Synthesis and biological evaluation of a radiolabeled analog of methyl 2-acetamido-2,4-dideoxy-β-D-xylo-hexopyranoside directed towards influencing cellular glycosaminoglycan biosynthesis

Article abstract of DOI:10.1016/S0008-6215(01)00285-3

Two methods are presented for the synthesis of methyl 2-acetamido-2,4-dideoxy-β-D-xylo-hexopyranoside. The first method employs the Barton-McCombie deoxygenation methodology, and the second method utilizes an oxidation-β-elimination methodology that allows for the incorporation of hydrogen isotopes into the title compound. Hence, methyl 2-acetamido-2,4-dideoxy-β-D-xylo-hexopyranoside (4) and methyl 2-acetamido-2,4-dideoxy-β-D-xylo-hexopyranoside-6-t (14) were synthesized and evaluated for their ability to inhibit hepatocyte, cell-surface glycosaminoglycan biosynthesis and to incorporate a [³H] radiolabel into isolated glycosaminoglycans, respectively. Compound 4, at a concentration of 1.0 mM, demonstrated a reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated glycosaminoglycans by 69 and 59%, of the control cultures, respectively. At 10 and 20 mM, 4 demonstrated a maximum inhibition of incorporation of both radiolabels to approximately 10% of the control cultures. Compound 14 demonstrated a maximum incorporation of a [³H] radiolabel into isolated cell-surface glycosaminoglycans at 10 and 20 mM. The mechanism of inhibition of glycosaminoglycan biosynthesis is due, in part, to the incorporation of a 4-deoxy moiety into glycosaminoglycan chains resulting in premature chain termination.

Full text of DOI:10.1016/S0008-6215(01)00285-3

Carbohydrate Research 337 (2002) 37–44
www.elsevier.com/locate/carres
Synthesis and biological evaluation of a radiolabeled analog of
-xylo-hexopyranoside directed
methyl 2-acetamido-2,4-dideoxy-b-
D
towards influencing cellular glycosaminoglycan biosynthesis
Ali Berkin,a Walter A. Szarek,a,* Robert Kisilevskyb
aDepartment of Chemistry, Queen’s Uni6ersity, Kingston, Ont., Canada K7L 3N6
bDepartments of Pathology and Biochemistry, Queen’s Uni6ersity, and The Syl and Molly Apps Research Center, Kingston General Hospital,
Kingston, Ont., Canada K7L 3N6
Received 5 July 2001; accepted 2 October 2001
Abstract
Two methods are presented for the synthesis of methyl 2-acetamido-2,4-dideoxy-b-D-xylo-hexopyranoside. The first method
employs the Barton–McCombie deoxygenation methodology, and the second method utilizes an oxidation–b-elimination
methodology that allows for the incorporation of hydrogen isotopes into the title compound. Hence, methyl 2-acetamido-2,4-
dideoxy-b-
D
-xylo-hexopyranoside (4) and methyl 2-acetamido-2,4-dideoxy-b-D-xylo-hexopyranoside-6-t (14) were synthesized and
evaluated for their ability to inhibit hepatocyte, cell-surface glycosaminoglycan biosynthesis and to incorporate a [3H] radiolabel
into isolated glycosaminoglycans, respectively. Compound 4, at a concentration of 1.0 mM, demonstrated a reduction of
D
-[3H]glucosamine and [35S]sulfate incorporation into isolated glycosaminoglycans by 69 and 59%, of the control cultures,
respectively. At 10 and 20 mM, 4 demonstrated a maximum inhibition of incorporation of both radiolabels to approximately 10%
of the control cultures. Compound 14 demonstrated a maximum incorporation of a [3H] radiolabel into isolated cell-surface
glycosaminoglycans at 10 and 20 mM. The mechanism of inhibition of glycosaminoglycan biosynthesis is due, in part, to the
incorporation of a 4-deoxy moiety into glycosaminoglycan chains resulting in premature chain termination. © 2002 Elsevier
Science Ltd. All rights reserved.
Keywords: Glycosaminoglycan; Methyl 2-acetamido-2,4-dideoxy-b-
D
-xylo-hexopyranoside; Radiolabel; Incorporation
monosaccharides in influencing proteoglycan biosynthe-
sis in culture.19–22
1. Introduction
Proteoglycans are very important constituents of cell-
surface membranes and have been demonstrated to
play a role in several cellular functions including cell
migration, differentiation, communication, and prolif-
eration.1 In a disease state, alterations in cellular pro-
teoglycan synthesis can affect the structure and
functions of these glycoconjugates. These proteoglycans
have been implicated in several pathologies including
those associated with Alzheimer’s disease,2–7 cancer,8–10
diabetes,11–15 and the herpes simplex virus.16–18 We
have launched a program to investigate the role of
The proteoglycans of interest are those which contain
heparan sulfate as the glycosaminoglycan (GAG) por-
tion and are localized on the cell-surface of mouse
hepatocytes.21,23 Heparan sulfate is composed of a re-
peating disaccharide unit of a
linked (1“4) to a uronic acid residue (either
curonic acid or -iduronic acid) and can be variably N-
D
-glucosamine residue
D
-glu-
L
and O-sulfonated. This GAG is linked to a core protein
through a conserved tetrasaccharide region consisting
of
-
D
-GlcA-b-(1“3)-
D
-Gal-b-(1“3)- -Gal-b-(1“4)-
D
D
-Xyl-b- attached to a serine or threonine residue of
the core protein. Monosaccharide analogs of the com-
ponents of the tetrasaccharide region including 3-de-
* Corresponding author. Tel.: +1-613-5332643; fax: +1-
613-5336532.
oxy-D
-xylo-hexopyranose (3-deoxy-D-galactose) and a
E-mail address: szarekw@chem.queensu.ca (W.A. Szarek).
