.
Hence, galactosylation of the GlcNAc primers to generate a
lactosamine prior to sialylation must have taken place. This is
supported by the electrospray ionization mass spectral results
(negative ion mode). For example, fragmentation of sialylated
GalGN H6F6 (m/z 1075.2) gave a pattern that clearly shows a
peak at m/z 784.1 corresponding to the galactosylated GN
H6F6 primer (lactosamine derivative) as shown in Figure 3.
Surprisingly, among the primers, both Lac H4F8 and GN
H4F8 having the same fluorous tag hardly gave any products.
Although HPTLC results showed that these primers are present
in the cell fraction, a band corresponding to a glycosylated

1
412 Bull. Chem. Soc. Jpn. Vol. 82, No. 11 (2009)
Fluorous-Tagged Saccharide Primers
A
Fluorous-tagged Lac primers
Endogenous GM3
Sialylated primer
Lac H8F4
Lac H6F6
Lac H4F8 Lac H2F8 Lac H3F8
Lac H2F10 Control
B
Fluorous-tagged Lac primers
Endogenous GM3
Sialylated primer
Lac H8F4
Lac H6F6
Lac H4F8
Lac H2F8
Lac H3F8 Lac H2F10 Control
C
GlcNAc primer
Galactosylated GlcNAc primer
Endogenous GM3
GN H8F4 GN H6F6 GN H2F8 GN H3F8 GNH4F8
D
GlcNAc primer
Endogenous GM3
Sialylated Gal GlcNAc primer
GN H8F4 GN H6F6 GN H2F8 GN H3F8 GNH4F8
Figure 2. HPTLC profile of lipids (A and C, cell fraction; B and D, culture medium fraction) obtained after incubation of B16 cells
with fluorous-tagged lactoside and GlcNAc primers.
Further investigation is currently undertaken to explain these
results.
modification except glycosylation. The fluorous tag did not
affect in any way the regio- and stereospecific glycosylation of
primers by cellular enzymes. The glycosylation products have
the same glycan structures as those biosynthesized by the cells
used. This is particularly true with the lactoside primers that
gave analogs of GM3, the major ganglioside produced by B16
cells.
Among the many advantages of the incorporation of fluorine
atoms in the saccharide primer structure include the ease of
separation of the glycosylated primer. Separation of the
glycosylated Lac H2F8 from the rest of the components of
the culture medium was accomplished by gradient elution with
methanol using fluorous solid phase extraction cartridges. As
Compounds with numerous fluorine atoms in the aglycon
unit did not pose any steric barrier to primer assimilation into
cells. The more the fluorine atoms present, the more likely that
they remain in the cells. Based on HPTLC results of the cell
fraction, the band corresponding to primers Lac H2F10 and Lac
H4F8, or Lac H3F8, are more intense than primers Lac H6F6 or
Lac H8F4 suggesting that the presence of more fluorine atoms
enhances primer retention in the cell. Results also confirmed
that the carbon­fluorine bond is stable.10 The fluorous-tagged
primers diffused in cells, were elongated by cellular enzymes
and then released to the culture medium without any structural

M. C. Z. Kasuya et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 11 (2009) 1413
A
SA Gal GN H6F6
OH
OH
O
HO
O
O
OC6H12C6F13
HOOC
HO
O
OH
NHAc
HO OH
HO
O
MW: 1076.8
OH
AcHN
B
GN H6F6
Gal GN H6F6
OH
O
OH
OH
O
HO
HO
O
HO
HO
OC6H12C6F13
O
HO
OC6H12C6F13
NHAc
OH
NHAc
MW: 623.4
MW: 785.5
Figure 3. Electrospray ionization mass spectrum (negative ion mode) of (A) SAGalGN H6F6 obtained after incubation of B16 cells
with GNH6F6 and (B) fragmentation of SAGalGN H6F6 gave 2 major peaks corresponding to GalGN H6F6 primer and GN H6F6.
Fluorous-tagged Lac primers
glycosylated primer
Lac H8F4 Lac H6F6 Lac H2F8 Lac H3F8 Lac H4F8 Lac H2F10
Figure 4. HPTLC profile of lipids from the culture medium fraction obtained after incubation of Vero cells with fluorous-tagged
6
lactoside primers. Vero cells (2 © 10 , 100 mm dish) were incubated for 48 h in serum free 1:1 DMEM­F12 (7 mL) supplemented
with transferrin and insulin (TI/DF) in the absence or presence of 50 ¯M primer.
shown in Figure 5, the glycosylated primers could be effec-
tively separated using 40% MeOH in water. Similarly, the rest
of the glycosylation products could be separated by gradient
elution using fluorous solid phase extraction cartridges.
