Thiosugar nucleotide analogs: Synthesis of 5′-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-α-D-galactopyranosyl diphosphate)

Article abstract of DOI:10.1016/S0008-6215(02)00210-0

The synthesis of a novel analog of uridine diphosphate galactose (UDP-Gal) is described. A sulfur atom was inserted into the 6-position of galactose to give uridine 5′-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-α-D-galactopyranosyl diphosphate). This peracetylated thiol analogue of UDP-Gal has been synthesized in nine steps starting from methyl α-D-galactopyranoside in an overall yield of 3%.

Full text of DOI:10.1016/S0008-6215(02)00210-0

Carbohydrate Research 337 (2002) 1935–1940
www.elsevier.com/locate/carres
Thiosugar nucleotide analogs: Synthesis of
5%-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a- -galactopyranosyl
D
diphosphate)
Jordan Elhalabi, Kevin G. Rice*
Department of Medicinal Chemistry, College of Pharmacy, Uni6ersity of Michigan, Ann Arbor, MI 48109-1065, USA
Received 19 March 2002; accepted 10 July 2002
Dedicated to Professor Derek Horton on the occasion of his 70th birthday
Abstract
The synthesis of a novel analog of uridine diphosphate galactose (UDP-Gal) is described. A sulfur atom was inserted into the
-galactopyranosyl diphosphate). This peracety-
6-position of galactose to give uridine 5%-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a-
D
lated thiol analogue of UDP-Gal has been synthesized in nine steps starting from methyl a-D-galactopyranoside in an overall yield
of 3%. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Thiosugars; UDP-galactose; Sugar nucleotides; b-(1“4)-Galactosyltransferase
1. Introduction
nating glycoconjugates to create a new b-(1“4)-glyco-
sidic linkage (Fig. 1). Morrison and Ebner5,6 have
determined the kinetic mechanism to be ordered se-
quential. The donor substrate specificity of b-(1“4)-
galactosyltransferase has been studied extensively. The
2-, 3-, 4-, and 6-deoxy-Gal analogs7–10 have been shown
to serve as substrates for the enzyme. Likewise, UDP-
arabinose (UDP-Ara), which lacks the 6-CH2OH arm,11
as well as the 6-fluorogalactose derivative,9 are used by
the enzyme, while the 2-deoxy-2-fluoro derivative was
found to be a competitive inhibitor.12 Even some O-
methylated galactose derivatives of UDP-Gal were used
as substrates.13 Several UDP-Gal analogs, where the
ring oxygen is replaced by either sulfur or a carbon
atom, have been prepared by different groups. Yuasa et
al.14 reported the synthesis of UDP-5S-Gal that was
found to be transferred to a GlcNAc acceptor at 5% of
the rate of the natural substrate. UDP-5S-GalNAc was
also prepared and was transferred at a 0.23% rate.15
However, the carbocyclic analog16 of UDP-Gal was an
inhibitor of b-(1“4)-GalT, which is consistent with the
proposed mechanism of this enzyme that proceeds
through an oxocarbonium ion-like transition state.
At least one of these analogs has been used to
prepare unnatural oligosaccharides. Kajihara et al.10
Glycosyltransferases are a group of enzymes that
participate in the biosynthesis of oligosaccharides. Nu-
merous glycosyltransferases have been cloned,1 and
very recently some of their crystal structures have been
elucidated.2–4 In addition to transferring natural sub-
strates, the specificity of these enzymes is sufficiently
flexible to allow the synthesis of unnatural or
‘‘modified’’ oligosaccharides. This has the advantage of
being both regio- and stereoselective, thereby avoiding
all the selective chemical protection and deprotection
steps that otherwise would be needed.