4-chloro-4-deoxy analog of
D
-glucuronic acid, namely
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00285-3

38
A. Berkin et al. / Carbohydrate Research 337 (2002) 37–44
methyl (methyl 4-chloro-4-deoxy-b-
D
-galactopyran-
genic cell growth by treatment separately with several
deoxy and deoxyfluoro analogs of 2-acetamido-2-de-
oxy-D-hexose. However, the mechanism of inhibition of
GAG synthesis is not known for these analogs. Herein
we describe the synthesis of methyl 2-acetamido-2,4-
osid)uronate, demonstrated impaired cellular GAG
chain elongation of primary hepatocytes in culture.21
However, no inhibition of heparan sulfate synthesis was
observed with mouse hepatocytes exposed to 4-deoxy-
dideoxy-b-D-xylo-hexopyranoside and the radiolabeled
L
-threo-pentose, although inhibition of neuronal and
astrocytic proteoglycans was observed.22 Monosaccha-
ride analogs of the components of the repeating disac-
charide unit of heparan sulfate, including 4-deoxy
analog, namely methyl 2-acetamido-2,4-dideoxy-b-D-
xylo-hexopyranoside-6-t, and their effects on GAG
biosynthesis.
analogs of 2-acetamido-2-deoxy-D
-glucose20 and 4-de-
oxy-4-fluoro analogs of 2-acetamido-2-deoxy-D-
hexose,19 have demonstrated significant inhibition of
GAG biosynthesis. Furthermore, Bernacki and co-
workers24–26 have described the inhibition of tumori-
2. Results and discussion
Chemical synthesis.—The 4-deoxy analogs of methyl
2-acetamido-2-deoxy-b-D-glucopyranoside were synthe-
sized as shown in Schemes 1 and 2. Several methods
exist for the deoxygenation of carbohydrates. These
have been reviewed by Hartwig,27 albeit many of the
conventional methods are applicable only to unhin-
dered primary alcohols. Following the Barton–Mc-
Combie deoxygenation procedure,28 compound 129 in
tetrahydrofuran was sequentially treated with sodium
hydride, carbon disulfide, and methyl iodide to afford
the 4-O-methylxanthyl derivative, namely methyl 2-
acetamido-3,6-di-O-benzyl-2-deoxy-4-O-[(methylthio)-
thiocarbonyl]-b- -glucopyranoside (2), in 93.5% yield
D
following column chromatography. 1H NMR spec-
troscopy of 2 showed that the signal for the H-4 proton
had shifted downfield by 2.3 ppm relative to the corre-
sponding signal in the spectrum of 1. Treatment of 2
with tri-n-butyltin hydride in toluene at refluxing tem-
perature afforded the deoxygenated product, namely
Scheme 1. Reagents and conditions: (i) NaH, THF, CS2, MeI
(93.5%); (ii) Bu3SnH, AIBN, toluene (70.0%); (iii) H2, Pd–C,
MeOH (86.5%).
methyl 2-acetamido-3,6-di-O-benzyl-2,4-dideoxy-b-D-
xylo-hexopyranoside (3), in 70.0% yield following
column chromatography and recrystallization. Struc-
tural confirmation of 3 was obtained by 1H NMR
spectroscopy; signals for two protons were observed at
1.4 and 2.2 ppm corresponding to the two protons at
C-4. Also, 13C NMR spectroscopy of 3 exhibited an
upfield shift by 43.6 ppm of the signal for C-4 relative
to the corresponding signal in the spectrum of 1.
Removal of the benzyl protecting groups of 3 was
accomplished using palladium-on-carbon (Pd–C) and
hydrogen to afford methyl 2-acetamido-2,4-dideoxy-b-
D
-xylo-hexopyranoside (4) in 86.5% yield following
column chromatography. Compound 4 was evaluated
for the inhibition of hepatocyte cell-surface GAG
synthesis.
The synthesis of a radiolabeled analog of 4 (Scheme
2) was begun by the treatment of methyl 2-acetamido-2-
deoxy-b-
chloride in pyridine to afford methyl 2-acetamido-2-de-
oxy-6-O-triphenylmethyl-b- -glucopyranoside (6) in
79.1% yield following crystallization of the crude reac-
tion product. Acetylation of 6 proceeded smoothly
using acetic anhydride in pyridine to afford methyl
D
-glucopyranoside (5)30 with triphenylmethyl
D
Scheme 2. Reagents and conditions: (i) TrCl, pyr (79.1%); (ii)
Ac2O, pyr (89.3%); (iii) HCO2H, Et2O (89.9%); (iv) SO3·pyr,
Et3N, Me2SO (87.0%); (v) NaBH4, MeOH (87.4%); (vi) H2,
Pd–C, MeOH (73.0%).