The presence of fluorine atom enhances primer hydro-
phobicity. However, the number of fluorine is a significant
consideration in the preparation of oligosaccharides by the
saccharide primer strategy. Cellular uptake and glycosylation
are affected by varying the number of fluorine atoms in the
aglycon unit. Incorporation of fluorine atom in the saccharide
primers does not only facilitate purification methods but more
importantly, may shed light on the glycosylation requirements
and processes in biological systems.
ration of oligosaccharides by the saccharide primer strategy.
The presence of numerous fluorine atoms did not affect the
regio- and stereospecific glycosylation by cells. However, the
uptake, release and the amount of glycosylated products vary
depending on the number of fluorine atoms present. Generally,
the more the fluorine atoms present, the more likely the primers
remained in the cells and the smaller the amount of product
released into the culture medium.
Experimental
General methods: 1H and 13C NMR spectra were recorded at a
00 MHz JEOL spectrometer in Me2SO or in MeOH-d4 or in
6
CDCl3 using tetramethylsilane as internal reference. High-reso-
lution mass spectra of the fluorous-tagged primers were recorded
with a JEOL JMS-700AM spectrometer. All chemical synthetic
reactions were monitored by thin layer chromatography on
Silica Gel 60 F-254 (Merck), with detection by UV light or by
Conclusion
The number of fluorine atoms in the aglycon unit of the
saccharide primers is an essential consideration in the prepa-

1
414 Bull. Chem. Soc. Jpn. Vol. 82, No. 11 (2009)
Fluorous-Tagged Saccharide Primers
OH
OH
HO
HO
O
F
F
F
F
F
F
F
O
F
F
F
F
F
F
O
HO
O
F
F
F F
OH
OH
OH
OH
HO
O
O
F
F
F
F
F
F
F
O
HO
O
F
F
F
F
F
F
F
F
HOOC
O
OH
OH
F F
HO OH
HO
AcHN
O
OH
before
separation
10 20 30 40 50 60 70 80 90 100 % MeOH
Figure 5. HPTLC of lipids obtained after separation by gradient elution using fluorous solid phase extraction (FSPE) cartridges.
6
2
© 10 B16 cells were seeded into 10 cm dish containing 7 mL of 10% FBS DMEM/F12. Then, 50 ¯M Lac H2F8 primer in 7 mL
ITS-X DMEM/F12 were administered into cells and incubated for 48 h. The culture medium was collected and the lipids were
extracted. Separation was carried out as follows: pre-treatment of the FSPE cartridges (2 g) with THF (4 mL) followed by water
(4 mL), then sample loading and washing with water (5 mL), and finally gradient elution with 5 mL MeOH/water.
visualizing by spraying with anisaldehyde­H2SO4 and heating.
Lipids from the cell homogenate and culture medium fractions
were analyzed by HPTLC with CHCl3:MeOH:0.25%KCl(aq) =
1H), 4.36 (d, J = 7.6 Hz, 1H); 13C NMR (CD3OD, 125 MHz): ¤
32.46 (t, J = 21.7 Hz), 61.80, 62.38, 62.56, 70.16, 72.41, 74.43,
74.71, 76.24, 76.40, 76.95, 80.50, 104.30, 104.96, 108.00­121.87
+
5
:4:1 (v/v/v) as developing solvent. HPTLC plates were sprayed
(m); HRMS (FAB) m/z calcd for C24H26F21O11 ([M + H] ):
with resorcinol and heated to detect the separated glycolipids that
were quantified by densitometry. Column chromatography was
performed on Silica Gel 60 (70­230 mesh, E. Merck, Darmstadt).
The mass spectrum of the products of cellular-enzyme-catalyzed
glycosylation was recorded on a Bruker Esquire HCT Ultra ESI
LC MS using MeOH:acetonitrile (1:1, v/v). Solid phase extraction
cartridges were by Fluorous Technologies Inc.
889.1140, found 889.1184.
