Synthesis of 4-deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose and their effects on cellular glycosaminoglycan biosynthesis

Article abstract of DOI:10.1016/S0008-6215(00)00049-5

4-Deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose were synthesized and evaluated as inhibitors of hepatic glycosaminoglycan biosynthesis. 2-Acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro-D-glucopyranose (16) exhibited a reduction of [³H]GlcN and [³⁵S]SO₄ incorporation into hepatocyte cellular glycosaminoglycans to 12 and 18%, respectively, of the control cells, at 1.0 mM. Similarly, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro-D-galactopyranose (31) exhibited a reduction of [³H]GlcN and [³⁵S]SO₄ incorporation to 1 and 9%, respectively, of the control cells, at 1.0 mM. Unlike 16, 31 exhibited a reduction of [¹⁴C]Leu incorporation into cellular protein to 57% of control cells, at 1.0 mM. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00049-5

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Carbohydrate Research 326 (2000) 250–263
Synthesis of 4-deoxy-4-fluoro analogues of
2-acetamido-2-deoxy-
D-glucose and
2-acetamido-2-deoxy- -galactose and their effects on
D
cellular glycosaminoglycan biosynthesis
Ali Berkin a, Walter A. Szarek a,*1, Robert Kisilevsky b,*2
a Department of Chemistry, Queen’s Uni6ersity, Kingston, Ont., Canada K7L 3N6
b Departments of Pathology and Biochemistry, Queen’s Uni6ersity, and The Syl and Molly Apps Research Center,
Kingston General Hospital, Kingston, Ont., Canada K7L 3N6
Received 4 January 2000; accepted 31 January 2000
Abstract
4-Deoxy-4-fluoro analogues of 2-acetamido-2-deoxy-
sized and evaluated as inhibitors of hepatic glycosaminoglycan biosynthesis. 2-Acetamido-1,3,6-tri-O-acetyl-2,4-
dideoxy-4-fluoro-
-glucopyranose (16) exhibited a reduction of [3H]GlcN and [35S]SO4 incorporation into hepatocyte
cellular glycosaminoglycans to 12 and 18%, respectively, of the control cells, at 1.0 mM. Similarly, 2-acetamido-1,3,6-
D-glucose and 2-acetamido-2-deoxy-D-galactose were synthe-
D
tri-O-acetyl-2,4-dideoxy-4-fluoro-
-galactopyranose (31) exhibited a reduction of [3H]GlcN and [35S]SO4 incorpora-
D
tion to 1 and 9%, respectively, of the control cells, at 1.0 mM. Unlike 16, 31 exhibited a reduction of [14C]Leu
incorporation into cellular protein to 57% of control cells, at 1.0 mM. © 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: Glycosaminoglycans; 2-Acetamido-2,4-dideoxy-4-fluoro-
fluoro- -galactopyranose derivatives
D-glucopyranose derivatives; 2-Acetamido-2,4-dideoxy-4-
D
1. Introduction
adult-onset diabetes [4], and Alzheimer’s
disease [5].
Cell-surface heparan sulfate (HS) proteo-
glycans play an important role in regulat-
ing several biological functions of a cell,
including cell–cell migration, adhesion,
growth and communication, and can serve
as receptors for various proteins. HS has
been identified as a receptor for herpes
simplex virus [1] and is implicated in tumor
invasion and metastasis [1,2], arthritis [3],
The availability of synthetic HS analogues
to perturb glycosaminoglycan (GAG) biosyn-
thesis could greatly affect the above-men-
tioned disorders. The GAG chain of HS is
composed of repeating disaccharides of (1“
4)-linked
D
-glucosamine and uronic acid
residues that are variably N- and O-sul-
fonated. This polysaccharide chain is attached
by a tetrasaccharide linkage region consisting
of
b-(1“4)-
residue is attached to the core protein. We
have shown [6] that 3-deoxy- -xylo-hexose
(‘3-deoxy- -galactose’) and a 4-chloro-4-de-
-D
-GlcA-b-(1“3)-
D
-Gal-b-(1“3)-
D
-Gal-
D
-Xyl-b- to
L
-serine, and the serine
1 *Corresponding author. Tel.: +1-613-5332643; fax: +1-
613-5336532; e-mail: szarekw@chem.queensu.ca
2 *Corresponding author.
D
D
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00049-5

A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
251
oxy analogue of a uronic acid, namely methyl
(methyl 4-chloro-4-deoxy-b- -galactopyran-
2-acetamido-2,4-dideoxy-4-fluoro-
pyranoses, directed towards influencing cellu-
lar GAG and total protein biosynthesis.
D
-galacto-
D
osid)uronate [6], significantly reduced
D-
[3H]glucosamine ([3H]GlcN) and [35S]sulfate
([35S]SO4) incorporation into cellular GAGs of
mouse hepatocytes in culture. The latter com-
pound also exhibited a significant reduction of
2. Results and discussion
L
-[14C]leucine ([14C]Leu) incorporation into to-
Chemical synthesis.—4-Fluoro analogues of
tal cellular protein synthesis in vitro. Recently,
we also found [7] that some 4-deoxy analogues
2-acetamido-2,4-dideoxy- -glucose (Scheme 1)
D
were synthesized by double inversion of the
configuration at C-4 of the corresponding 3,6-
di-O-benzoyl analogues. The 3,6-di-O-benzoyl
analogues 1–3 were prepared from the corre-
sponding methyl and benzyl glycosides in
a one-step fashion following the procedure
of Wang and Lee [10]. Briefly, methyl 2-
of 2-acetamido-2-deoxy- -glucose were signifi-
D
cant inhibitors of both protein and glycocon-
jugate biosynthesis.
Fluorinated carbohydrates have significant
biological activities [8] and are of interest as
biological probes, antitumor agents, and in-
hibitors of hexokinases. The altered hydrogen-
bonding properties of fluorine and its atomic
size relative to that of hydrogen make it of
interest in the synthesis of other HS ana-
logues. Sharma et al. [9] have evaluated a
series of 4- and 6-fluoro derivatives of 2-ac-
acetamido-2-deoxy-a-
methyl 2-acetamido-2-deoxy-b-
oside [11] and benzyl 2-acetamido-2-deoxy-a-
-glucopyranoside [12] were each treated with
D
-glucopyranoside [11],
D
-glucopyran-
D
benzoyl chloride in pyridine at −45 °C to
afford 1 (82.4%), 2 [10] (70.9%), and 3 [13]
(76.9%), respectively, following flash chro-
matography. Compounds 1, 2, and 3 were
each treated with trifluoromethanesulfonic an-
hydride in pyridine to afford the 4-O-triflyl
derivatives, which were isolated and immedi-
ately treated with sodium nitrite in DMF to
produce the inversion products 4 [10] (58.8%),
etamido-2-deoxy- -hexopyranoses on L1210
D
leukemia cell growth in vitro and found sig-
nificant inhibition of both total protein and
glycoconjugate biosynthesis.
Herein we report the synthesis and biologi-
cal evaluation of a series of 2-acetamido-2,4-
dideoxy-4-fluoro- -glucopyranoses and of
D
Scheme 1. Synthesis of 4-fluoro analogues of 2-acetamido-2,4-dideoxy-
D-glucopyranose. Reagents: (i) Tf2O, pyr; (ii) NaNO2,
DMF; (iii) DAST, CH2Cl2; (iv) KOH, MeOH; (v) H2, Pd–C, AcOH; (vi) Ac2O, pyr.

252
A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
Scheme 2. Synthesis of 4-fluoro analogues of 2-acetamido-2,4-dideoxy-
MeCN; (iii) H2, Pd–C, AcOH; (iv) Ac2O, pyr.
D-galactopyranose. Reagents: (i) MsCl, pyr; (ii) TBAF,
5 [10] (50.8%), and 6 [14] (58.2%), respectively.
1H NMR spectroscopy provided evidence for
the inversion of configuration at C-4 for each
of compounds 4–6 by the disappearance of a
discernible H-4–H-5 coupling, an observation
indicative of the galacto configuration, which
was confirmed by NOE experiments.
copyranose [9a] (16, 93.2%). 1H NMR
spectroscopy of 13 indicated a mixture of a
and b anomers in a ratio of 5.7:1, respectively.
Compounds 10 and 11 were each acetylated,
as above, to afford methyl 2-acetamido-3,6-di-
O-acetyl-2,4-dideoxy-4-fluoro-a- -glucopyran-
D
oside (14, 88.2%) and methyl 2-ace-
tamido-3,6-di-O-acetyl-2,4-dideoxy-4-fluoro-b-
Treatment of each of compounds 4, 5,
and 6 with (diethylamino)sulfur trifluoride
(DAST) afforded 7 (81.4%), 8 (40.2%), and 9
(85.2%), respectively. The low yield for the
conversion of 5 into 8 is a result of the forma-
tion of two unknown side-products, one of
which was a fluorinated compound. The inver-
sion of configuration at C-4 for each of com-
D
-glucopyranoside (15, 86.8%), respectively.
The synthesis of 4-fluoro analogues of 2-ac-
etamido-2,4-dideoxy- -galactose (Scheme 2)
D
was attempted from 1–3 using DAST, but
was unsuccessful. An alternative route em-
ploying the 3,6-di-O-benzyl analogues, 17–19,
was investigated. Compounds 17 [16], 18 [17],
and 19 [18] were each prepared according to
literature procedures and were treated with
methanesulfonyl chloride in pyridine to afford
20 (75.2%), 21 (73.6%), and 22 [9a,19] (77.5%),
respectively. Compounds 20, 21, and 22 were
each treated with anhydrous tetra-n-butylam-
monium fluoride (TBAF) [20] in MeCN to
afford 23 (54.3%), 24 (64.0%), and 25 [9a,19]
(62.0%), respectively. The difficulty of fluorine
substitution at C-4 of 20–22 is evident by the
low yields and long reaction times (MeCN at
reflux temperature, 4–6 days). Notably, the
1
pounds 7–9 was determined by H NMR
19
spectroscopy and NOE experiments. Also, F
NMR spectroscopy of 7–9 revealed in each
case a doublet of doublets at l ꢀ197 (Jꢀ14
and 51 Hz), a result indicative of an equatori-
ally disposed fluorine atom at C-4 [15].
Compounds 7, 8, and 9 were each O-deben-
zoylated using KOH in MeOH to afford 10
(90.2%), 11 (81.4%), and 12 (92.5%), respec-
tively. Removal of the benzyl protecting group
of 12 by catalytic hydrogenolysis in the pres-
ence of 10% Pd–C and hydrogen afforded 13
[9a] (89.1%), which was acetylated with acetic
anhydride and pyridine to afford 2-acetamido-
1
TBAF must be completely dry before use. H
NMR spectroscopy and NOE experiments
1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro- -glu-
D