A. Berkin et al. / Carbohydrate Research 337 (2002) 37–44
39
with a mixture of Me2SO, sulfur trioxide–pyridine
complex, and triethylamine to afford methyl 2-acetam-
ido-3-O-acetyl-2,4-dideoxy-a-L-threo-hex-4-enodialdo-
1,5-pyranoside (9) in 87.0% yield. In the 1H NMR
spectrum of 9, the H-4 signal appeared as a doublet of
doublets rather than as simply a doublet, owing to
long-range coupling (J 1.0 Hz) to H-2, an observation
that was confirmed by NOE experiments. Perlin et al.34
also described long-range coupling between H-2 and
H-4 in similar 4-enopyranoside systems, and the pre-
ferred conformation of 9 may be similar to the envelope
form suggested by Perlin et al. for methyl 2,3-di-O-ace-
Fig. 1. Possible molecular structure of 9 as proposed by Perlin
and co-workers34 for an analog of 9.
tyl-4-deoxy-a-L-threo-hexo-4-enodialdo-1,5-pyranoside
(Fig. 1). Compound 9 was treated with sodium borohy-
dride in MeOH for 20 min to afford methyl 2-acetam-
ido-2,4-dideoxy-a- -threo-hex-4-enopyranoside (10) in
L
87.4% yield. Compound 10 was subjected to a hydro-
gen-balloon pressure in the presence of Pd–C for 3
days. TLC showed the formation of a major and a
minor component; the major component was isolated
by column chromatography and characterized as the
desired product 4 in a yield of 73.0%. The minor
component was not isolated but was presumed to be
the C-5 epimer.
Fig. 2. Effect of increasing concentration of methyl 2-acetam-
ido-2,4-dideoxy-b-
cosaminoglycan biosynthesis. Hepatocyte cultures were
incubated with
-[3H]glucosamine and [35S]sulfate for 24 h in
D-xylo-hexopyranoside (4) on cellular gly-
D
the absence (control) or presence of compound 4 at 1, 10, and
20 mM. The values represent the mean9S.D. of triplicate
cultures. Statistical analyses using an unpaired t-test revealed
that control vs. 1 mM, PB0.01; control vs. 10 mM, PB0.01;
control vs. 20 mM, PB0.01.
For the incorporation of hydrogen isotopes into the
title compound, 9 was treated with sodium borodeute-
ride, which allowed for the incorporation of deuterium
1
2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-triphenyl-
at C-6 to afford 11. In the H NMR spectrum, the H-6
methyl-b-
D
-glucopyranoside (7) in 89.3% yield follow-
proton appeared as a one-proton singlet at 3.84 ppm;
also, the 13C NMR spectrum of 11 exhibited the ex-
pected triplet for C-6, and the 2H NMR spectrum
exhibited a singlet at 3.14 ppm. Hydrogenation of 11 in
the presence of Pd–C afforded 13 (49.4% from 9); the
13C NMR spectrum exhibited the expected triplet for
C-6.
For the synthesis of the radiolabeled analog of 4,
compound 9 was treated with sodium borotritiide (1
mCi) followed by sodium borohydride. The crude
product was subjected to a hydrogen-balloon pressure
in the presence of Pd–C until TLC showed the com-
plete disappearance of the starting material (3 weeks).
Compound 14 was isolated as a chromatographically
homogeneous product and was used as a probe in cell
cultures to determine the mechanism of biological inhi-
bition of cellular GAGs.
ing crystallization of the crude reaction product. In a
separate experiment, 5 was converted into 7 in a one-
pot fashion in 91.4% yield.
Removal of the 6-O-triphenylmethyl group by acid-
catalyzed hydrolysis was considered to be possibly
problematic owing to the potential for acetyl group
migration. Randazzo et al.31 have described the use of
metal ions for the selective removal of triphenylmethyl
groups under conditions that avoided hydrolysis or
migration of other groups. Using similar reaction con-
ditions, 7 was treated with anhydrous copper sulfate in
toluene at reflux temperature for 1 week; however, TLC
indicated that no reaction had occurred.
A promising method for the selective detritylation of
protected primary hydroxyl groups in carbohydrates
was developed by Bessodes et al.32 and involves the use
of formic acid in Et2O. The advantage of this method is
the short reaction time (10–15 min), which allows for
the presence of other acid-labile protecting groups. In
the present investigation, 7 was treated with formic acid
in Et2O for 1.5 h, at which time TLC showed the
presence of only one new component, namely methyl
Biological e6aluation.—Hepatocyte cultures treated
with
D
-[3H]glucosamine (GlcN) and [35S]sulfate (SO4)
are known to incorporate and express both radiolabels
into cellular heparan sulfate glycosaminoglycans. Com-
pound 4 was evaluated for its inhibitory effect on
hepatocyte GAG synthesis at 1, 10, and 20 mM (Fig.
2). The addition of 4 to hepatocyte cultures at 1.0 mM
caused a decrease in both [3H]GlcN and [35S]SO4 incor-
poration to 69 and 59% of the control cells, respec-
tively. At 10 and 20 mM, the incorporation of both
radioisotopes was decreased in each case to approxi-
2-acetamido-3,4-di-O-acetyl-2-deoxy-b-D-glucopyran-
oside (8), that was isolated in 89.9% yield by column
chromatography.