One of the late-acting enzymes in N-glycan biosyn-
thesis is b-(1“4)-galactosyltransferase (GalT, EC
2.4.1.90) that transfers a galactose moiety from the
sugar nucleotide donor, uridine diphosphate galactose
(UDP-Gal), to 2-acetamido-2-deoxy-D-glucose-termi-
* Corresponding author. Present address: Division of
Medicinal and Natural Products Chemistry, College of Phar-
macy, The University of Iowa, Iowa City, IA 52242, USA;
tel.: +1-319-3359903; fax: +1-319-3358766
E-mail address: kevin-rice@uiowa.edu (K.G. Rice).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00210-0

1936
J. Elhalabi, K.G. Rice / Carbohydrate Research 337 (2002) 1935–1940
2. Discussion and results
transferred UDP-6-deoxy-Gal to asialo agalacto a1-acid
glycoprotein, showing the potential application of un-
natural sugar nucleotides to remodel the N-glycans on
a glycoprotein. The synthesis and utilization of differ-
ent sugar nucleotide analogs by different glycosyltrans-
ferases has been extensively reviewed.17
The chemical synthesis of uridine 5%-(2,3,4-tri-O-ace-
tyl-6-S-acetyl-6-thio-a-
(8) is shown in Scheme 1. Starting with the commer-
cially available methyl a- -galactopyranoside (1), selec-
D-galactopyranosyl diphosphate)
D
tive primary hydroxyl group tosylation, acetylation and
introduction of the thiol group at the 6-position with
potassium thioacetate at reflux temperature gave
methyl 2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a-galactopy-
ranoside (2) in 53% yield. An attempt to replace the
O-methyl group with a bromo group using dimethyl-
boron bromide18 was only partially successful. Instead,
the O-methyl group was converted into an O-acetyl
group using low temperature acetolysis to give 1,2,3,4-
tri-O-acetyl-6-S-acetyl-6-thio-a-galactopyranose (3) in
89% yield. A more efficient synthesis of 3 involved the
use of the commercially available 1,2:3,4-di-O-iso-
To study further the functional and biological prop-
erties of remodeled N-glycans, we propose to introduce
a thiol into the C-6 position of terminal Gal residues.
This thiol moiety can then be used as a unique and
flexible derivatization site. Toward this aim, the synthe-
sis of a novel modified UDP-Gal possessing a thiol
group at the 6-position of Gal is described.
propylidene-a- -galactopyranose (9) (Scheme 2). The
D
thioacetate group was introduced using Mitsunobo
conditions19 to give 6-S-acetyl-1,2,3,4-di-O-isopropyli-
dene-6-thio-a- -galactopyranose (10), which in turn
D
was converted to 3 in 47% overall yield using the same
acetolysis conditions as shown in Scheme 2.
Fig. 1. Enzymatic synthesis of oligosaccharides by galactosyl-
transferase (b-(1“4)-GalT).
Hydrolysis of the anomeric acetate using benzyl-
amine at room temperature produced a mixture of
products. Alternatively, selective hydrolysis using
glacial acetic acid–water7 at 75 °C provided 2,3,4-tri-O-
acetyl-6-S-acetyl-6-thio-a/b-
b) (5), but in poor yield (26%). Instead, 3 was converted
into 2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a- -galactopy-
D-galactopyranose (2:1 a/
D
ranosyl bromide (4) using TiBr4. The glucosyl bromide
was then hydrolyzed to give 5 as a (3:1) a/b mixture in
56% yield from 3.
Phosphorylation of 5 was achieved using n-butyl-
lithium and tetrabenzyl pyrophosphate (TBPP). This
gave the unstable dibenzyl 2,3,4-tri-O-acetyl-6-S-acetyl-
6-thio-a- -galactopyranosyl phosphate (6) which was
D
debenzylated by transfer hydrogenolysis using 1,4-cy-
clohexadiene and palladium-on-charcoal catalyst20 to
give 2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a-D-galactopy-
ranosyl 1-phosphate mono-triethylammonium salt (7).
The hydrogenolysis reaction was sluggish and required
excess catalyst, presumably due to sulfur poisoning.
Alternatively, the benzyl groups were removed using
bromotrimethylsilane21 in the presence of triethylamine,
followed by water hydrolysis. Product 7 was in turn
coupled to UMP-morpholidate22 to produce uridine
Scheme 1.