A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
253
confirmed the galacto configuration for each
of 23–25. Furthermore, the 19F NMR spec-
trum for each of 23–25 exhibited a doublet of
triplets at l ꢀ220 (Jꢀ28 and 50 Hz) indica-
tive of an axially disposed fluorine atom at
C-4 [9a,19].
[14C]Leu in the presence and absence of sugar
analogues for 24 h. Peracetylated sugars are
more lipophilic than their non-acetylated
counterparts, a feature that allows for in-
creased cellular uptake by passive diffusion
[21]. Therefore, the fluorinated analogues were
fully acetylated for in vitro testing.
No significant inhibitory effect was ob-
served (results not shown) for the 4-fluoro
analogues of methyl 2-acetamido-2,4-dideoxy-
Removal of the benzyl protecting groups of
each of 23–25 by catalytic hydrogenolysis in
the presence of 10% Pd–C and hydrogen af-
forded 26 (98.2%), 27 (86.5%), and 28 [9a]
(87.6%), respectively, which were each acety-
lated to afford compounds 29 (84.1%), 30
D-glucopyranoside (14 and 15), whereas the
analogue 16 displayed a marked reduction of
[3H]GlcN and [35S]SO4 incorporation into hep-
atocyte cellular GAGs to 12 and 18%, respec-
tively, of the control cells, at 1.0 mM (Fig.
1(A)). The 4-fluoro analogues of methyl 2-ac-
1
(88.9%), and 31 [9a] (quant), respectively. H
NMR spectroscopy of 31 indicated a mixture
of a and b anomers in a ratio of 3.2:1,
respectively.
Biological e6aluation.—Murine hepatocytes
were isolated and placed in culture as de-
scribed previously [6], and these were then
treated with [3H]GlcN and [35S]SO4 or with
etamido-2,4-dideoxy- -galactopyranoside (29
D
and 30) exhibited no significant inhibitory ef-
fects on [3H]GlcN and [35S]SO4 incorporation
into isolated GAGs up to 1.0 mM (results not
shown). The corresponding analogue 31 ex-
hibited a large inhibition of cellular GAG
synthesis by a reduction of [3H]GlcN and
[35S]SO4 incorporation to 1 and 9%, respec-
tively, of the control cells, at 1.0 mM (Fig.
1(B)). It is possible that the methyl glycosides
14, 15, 29, and 30 are poor substrates for
lysosomal glycosidases as has been observed
for 4-deoxy-
4-fluoro- -galactopyranosides [22,23].
The 4-fluoro analogues of N-acetyl-
cosamine and N-acetyl- -galactosamine (16
D
-glucopyranosides and 4-deoxy-
D
D
-glu-
D
and 31, respectively) were also evaluated for
total protein inhibition by the measurement of
[14C]Leu incorporation into isolated glycocon-
jugates. Compound 16 exhibited no inhibition
of cellular protein synthesis up to 1.0 mM
(Fig. 2(A)); however, 31 exhibited a reduction
in [14C]Leu incorporation into cellular protein
to 57% of the control cells, at 1.0 mM (Fig.
2(B)). The reduction in total protein synthesis
observed for 31 may not be surprising since
D
-galactosamine is known to be rapidly con-
Fig. 1. Effects of increasing concentrations of compounds 16
and 31 on hepatocyte cellular GAG synthesis. Hepatocyte
cultures were incubated with D-[3H]glucosamine and
[35S]sulfate for 24 h in the absence (control) and presence of
compounds 16 (panel A) and 31 (panel B) at 0.01, 0.1, and 1.0
mM. The values represent the mean9S.D. of triplicate cul-
tures. Statistical analyses using an unpaired t-test revealed
that in panel A, control vs. 0.01 or 0.1 mM, not significant;
control vs. 1.0 mM, PB0.01. In panel B, control vs. 0.01
mM, not significant; control vs. 0.1 mM, PB0.01; control vs.
1.0 mM, PB0.01.
verted into a UDP-analogue, a process which
acts to trap uridine and deplete intracellular
UDP pools, and which has been shown to
inhibit protein biosynthesis [24,25].
Sharma et al. [9a] have shown that com-
pounds 16 and 31 demonstrated ID50 values of
0.035 and 0.034 mM, respectively, for the
inhibition of L1210 leukemia cell growth. Fur-

254
A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
protons in the deuterated solvents were used
as internal standards. Chemical shifts (l) are
1
reported in ppm downfield from Me4Si for H
and 13C NMR spectra and downfield from
19
CFCl3 for F NMR spectra. Thin-layer chro-
matography (TLC) was performed using glass
plates precoated with EM Science Silica Gel
60 F254. Flash chromatography was performed
using EM Science Silica Gel 60 (230–400
mesh).
D
-(1,6-3H2)Glucosamine·HCl
(42.3
Ci/
mmol),
L
-[14C]leucine (320 mCi/mmol), and
Na325SO4 (867 mCi/ml) were purchased from
either Dupont or ICN Biomedicals. Labora-
tory chemicals of reagent grade were pur-
chased from Sigma–Aldrich, Fisher Scientific,
or BDH Chemicals. Williams’ Medium E with
L
-glutamine, 10% fetal bovine serum, and an-
tibiotic–antimycotic mixtures were supplied
by Gibco. Fibronectin, collagenase, papain,
and the GAG reference standards (chon-
droitin sulfate, hyaluronic acid, heparin) as
sodium salts were purchased from Sigma–
Aldrich.
Fig. 2. Effects of increasing concentrations of compounds 16
and 31 on hepatocyte cellular total protein synthesis. Hepato-
cyte cultures were incubated with L-[14C]leucine for 24 h in the
presence of compounds 16 (panel A) and 31 (panel B) at 0.01,
0.1, and 1.0 mM. The values represent the mean9S.D. of
triplicate cultures. Statistical analyses using an unpaired t-test
revealed that in panel A, control vs. 0.01, 0.1, or 1.0 mM, not
significant. In panel B, control vs. 0.01 or 0.1 mM, not
significant; control vs. 1.0 mM, PB0.01.
Preparation of methyl 2-acetamido-3,6-di-
O-benzoyl-2-deoxy-h- -galactopyranoside
D
(4).—To an ice-cold solution of 1 [7] (3.75 g,
8.46 mmol) in CH2Cl2 (50 mL) and pyr (7.0
mL) was added dropwise trifluoromethanesul-
fonic anhydride (4.76 g, 2 equiv). The solution
was stirred for 20 min, diluted with CH2Cl2
(200 mL), and washed successively with ice-
cold, aq solutions of KHSO4 (10%) and
NaHCO3 (satd), and water, dried (MgSO4),
and concentrated under reduced pressure. The
crude triflate was used without further purifi-
cation. To a stirred solution in DMF (100
mL) was added NaNO2 (1.75 g, 25.4 mmol),
and after 40 h at rt, the mixture was concen-
trated and the residue was coevaporated with
toluene under reduced pressure to afford an
oil that was subjected to flash chromatogra-
phy on silica gel (1:1 EtOAc–hexanes). Com-
pound 4 was isolated as tiny, white needles
(2.20 g, 58.8%): Rf 0.31 (3:1 EtOAc–hexanes);
mp 202–205 °C, lit. 188–189 °C [10]; [h]D
thermore, in their study, compound 31 re-
duced the incorporation of [3H]GlcN and
[14C]Leu into the total biosynthesized glyco-
conjugates of L1210 leukemia cells to 48 and
74%, respectively, of control cells at 1.0 mM
[9a].
The mechanism of action of the monosac-
charide derivatives described in the present
work is under investigation.
3. Experimental
General methods and materials.—Melting
points were determined on a Fisher–Johns
apparatus and are uncorrected. Optical rota-
tions were measured with a Perkin–Elmer 241
polarimeter for solutions in a 1 dm cell at
1
+85.1° (c 1, CHCl3); H NMR (CDCl3): l
1
13
19
1.86 (s, 3 H, NAc), 3.42 (s, 3 H, OMe), 4.22
(ddꢀt, 1 H, J5,6=J5,6% 6.4 Hz, H-5), 4.26 (d, 1
H, H-4), 4.54 and 4.61 (dq, 2 H, Jgem 11.4 Hz,
H-6, H-6%), 4.82 (d, 1 H, J1,2 3.6 Hz, H-1), 4.90
room temperature (rt). H, C, and F NMR
spectra were recorded on a Bruker AM 400
spectrometer at 400.1, 100.6, and 376.5 MHz,
respectively. The signals arising from residual