The method of Cree et al.33 was followed for the
oxidation–b-elimination process in which 8 was treated

40
A. Berkin et al. / Carbohydrate Research 337 (2002) 37–44
mately 10% of the control cells. These results demon-
strate that 4, at varying concentrations, perturbs cellu-
lar GAG synthesis. The mechanism of action of 4 on
cellular GAG synthesis may involve the following: (1)
inhibition of various enzymatic steps which would
transform 4 into a UDP-analog; (2) inhibition of the
corresponding UDP-transferase which would affect
GAG chain-elongation; (3) incorporation of 4, or a
metabolite derived from 4, into GAGs causing prema-
ture chain termination owing to the 4-deoxy moiety of
4.
To determine the mechanism of inhibition of cellular
GAG synthesis, the [3H] radiolabeled analog of 4,
namely 14, was evaluated for its inhibitory effect on
hepatocyte GAG synthesis. Compound 14 was evalu-
ated at 1.0, 10, and 20 mM in the absence of radiola-
beled GAG precursors (i.e., [3H]GlcN and [35S]SO4).
Fig. 3 demonstrates that addition of 14 to hepatocyte
cultures caused a concentration-dependant incorpora-
tion of a [3H] radiolabel into isolated GAGs. The
maximum incorporation of a [3H] radiolabel occurred
at concentrations of 10 and 20 mM, a result which
reflects the ability of 4, at 10 and 20 mM, to have a
maximum effect on the inhibition of [3H]GlcN and
[35S]SO4 into isolated GAGs.
deoxy-4-fluoro derivatives19,25,36 of 2-acetamido-2-
deoxy- -hexopyranoses.
D
3. Experimental
General methods.—Melting points were determined
on a Fisher–Johns apparatus and are uncorrected.
Optical rotations were measured with a Perkin–Elmer
1
241 polarimeter for solutions in a 1-dm cell at rt. H
and 13C NMR spectra were recorded on a Bruker
Avance 400 spectrometer at 400.1 and 100.6 MHz,
respectively. The solvent was CDCl3, unless stated oth-
erwise. The signals owing to residual protons in the
deuterated solvents were used as internal standards.
Chemical shifts (l) are reported in ppm downfield from
Me4Si for 1H and 13C NMR spectra. Infrared (IR)
spectra were recorded on a Bomem MB series FTIR
spectrophotometer. Thin-layer chromatography (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).
Methyl
2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-
-glucopyranoside (2).—A
[(methylthio)thiocarbonyl]-i-
D
mixture of 129 (0.098 g, 0.24 mmol), NaH (60% oily
suspension, 3.3 equiv, 0.032 g), and three crystals of
imidazole in THF (2 mL) was stirred for 1 h, and then
CS2 (10 equiv, 0.146 mL) was added. After 1 h, MeI (3
equiv, 0.045 mL) was added, and the reaction mixture
was stirred overnight. The mixture was concentrated
under reduced pressure to afford a yellowish oil that
was subjected to flash chromatography (10:1 CH2Cl2–
MeOH) to afford 2 (0.112 g, 93.5%) as a yellowish
solid: Rf 0.45 (1:2 EtOAc–Et2O); mp 133–134 °C; [h]D
+63.3° (c 1, CHCl3); 1H NMR (CDCl3): l 1.89 (s, 3 H,
NAc), 2.54 (s, 3 H, SMe), 3.29 (m, 1 H, H-2), 3.51 (s,
3 H, OMe), 3.62 (m, 2 H, H-6, H-6%), 3.84 (m, 1 H,
H-5), 4.41 (dd, 1 H, J3,2=J3,4 8.9 Hz, H-3), 4.48–4.70
(m, 4 H, 2 PhCH2), 4.95 (d, 1 H, J1,2 8.0 Hz, H-1), 5.65
(d, 1 H, NH), 5.98 (dd–t, 1 H, J4,5 9.0 Hz, H-4),
7.24–7.34 (m, 10 H, 2 Ph); 13C NMR (CDCl3): l 19.5
(SMe), 23.6 (NCOCH3), 57.1 (OMe), 57.3 (C-2), 69.4
(C-6), 73.5 (C-3), 73.6 and 74.2 (2 PhCH2), 77.6 (C-4),
79.9 (C-5), 100.5 (C-1), 127.6–128.5 (Ph), 172.0
(NCꢀO), 201.0 (OCꢀS). Anal. Calcd for C25H31NO6S2:
C, 59.38; H, 6.18; N, 2.77; S, 12.68. Found: C, 59.30;
H, 6.18; N, 2.79; S, 12.46.