5%-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a-D-galactopy-
ranosyl diphosphate) (8). Purification of the final per-
acetylated product was achieved by preparative C18
RP-HPLC to give 8 as a bis-morpholinium salt. The
final purification yielded micromole quantities of 8 and
has the advantage of speed versus a lengthy low-pres-
sure ion-exchange column, followed by repeated desalt-
ing using gel filtration.
Scheme 2.

J. Elhalabi, K.G. Rice / Carbohydrate Research 337 (2002) 1935–1940
1937
Compound 8 is the acetate-protected form of the
sugar nucleotide donor analog UDP-6S-Gal. Removal
of the acetyl groups of 8 under basic conditions led to
decomposition due to hydrolysis of the phosphodiester
linkage. Alternatively, the thiol group could be trapped
during the O,S-deacetylation using 2,2%-dithioldi-
pyridine, resulting in a stable, reducible intermediate
that is readily deprotected by reduction prior to enzyme
transfer. The full details of this procedure will be
reported separately along with the results of
chemoenyzymatic synthesis using this and related
analogues.
toluenesulfonyl chloride (TsCl, 1.05 g) at rt. The reac-
tion was monitored by TLC using acetone–toluene as
the mobile phase, which resolved the starting material 1
(Rf 0) from the tosylated Gal derivative (Rf 0.1). After
24 h, an additional 0.1 equiv (0.17 g) of TsCl was added
and allowed to react with stirring for 2 h. Ac2O (10
mL) was then added, and the reaction was stirred at rt
for 4 h.
Et2O (100 mL) and water (50 mL) were added to the
reaction in an ice-bath, and the organic layer was
separated and washed twice with 1 N HCl (50 mL),
satd aq NaHCO3 (2×30 mL) and water (2×30 mL).
The organic layer was dried over MgSO4 and, after
filtration, was concentrated to dryness using a water
aspirator for 12 h.
3. Experimental
The oily residue was dissolved in CH3CN (50 mL).
KSAc (2.98 g, 4.75 equiv) was added, and the mixture
was refluxed at 70 °C for 5 h while monitoring the
reaction progress on TLC using 2:1 hexane–EtOAc.
Product 2 migrated with Rf 0.6 relative to the acety-
lated, tosylated derivative with Rf 0.4. The reaction was
diluted with CHCl3 (70 mL), filtered, washed with satd
aq NaCl (3×100 mL) and water (3×100 mL), and the
organic extract was then concentrated. The dark oily
residue was applied to a silica gel column (4×20 cm)
using 3:1 hexane–EtOAc as the mobile phase. The
fractions corresponding to the product were collected,
and the solvent was evaporated to yield 2 as a brown
General methods.—1H and 13C NMR spectra were
recorded at ambient temperature on a Bruker NMR
(ADVANCE DRX 500 or ADVANCE DPX 300).
Samples in CDCl3 used 1% TMS as an internal stan-
dard, whereas samples prepared in MeOH or D2O used
acetone as the internal standard. 31P NMR spectra were
recorded using neat H3PO4 as an external standard.
Chemical shifts are expressed in l (ppm) and coupling
constants (J) in Hz. High-resolution mass spectra
(HRMS) were recorded using a magnetic sector (Micro-
mass, model 70-250S) by chemical ionization with am-
monia. All reactions were monitored by TLC on
aluminum sheets precoated with Silica Gel 60 F254
(Alltech) (0.2 mm thickness) visualized by UV light or
by charring with 10% H2SO4 in MeOH. Flash column
chromatography was carried out using silica gel (40 mm,
Scientific Adsorbents). All reaction solvents were dried
prior to use according to standard procedures. Organic
solvents were removed on a rotary evaporator under
water aspiration vacuum with bath temperature of
40 °C unless specified otherwise. Whenever anhydrous
conditions were required, the reactions were conducted
under dry nitrogen, and reagent transfer was performed
using hypodermic syringes. Silica gel chromatography
solvents were of HPLC grade. All reagents were pur-
chased from Aldrich Chemical Co. with the exception
1
solid (1.75 g, 53%). H NMR (CDCl3, 1% TMS 300
MHz): l 5.48 (dd, 1 H, H-4), 5.32 (dd, 1 H, H-3), 5.13
(dd, 1 H, H-2), 4.98 (d, 1 H, H-1), 3.99 (dt, 1 H, H-5),
3.43 (s, 3 H, OMe), 2.91–3.10 (m, 2 H, H-6, H-6%), 2.36
(s, 3 H, SAc), 2.18 (s, 3 H, OAc), 2.10 (s, 3 H, OAc),
1.99 (s, 3 H, OAc). 13C NMR (CDCl3, ext. TMS, 120
MHz): l 194.62 (SAc), 170.45, 170.34, 169.93 (OAc),
98.48, 69.01, 68.04, 67.71, 67.45, 55.42, 30.48 (OMe),
28.63 (SAc), 20.86, 20.72, 20.66 (OAc). HRMS: Calcd
for C15H22O9S, 378.0985. Found, m/z 378.0985.