2-(Perfluorooctyl)ethyl
4-O-(¢-D-Galactopyranosyl)-¢-D-
1
glucopyranoside (Lac H2F8); H NMR (CD3OD, 600 MHz): ¤
2.55­2.63 (m, 2H), 3.25 (t, J = 8.6 Hz, 1H), 3.40­3.45 (m, 1H),
3.47 (dd, J = 9.6 Hz, 3.4 Hz, 1H), 3.50­3.59 (m, 3H), 3.53
(dd, J = 9.6 Hz, 2.1 Hz, 1H), 3.68 (dd, J = 11.3 Hz, 4.5 Hz, 1H),
3.77 (dd, J = 11.7 Hz, 7.6 Hz, 1H), 3.78­3.93 (m, 4H), 4.17
(dt, J = 10.3 Hz, 6.9 Hz, 1H), 4.32 (d, J = 7.6 Hz, 1H), 4.34
Chemical Synthesis of Saccharide Primers.
General
13
Procedure: Glycosylation of the perfluoroalcohols was carried
(d, J = 8.2 Hz, 1H); C NMR (CD3OD, 125 MHz): ¤ 32.52
(t, J = 21.7 Hz), 61.85, 62.46, 62.62, 70.26, 72.51, 74.55, 74.80,
76.34, 76.51, 77.04, 80.53, 104.40, 105.05, 108.89­119.93 (m);
5
,6
out at room temperature using peracetyl lactose
(for the
preparation of Lac H2F10 and Lac H2F8) or peracetyl lactosyl
bromide (for the preparation of Lac H6F6, Lac H3F8, Lac H4F8,
and Lac H8F4) in dichloromethane with Lewis acid or silver
carbonate and silver perchlorate as promoter, respectively. Usual
work-up procedures followed by silica gel column chromato-
graphic separation afforded the desired products of glycosylation:
AceLac H8F4 (36%), AceLac H6F6 (24%), AceLac H4F8 (16%),
AceLac H2F8 (75%), and AceLac H2F10 (71%). On the other
hand, all the GlcNAc primers were prepared via glycosylation
+
HRMS (FAB) m/z calcd for C22H26F17O11 ([M + H] ): 789.1204
found 789.1179.
3-(Perfluorooctyl)propyl 4-O-(¢-D-Galactopyranosyl)-¢-D-
glucopyranoside (Lac H3F8); H NMR (CD3OD, 600 MHz): ¤
1
1.87­1.95 (m, 2H), 2.25­2.40 (m, 2H), 3.28 (dd, J = 8.9 Hz,
7.6 Hz, 1H), 3.40­3.43 (m, 1H), 3.48 (dd, J = 9.6 Hz, 3.4 Hz, 1H),
3.53­3.59 (m, 4H), 3.66 (dt, J = 9.6 Hz, 5.5 Hz, 1H), 3.70 (dd,
J = 11.3 Hz, 5.0 Hz, 1H), 3.76­3.87 (m, 3H), 3.90 (dd, J =
12.4 Hz, 5.0 Hz, 1H), 3.98 (dt, J = 10.3 Hz, 5.0 Hz, 1H), 4.30 (d,
J = 8.2 Hz, 1H), 4.36 (d, J = 7.6 Hz, 1H); 13C NMR (CD3OD,
125 MHz): ¤ 21.84, 28.85 (t, J = 21.7 Hz), 61.92, 62.40, 69.16,
70.14, 72.42, 74.49, 74.71, 76.29, 76.94, 80.70, 104.02, 104.96,
109.26­119.93 (m); HRMS (FAB) m/z calcd for C23H28F17O11
8
using the oxazoline derivative and camphor sulfonic acid (CSA)
in dichloroethane at 90 °C to afford AceGN H8F4 (17%), AceGN
H6F6 (49%), AceGN H4F8 (60%), AceGN H3F8 (42%), and
AceGN H2F8 (44%).
Deacylation was carried out using methanol and sodium
methoxide. Stirring at room temperature followed by neutralization
with cation-exchange resin afforded the fluorous-tagged saccharide
primers and the respective yields: Lac H8F4 (68%), Lac H6F6
+
([M + H] ): 803.1360, found 803.1309.
4-(Perfluorooctyl)butyl
glucopyranoside (Lac H4F8);
4-O-(¢-D-Galactopyranosyl)-¢-D-
1
H NMR (CD3OD, 600 MHz):
(
98%), Lac H4F8 (47%), Lac H2F8 (96%), Lac H2F10 (quant) and
GN H8F4 (97%), GN H6F6 (88%), GN H4F8 (92%), GN H3F8
93%), GN H2F8 (85%).