These results suggest that the mechanism of action of
4 is due, in part, to the incorporation of a metabolite
derived from 4 into GAG chains causing premature
chain termination. The methyl aglycone of 4 can be
hydrolyzed by glycosidases, and the resulting free sugar
would be rapidly converted into a UDP-metabolite and
incorporated into growing GAG chains. Incorporation
of the 4-deoxy moiety into GAG chains would result in
premature GAG-chain termination, and the resulting
GAGs would have a decreased level of [3H]GlcN and
[35S]SO4 incorporation. Similar reductions of incorpora-
tions of isotopic labels were observed with 4-deoxy
derivatives,20 a 3-deoxy-3-fluoro derivative,35,36 and 4-
Fig. 3. Effect of increasing concentration of methyl 2-acetam-
ido-2,4-dideoxy-b-D-xylo-hexopyranoside-6-t (14) on hepato-
Methyl 2-acetamido-3,6-di-O-benzyl-2,4-dideoxy-i-
D
-xylo-hexopyranoside (3).—A solution of 2 (0.400 g,
cyte cellular glycosaminoglycan synthesis. Hepatocyte
cultures were incubated for 24 h in the absence (control) or
presence of compound 14 at 1, 10, and 20 mM. The values
represent the mean9S.D. of triplicate cultures. Statistical
analyses using an unpaired t-test revealed that control vs. 1
mM, PB0.01; control vs. 10 mM, PB0.01; control vs. 20
mM, PB0.01.
0.79 mmol), AIBN (0.8 equiv, 0.090 g), and Bu3SnH
(15 equiv, 3.18 mL) in toluene (30 mL) was heated at
reflux temperature for 20 min. The solvent was re-
moved under reduced pressure, and the crude product
was purified by column chromatography on silica gel

A. Berkin et al. / Carbohydrate Research 337 (2002) 37–44
41
(hexanes flush, then EtOAc–Et2O gradient). Recrystal-
lization from EtOAc–hexanes afforded pure 3 (0.221 g,
70.0%) as a white, crystalline solid: Rf 0.20 (1:2
EtOAc–Et2O); mp 160–161 °C; [h]D +17.8° (c 1,
CH2Cl2); mp 194–196 °C, lit. 182–184 °C;37 [h]D
−29.9° (c 1, MeOH), lit. −49.5° (c 1, CHCl3);37 1H
NMR (CDCl3): l 1.91 (s, 3 H, NAc), 3.23 (m, 1 H,
H-6), 3.30–3.25 (m, 4 H, H-3, H-4, H-5, H-6%), 3.48 (s,
3 H, OMe), 3.64 (m, 1 H, H-2), 4.30 (d, 1 H, J1,2 8.4
Hz, H-1), 7.12–7.43 (m, 15 H, Ph); 13C NMR (CDCl3):
l 23.0 (NCOCH3), 56.7 (C-2), 57.3 (OMe), 64.7 (C-6),
72.4, 76.4, and 76.9 (C-3, C-4, C-5), 87.6 (CPh3), 103.4
(C-1), 128.0–130.0 (Ph), 145.5 (Ph), 173.8 (NCꢀO).
Anal. Calcd for C28H31NO6: C, 70.42; H, 6.54; N, 2.93.
Found: C, 70.29; H, 6.40; N, 2.87.
1
CHCl3); H NMR (CDCl3): l 1.43 (apparent q, 1 H,
J4ax,3=J4ax,5=J4ax,4eq 11.4 Hz, H-4ax), 1.94 (s, 3 H,
NAc), 2.20 (ddd, 1 H, J4eq,3 4.7, J4eq,5 1.5 Hz, H-4eq),
3.19 (m, 1 H, H-2), 3.48 (s, 3 H, OMe), 3.49–3.75 (m,
3 H, H-5, H-6, H-6%), 4.04 (ddd, 1 H, J3,2 10.6 Hz, H-3),
4.44 and 4.59 (2 d, 2 H, J 11.7 Hz, PhCH2), 4.58 (s, 2
H, PhCH2), 4.72 (d, 1 H, J1,2 8.2 Hz, H-1), 5.63 (dd, 1
H, JNH,2 6.8 Hz, NH), 7.29–7.36 (m, 10 H, Ph); 13C
NMR (CDCl3): l 23.8 (NCOCH3), 34.1 (C-4), 56.6
(OMe), 58.3 (C-2), 71.1 (C-3, 6), 72.5 and 73.5 (2
PhCH2), 74.7 (C-5), 101.1 (C-1), 127.7–128.5, 139.4,
and 139.5 (Ph), 170.5 (NCꢀO). Anal. Calcd for
C23H29NO5: C, 69.15; H, 7.32; N, 3.51. Found: C,
69.03; H, 7.23; N, 3.53.