1,2,3,4-Tetra-O-acetyl-6-S-acetyl-6-thio-h-
pyranose (3).—Methyl 2,3,4-tri-O-acetyl-6-S-acetyl-6-
thio-a- -galactopyranoside (2) (1.50 g, 3.96 mmol) was
D-galacto-
D
dissolved in Ac2O (70 mL) and HOAc (70 mL glacial)
at 0 °C. Concentrated H2SO4 (1.5 mL) was added drop-
wise while stirring for 30 min at 0 °C. The reaction
proceeded for 20 h during which time it was allowed to
warm to rt. TLC (2:1 hexane–EtOAc) showed the
complete conversion of 2 (Rf 0.6) to product 3 (Rf 0.55).
The reaction was poured over ice-water and extracted
with CH2Cl2 (3×100 mL). The combined organic lay-
ers were washed with ice-cold water (50 mL) and satd
aq NaHCO3 (50 mL), and the solvent was evaporated
of the following: methyl a-D-galactopyranoside and
uridine 5-monophosphomorpholidate were purchased
from Sigma Chemical Co. Thioacetic acid was pur-
chased from Lancaster Synthesis, Inc. Analytical RP-
HPLC experiments were carried out using a C18 column
(Microsorb-MW, 25 cm×4.6 mm) on an Isco model
2350 pump equipped with a 2360 gradient programmer,
while preparative RP-HPLC purification steps were
carried out using a Prosphere C18 column (Alltech, 25
cm×22 mm) on a Isco model 2350 dual pump system.
1
to yield 1.44 g of 3 (89%). H NMR (CDCl3, 1% TMS
Methyl 2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-h-
D
-gal-
300 MHz): l 6.36 (d, 1 H, H-1, J1,2 3.7 Hz), 5.52
(pseudo d, 1 H, H-4), 5.30–5.35 (m, 2 H, H-2, H-3),
4.14 (pseudo t, 1 H, H-5), 3.06 (dd, 1 H, H-6), 2.98 (dd,
1 H, H-6%), 2.34 (s, 3 H, SAc), 2.19, 2.16, 2.03, 2.01
actopyranoside (2).—Methyl a-
D
-galactopyranoside (1)
(1.0 g, 5.5 mmol) was dissolved in anhyd pyridine (50
mL) and reacted with stirring with one equiv of p-

1938
J. Elhalabi, K.G. Rice / Carbohydrate Research 337 (2002) 1935–1940
(each s, each 3 H, each OAc). 13C NMR, CDCl3, (120
MHz): l 194.39 (SAc), 170.24, 170.11, 169.91, 168.99,
89.72 (C-1), 70.17, 68.10, 67.62, 66.35, 30.45, 28.17,
20.88, 20.67, 20.63, 20.54. HRMS: Calcd for
C16H22O10S, 406.0934. Found, m/z 406.0922.