A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
255
(ddd, 1 H, H-2), 5.32 (dd, 1 H, J3,4 3.0, J3,2
11.1 Hz, H-3), 5.79 (d, 1 H, JNH,2 9.8 Hz,
NH), 7.38–7.55 (m, 6 H, Ph), 8.01–8.03 (m, 4
Preparation of benzyl 2-acetamido-3,6-di-O-
benzoyl-2-deoxy-h- -galactopyranoside (6).—
D
To an ice-cold solution of 3 [7,13] (4.39 g, 8.45
mmol) in CH2Cl2 (150 mL) and pyr (10 mL)
was added dropwise trifluoromethanesulfonic
anhydride (5.20 g, 2.2 equiv). The mixture was
stirred for 20 min, diluted with CH2Cl2 (200
mL), and washed sequentially with ice-cold,
aq solutions of KHSO4 (10%) and NaHCO3
(satd), and water, dried (MgSO4), and concen-
trated to afford the 4-triflate, which was used
without further purification. To a stirred solu-
tion in DMF (50 mL) was added NaNO2 (1.75
g, 25.4 mmol), and after 16 h at rt, the mix-
ture was concentrated, and the residue was
coevaporated with toluene under reduced
pressure to afford an oil that was subjected to
flash chromatography on silica gel (1:2
EtOAc–hexanes). Compound 6 was isolated
and recrystallized (EtOAc–hexanes) to afford
white needles (2.56 g, 58.2%): Rf 0.48 (1:1
EtOAc–hexanes); mp 187–189 °C, lit. 180 °C
[14]; [h]D +100.2° (c 1.25, CHCl3), lit.+86.4°
(c 1, CHCl3) [14]; 1H NMR (CDCl3): l 1.82 (s,
3 H, NAc), 2.65 (br s, 1 H, OH), 4.27 (d, 1 H,
J4,3 2.5 Hz, H-4), 4.32 (ddꢀt, 1 H, J5,6 =J5,6%
6.4 Hz, H-5), 4.52 and 4.76 (2 d, 2 H, J 11.7
Hz, PhCH2), 4.53 and 4.63 (dq, 2 H, Jgem 11.4
Hz, H-6, H-6%), 4.91 (m, 1 H, H-2), 5.02 (d, 1
H, J1,2 3.7 Hz, H-1), 5.36 (dd, 1 H, H-3), 5.72
(d, 1 H, JNH,2 9.8 Hz, NH), 7.29–8.05 (m, 15
H, Ph); 13C NMR (CDCl3):
l
23.3
(NCOCH3), 47.3 (C-2), 55.3 (OMe), 63.7 (C-
6), 67.4 (C-4), 68.2 (C-5), 72.1 (C-3), 98.8
(C-1), 128.4–133.4 (Ph), 166.4 and 166.6 (2
PhCꢀO), 170.2 (NCꢀO). Anal. Calcd for
C23H25NO8: C, 62.30; H, 5.68; N, 3.16. Found:
C, 62.09; H, 5.84; N, 3.16.
Preparation of methyl 2-acetamido-3,6-
di-O-benzoyl-2-deoxy-i- -galactopyranoside
D
(5).—To a solution of trifluoromethanesul-
fonic anhydride (1.28 mL, 7.62 mmol) in
CH2Cl2 (25 mL) at −15 °C was added drop-
wise a solution of pyr (1.24 mL, 15.3 mmol) in
CH2Cl2 (4.0 mL); a solution of 2 [7,10] (2.11 g,
5.09 mmol) in CH2Cl2 (10 mL) was added,
and the mixture was stirred at −15 °C for 1
h. The reaction mixture was diluted with
CH2Cl2 (50 mL), washed successively with
cold 5% HCl, an aq solution of NaHCO3
(satd), and water, dried (MgSO4), and concen-
trated under reduced pressure. The crude tri-
flate was used without further purification. To
a stirred solution in DMF (10 mL) was added
NaNO2 (3.51 g, 50.9 mmol), and after 10 h at
rt, the mixture was diluted with CH2Cl2 (150
mL), washed with brine, and concentrated,
and the residue was coevaporated with toluene
under reduced pressure to afford an oil that
was subjected to flash chromatography on
silica gel (2:1 EtOAc–hexanes). Compound 5
was isolated as tiny, white needles (1.07 g,
50.8%): Rf 0.30 (3:1 EtOAc–hexanes); mp
249–252 °C, lit. 237–242 °C [10]; [h]D +13.6°
H, Ph); 13C NMR (CDCl3):
l
23.2
(NCOCH3), 47.3 (C-2), 63.5 (C-6), 67.5 (C-4),
68.5 (C-5), 69.8 (PhCH2), 72.0 (C-3), 97.0
(C-1), 128.1–136.7 (Ph), 166.3 and 166.5 (2
PhCꢀO), 170.0 (NCꢀO). Anal. Calcd for
C29H29NO8: C, 67.04; H, 5.63; N, 2.70. Found:
C, 67.10; H, 5.71; N, 2.61.
1
{c 1, (1:1 MeOH–CHCl3)}; H NMR (1:1
CD3OD–CDCl3): l 1.96 (s, 3 H, NAc), 3.61
(s, 3 H, OMe), 4.11 (dd, 1 H, J5,6=J5,6% 6.3
Hz, H-5), 4.34 (d, 1 H, H-4), 4.53 (dd, 1 H,
J2,3 11.0 Hz, H-2), 4.64 (d, 1 H, J1,2 8.6 Hz,
H-1), 4.65 and 4.70 (dq, 2 H, J 11.2 Hz, H-6,
H-6%), 5.22 (dd, 1 H, J3,4 3.2 Hz, H-3), 7.52–
8.15 (m, 10 H, Ph); 13C NMR (1:1 CD3OD–
CDCl3): l 22.9 (NCOCH3), 50.8 (C-2),
56.9 (OMe), 64.2 (C-6), 66.8 (C-4), 73.3
(C-5), 75.2 (C-3), 102.9 (C-1), 129.1–134.0
(Ph), 167.2 and 167.4 (2 PhCꢀO), 173.0
(NCꢀO). Anal. Calcd for C23H25NO8: C,
62.30; H, 5.68; N, 3.16. Found: C, 62.18; H,
5.48; N, 3.05.
Methyl 2-acetamido-3,6-di-O-benzoyl-2,4-
dideoxy-4-fluoro-h- -glucopyranoside (7).—
D
To a solution of DAST (5.70 g, 35.3 mmol) in
CH2Cl2 (20 mL) in a Teflon Erlenmeyer flask
was added dropwise a solution of 4 (1.84 g,
4.16 mmol) in CH2Cl2 (40 mL) at −5 °C. The
reaction mixture was allowed to warm to rt
overnight and then cooled to −5 °C.
Methanol (20 mL) was added dropwise to
destroy the excess of DAST, and the solution
was concentrated under reduced pressure to
afford a yellowish oil that was subjected to
flash chromatography on silica gel (3:2