Methyl
triphenylmethyl-i-
2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-
-glucopyranoside (7).—Compound
D
6 (1.83 g, 3.83 mmol) was treated with Ac2O (15 mL) in
pyridine (20 mL) at 0 °C, and the reaction mixture was
stirred overnight at rt. The solution was concentrated,
and the residue was coevaporated with toluene under
reduced pressure to afford a yellowish solid that was
recrystallized from EtOAc to afford 7 (1.92 g, 89.3%) as
tiny, white needles: Rf 0.72 (5:1 EtOAc–toluene); mp
253–255 °C; [h]D +24.3° (c 1, CHCl3); 1H NMR
(CDCl3): l 1.71 (s, 3 H, OAc), 1.95 and 2.01 (2 s, 6 H,
OAc, NAc), 3.07 and 3.45 (dq, 2 H, J6,5 4.8, Jgem 10.4,
J6%,5 2.1 Hz, H-6, H-6%), 3.54 (m, 1 H, H-5), 3.56 (s, 3 H,
OMe), 4.00 (m, 1 H, H-2), 4.52 (d, 1 H, J1,2 8.3 Hz,
H-1), 5.15 (dd–t, 1 H, J3,2=J3,4 9.3 Hz, H-3), 5.17
(dd–t, J4,5 10.0 Hz, H-4), 5.59 (d, 1 H, JNH,2 9.0 Hz,
NH), 7.19–7.46 (m, 15 H, Ph); 13C NMR (CDCl3): l
20.4 and 20.7 (2 OCOCH3), 23.4 (NCOCH3), 54.5
(C-2), 56.3 (OMe), 62.0 (C-6), 68.8 (C-4), 72.8 (C-3),
73.3 (C-5), 86.5 (CPh3), 101.6 (C-1), 126.9–128.6 (Ph),
143.6 (Ph), 168.9, 170.2, and 171.2 (2 OCOCH3,
NCꢀO). Anal. Calcd for C32H35NO8: C, 68.44; H, 6.28;
N, 2.49. Found: C, 68.60; H, 6.19; N, 2.55.
Preparation of methyl 2-acetamido-2,4-dideoxy-i-D-
xylo-hexopyranoside (4).—A mixture of 3 (0.120 g,
0.300 mmol) and 5% Pd–C (0.120 g) in ꢀ2% hydrogen
chloride in MeOH (7 mL) was subjected to a hydrogen
pressure (50 psig) for 12 h. The mixture was neutralized
with Amberlite IRA-400 (OH−) resin and filtered
through Celite 521 (Aldrich), and the filtrate was con-
centrated under reduced pressure to yield a crude solid.
Flash chromatography on silica gel (1:9 MeOH–
CHCl3) afforded 4 as a solid that was recrystallized
from MeOH–EtOAc to afford pure 420 (0.057 g,
86.5%) as a white solid: Rƒ 0.18 (1:20 MeOH–CH2Cl2);
mp 200–201 °C, lit. 200 °C;20 [h]D −33.2° (c 0.16,
MeOH), lit. −33° (c 0.16, MeOH);20 1H NMR (D2O):
l 1.28 (apparent q, 1 H, J3,4ax=J4ax,4eq=J4ax,5 11.8 Hz,
H-4ax), 1.85–1.91 (m, 1 H, H-4eq), 1.88 (s, 3 H, NAc),
3.35 (s, 3 H, OMe), 3.35–3.40 (m, 1 H, H-2), 3.48–3.57
(m, 3 H, H-5, H-6, H-6%), 3.63 (apparent dt, 1 H, J2,3
10.8, J3,4eq 5.0 Hz, H-3), 4.20 (d, 1 H, J1,2 8.5 Hz, H-1);
13C NMR (D2O): l 24.7 (NCOCH3), 37.0 (C-4), 59.5
(C-2, OMe), 66.1 (C-6), 71.4 (C-3), 75.1 (C-5), 104.8
(C-1), 177.3 (NCꢀO). Anal. Calcd for C9H17NO5: C,
49.30; H, 7.82; N, 6.39. Found: C, 49.44; H, 7.86; N,
6.28.
Methyl
2-acetamido-3,4-di-O-acetyl-2-deoxy-i-D-
glucopyranoside (8).—To compound 7 (1.50 g, 2.67
mmol) was added a solution of 1:1 HCO2H–Et2O (25
mL), and the mixture was stirred for 1.5 h at rt. The
mixture was concentrated under reduced pressure, and
the residue was coevaporated with toluene to afford a
white solid that could not be recrystallized. Flash chro-
matography (EtOAc) on silica gel afforded 8 (0.767 g,
89.9%) as a white, amorphous solid: Rf 0.16 (EtOAc);
mp 196–197 °C; [h]D −14.3° (c 1, MeOH); IR (KBr):
Preparation of methyl 2-acetamido-2-deoxy-6-O-
1
w 3426, 1746, 1721, 1654, and 1555 cm−1; H NMR
triphenylmethyl-i-
tion of methyl 2-acetamido-2-deoxy-b-
D
-glucopyranoside (6).—To a solu-
-glucopyrano-
D
(CDCl3): l 1.94 (s, 3 H, NAc), 2.02 and 2.03 (2 s, 6 H,
2 OAc), 3.46–3.61 (m, 2 H, H-5, H-6), 3.49 (s, 3 H,
OMe), 3.74 (dd, 1 H, J6%,5 2.3, Jgem 12.5 Hz, H-6%), 3.89
(ddd, 1 H, H-2), 4.58 (d, 1 H, J1,2 8.4 Hz, H-1), 5.02
(dd–t, 1 H, J4,3=J4,5 9.6 Hz, H-4), 5.28 (dd–t, 1 H, J3,2
10.1 Hz, H-3), 5.90 (d, 1 H, JNH,2 8.9 Hz, NH); 13C
NMR (CDCl3): l 20.7, 23.3, and 23.4 (2 OCOCH3,
NCOCH3), 54.5 (C-2), 56.9 (OMe), 61.3 (C-6), 69.0
(C-4), 72.4 (C-3), 74.0 (C-5), 101.7 (C-1), 170.2, 170.5,
and 171.1 (2 OCOCH3, NCꢀO). Anal. Calcd for
C13H21NO8·H2O: C, 47.56; H, 6.75; N, 4.27. Found: C,
47.90; H, 6.41; N, 4.38.
side (5)30 (1.50 g, 6.38 mmol) in anhyd pyridine (20 mL)
was added freshly prepared Ph3CCl (1.1 equiv, 1.96 g).