-(Perfluorodecyl)ethyl
glucopyranoside (Lac H2F10);
¤ 1.70­1.77 (m, 4H), 2.15­2.23 (m, 2H), 3.24­3.30 (m, 1H),
3.25 (t, J = 8.6 Hz, 1H), 3.30­3.42 (m, 4H), 3.53 (t, J = 8.9 Hz,
1H), 3.55 (dd, J = 15.8 Hz, 8.9 Hz, 1H), 3.58­3.63 (m, 1H),
3.68 (dd, J = 11.7 Hz, 5.5 Hz, 1H), 3.85­3.92 (m, 3H), 3.92­
3.97 (m, 1H), 4.30 (d, J = 7.6 Hz, 1H), 4.41 (d, J = 7.6 Hz, 1H);
(
2
4-O-(¢-D-Galactopyranosyl)-¢-D-
1
H NMR (CD3OD, 600 MHz):
13
¤
2.15­2.25 (m, 2H), 3.28 (t, J = 8.3 Hz, 1H), 3.38­3.46 (m, 1H),
C NMR (CD3OD, 125 MHz): ¤ 16.93, 28.80, 30.25 (t, J =
3
.49 (dd, J = 9.6 Hz, 3.4 Hz, 1H), 3.52­3.63 (m, 4H), 3.71 (dd,
22.4 Hz), 60.73, 61.19, 68.82, 68.97, 71.24, 73.38, 73.51, 75.12,
J = 11.3 Hz, 4.5 Hz, 1H), 3.76­3.98 (m, 6H), 4.30 (d, J = 7.6 Hz,
75.16, 75.76, 79.54, 102.89, 103.78, 106.26­121.59 (m); HRMS

M. C. Z. Kasuya et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 11 (2009) 1415
+
6.2 Hz, 1H), 4.28 (d, J = 8.2 Hz, 1H); 13C NMR (CD3OD,
125 MHz): ¤ 18.19, 22.86, 29.96, 31.49 (t, J = 21.7 Hz), 57.40,
62.86, 69.60, 72.22, 76.09, 78.02, 102.64, 110.02­123.55 (m),
173.65; HRMS (FAB) m/z calcd for C20H27F13NO6 ([M + H] ):
624.1631, found 624.1600.
(
8
FAB) m/z calcd for C24H30F17O11 ([M + H] ): 817.1517, found
17.1497.
-(Perfluorohexyll)hexyl 4-O-(¢-D-Galactopyranosyl)-¢-D-
6
1
+
glucopyranoside (Lac H6F6); H NMR (CD3OD, 600 MHz): ¤
.40­1.48 (m, 4H), 1.58­1.70 (m, 4H), 2.08­2.19 (m, 2H), 3.27 (t,
J = 8.6 Hz, 1H), 3.35­3.42 (m, 1H), 3.49 (dd, J = 9.6 Hz, 3.4 Hz,
H), 3.51­3.60 (m, 5H), 3.71 (dd, J = 11.3 Hz, 4.5 Hz, 1H), 3.76­
.95 (m, 5H), 4.29 (d, J = 7.6 Hz, 1H), 4.36 (d, J = 7.6 Hz, 1H);
C NMR (CD3OD, 125 MHz): ¤ 21.09, 26.61, 29.76, 30.35, 31.62
t, J = 22.4 Hz), 61.90, 62.37, 70.13, 70.62, 72.41, 74.56, 74.68,
6.24, 76.31, 76.92, 80.70, 104.06, 104.96, 108.05­120.20 (m);
HRMS (FAB) m/z calcd for C24H34F13O11 ([M + H] ): 745.1894,
found 745.1880.