acid (1.1 mL), and the reaction was carried out at rt for
6 h while monitoring by TLC (3:1 hexane–EtOAc) (Rf
0.8). At reaction completion, the solvent was evapo-
rated, and the residue was dissolved in Et2O (150 mL),
cooled to −10 °C and filtered. The cold filtrate was
washed with cold dilute M HCl (50 mL), satd aq
NaHCO3 (50 mL) and water (50 mL). The organic
extract was concentrated to dryness, and the crude
product was then flash chromatographed on silica gel
(4×10 cm) using 4:1 hexane–EtOAc to give 10 (4.11 g,
67%) that was converted to 3 in 70% yield following the
2,3,4-Tri-O-acetyl-6-S-acetyl-6-thio-h,i-
pyranose (5).—1,2,3,4-Tetra-O-acetyl-6-S-acetyl-6-thio-
a- -galactopyranose (3) (1.00 g, 2.46 mmol) was dis-
D-galacto-
D
solved in a mixture of 1:1 AcOH–water (40 mL) and
heated at 75 °C for 18 h. The reaction mixture was then
evaporated to dryness using a vacuum pump, and the
crude product was purified by flash chromatography on
silica gel (4×10 cm) eluting with 2:1 hexane–EtOAc to
give 240 mg of 5 in 26% yield (2:1 a/b ratio).
1
procedure described above for 2“3. Product 10: H
NMR (CDCl3, 1% TMS 300 MHz): l 5.51 (d, 1 H,
H-1), 4.61 (dd, 1 H, H-3). 4.24–4.31(m, 2 H, H-2, H-4),
3.85 (pseudo t, 1 H, H-5), 3.17 (dd, 1 H, H-6), 3.03 (dd,
1 H, H-6%), 2.34 (s, 3 H, SAc), 1.48, 1.46, 1.35, 1.32
(each s, each 3 H, each ꢀCH3). 13C NMR (CDCl3) (120
MHz): l 196.26 (SAc), 109.86, 109.19, 96.92, 72.42,
71.31, 70.88, 67.19, 30.95, 30.08, 26.34, 25.39, 24.82.
HRMS: Calcd for C14H12O6S, 318.1137. Found, m/z
318.1122.
Alternatively, 1.3 g (3.2 mmol) of 3 was dissolved in
CH2Cl2 (20 mL) and EtOAc (2 mL). Titanium
tetrabromide23 (3.12 g, 2.7 equiv) was added, and the
reaction was stirred at rt under nitrogen for 16 h. TLC
2:1 (hexane–EtOAc) demonstrated the complete con-
version of the a-acetate 3 (Rf 0.55) into 4 (Rf 0.8).
NaOAc (1.2 g) was added and stirred for 15 min, then
filtered through Celite, and the Celite pad was washed
with CH2Cl2 (10 mL). The organic filtrate was washed
with cold water (100 mL) and evaporated to dryness.
The crude mixture was dissolved in acetone (30 mL)
containing water (1 mL). Ag2CO3 (2.0 g) was added,
and the mixture was stirred for 12 h at rt in the dark,
after which time TLC (2:1 hexane–EtOAc) identified
the major product 5 (Rf 0.2). The reaction was filtered,
the filter paper was washed with acetone (15 mL), and
the solvent was evaporated to dryness using a water
aspirator. A final purification was performed by flash
chromatography on silica gel (4×10 cm) using 2:1
hexane–EtOAc to give of 5 (650 mg, 56%) as an a/b
2,3,4-Tri-O-acetyl-6-S-acetyl-6-thio-h-D-galactopyran-
osyl-phosphate mono-triethylammonium salt (7).—Tet-
rabenzylpyrophospahte (TBPP) was prepared22 by
dissolving dibenzyl phosphate (2.24 g, 8.05 mmol) with
N,N-dicyclohexylcarbodiimide (DCC, 830 mg 4.02
mmol) in toluene with stirring at rt for 5 h. The
N,N-dicyclohexylurea was removed by filtration. The
toluene was evaporated using a vacuum pump to give
an oily residue that solidified as a white powder upon
freezing to yield TBPP (2.05 g, 95%), which was used
immediately in the next step without further
purification.