The mixture was stirred at rt until TLC indicated the
complete disappearance of 5 (4 days). The mixture was
added slowly to ice–water and extracted with CH2Cl2;
the extracts were washed sequentially with aq NaHCO3
(satd) and H2O, dried (Na2SO4), and concentrated un-
der reduced pressure. The residue was coevaporated
with toluene to afford a creamy, white solid. Recrystal-
lization of the residue from EtOH–hexanes afforded
crystalline 6 (2.41 g, 79.1%): Rƒ 0.40 (1:9 MeOH–

42
A. Berkin et al. / Carbohydrate Research 337 (2002) 37–44
Methyl
2-acetamido-3-O-acetyl-2,4-dideoxy-h-
L
-
3.98 (m, 1 H, H-2), 4.70 (d, 1 H, H-1), 4.98 (d, 1 H,
H-4); 13C NMR (MeOH-d4): l 22.5 (NCOCH3), 53.5
(C-2), 56.6 (OMe), 62.1 (t, C-6), 65.8 (C-3), 100.6 (C-4),
100.9 (C-1), 152.6 (C-5), 173.3 (NCꢀO); 2H NMR
(MeOH-d4): l 3.14. The compound decomposed on
standing.
In an analogous manner, 9 (0.200 g, 0.777 mmol) was
treated with sodium borotritide (NaB3H4, 1 mCi) in
MeOH at 0 °C. The mixture was stirred for 5 min
followed by the addition of NaB1H4 (1 equiv, 0.029 g).
After 30 min at rt, the mixture was concentrated under
reduced pressure, and the residue was coevaporated
several times with MeOH to afford crude 12.
threo-hex-4-enodialdo-1,5-pyranoside (9).—To a mix-
ture of 8 (6.30 g, 19.7 mmol) in Me2SO (80 mL) was
added Et3N (36 mL) at 0 °C. To this stirred solution
was added dropwise a solution of sulfur trioxide–pyri-
dine complex (6.6 equiv, 20.9 g) in Me2SO (160 mL)
over a 1.5-h period. The mixture was diluted with
CH2Cl2 (400 mL) and washed sequentially with ice-
cooled aq tartaric acid (satd), aq NaHCO3 (satd), and
H2O, dried (Na2SO4), and concentrated under reduced
pressure to afford a residue. The residue was subjected
to flash chromatography on silica gel (3:1 EtOAc–hex-
anes) to afford 8 (4.42 g, 87.0%) as a clear, bubbly oil
that could not be crystallized and that decomposed
upon standing: Rf 0.46 (EtOAc); [h]D +32.9° (c 1,
CHCl3); IR (KBr): w 3470, 1738 (ester), 1705 (alde-
Preparation of methyl 2-acetamido-2,4-dideoxy-i-D-
xylo-hexopyranoside (4) from 10 and the preparation of
the deuterated analog (13) and the radiolabeled analog
(14).—A mixture of 10 (0.205 g, 0.944 mmol) and 10%
Pd–C (0.250 g) in MeOH (7 mL) was subjected to a
hydrogen-balloon pressure for 3 days. The mixture was
filtered through Celite 521 (Aldrich), the residue was
washed with MeOH, and the combined filtrate and
washings were concentrated under reduced pressure to
give a residue which was purified by flash chromatogra-
phy on silica gel (1:6 MeOH–CH2Cl2) to afford 4
(0.151 g, 73.0%) as a white solid. The 1H and 13C NMR
spectra were identical to those obtained for a sample of
4 obtained by the route shown in Scheme 1.
hyde), 1660 (CꢀC), and 1544 cm−1; H NMR (CDCl3):
1
l 1.97 (s, 3 H, NAc), 2.09 (s, 3 H, OAc), 3.46 (s, 3 H,
OMe), 4.46 (m, 1 H, H-2), 5.10 (d, 1 H, J1,2 2.4 Hz,
H-1), 5.14 (dd, 1 H, J3,2 2.0, J3,4 4.6 Hz, H-3), 5.83 (d,
1 H, JNH,2 9.3 Hz, NH), 6.08 (dd, 1 H, J4,2 1.0 Hz,
H-4), 9.25 (s, 1 H, CHꢀO); 13C NMR (CDCl3): l 20.9
and 23.0 (OCOCH3, NCOCH3), 49.2 (C-2), 57.0
(OMe), 64.7 (C-3), 99.2 (C-1), 116.7 (C-4), 149.2 (C-5),
169.6 and 170.1 (OCOCH3, NCꢀO), 186.6 (CHꢀO);
FABMS (positive-ion): Calcd for C11H16NO6: 258.0978.
Found: 258.0975.