1
8-(Perfluorobutyl)octyl
2-Acetamido-2-deoxy-¢-D-gluco-
1
1
pyranoside (GN H8F4); H NMR (CD3OD, 600 MHz): ¤ 1.30­
1.42 (m, 8H), 1.48­1.61 (m, 4H), 1.95 (s, 3H), 2.08­2.20 (m, 2H),
3.21­3.24 (m, 1H), 3.26­3.31 (m, 1H), 3.39­3.46 (m, 2H), 3.60
(dd, J = 10.3 Hz, 8.3 Hz, 1H), 3.65 (dd, J = 12.1 Hz, 5.9 Hz, 1H),
3.83­3.88 (m, 2H), 4.36 (d, J = 8.3 Hz, 1H); 13C NMR (CD3OD,
125 MHz): ¤ 21.25, 22.98, 27.04, 30.06, 30.24, 30.38, 30.63,
31.63 (t, J = 22.4 Hz), 57.47, 62.85, 70.55, 72.21, 76.10, 77.97,
102.74, 107.02­120.05 (m), 173.62; HRMS (FAB) m/z calcd for
3
1
3
(
7
+
8
-(Perfluorobutyl)octyl
glucopyranoside (Lac H8F4); H NMR (CD3OD, 600 MHz): ¤
.30­1.48 (m, 8H), 1.55­1.68 (m, 4H), 2.05­2.15 (m, 2H), 3.28 (t,
4-O-(¢-D-Galactopyranosyl)-¢-D-
1
+
C20H30F9NO6Na ([M + Na] ): 574.1827, found 574.1847.
1
Cellular Uptake of Glycoside Primers.
Cell culture,
J = 8.6 Hz, 1H), 3.40 (dt, J = 6.9 Hz, 2.7 Hz, 1H), 3.49 (dd,
J = 9.6 Hz, 3.4 Hz, 1H), 3.51­3.61 (m, 5H), 3.71 (dd, J = 11.7 Hz,
incubation of cells with primer and identification of glycosylated
product were carried out according to the literature cited in the
text.1
,6
4.1 Hz, 1H), 3.81 (dd, J = 11.7 Hz, 7.6 Hz, 1H), 3.81­3.92 (m,
H), 4.29 (d, J = 7.6 Hz, 1H), 4.36 (d, J = 7.6 Hz, 1H); 13C NMR
4
(
(
7
CD3OD, 125 MHz): ¤ 20.98, 26.72, 29.84, 30.06, 30.47, 31.50
t, J = 22.4 Hz), 61.85, 62.27, 70.01, 70.78, 72.29, 74.43, 74.57,
The authors would like to thank Associate Professor Koji
Matsuoka and Mr. Tetuo Koyama of Saitama University for the
mass spectral measurements of all the saccharide primers. This
work was supported partially by a grant for “Development of
Novel Diagnostic and Medical Applications through Elucida-
tion of Sugar Chain Functions” from the New Energy and
Industrial Technology Development Organization (NEDO).
6.10, 76.20, 76.79, 80.69, 103.95, 104.84, 107.61­121.87 (m);
+
HRMS (FAB) m/z calcd for C24H38F9O11 ([M + H] ): 673.2270,
found 673.2304.
4
-(Perfluorooctyl)butyl
2-Acetamido-2-deoxy-¢-D-gluco-
1
pyranoside (GN H4F8);
H NMR (CD3OD, 600 MHz): ¤
.62­1.70 (m, 4H), 1.96 (s, 3H), 2.13­2.21 (m, 2H), 3.24­3.29 (m,
1
1
3
1
H), 3.30­3.37 (m, 1H), 3.44 (dd, J = 10.3 Hz, 8.9 Hz, 1H), 3.48­
.53 (m, 1H), 3.62­3.72 (m, 2H), 3.88 (dd, J = 11.7 Hz, 2.1 Hz,
H), 3.90­3.98 (m, 1H), 4.40 (d, J = 8.2 Hz, 1H); 13C NMR
References
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T. Sato, K. Hatanaka, H. Hashimoto, T. Yamagata, Trends
(
5
CD3OD, 125 MHz): ¤ 18.19, 22.86, 29.96, 31.50 (t, J = 22.4 Hz),
7.40, 62.86, 69.60, 72.23, 76.09, 78.02, 102.64, 105.53­119.05
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(
m), 173.65; HRMS (FAB) m/z calcd for C20H23F17NO6
+
(
[M + H] ): 696.1254, found 696.1278.
3
-(Perfluorooctyl)propyl 2-Acetamido-2-deoxy-¢-D-gluco-
1
pyranoside (GN H3F8);
.81­1.87 (m, 2H), 1.96 (s, 3H), 2.00­2.32 (m, 2H), 3.22­3.29
m, 1H), 3.29­3.35 (m, 1H), 3.44 (dd, J = 10.7 Hz, 8.6 Hz, 1H),
H NMR (CD3OD, 600 MHz): ¤
1
(
2
H. H. Freeze, D. Sampath, A. Varki, J. Biol. Chem. 1993,
268, 1618; A. K. Sarkar, J. R. Brown, J. D. Esko, Carbohydr. Res.
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Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3323.