2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-a,b-D-galactopy-
1
(3:1) mixture. H NMR (CDCl3, 1% TMS, 1 drop of
ranose (5) (550 mg, 1.51 mmol) was dissolved in dry
THF (10 mL) at −78 °C. Butyllithium (1.0 mL, 1.6
mmol, in hexane) was added to 5 at −78 °C. The
reaction was stirred for 5 min, after which time, TBPP
(1.63 g, 3.02 mmol) in dry THF (3 mL) was added
dropwise over 5 min. The reaction mixture was brought
to −60 °C and allowed to react with stirring for 30
min. The reaction mixture was brought to rt, diluted
with 30 mL of Et2O, washed with satd aq NaHCO3
(2×10 mL) and water (2×10 mL) and evaporated to
dryness. The residue was then applied to a silica gel
column (4×10 cm) using 2:1 hexane–EtOAc, contain-
ing 1% Et3N as the mobile phase. The fractions corre-
sponding to the product (monitored by both TLC
charring and UV detection) were collected, and the
solvent was evaporated to give 6 (290 mg, 31% yield).
1H NMR (CDCl3, TMS 300 MHz): l 7.34–7.40 (m, 10
H, Ar), 5.95 (dd, 1 H, H-1, J1,2 3.6, J1,P 7.7 Hz), 5.48 (d,
1 H, H-4), 5.30 (dd, 1 H, H-3), 5.20 (dt, 1 H, H-2), 5.10
(pseudo d, 4 H, benzylic), 4.13 (pseudo t, 1 H, H-5),
2.97 (m, 2 H, H-6, H-6%), 2.23, 2.17, 2.00, 1.90 (each s,
each 3 H, 3×OAc, SAc). 13C NMR (CDCl3, TMS,
D2O, 300 MHz): l 5.48–5.52 (m, 2 H, H-1 (a), H-4
(a)), 5.44 (dd, 0.5 H, H-4 (b)), 5.40 (dd, 1 H, H-3 (a)),
5.16 (dd, 1 H, H-2 (a), J2,3 10.5, J1,2 3.7 Hz), 5.02–5.08
(m, 1 H, H-2 (b), H-3 (b)), 4.68 (d, 0.5 H, H-1 (b)), 4.27
(pseudo t, 1 H, H-5 (a)), 3.75 (pseudo t, 0.5 H, H-5 (b)),
2.95–3.17 (m, 3 H, H-6 (a/b), H-6%(a/b)), 2.36 (s, 4.5 H,
SAc), 2.20, 2.12, 2.01 (each s, each 1.5 H, each OAc
(b)), 2.19, 2.11, 2.00 (each s, each 3 H, each OAc (a)).
13C NMR (CDCl3, 1 drop D2O, 120 MHz): l (a-
anomer only) 195.04, 170.77, 170.76, 170.65, 91.14,
71.43, 69.31, 68.60, 68.05, 67.85, 30.91, 28.87, 21.28,
21.15, 21.09. HRMS: Calcd for C14H20O9S, 364.0828.
Found, m/z 364.0836.
1,2,3,4-Tetra-O-acetyl-6-S-acetyl-6-thio-h-
D-galacto-
pyranose (3) from 9.—1:2,3:4-Di-O-isopropylidene-a-
D-
galactopyranose (9) (5.00 g, 19.2 mmol) was dissolved
along with (p-dimethylamino)phenyl diphenylphos-
phine (5.0 g, 1.2 equiv) in THF (50 mL) with stirring at
−10 °C under nitrogen according to the established
procedure.21 Diethyl azodicarboxylate (DEAD, 3.45
mL, 17.5 mmol) was added, followed by of thioacetic

J. Elhalabi, K.G. Rice / Carbohydrate Research 337 (2002) 1935–1940
1939
(120 MHz): l 194.40 (SAc), 170.59, 170.49, 170.30 (3
OAc), 128.22–129.12, 94.95 (d, C1), 70.26, 70.06 (d,
benzylic), 69.95 (d, benzylic), 68.45, 67.56, 67.67, 67.32,
30.75, 28.44, 21.09, 21.04, 20.87. 31P NMR (120 MHz):
l −1.52. HRMS: Calcd for C28H33O12PS, 624.1430.