256
A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
166.8 (2 PhCꢀO), 170.0 (NCꢀO); 19F NMR
(CDCl3): l 197.0 (dd, JF,H3 13.6, JF,H4 51.2
Hz). Anal. Calcd for C23H24FNO7: C, 62.02;
H, 5.43; F, 4.26; N, 3.14. Found: C, 62.12; H,
5.42; F, 4.43; N, 3.18.
EtOAc–hexanes). Compound 7 was isolated
and recrystallized from EtOAc–hexanes to af-
ford tiny, white needles (1.51 g, 81.4%): Rf
0.45 (3:1 EtOAc–hexanes); mp 149–152 °C;
1
[h]D +87.2° (c 1, CHCl3); H NMR (CDCl3):
Benzyl 2-acetamido-3,6-di-O-benzoyl-2,4-
l 1.86 (s, 3 H, NAc), 3.45 (s, 3 H, OMe), 4.18
(m, 1 H, H-5), 4.48 (ddd, 1 H, J2,1 3.6, J2,3
10.3, J2,NH 9.9 Hz, H-2), 4.56 (ddd, 1 H, J6,5
4.7, J6,F 1.3, Jgem 12.1 Hz, H-6), 4.67 (m, 1.5
H, H-4, H-6%), 4.79 (m, 1.5 H, H-1, H-4), 5.63
(dddd, 1 H, J3,4 9.0, J3,F 13.7 Hz, H-3), 5.90
(br d, 1 H, NH), 7.43–7.61 (m, 6 H, Ph),
dideoxy-4-fluoro-h- -glucopyranoside (9).—
D
To a solution of DAST (1.39 g, 8.63 mmol) in
CH2Cl2 (5 mL) in a Teflon Erlenmeyer flask
was added dropwise a solution of 6 (0.500 g,
1.02 mmol) in CH2Cl2 (10 mL) at −5 °C. The
reaction mixture was allowed to warm to rt
overnight and then cooled to −5 °C.
Methanol (5 mL) was added dropwise to de-
stroy the excess of DAST, and the solution
was concentrated under reduced pressure to
afford an oil which was subjected to flash
chromatography on silica gel (1:2 EtOAc–
hexanes). Compound 9 was isolated as a clear,
colorless oil (0.426 g, 85.2%): Rf 0.30 (1:2
EtOAc–hexanes); [h]D +101.2° (c 1.7,
13
8.02–8.07 (m, 4 H, Ph); C NMR (CDCl3): l
23.0 (NCOCH3), 51.7 (J 6.8 Hz, C-2), 55.5
(OMe), 62.5 (C-6), 67.5 (J 23.2 Hz, C-5), 71.7
(J 18.4 Hz, C-3), 87.0 (J 187.7 Hz, C-4), 98.3
(C-1), 128.4–133.4 (Ph), 166.1 and 166.8 (2
19
PhCꢀO), 170.0 (NCꢀO); F NMR (CDCl3): l
197.0 (dd, JF,H3 13.6, JF,H4 51.2 Hz). Anal.
Calcd for C23H24FNO7: C, 62.02; H, 5.43; F,
4.26; N, 3.14. Found: C, 61.82; H, 5.57; F,
4.07; N, 3.12.
1
CHCl3); H NMR (CDCl3): l 1.80 (s, 3 H,
Methyl 2-acetamido-3,6-di-O-benzoyl-2,4-
NAc), 4.24 (m, 1 H, H-5), 4.47–4.63 (m, 3 H,
H-2, H-6, H-6%), 4.56 and 4.77 (2 d, 2 H, J
11.8 Hz, PhCH2), 4.76 (dddꢀdt, 1 H, J4,5 9.4,
J4,F 51.0 Hz, H-4), 4.98 (ddꢀt, 1 H, J1,2 =J1,F
3.3 Hz, H-1), 5.68 (dddd, 1 H, J3,4 9.0, J3,2
10.9, J3,F 13.6 Hz, H-3), 5.93 (d, 1 H, JNH,2 9.6
Hz, NH), 7.32–7.59 (m, 9 H, Ph), 8.02–8.09
(m, 6 H, Ph); 13C NMR (CDCl3): l 22.9
(NCOCH3), 51.7 (J 6.8 Hz, C-2), 62.4 (C-6),
67.8 (J 22.7 Hz, C-5), 70.1 (PhCH2), 71.6 (J
18.7 Hz, C-3), 87.0 (J 187.7 Hz, C-4), 96.5
(C-1), 128.1–136.3 (Ph), 166.0 and 166.7 (2
dideoxy-4-fluoro-i- -glucopyranoside (8).—
D
To a mixture of 5 (0.875 g, 2.11 mmol) in
CH2Cl2 (20 mL) in a Teflon Erlenmeyer flask
was added dropwise DAST (2.89 g, 17.9
mmol) at −40 °C. The reaction mixture was
allowed to warm to rt overnight and then
cooled to −5 °C. Methanol (5 mL) was
added dropwise to destroy the excess of
DAST, and the solution was concentrated un-
der reduced pressure to afford a yellowish oil
that was subjected to flash chromatography
on silica gel (1:1 EtOAc–hexanes). Compound
19
PhCꢀO), 170.0 (NCꢀO); F NMR (CDCl3): l
8
was isolated and recrystallized from
197.0 (dd, JF,H3 13.6, JF,H4 51.2 Hz); ESIMS
(positive-ion): Expected for C29H28FNO7
[M+ H]+: 522.2. Found: 522.3. Anal. Calcd
for C29H28FNO7·H2O: C, 64.55; H, 5.60; F,
3.52; N, 2.60. Found: C, 64.79; H, 5.59; F,
3.26; N, 2.51.
EtOAc–hexanes to afford an amorphous,
white solid (0.353 g, 40.2%): Rf 0.62 (EtOAc);
mp 173–175 °C; [h]D −7.5° (c 2, CHCl3); H
1
NMR (CDCl3): l 1.86 (s, 3 H, NAc), 3.45 (s,
3 H, OMe), 4.18 (m, 1 H, H-5), 4.48 (ddd, 1
H, J2,1 3.6, J2,3 10.3, J2,NH 9.9 Hz, H-2), 4.56
(ddd, 1 H, J6,5 4.7, J6,F 1.3, Jgem 12.1 Hz, H-6),
4.67 (m, 1.5 H, H-4, H-6%), 4.79 (m, 1.5 H,
H-1, H-4), 5.63 (dddd, 1 H, J3,4 9.0, J3,F 13.7
Hz, H-3), 5.90 (br d, 1 H, NH), 7.43–7.61 (m,
6 H, Ph), 8.02–8.07 (m, 4 H, Ph); 13C NMR
(CDCl3): l 23.0 (NCOCH3), 51.7 (J 6.8 Hz,
C-2), 55.5 (OMe), 62.5 (C-6), 67.5 (J 23.2 Hz,
C-5), 71.7 (J 18.4 Hz, C-3), 87.0 (J 187.7 Hz,
C-4), 98.3 (C-1), 128.4–133.4 (Ph), 166.1 and
Methyl 2-acetamido-2,4-dideoxy-4-fluoro-h-
-glucopyranoside (10).—To a solution of
D
compound 7 (0.35 g, 0.78 mmol) in MeOH (20
mL) was added KOH (0.5 g), and the mixture
was stirred for 30 min at rt. The mixture was
neutralized with Amberlite IR-120 (H+) resin
and filtered, and the filtrate was concentrated
under reduced pressure to give a crude
product that was recrystallized from absolute
EtOH to afford 10 (0.18 g, 90.2%) as white

A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
257
Rf 0.38 (1:9 MeOH–CH2Cl2); mp 197–198 °C;
[h]D +206.3° (c 1, MeOH); 1H NMR
(CD3OD): l 1.85 (s, 3 H, NAc), 3.61 (ddd, 1
H, J6,5 4.9, Jgem 12.3, J6,F 1.5 Hz, H-6), 3.67–
3.73 (m, 2 H, H-5, H-6%), 3.81–3.91 (m, 2 H,
H-2, H-3), 4.22 (ddd, 1 H, J4,3 8.1, J4,5 9.8, J4,F
50.9 Hz, H-4), 4.43 and 4.65 (2 d, 2 H, J 12.1
Hz, PhCH2), 4.76 (d, 1 H, J1,2 3.7 Hz, H-1),
needles: Rf 0.65 (1:3 MeOH–CHCl3); mp
1
189–190 °C; [h]D +139.0° (c 1, MeOH); H
NMR (CD3OD): l 1.92 (s, 3 H, NAc), 3.32 (s,
3 H, OMe), 3.64 (m, 2 H, H-5, H-6), 3.73 (m,
1 H, J6%,F 2.1, Jgem 10.0, H-6%), 3.78–3.91 (m, 2
H, J2,3 10.8 Hz, H-2, H-3), 4.23 (dddꢀdt, 1
H, J4,3=J4,5 8.1, J4,F 51.1 Hz, H-4), 4.60 (t, 1
H, J1,2 =J1,F 3.2 Hz, H-1); 13C NMR
(CD3OD): l 22.5 (NCOCH3), 54.9 (J 8.2 Hz,
C-2), 55.7 (OMe), 61.7 (C-6), 70.9 (J 18.9 Hz,
C-3), 71.1 (J 24.6 Hz, C-5), 91.3 (J 180.1 Hz,
C-4), 99.6 (C-1), 173.7 (NCꢀO); 19F NMR
(CD3OD): l 197.1 (dd, JF,H3 14.5, JF,H4 51.8
Hz). Anal. Calcd for C9H16FNO5: C, 45.57; H,
6.37; F, 8.01; N, 5.90. Found: C, 45.72; H,
6.50; F, 8.27; N, 5.89.
13
7.23 (m, 5 H, Ph); C NMR (CD3OD): l 22.5
(NCOCH3), 54.9 (J 8.4 Hz, C-2), 61.7 (C-6),
70.4 (PhCH2), 70.6 (J 18.8 Hz, C-5), 71.5 (J
24.7 Hz, C-3), 91.4 (J 180.0 Hz, C-4), 97.3
(C-1), 128.9–138.7 (Ph), 173.6 (NCꢀO); 19F
NMR (CD3OD): l 169.6 (dd, JF,H3 14.4, JF,H4
51.6 Hz). Anal. Calcd for C15H20FNO5: C,
57.50; H, 6.43; F, 6.06; N, 4.47. Found: C,
57.42; H, 6.50; F, 5.93; N, 4.37.
Methyl 2-acetamido-2,4-dideoxy-4-fluoro-
-glucopyranoside (11).—To a solution of 8
i-D
Preparation of 2-acetamido-2,4-dideoxy-4-
(0.196 g, 0.440 mmol) in MeOH (15 mL) was
added KOH (0.2 g), and the mixture was
stirred for 30 min at rt. The mixture was
neutralized with Amberlite IR-120 (H+) resin
and filtered, and the filtrate was concentrated
under reduced pressure to afford a crude
product that was recrystallized from absolute
EtOH to afford 11 (0.085 g, 81.4%) as an
amorphous solid: Rf 0.46 (1:3 MeOH–
CHCl3); mp 192–194 °C; [h]D −50.0° (c 0.61,
fluoro- -glucopyranose (13).—A mixture of 12
D
(0.58 g, 1.85 mmol), acetic acid (15 mL), and
10% Pd–C (0.5 g) was subjected to a hydro-
gen pressure (55 psig) for 3 days. The mixture
was filtered through Celite 521 (Aldrich) and
the residue washed with MeOH. The filtrate
and washings were combined and concen-
trated under reduced pressure to afford an oil
that was purified by flash chromatography on
silica gel (1:9 to 1:5 MeOH–CH2Cl2) to yield
13 (0.37 g, 89.1%) as a mixture of a and b
anomers in a ratio of 5.7: 1 as determined by
1H NMR spectroscopy. Rf 0.25 and 0.31 (1:3
MeOH–CHCl3); mp 176–180 °C (dec), lit.
174–175 °C [9a]; [h]D +59.3° (equil) (c 1,
MeOH), lit.+61.5° (c 1, MeOH) [9a]. a
1
MeOH); H NMR (CD3OD): l 1.89 (s, 3 H,
NAc), 3.35–3.42 (m, 1 H, H-5), 3.38 (s, 3 H,
OMe), 3.57 (m, 1 H, H-2), 3.65 (m, 1 H, H-6),
3.69 (m, 1 H, H-3), 3.75 (m, 1 H, Jgem 12.3 Hz,
H-6%), 4.18 (ddd, 1 H, J4,3=J4,5 8.7, J4,F 50.8
Hz, H-4), 4.29 (d, 1 H, J1,2 8.3 Hz, H-1); 13C
NMR (CD3OD): l 22.9 (NCOCH3), 57.0 (J
8.3 Hz, C-2), 57.1 (OMe), 61.7 (C-6), 73.7 (J
18.6 Hz, C-3), 75.3 (J 24.6 Hz, C-5), 91.0 (J
181.0 Hz, C-4), 103.4 (C-1), 173.8 (NCꢀO);
19F NMR (CD3OD): l 197.0 (dd, JF,H3 16.1,
JF,H4 50.6 Hz); FABMS (positive-ion): Ex-
pected for C9H17FNO5: 238.1091. Found:
238.1080.
1
Anomer: H NMR (CD3OD): l 1.91 (s, 3 H,
NAc), 3.52–3.75 (m, 2 H, H-6, H-6%), 3.78–
3.94 (m, 3 H, H-2, H-3, H-5), 4.24 (m, J4,F
46.3 Hz, H-4), 5.03 (m, 1 H, J1,2 3.2 Hz, H-1);
13C NMR (CD3OD): l 22.6 (NCOCH3), 55.5
(J 8.0 Hz, C-2), 61.8 (C-6), 70.5 and 70.7 (C-3,
C-5), 91.5 (J 182.8 Hz, C-4), 92.4 (C-1), 173.7
(NCꢀO); 19F NMR (CD3OD): l 199.6 (dd,
JF,H3 14.3, JF,H4 50.6 Hz). Anal. Calcd for
C8H14FNO5: C, 43.05; H, 6.32; F, 8.51; N,
6.28. Found: C, 42.86; H, 6.37; F, 8.61; N,
6.22.
Benzyl 2-acetamido-2,4-dideoxy-4-fluoro-h-
-glucopyranoside (12).—To a solution of
D
compound 9 (1.45 g, 2.94 mmol) in MeOH (40
mL) was added KOH (1.0 g), and the mixture
was stirred for 30 min at rt. The mixture was
neutralized with 1N HCl and concentrated
under reduced pressure to give a solid that
was recrystallized from absolute EtOH to af-
ford 12 (0.81 g, 92.5%) as tiny, white crystals:
Methyl
dideoxy-4-fluoro-h-
2-acetamido-3,6-di-O-acetyl-2,4-
-glucopyranoside (14).—
D
Compound 10 (0.10 g, 0.42 mmol) was treated
with Ac2O (1.0 mL) in pyr (2.0 mL) at 0 °C,
and the mixture was stirred overnight at rt.