3
1
2
8
2
1
.55 (dt, J = 9.6 Hz, 6.2 Hz, 1H), 3.64 (dd, J = 10.3 Hz, 8.2 Hz,
H), 3.68 (dd, J = 11.7 Hz, 5.5 Hz, 1H), 3.88 (dd, J = 11.7 Hz,
.1 Hz, 1H), 3.98 (dt, J = 10.3 Hz, 5.5 Hz, 1H), 4.39 (d, J =
3
J. C. Paulson, K. J. Colley, J. Biol. Chem. 1989, 264,
13
.3 Hz, 1H); C NMR (CD3OD, 125 MHz): ¤ 21.86, 22.83,
8.78 (t, J = 21.7 Hz), 57.33, 62.83, 68.75, 72.16, 75.94, 78.03,
17615; K. J. Colley, Glycobiology 1997, 7, 1; A. Varki, Trends Cell
Biol. 1998, 8, 34.
02.70, 106.66­122.55 (m), 173.60; HRMS (FAB) m/z calcd
4
H. Nakajima, Y. Miura, T. Yamagata, J. Biochem. 1998,
+
for C19H21F17NO6 ([M + H] ): 682.1097, found 682.1122.
124, 148.
2
-(Perfluorooctyl)ethyl
2-Acetamido-2-deoxy-¢-D-gluco-
pyranoside (GN H2F8); H NMR (CD3OD, 600 MHz): ¤ 1.93
s, 3H), 2.35­2.53 (m, 2H), 3.26­3.30 (m, 2H), 3.42 (dd, J =
5
M. C. Kasuya, A. Ito, K. Hatanaka, J. Fluorine Chem.
1
2007, 128, 562.
(
6
M. C. Z. Kasuya, R. Cusi, O. Ishihara, A. Miyagawa,
1
6
0.7 Hz, 8.6 Hz, 1H), 3.60­3.64 (m, 1H), 3.66 (dd, J = 12.1 Hz,
.2 Hz, 1H), 3.79 (dt, J = 10.3 Hz, 5.9 Hz, 1H), 3.86 (dd,
K. Hashimoto, T. Sato, K. Hatanaka, Biochem. Biophys. Res.
Commun. 2004, 316, 599.
J = 11.7 Hz, 2.1 Hz, 1H), 4.13 (dt, J = 10.3 Hz, 6.2 Hz, 1H),
7
Z. Luo, Q. Zhang, Y. Oderaotoshi, D. Curran, Science 2001,
1
3
4
3
1
.42 (d, J = 8.3 Hz, 1H); C NMR (CD3OD, 125 MHz): ¤ 22.83,
2.33 (t, J = 20.1 Hz), 57.21, 62.30, 62.79, 72.13, 75.91, 78.11,
291, 1766; J. A. Gladysz, D. P. Curran, I. T. Horvath, Handbook of
Fluorous Chemistry, 2004.
02.63, 108.02­120.05 (m), 173.75; HRMS (FAB) m/z calcd for
8
S. Nakabayashi, C. D. Warren, R. W. Jeanloz, Carbohydr.
+
C18H19F17NO6 ([M + H] ): 668.0941, found 668.0969.
Res. 1986, 150, c7; S. Nishimura, K. Matsuoka, T. Furuike, S.
Ishii, K. Kurita, K. M. Nishimura, Macromolecules 1991, 24,
4236.
6
-(Perfluorohexyl)hexyl
2-Acetamido-2-deoxy-¢-D-gluco-
1
pyranoside (GN H6F6); H NMR (CD3OD, 600 MHz): ¤
.25­1.35 (m, 4H), 1.45­1.56 (m, 4H), 1.87 (s, 3H), 2.00­2.13 (m,
H), 3.13­3.17 (m, 1H), 3.18­3.23 (m, 1H), 3.31­3.40 (m, 2H),
.53 (dd, J = 10.3 Hz, 8.3 Hz, 1H), 3.58 (dd, J = 12.0 Hz, 5.8 Hz,
H), 3.78 (dd, J = 11.7 Hz, 2.1 Hz, 1H), 3.80 (dt, J = 9.6 Hz,
1
2
3
1
9
M. C. Z. Kasuya, M. Ikeda, K. Hashimoto, T. Sato, K.
Hatanaka, J. Carbohydr. Chem. 2005, 24, 705.
10 B. E. Smart, J. Fluorine Chem. 2001, 109, 3.