Found, 624.1419. Compound 6 (290 mg) was debenzy-
lated in EtOH (10 mL) with 150 mg Pd-on-charcoal
(10% 150 mg), Et3N (160 mL) and 1,4-cyclohexadiene
(0.2 mL). The reaction was followed by TLC using 1:1
EtOAc–hexane to monitor the removal of the first
benzyl group and using 60:35:4, CHCl3–MeOH–water
to monitor the removal of the second benzyl group with
the addition of catalyst (40–60 mg) every 2 h until the
reaction was complete. The mixture was filtered, the
filter paper was washed with EtOH (5 mL), and the
solvent was evaporated to give 7 (185 mg, 67% yield).
Alternatively, compound 6 (40 mg, 0.064 mmol) was
dissolved in CH2Cl2 (5 mL) at 0 °C with Et3N (9 mL, 65
mmol) and bromotrimethylsilane (52 mL, 0.38 mmol).
The reaction was stirred at rt for 6 h after which time
water (200 mL) was added, and the mixture was stirred
vigorously for an additional 20 min. The mixture was
dried using a vacuum pump, and the crude product was
then loaded on a silica gel column (5×0.5 cm) and
eluted with EtOAc (100 mL), followed by MeOH (50
mL), to remove side products. The methanolic fraction
was collected and dried to give 7 (25 mg, 72% yield). 1H
NMR (D2O, acetone as int. std., 300 MHz): l 5.58 (dd,
1 H, H-1, J1,2 3.6, J1,P 7.8 Hz), 5.40 (d, 1 H, H-4), 5.24
(dd, 1 H, H-3), 5.07 (dt, 1 H, H-2), 4.32 (pseudo t, 1 H,
H-5), 3.06 (m, 2 H, H-6, H-6%), 2.24 (s, 3 H, SAc), 2.12,
2.01, 1.91 (each s, each 3 H, 3×OAc). 31P (120 MHz):
l −0.52 ppm. ESIMS (negative-ion mode): Calcd for
C14H21O12PS, 444.05. Found, m/z 443.1.
purified product on analytical RP-HPLC revealed a
single peak eluting at 19 min. The final coupling and
purification gave 4.5 mmol of 8 (3.36 mg, 24.6% yield)
as the bis-morpholinium salt based on the absorbance
spectrum (m262nm of 9000 M−1 cm−1 for uridine). The
1
product was stable when stored frozen at −30 °C. H
NMR (D2O, acetone internal std, 500 MHz): l 7.90 (d,
1 H, H-6 of Ur), 5.85–5.91 (not resolved) (m, 2 H, H-5
of Ur, H-1 ribose), 5.69 (dd, 1 H, H-1 pyranose), 5.38
(d, 1 H, H-4 pyranose), 5.23 (dd, 1 H, H-3 pyranose),
5.15 (dt, 1 H, H-2 pyranose), 4.35 (pseudo t, 1 H, H-5
pyranose), 4.25 (m, 2 H, H-2, H-3 furanose), 4.18 (m, 2
H, H-5, H-5%furanose), 4.10 (m, 1 H, H-4 furanose),
3.05 (m, 2 H, H-6, H-6% pyranose), 2.24 (s, 3 H, SAc),
2.12, 2.04, 1.90 (each s, each 3 H, 3×OAc), morpho-
line signals at 3.69, 3.34 (each t, each 8 H). 31P NMR
(120 MHz): l −10.18, −12.27 (each d, J 20.0 Hz).
ESIMS (negative-ion): Calcd for C23H30N2O20P2S
749.0, [M+H]−. Found, m/z 749.2.
Acknowledgements
The authors gratefully acknowledge the helpful com-
ments from Professor Leroy B. Townsend during the
preparation of this manuscript. Financial support from
NIH AI33189, GM48049 and the College of Pharmacy
are gratefully acknowledged.
References
1. Schachter, H. Molecular Cloning of Glycosyltransferases.
In Molecular Glycobiology; Fukuda, A.; Hindsgaul, O.,
Eds.; Oxford University Press: Oxford, 1994; pp 88–162.
2. Vrielink, A.; Ruger, W.; Driessen, H.; Freemont, P.
EMBO 1994, 13, 3413–3422.