Methyl
2-acetamido-2,4-dideoxy-h-
L
-threo-hex-4-
In a separate experiment, a mixture of 11 (0.347 g,
obtained in a crude form from 9) and 10% Pd–C (0.300
g) in MeOH (10 mL) was subjected to a hydrogen-bal-
loon pressure for 4 days. The reaction mixture was
processed as described above for the preparation of 4
from 10 to afford 13 (0.107 g, 49.4% from 9) as a white
solid: Rƒ 0.37 (1:3 MeOH–CHCl3); mp 198–202 °C;
enopyranoside (10) and the preparation of the deuterated
analog (11) and the radiolabeled analog (12).—To a
solution of 9 (0.237 g, 0.921 mmol) in MeOH (7 mL)
was added NaBH4 (1 equiv, 0.035 g). The mixture was
stirred for 20 min at rt and concentrated under reduced
pressure, and the residue was coevaporated several
times with MeOH. The resulting residue was subjected
to flash chromatography on silica gel (1:9 MeOH–
CH2Cl2) to afford 10 (0.175 g, 87.4%) as a clear,
colorless oil which could not be crystallized and which
decomposed upon standing: [h]D −9.9° (c 1, MeOH);
1
[h]D − 32.0° (c 1, MeOH); H NMR (MeOH-d4): l
1.30 (apparent q, 1 H, J3,4ax=J4ax,4eq=J4ax,5 11.8 Hz,
H-4ax), 1.87–1.91 (m, 1 H, H-4eq), 1.90 (s, 3 H, NAc),
3.37 (s, 3 H, OMe), 3.40–3.46 (m, 2 H, H-2, H-5), 3.48
(m, 1 H, H-6), 3.57 (apparent dt, 1 H, J2,3 10.9, J3,4eq
5.0 Hz, H-3), 4.15 (d, 1 H, J1,2 8.3 Hz, H-1); 13C NMR
(MeOH-d4): l 23.0 (NCOCH3), 37.0 (C-4), 56.9 (C-2),
58.8 (OMe), 65.2 (t, C-6), 70.8 (C-3), 73.9 (C-5), 103.9
(C-1), 174.0 (NCꢀO).
In an analogous manner, 12 (obtained in a crude
form from 9) was converted into the 6-tritio analog 14
by treatment with 10% Pd–C (0.200 g) in MeOH (10
mL) and a hydrogen-balloon pressure for 3 weeks.
Compound 14 (0.062 g, 36.5%) was isolated as de-
scribed for the preparation of 13.
IR (KBr): w 3290, 1647, and 1548 cm−1 1H NMR
;
(MeOH-d4): l 1.90 (s, 3 H, NAc), 3.39 (s, 3 H, OMe),
3.83 (dd, 1 H, H-3), 3.91 (s, 2 H, H-6, H-6%), 4.04 (dd,
1 H, J2,3 3.9 Hz, H-2), 4.87 (d, 1 H, J1,2 4.2 Hz, H-1),
5.00 (d, 1 H, J4,3 4.2 Hz, H-4); 13C NMR (MeOH-d4):
l 22.6 (NCOCH3), 53.5 (C-2), 56.6 (OMe), 62.4 (C-6),
65.8 (C-3), 100.5 (C-4), 100.8 (C-1), 152.6 (C-5), 173.2
(NCꢀO); FABMS (positive-ion): Calcd for C9H16NO5:
218.1029. Found: 218.1006.
In a separate experiment, sodium borodeuteride
(NaB2H4, 1 equiv, 0.037 g) was added to a solution of
9 (0.256 g, 0.995 mmol) in MeOH (7 mL). The mixture
was stirred for 20 min at rt and concentrated under
reduced pressure, and the residue was coevaporated
several times with MeOH to afford crude 11 (0.347 g):
1H NMR (MeOH-d4): l 1.82 (s, 3 H, NAc), 3.30 (s, 3
H, OMe), 3.78 (m, 1 H, H-3), 3.84 (s, 1 H, H-6, H-6%),
Biological e6aluation
Materials.
D
-(6-3H)Glucosamine–HCl (25.6 Ci/
mmol), Na325SO4 (867 mCi/mL), and NaB3H4 (204.1
mCi/mmol) were purchased from either Dupont or ICN
Biomedicals. EGTA, D-glucosamine hydrochloride, and
Hepes buffer were of reagent grade and were obtained
from Sigma Chemical Co., Fisher Scientific Co., or

A. Berkin et al. / Carbohydrate Research 337 (2002) 37–44
43
BDH Chemicals. Williams’ Medium E with
L
-glu-
Research Council of Canada (Grant MT-3153) for
tamine, 10% fetal bovine serum, and antibiotic–antimy-
cotic mixtures were supplied by Gibco. Fibronectin and
collagenase were purchased from Sigma Chemical Co.
Hepatocyte isolation and cell culture. Hepatocytes
were obtained from 6–8-week-old, female Swiss white
mice (Charles River Canada, St. Constant, Quebec) by
the procedure described previously.38,39 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 hepa-
tocytes were separated from the capsule by gentle teas-
ing. 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
hemocytometer to determine the viability and cell num-
ber. The viability was usually greater then 85%. The
cells were plated in triplicate on fibronectin-coated tis-
sue culture dishes (Falcon 35×10 mm) at a density of
2–2.5×106 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 treated with fresh
financial support.
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