258
A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
The solution was concentrated, and the
residue was coevaporated with toluene under
reduced pressure to afford a clear oil which
was crystallized from EtOAc–hexanes to give
14 (0.13 g, 88.2%) as white crystals: Rf 0.64
(EtOAc); mp 112–113 °C; [h]D +58.9° (c 1,
2-Acetamido-1,3,6-tri-O-acetyl-2,4-dide-
oxy-4-fluoro- -glucopyranose (16).—Com-
D
pound 13 (0.10 g, 0.45 mmol) was treated with
acetic anhydride (1.0 mL) in pyr (2.0 mL) at
0 °C, and the mixture was stirred overnight at
rt. The solution was concentrated, and the
residue was coevaporated with toluene under
reduced pressure to produce an opaque oil
that was crystallized from EtOAc–hexanes to
give the b anomer of 16 (0.032 g) as colorless
rods. The remainder of 16 crystallized as a
mixture of a and b anomers (0.125 g, 93.2%).
b Anomer: Rf 0.38 (EtOAc); mp 188–189 °C;
1
CHCl3); H NMR (CDCl3): l 1.95 (s, 3 H,
NAc), 2.08 and 2.10 (2 s, 6 H, 2 OAc), 3.40 (s,
3 H, OMe), 3.95 (m, 1 H, H-5), 4.23–4.29 (m,
2 H, H-2, H-6), 4.39 (m, 1 H, H-6%), 4.49
(dddꢀdt, 1 H, J4,3=J4,5 9.5, J4,F 51.0 Hz,
H-4), 4.68 (t, 1 H, J1,2 =J1,F 3.3 Hz, H-1), 5.31
(ddd, 1 H, J3,2 9.0, J3,4 10.8, J3,F 14.2 Hz, H-3),
5.72 (d, 1 H, JNH,2 9.5 Hz, NH); 13C NMR
(CDCl3): l 20.8 (NCOCH3), 51.7 (J 6.8 Hz,
C-2), 55.5 (OMe), 62.0 (C-6), 67.2 (J 23.6 Hz,
C-5), 71.2 (J 18.6 Hz, C-3), 86.6 (J 186.5 Hz,
C-4), 98.2 (C-1), 170.0, 170.6, and 171.3 (2
OCOCH3, NCꢀO); 19F NMR (CDCl3): l
227.3 (dd, JF,H3 13.8, JF,H4 51.2 Hz). Anal.
Calcd for C13H20FNO7: C, 48.60; H, 6.27; F,
5.91; N, 4.36. Found: C, 48.18; H, 6.35; F,
5.69; N, 4.42.
1
[h]D −29.2° (c 1, CHCl3); H NMR (CDCl3):
l 1.92 (s, 3 H, NAc), 2.09 and 2.11 (3 s, 9 H,
3 OAc), 3.83 (m, 1 H, H-5), 4.21–4.31 (m, 2
H, H-2, H-6), 4.40 (d, 1 H, Jgem 12.3 Hz,
H-6%), 4.53 (dt, 1 H, J4,3=J4,5 9.3, J4,F 50.4
Hz, H-4), 5.23 (ddd, 1 H, J3,2 8.9, J3,4 10.7, J3,F
14.2 Hz, H-3), 5.67 (d, 1 H, J1,2 8.8 Hz, H-1),
5.77 (d, 1 H, JNH,2 9.5 Hz, NH); 13C NMR
(CDCl3): l 20.8 and 20.9 (3 OCOCH3), 23.2
(NCOCH3), 52.6 (J 7.4 Hz, C-2), 61.8 (C-6),
72.1 (J 24.1 Hz, C-5), 86.1 (J 187.0 Hz, C-4),
92.5 (C-1), 169.5, 170.3, 170.6, and 171.2 (3
OCOCH3, NCꢀO); 19F NMR (CDCl3): l
200.0 (dd, JF,H3 13.5, JF,H4 50.1 Hz). Anal.
Calcd for C14H20FNO8: C, 48.14; H, 5.77; F,
5.44; N, 4.01. Found: C, 47.90; H, 5.52; F,
5.63; N, 3.99.
Methyl
2-acetamido-3,6-di-O-acetyl-2,4-
-glucopyranoside (15).—
dideoxy-4-fluoro-i-
D
Compound 11 (0.085 g, 0.357 mmol) was
treated with Ac2O (1.5 mL) in pyr (2.5 mL) at
0 °C, and the mixture was stirred overnight at
rt. The solution was concentrated, and the
residue was coevaporated with toluene under
reduced pressure to afford a crude solid that
was recrystallized from EtOAc–Et2O to af-
ford 15 (0.100 g, 86.8%) as clear, colorless
rods: Rf 0.30 (EtOAc); mp 174–176 °C; [h]D
Methyl 2-acetamido-3,6-di-O-benzyl-2-de-
oxy-4-O-mesyl-h-
To
3,6-di-O-benzyl-a-
D
-glucopyranoside (20).—
solution of methyl 2-acetamido-
-glucopyranoside [16] (17,
a
1
D
−61.4° (c 1, MeOH); H NMR (CDCl3): l
4.75 g, 11.4 mmol) in pyr (60 mL) was added
a solution of methanesulfonyl chloride (5 mL)
in pyr (7 mL) with stirring at 0 °C. The solu-
tion was kept at 0 °C for 11 h and then
poured into ice–water. The resulting solid was
collected by filtration and dissolved in CHCl3;
the solution was washed with H2O, dried
(MgSO4), and concentrated to a solid that was
recrystallized from EtOAc–hexanes to give 20
(4.24 g, 75.2%) as short, glassy needles: Rf
0.39 (3:1 EtOAc–hexanes); mp 194–195 °C
1.80 (s, 3 H, NAc), 1.96 and 1.99 (2 s, 6 H, 2
OAc), 3.40 (s, 3 H, OMe), 3.95 (m, 1 H, H-5),
4.23–4.29 (m, 2 H, H-2, H-6), 4.39 (m, 1 H,
H-6%), 4.49 (dddꢀdt, 1 H, J4,3=J4,5 9.5, J4,F
51.0 Hz, H-4), 4.68 (t, 1 H, J1,2 =J1,F 3.3 Hz,
H-1), 5.31 (ddd, 1 H, J3,2 9.0, J3,4 10.8, J3,F
14.2 Hz, H-3), 5.72 (d, 1 H, JNH,2 9.5 Hz,
NH); 13C NMR (CDCl3): l 20.6 and 22.7
(NCOCH3, 2 OCOCH3), 55.2 (J 5.9 Hz, C-2),
57.3 (OMe), 63.4 (C-6), 72.4 (J 23.6 Hz, C-5),
74.1 (J 18.8 Hz, C-3), 88.7 (J 184.3 Hz, C-4),
102.9 (C-1), 171.8, 172.4, and 173.5 (2
OCOCH3, NCꢀO); 19F NMR (CDCl3): l
227.2 (dd, JF,H3 14.9, JF,H4 49.4 Hz). Anal.
Calcd for C13H20FNO7: C, 48.60; H, 6.27; F,
5.91; N, 4.36. Found: C, 48.74; H, 6.42; F,
6.16; N, 4.43.
1
(dec); [h]D +90.2° (c 1, CHCl3); H NMR
(CDCl3): l 1.87 (s, 3 H, NAc), 2.84 (s, 3
H, SMe), 3.37 (s, 3 H, OMe), 3.70 (dd, 1
H, J6,5 5.2, Jgem 11.0 Hz, H-6), 3.79–3.84 (m,
2 H, H-3, H-6%), 3.88 (m, 1 H, H-5), 4.41
(dddꢀdt, 1 H, J2,1 3.6, J2,3 =J2,NH 10.0 Hz,