Uridine 5%-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-h-
galactopyranosyl diphosphate) (8).—Uridine 5%-
monophosphomorpholidate (4-morpholine-N%,N-di-
D-
cyclohexylcarboxamidine salt) (100 mg, 0.14 mmol) was
coevaporated with dry DMF (3×3 mL) and added to
a solution of 7 (10 mg, 18.3 mmol) in dry DMF (3 mL),
which was previously coevaporated with dry DMF
(3×3 mL). The coupling was performed at rt under
nitrogen atmosphere for 10 h. The reaction was moni-
tored by injecting 100 mL (1 mL reaction diluted into 1
mL of water) onto analytical C18 RP-HPLC while
eluting at 1 mL/min using a mobile phase of 10 mM
NH4OAc (pH 5.0) and a gradient of MeCN (0–35%
over 30 min) while monitoring Abs262nm. The reaction
mixture was dried under vacuum, dissolved in water (3
mL) and injected (500 mL, 3 mmol) onto a C18 RP-
HPLC (2×25 cm) column eluted at 10 mL/min with 10
mM NH4OAc and a gradient of 0–35% MeCN over 30
min. The peak detected by Abs262nm eluting at 19 min
was collected and concentrated repeatedly under vac-
uum at 30 °C. Final traces of NH4OAc were removed
by repeated freeze-drying. Re-chromatography of the
3. Gastinel, L. N.; Cambillau, C.; Bourne, Y. EMBO 1999,
18, 3546–3557.
4. Unligil, U. M.; Rini, J. M. Cur. Opin. Struct. Biol. 2000,
10, 510–517.
5. Morrison, J. F.; Ebner, K. E. J. Biol. Chem. 1971, 264,
3977–3984.
6. Morrison, J. F.; Ebner, K. E. J. Biol. Chem. 1971, 264,
3985–3991.
7. Srivastava, G.; Hindsgaul, O.; Palcic, M. M. Carbohydr.
Res. 1993, 245, 137–144.
8. Palcic, M. M.; Hindsgaul, O. Glycobiology 1991, 1, 205–
209.
9. Kodama, H.; Kajihara, Y.; Endo, T.; Hashimoto, H.
Tetrahedron Lett. 1993, 34, 6419–6422.
10. Kajihara, Y.; Endo, T.; Ogasawara, H.; Kodama, H.;
Hashimoto, H. Carbohydr. Res. 1995, 269, 273–294.
11. Berliner, L. J.; Robinson, R. D. Biochemistry 1982, 21,
6340–6343.
12. Hayashi, T.; Murray, B. W.; Wang, R.; Wong, C.-H.
Bioorg. Med. Chem. 1997, 5, 497–500.
13. Endo, T.; Kajihara, Y.; Kodama, H.; Hashimoto, H.
Bioorg. Med. Chem. 1996, 4, 1939–1948.

1940
J. Elhalabi, K.G. Rice / Carbohydrate Research 337 (2002) 1935–1940
14. Yuasa, H.; Hindsgaul, O.; Palcic, M. M. J. Am. Chem.
Soc. 1992, 114, 5891–5892.
15. Tsuruta, O.; Shinohara, G.; Yuasa, H.; Hashimoto, H.
Bioorg. Med. Chem. Lett. 1997, 7, 2523–2526.
16. Yuasa, H.; Palcic, M. M.; Hindsgaul, O. Can. J. Chem.
1995, 73, 2190–2195.
17. Elhalabi, J. M.; Rice, K. G. Cur. Med. Chem. 1999, 6,
93–116.
18. Guindon, Y.; Yoakim, C.; Morton, H. E. J. Org. Chem.
1984, 49, 3912–3920.
19. von Itzstein, M.; Jenkins, M. J.; Mocerino, M. Carbo-
hydr. Res. 1990, 208, 287–288.
20. Felix, A. M.; Heimer, E. P.; Lambros, T. J.; Tzou-
graki, C.; Meienhofer, J. J. Org. Chem. 1978, 43, 4194–
4196.
21. Lazar, S.; Guillaumet, G. Synth. Commun. 1992, 22,
923–931.
22. Moffatt, J. G. Methods Enzym. 1956, 15, 136–142.
23. Hallgren, C.; Hindsgaul, O. J. Carbohydr. Chem. 1995,
14, 453–464.