A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
259
+103.2° (c 1, CHCl3), lit.+98° (c 0.8,
CHCl3) [19]; 1H NMR (CDCl3): l 1.84 (s, 3 H,
NAc), 2.86 (s, 3 H, SMe), 3.73 (dd, 1 H, J6,5
5.1, Jgem 11.0 Hz, H-6), 3.81 (dd, 1 H, J6%,5 2.4
Hz, H-6%), 3.89 (ddꢀt, 1 H, J3,4 9.1 Hz, H-3),
4.01 (m, 1 H, H-5), 4.45 (dddꢀdt, 1 H,
J2,3=J2,NH 9.7 Hz, H-2), 4.49 and 4.74 (2 d, 2
H, J 11.6 Hz, PhCH2), 4.58 and 4.65 (2 d, 2
H, J 11.8 Hz, PhCH2), 4.69 (d, 2 H, J 2.1 Hz,
PhCH2), 4.74 (m, 1 H, H-4), 4.92 (d, 1 H, J1,2
3.4 Hz, H-1), 5.52 (d, 1 H, NH), 7.28–7.41
(m, 15 H, Ph); 13C NMR (CDCl3): l 23.3
(NCOCH3), 38.5 (SMe), 52.2 (C-2), 68.3 (C-
6), 69.7 (C-5), 69.8 (PhCH2), 73.6 and 73.9 (2
PhCH2), 78.0 (C-4), 78.1 (C-3), 96.6 (C-1),
127.5–128.6, 136.6, 137.1, and 137.9 (Ph),
169.6 (NCꢀO).
H-2), 4.55 and 4.62 (2 d, 2 H, J 11.8 Hz,
PhCH2), 4.67–4.72 (m, 4 H, H-1, H-4,
PhCH2), 5.56 (d, 1 H, NH), 7.25–7.36 (m, 10
H, Ph); 13C NMR (CDCl3):
l
23.3
(NCOCH3), 38.5 (SMe), 52.1 (C-2), 55.3
(OMe), 68.4 (C-6), 69.2 (C-5), 73.5 and 73.9 (2
PhCH2), 78.0 (C-3, C-4), 98.3 (C-1), 127.5–
128.6, 137.1, and 137.8 (Ph), 169.7 (NCꢀO).
Anal. Calcd for C24H31NO8S: C, 58.40; H,
6.33; N, 2.84; S, 6.50. Found: C, 58.44; H,
6.57; N, 2.82; S, 6.61.
Methyl 2-acetamido-3,6-di-O-benzyl-2-de-
oxy-4-O-mesyl-i- -glucopyranoside (21).—
D
To a solution of methyl 2-acetamido-3,6-
di-O-benzyl-b- -glucopyranoside [17] (18,
D
2.00 g, 4.81 mmol) in pyr (30 mL) was added
a solution of methanesulfonyl chloride (3 mL)
in pyr (5 mL) with stirring at 0 °C. The solu-
tion was kept at 0 °C for 24 h and processed
as described for the preparation of 20 to af-
ford 21 (1.75 g, 73.6%) as white rods: Rf 0.31
(2:1 EtOAc–hexanes); mp 170–171 °C (dec);
Methyl 2-acetamido-3,6-di-O-benzyl-2,4-di-
deoxy-4-fluoro-h- -galactopyranoside (23).—
D
To a solution of 20 (2.1 g, 4.25 mmol) in
anhyd MeCN (30 mL) was added a solution
of anhyd tetra-n-butylammonium fluoride
[20] (10.9 g, 9.8 equiv) in anhyd MeCN (15
mL), and the solution was heated at reflux
temperature for 6 days. The solvent was re-
moved under reduced pressure, and the
residue was dissolved in CH2Cl2; the solution
was washed with H2O and concentrated to a
brownish solid. The aqueous layer was ex-
tracted with CH2Cl2, and the extracts were
dried (MgSO4) and concentrated to a brown-
ish solid. The combined samples of crude
brownish solids were recrystallized from
EtOAc–hexanes to afford 23 (0.96 g, 54.3%)
as tiny, white needles: Rf 0.47 (2:1 EtOAc–
hexanes); mp 203–204 °C; [h]D +131.7° (c 1,
1
[h]D +24.0° (c 1, CHCl3); H NMR (CDCl3):
l 1.89 (s, 3 H, NAc), 2.87 (s, 3 H, SMe), 3.40
(m, 1 H, H-2), 3.49 (s, 3 H, OMe), 3.72 (m, 2
H, H-5, H-6), 3.86 (dd, 1 H, J6%,5 3.9, Jgem 12.8
Hz, H-6%), 4.33 (dd, 1 H, J3,2 9.4 Hz, H-3),
4.61 (d, 2 H, J 1.6 Hz, PhCH2), 4.64 (m, 1 H,
H-4), 4.67 and 4.78 (2 d, 2 H, J 11.1 Hz,
PhCH2), 4.83 (d, 1 H, J1,2 7.9 Hz, H-1), 5.84
(d, 1 H, JNH,2 7.6 Hz, NH), 7.27–7.38 (m, 10
H, Ph); 13C NMR (CDCl3):
l
23.5
(NCOCH3), 38.6 (SMe), 56.9 (OMe), 57.6 (C-
2), 68.8 (C-6), 73.3 (C-5), 73.6 (PhCH2), 74.0
(PhCH2), 78.2 (C-3), 78.5 (C-4), 100.3 (C-1),
127.7–128.6, 137.6, and 137.9 (Ph), 171.5
(NCꢀO). Anal. Calcd for C24H31NO8S: C,
58.40; H, 6.33; N, 2.84; S, 6.50. Found: C,
58.32; H, 6.51; N, 2.87; S, 6.73.
1
CHCl3); H NMR (CDCl3): l 1.93 (s, 3 H,
NAc), 3.32 (s, 3 H, OMe), 3.55 (dddd, 1 H,
J3,4 2.3, J3,2 11.0, J3,F 27.8 Hz, H-3), 3.64
(dddd, 2 H, J6,F 1.4, Jgem 9.4 Hz, H-6, H-6%),
3.72 (m, 1 H, H-2), 3.85 (dddꢀdt, 1 H,
J5,6=J5,6% 6.6, J5,F 28.8 Hz, H-5), 4.47 and
4.76 (2 d, 2 H, J 12.3 Hz, PhCH2), 4.57 (d, 2
H, J 3.3 Hz, PhCH2), 4.77 (overlapping d, 1
H, H-1), 4.92 (dd, 1 H, J4,F 50.2 Hz, H-4),
5.34 (d, 1 H, JNH,2 8.8 Hz, NH), 7.29–7.35 (m,
10 H, Ph); 13C NMR (CDCl3): l 23.4
(NCOCH3), 48.4 (C-2), 55.4 (OMe), 68.0 (J
6.5 Hz, C-6), 68.2 (J 17.9 Hz, C-5), 70.7 and
73.7 (2 PhCH2), 74.0 (J 18.6 Hz, C-3), 85.3 (J
184.9 Hz, C-4), 98.7 (C-1), 127.7–128.5, 137.1,
and 137.8 (Ph), 169.8 (NCꢀO); 19F NMR
Preparation of benzyl 2-acetamido-3,6-di-O-
benzyl-2-deoxy-4-O-mesyl-h- -glucopyrano-
D
side (22).—To a solution of benzyl 2-ac-
etamido-3,6-di-O-benzyl-a- -glucopyranoside
D
[18] (19, 0.390 g, 0.793 mmol) in pyr (5 mL)
was added a solution of methanesulfonyl chlo-
ride (0.5 mL) in pyr (0.7 mL) with stirring at
0 °C. The solution was kept at 0 °C for 24 h
and processed as described for the preparation
of 20 to afford 22 (0.45 g, 77.5%) as tiny,
white needles: Rf 0.62 (2:1 EtOAc–hexanes);
mp 172–173 °C, lit. 168–170 °C [19]; [h]D

260
A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
25 (0.96 g, 62.0%) as an amorphous, white
solid: Rf 0.22 (1:1 EtOAc–hexanes); mp 180–
182 °C, lit. 186–187 °C [9a]; [h]D +137.8° (c
(CDCl3): l 219.9 (dt, JF,H3ꢀJF,H5 28.2, JF,H4
50.3 Hz). Anal. Calcd for C23H28FNO5: C,
66.17; H, 6.76; F, 4.55; N, 3.36. Found: C,
65.95; H, 6.57; F, 4.39; N, 3.26.
1
1, CHCl3), lit.+117° (c 1, CHCl3) [9a]; H
NMR (CDCl3): l 2.00 (s, 3 H, NAc), 3.65 (m,
2 H, H-3, H-6), 3.72 (m, 1 H, H-6), 3.94
(dddꢀdt, 1 H, J5,6 =J5,6% 6.5, J5,F 29.2 Hz,
H-5), 4.45 and 4.67 (2 d, 2 H, J 11.8 Hz,
PhCH2), 4.48 and 4.75 (2 d, 2 H, J 12.2 Hz,
PhCH2), 4.56 (s, 2 H, PhCH2), 4.57 (m, 1 H,
H-2), 4.95 (dd, 1 H, J4,3 2.0, J4,F 50.1 Hz,
H-4), 5.00 (d, 1 H, J1,2 3.7 Hz, H-1), 5.33 (d,
1 H, JNH,2 8.9 Hz, NH), 7.24–7.38 (m, 15 H,
Ph); 13C NMR (CDCl3): l 23.3 (NCOCH3),
48.4 (C-2), 67.9 (J 5.1 Hz, C-6), 68.6 (J 18.6
Hz, C-5), 69.9 (PhCH2), 70.8 and 73.7 (2
PhCH2), 74.2 (J 18.5 Hz, C-3), 85.3 (J 185.2
Hz, C-4), 97.2 (C-1), 127.7–128.6, 137.1, and
Methyl 2-acetamido-3,6-di-O-benzyl-2,4-di-
deoxy-4-fluoro-i- -galactopyranoside (24).—
D
To a solution of 21 (0.80 g, 1.62 mmol) in
anhyd MeCN (25 mL) was added a solution
of anhyd tetra-n-butylammonium fluoride [20]
(6.36 g, 15 equiv) in anhyd MeCN (7.5 mL),
and the mixture was heated at reflux tempera-
ture for 4 days. The reaction mixture was
processed as described for the preparation of
23 to afford a brownish solid that was sub-
jected to flash chromatography on silica gel
(3:1 EtOAc–hexanes). The resulting solid was
recrystallized from EtOAc–hexanes to afford
24 (0.43 g, 64.0%) as an amorphous, white
solid: Rf 0.38 (2:1 EtOAc–hexanes); mp 208–
19
137.8 (Ph), 169.8 (NCꢀO); F NMR (CDCl3):
1
l 220.3 (dt, JF,H3=JF,H5 28.6, JF,H4 50.9 Hz).
Anal. Calcd for C29H32FNO5: C, 70.57; H,
6.53; F, 3.85; N, 2.84. Found: C, 70.44; H,
6.49; F, 4.04; N, 2.86.
210 °C; [h]D +29.9° (c 1.1, CHCl3); H NMR
(CDCl3): l 1.93 (s, 3 H, NAc), 3.31 (m, 1 H,
H-2), 3.49 (s, 3 H, OMe), 3.66–3.76 (m, 3 H,
H-5, H-6, H-6%), 4.38 (dddd, 1 H, J3,4 2.4, J3,2
10.9, J3,F 28.1 Hz, H-3), 4.52 and 4.70 (2 d, 2
H, J 11.6 Hz, PhCH2), 4.57 (s, 2 H, PhCH2),
4.88 (dd, 1 H, J4,F 50.2 Hz, H-4), 4.99 (d, 1 H,
J1,2 8.2 Hz, H-1), 5.75 (br s, 1 H, NH), 7.26–
Methyl 2-acetamido-2,4-dideoxy-4-fluoro-h-
-galactopyranoside (26).—A mixture of 23
D
(0.83 g, 1.5 mmol), acetic acid (20 mL), and
10% Pd–C (1.0 g) was subjected to a hydro-
gen pressure (55 psig) for 4 days. The mixture
was diluted with MeOH, neutralized with Am-
berlite IRA-400 (OH−) resin, filtered through
Celite 521 (Aldrich), and the filtrate was con-
centrated under reduced pressure to a white
solid that was recrystallized (EtOH–acetone)
to afford 26 (0.30 g, 80.6%) as an amorphous,
white solid: Rf 0.50 (1:3 MeOH–CHCl3); mp
13
7.37 (m, 10 H, Ph); C NMR (CDCl3): l 23.7
(NCOCH3), 55.2 (C-2), 56.9 (OMe), 67.7 (C-
6), 71.8 (PhCH2), 72.0 (J 18.5 Hz, C-5), 73.6
(PhCH2), 74.5 (J 18.9 Hz, C-3), 85.1 (J 184.7
Hz, C-4), 100.2 (C-1), 127.3–128.5, and 137.6
(Ph), 171.0 (NCꢀO); 19F NMR (CDCl3): l
220.1 (dt, JF,H3 =JF,H5 28.2, JF,H4 50.5 Hz).
Anal. Calcd for C23H28FNO5: C, 66.17; H,
6.76; F, 4.55; N, 3.36. Found: C, 66.32; H,
7.00; F, 4.34; N, 3.41.
1
227–228 °C; [h]D +173.4° (c 1, MeOH); H
NMR (CD3OD): l 1.98 (s, 3 H, NAc), 3.32 (s,
3 H, OMe), 3.64 (m, 1 H, H-6), 3.69 (m, 1 H,
H-6%), 3.75–3.83 (m, 2 H, J3,4 2.6 Hz, H-3,
H-5), 4.18 (dd, 1 H, J2,3 11.2 Hz, H-2), 4.64 (d,
1 H, J1,2 3.6 Hz, H-1), 4.70 (dd, 1 H, J4,3 2.6,
J4,F 47.4 Hz, H-4); 13C NMR (CD3OD): l 22.6
(NCOCH3), 51.7 (C-2), 55.8 (OMe), 61.4 (J
5.2 Hz, C-6), 68.2 (J 19.4 Hz, C-3), 71.3 (J
18.5 Hz, C-5), 90.4 (J 180.7 Hz, C-4), 100.0
(C-1), 173.8 (NCꢀO); 19F NMR (CD3OD): l
221.2 (dt, JF,H3=JF,H5 29.0, JF,H4 50.6 Hz).
Anal. Calcd for C9H16FNO5: C, 45.57; H,
6.80; F, 8.01; N, 5.90. Found: C, 45.39; H,
6.88; F, 8.24; N, 5.72.
Benzyl 2-acetamido-3,6-di-O-benzyl-2,4 - di-
deoxy-4-fluoro-h- -galactopyranoside (25).—
D
To a solution of 22 (1.78 g, 3.10 mmol) in
anhyd MeCN (60 mL) was added a solution
of anhyd tetra-n-butylammonium fluoride [20]
(9.41 g, 11.6 equiv) in anhyd MeCN (40 mL),
and the mixture was heated at reflux tempera-
ture for 4 days. The reaction mixture was
processed as described for the preparation of
23 to afford a brownish solid that was sub-
jected to flash chromatography on silica gel
(1:1 EtOAc–hexanes). The resulting solid was
recrystallized from EtOAc–hexanes to afford

A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
261
Methyl 2-acetamido-2,4-dideoxy-4-fluoro-
i- -galactopyranoside (27).—A mixture of 24
treated with acetic anhydride (1.5 mL) in pyr
(2.5 mL) at 0 °C, and the mixture was stirred
overnight at rt. The solution was concen-
trated, and the residue was coevaporated with
toluene under reduced pressure to obtain a
crude product that was recrystallized from
EtOAc–hexanes to afford 29 (0.23 g, 84.1%)
as white needles: Rf 0.18 (3:1 EtOAc–hex-
anes); mp 166–168 °C; [h]D +101.6° (c 1,
D
(0.448 g, 0.833 mmol), acetic acid (20 mL),
and 10% Pd–C (0.6 g) was subjected to a
hydrogen pressure (55 psig) for 4 days. The
mixture was processed as described for 26 to
afford a solid that was recrystallized from
EtOH–hexanes to give 27 (0.17 g, 86.5%) as
tiny, colorless crystals: Rf 0.35 (1:3 MeOH–
CHCl3); mp 209–211 °C; [h]D −24.8° (c 1,
1
CHCl3); H NMR (CDCl3): l 1.95 (s, 3 H,
1
MeOH); H NMR (CD3OD): l 1.97 (s, 3 H,
NAc), 2.06 and 2.07 (2 s, 6 H, 2 OAc), 3.38 (s,
3 H, OMe), 3.98 (dddꢀdt, 1 H, J5,6 6.5, J5,F
28.4 Hz, H-5), 4.22 (dd, 1 H, Jgem 11.3 Hz,
H-6), 4.29 (ddd, 1 H, J6%,5 6.8, J6%,F 0.7 Hz,
H-6%), 4.61 (m, 1 H, H-2), 4.75 (d, 1 H, J1,2 3.5
Hz, H-1), 4.77 (dd, 1 H, J4,3 2.3, J4,F 50.7 Hz,
H-4), 5.09 (dddd, 1 H, J3,2 11.3, J3,F 27.8 Hz,
H-3), 5.63 (d, 1 H, JNH,2 9.6 Hz, NH); 13C
NMR (CDCl3): l 20.7 and 20.8 (2 OCOCH3),
23.3 (NCOCH3), 47.7 (C-2), 55.5 (OMe), 61.9
(J 5.3 Hz, C-6), 67.0 (J 18.0 Hz, C-5), 68.8 (J
17.4 Hz, C-3), 86.4 (J 186.0 Hz, C-4), 98.6
(C-1), 169.9, 170.4, and 171.2 (2 OCOCH3,
NCꢀO); 19F NMR (CDCl3): l 219.2 (dt,
NAc), 3.45 (s, 3 H, OMe), 3.58 (dddꢀdt, 1
H, J5,6 =J5,6% 6.6, J5,F 27.6 Hz, H-5), 3.72
(dddd, 1 H, J3,2 10.9, J3,4 2.5 Hz, H-3), 3.73 (d,
2 H, H-6, H-6%), 3.86 (dd, 1 H, H-2), 4.36 (dd,
1 H, J1,2 8.3, J1,F 0.8 Hz, H-1), 4.72 (dd, 1 H,
13
H-4); C NMR (CD3OD): l 23.0 (NCOCH3),
54.4 (C-2), 57.0 (OMe), 61.1 (J 5.1 Hz, C-6),
71.7 (J 19.3 Hz, C-3), 75.2 (J 18.1 Hz, C-5),
89.6 (J 181.0 Hz, C-4), 103.6 (C-1), 174.0
(NCꢀO); 19F NMR (CD3OD): l 217.1 (dt,
J
F,H3 =JF,H5 28.3, JF,H4 50.6 Hz). Anal. Calcd
for C9H16FNO5: C, 45.57; H, 6.80; F, 8.01; N,
5.90. Found: C, 45.70; H, 6.94; F, 8.19; N,
5.86.
J
F,H3=JF,H5 28.1, JF,H4 50.7 Hz). Anal. Calcd
2-Acetamido-2,4-dideoxy-4-fluoro-
D
-galac-
for C13H20FNO7: C, 48.60; H, 6.27; F, 5.91;
N, 4.40. Found: C, 48.69; H, 6.05; F, 6.12; N,
4.43.
topyranose (28).—A mixture of 25 (0.600 g,
1.22 mmol), acetic acid (15 mL), and 10%
Pd–C (1.0 g) was subjected to a hydrogen
pressure (55 psig) for 4 days. The mixture was
processed as described for 26 to afford a solid
that was recrystallized from EtOH–Et2O to
obtain 28 (0.27 g, 86.7%) as tiny, white
needles: Rf 0.17 (1:3 MeOH–CHCl3); mp
205–209 °C, lit. 195 °C [9a]; [h]D +87.9°
(equil) (c 1, MeOH), lit.+85.5° (c 1, MeOH)
[9a]. a Anomer: 1H NMR (CD3OD): l 1.93 (s,
3 H, NAc), 3.58 (dddd, 1 H, J6,5 6.7, J6,F 1.0,
Jgem 11.0 Hz, H-6), 3.65 (dd, 1 H, H-6%), 3.84
(ddd, 1 H, J3,2 11.1, J3,4 2.5, J3,F 28.8 Hz, H-3),
4.00 (dddꢀdt, 1 H, J5,6% 6.8, J5,F 30.3 Hz,
H-5), 4.13 (dd, 1 H, H-2), 4.71 (dd, 1 H, H-4),
5.07 (d, 1 H, J1,2 3.4 Hz, H-1); 13C NMR
(CD3OD): l 22.6 (NCOCH3), 52.2 (C-2), 61.3
(C-6), 68.0 (J 18.9 Hz, C-3), 70.6 (J 17.5 Hz,
C-5), 90.5 (J 179.6 Hz, C-4), 92.8 (C-1), 173.9
(NCꢀO); 19F NMR (CD3OD): l 221.1 (dt,
Methyl
dideoxy-4-fluoro-i-
2-acetamido-3,6-di-O-acetyl-2,4-
-galactopyranoside (30).
D
—Compound 27 (0.10 g, 0.42 mmol) was
treated with Ac2O (1.0 mL) in pyr (2.0 mL) at
0 °C, and the mixture was stirred overnight at
rt. The solution was processed as described for
29 to afford a solid that was recrystallized
from EtOAc to obtain 30 (0.12 g, 88.9%) as
tiny, white needles: Rf 0.20 (EtOAc); mp 235–
236 °C; [h]D −19.4° {c 1, (1:1 MeOH–
1
CHCl3)}; H NMR (1:1 CD3OD–CDCl3): l
1.97 (s, 3 H, NAc), 2.13 and 2.12 (2 s, 6 H, 2
OAc), 3.52 (s, 3 H, OMe), 3.91 (dddꢀdt, 1 H,
J5,6 6.5, J5,F 26.4 Hz, H-5), 4.09 (dd, 1 H, J2,3
11.1 Hz, H-2), 4.26 (dd, 1 H, Jgem 11.3 Hz,
H-6), 4.37 (ddd, 1 H, J6%,5 6.7, J6%,F 0.8 Hz,
H-6%), 4.59 (dd, 1 H, J1,2 8.4, J1,F 1.0 Hz, H-1),
4.84 (dd, 1 H, J4,3 2.5, J4,F 50.6 Hz, H-4), 5.13
(dddd, 1 H, J3,F 27.7 Hz, H-3); 13C NMR
(1:1 CD3OD–CDCl3): l 20.0 and 20.1 (2
OCOCH3), 22.2 (NCOCH3), 50.2 (C-2), 56.2
(OMe), 61.5 (J 5.3 Hz, C-6), 70.5 (C-3, C-5),
85.3 (J 185.3 Hz, C-4), 101.1 (C-1), 170.5,
J
F, H3=JF,H5 29.5, JF,H4 50.8 Hz).
Methyl 2-acetamido-3,6-di-O-acetyl-2,4-
dideoxy-4-fluoro-h- -galactopyranoside (29).
—Compound 26 (0.200 g, 0.843 mmol) was
D

262
A. Berkin et al. / Carbohydrate Research 326 (2000) 250–263
170.7, and 171.7 (2 OCOCH3, NCꢀO); 19F
NMR (1:1 CD3OD–CDCl3): l 219.2 (dt, JF,
H3 =JF,H5 28.1, JF,H4 50.7 Hz). Anal. Calcd for
C13H20FNO7: C, 48.60; H, 6.27; F, 5.91; N,
4.40. Found: C, 48.65; H, 5.91; F, 5.77; N,
4.36.
The viability was usually greater than 85%.
The cells were plated in triplicate on
fibronectin-coated tissue culture dishes (Fal-
con 35×10 mm) at a density of 2×106 cells
per plate. They were incubated in 2 mL of
Williams’ Medium E containing 10% Fetal
bovine serum and 1% antibiotic–antimycotic
mixtures. After 2 h, the non-adherent cells
were removed, and the attached cells were fed
with fresh plating medium for a 24-h period.
The cells were then provided with fresh
medium containing the necessary isotopes
and/or monosaccharide derivative. For GAG
labeling, [3H]GlcN (2 mCi/mL) and [35S]SO4 (4
mCi/mL) were included in the medium. For
experiments monitoring protein synthesis, the
cells were incubated with [14C]Leu (0.5 mCi/
mL). The labeled cellular GAGs, or total
proteins, were harvested 24 h later.
GAG isolation.—After the 24-h labeling pe-
riod, the medium was separated from the cells,
and the cells were solubilized in 4 M
guanidine·HCl, 2% Triton X-100 in 0.05 M
acetate buffer (pH 6.0). A GAG carrier (1
mg/mL each of chondroitin sulfate, hyaluro-
nan, and heparin) was added to all of the
samples. The isolation of radioactive GAGs
was based on the cetylpyridinium chloride
(CPC) precipitation technique described by
Hronowski and Anastassiades [28]. Briefly,
media and cell fractions were each subjected
to papain digestion, and the GAGs were pre-
cipitated as sodium salts by CPC. After
lyophilization, the samples were dissolved in a
known volume of water for analysis. Media
GAGs were analyzed only during preliminary
experiments. Cellular GAGs served as the as-
say material for the effects of the sugar
analogs.
Preparation of 2-acetamido-1,3,6-tri-O-
acetyl-2,4-dideoxy-4-fluoro- -galactopyranose
D
(31).—Compound 28 (0.10 g, 0.42 mmol) was
treated with Ac2O (1.0 mL) in pyr (2.0 mL) at
0 °C, and the mixture was stirred overnight at
rt. The solution was processed as described for
29 to afford a solid that was recrystallized
from EtOAc–hexanes to obtain 31 (0.16 g,
quant) as clear, colorless rods: Rf 0.13 and
0.19 (3:1 EtOAc–hexanes); mp 140–142 °C,
lit. 155−158 °C [9a]; [h]D +114.0° (c 1,
CHCl3), lit.+144° (c 1, CHCl3) [9a]. a
1
Anomer: H NMR (CDCl3): l 1.93 (s, 3 H,
NAc), 2.12 and 2.15 (2 s, 9 H, 3 OAc), 4.09
(dddꢀdt, 1 H, J5,6 6.6, J5,F 27.8 Hz, H-5),
4.19 (dd, 1 H, Jgem 11.2 Hz, H-6), 4.37 (ddd, 1
H, J6%,5 6.7, J6%,F 0.8 Hz, H-6%), 4.75 (m, 1 H,
H-2), 4.83 (dd, 1 H, J4,3 2.2, J4,F 50.0 Hz,
H-4), 5.16 (dddd, 1 H, J3,2 11.6, J3,F 26.9 Hz,
H-3); 5.57 (d, 1 H, H-2), 6.20 (d, 1 H, J1,2 3.6
Hz, H-1); 13C NMR (CDCl3): l 20.7, 20.8,
and 20.9 (3 OCOCH3), 23.1 (NCOCH3), 46.8
(C-2), 61.4 (J 6.2 Hz, C-6), 68.0 (J 18.4 Hz,
C-5), 68.9 (J 18.2 Hz, C-3), 85.8 (J 186.7 Hz,
C-4), 91.2 (C-1), 168.7, 170.0, 170.4, and 171.4
(3 OCOCH3, NCꢀO); 19F NMR (CDCl3): l
219.0 (dt, JF,H3 =JF,H5 27.4, JF,H4 50.2 Hz).
Biological e6aluation.—Hepatocytes were
obtained from 6–8-week-old, female Swiss
white mice (Charles River Canada, St. Con-
stant, Quebec) by the procedure described pre-
viously [26,27]. Briefly, the liver was perfused
with 50 mL of 0.01 M Hepes buffer (pH 7.4)
containing 0.5 mM EGTA, followed by 50
mL of a collagenase type-IV solution (0.5
mg/mL) in 0.1 M Hepes (pH 7.6). The liver
was removed, and the hepatocytes separated
from the capsule by gentle teasing. The pooled
cells were centrifuged at 200 rpm at 5 °C for 5
min and washed once with fresh medium.
After resuspension in 20 mL of Williams’
Medium E and filtration through a Nitex 110
nylon membrane, the cells were exposed to
Trypan Blue and counted on a hemocytome-
ter to determine the viability and cell number.
Total protein isolation.—Total cell protein
synthesis was determined by measuring
[14C]Leu incorporation. After the cells had
been incubated in the labeling medium for the
specified period of time, the culture medium
was removed, and the cell fractions were solu-
bilized by treatment with 1% sodium dodecyl
sulfate and then combined with the medium.
The proteins were precipitated by the addi-
tion of 3 mL of a 10% aq solution of
trichloroacetic acid (TCA) containing 10 mg/
mL of DL-leucine. The samples were heated at

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90 °C for 30 min to destroy leucine tRNA and
then cooled at 4 °C for 30 min. Using a Mil-
lipore 1225 sampling manifold, each sample
was passed through a glass microfiber filter
(Whatman 934-AH), and the filter was washed
three times with 5 mL of a cold, 5% aq
solution of TCA containing 10 mg/mL of
DL-leucine and once with 3 mL of EtOH. The
filters were air-dried and immersed in 5 mL of
scintillation solution, and the radioactivity
was measured. Nonspecific binding of [3H]Leu
was not more than 5% of the total incorpo-
rated radioactivity.
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the Medical Research Council of Canada
(Grant MT-3153) for financial support.
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