Synthesis of GDP-5-thiosugars and their use as glycosyl donor substrates for glycosyltransferases

Article abstract of DOI:10.1021/jo0300035

Two thiopyranoside analogues of GDP-sugars, GDP-5-thio-D-mannose (14) and GDP-5-thio-L-fucose (15), were synthesized. The syntheses included the phosphorylations of tetra-O-acetyl-5-thio-D-mannosyl bromide (4) and tri-O-benzoyl-L-fucosyl bromide (6) with silver dibenzyl phosphate, deprotection of the phosphate groups, and condensation of the deprotected phosphates with GMP-imidazolidate (13) in the presence of MgCl₂. These GDP-sugar analogues were found to be donor substrates for α(1,2)mannosyltransferase and α(1,3)fucosyltransferase, affording a 5-thiomannose-containing disaccharide (18) and a 5-thiofucose-containing trisaccharide (21), respectively. The conformation of the disaccharide analogue 18 was similar to that of its native counterpart by ROESY. These findings for GDP-5-thiosugars together with previous demonstrations of enzymatic transfer from UDP-5-thiosugars will allow the production of panels of oligosaccharide analogues with hydrolase-resistant properties.

Full text of DOI:10.1021/jo0300035

Syn th esis of GDP -5-th iosu ga r s a n d Th eir Use a s Glycosyl Don or
Su bstr a tes for Glycosyltr a n sfer a ses
Osamu Tsuruta,† Hideya Yuasa,*,† Hironobu Hashimoto,† Keiko Sujino,‡ Albin Otter,‡
Hong Li,‡ and Monica M. Palcic‡
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of
Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, J apan, and Department of Chemistry,
University of Alberta, Edmonton, Alberta T6G 2G2, Canada
hyuasa@bio.titech.ac.jp
Received J anuary 6, 2003
Two thiopyranoside analogues of GDP-sugars, GDP-5-thio-D-mannose (14) and GDP-5-thio-L-fucose
(15), were synthesized. The syntheses included the phosphorylations of tetra-O-acetyl-5-thio-D-
mannosyl bromide (4) and tri-O-benzoyl-L-fucosyl bromide (6) with silver dibenzyl phosphate,
deprotection of the phosphate groups, and condensation of the deprotected phosphates with GMP-
imidazolidate (13) in the presence of MgCl2. These GDP-sugar analogues were found to be donor
substrates for R(1,2)mannosyltransferase and R(1,3)fucosyltransferase, affording a 5-thiomannose-
containing disaccharide (18) and a 5-thiofucose-containing trisaccharide (21), respectively. The
conformation of the disaccharide analogue 18 was similar to that of its native counterpart by ROESY.
These findings for GDP-5-thiosugars together with previous demonstrations of enzymatic transfer
from UDP-5-thiosugars will allow the production of panels of oligosaccharide analogues with
hydrolase-resistant properties.
In tr od u ction
Alternatively, 5S-glycosides, the ring sulfur analogues
of native glycosides, are also resistant to glycosidases.5-7
Since 5S-glycosides can be synthesized by the usual
glycosylation methods with various 5S-glycosyl donors,
library construction appears as feasible as that of their
corresponding native counterparts. However, chemical
glycosylation with 5S-glycosyl donors gives mainly
R-glycosides7-13 even in the presence of an equatorial
acetoxy group that would generally promote â-glycoside
formation by anchimeric assistance. Therefore, it would
be difficult to chemically synthesize 5S-analogues with
the â-gluco or â-galacto configurations that are ubiqui-
tous in natural oligosaccharides.
Glycosidase-resistant oligosaccharide analogues are
expected to survive for a longer time in vivo than their
native counterparts. As such, they would be able to confer
metabolic stability to oligosaccharide-based drugs similar
to drugs based on peptide mimetics, e.g., buserelin and
octreotide.1 Glycosidase-resistant oligosaccharides such
as S-glycosides2 and C-glycosides3 can also be used for
biochemical studies. For example, in the X-ray analysis
of the complex between endoglucanase-I and a cellopen-
toside analogue in which all the interglycosidic bonds are
sulfides and therefore hydrolase-resistant, a stable con-
formation was captured in the enzyme-substrate com-
plex.4 The availability of glycosidase-resistant oligosac-
charide analogues would be valuable for the discovery of
biologically active oligosaccharide ligands, even when the
target receptor is a hydrolase or the assay system
involves administration to cells or organisms rich in
hydrolases. However, it may be difficult to construct a
library containing S- or C-glycoside in all possible posi-
tions. In a simple case such as the construction of an
S-linked glucose-glucose disaccharide library using a
normal glucosyl donor, one needs to elaborate four
intricate glucose analogues, each having a thiol group in
place of a hydroxyl group from OH-2 to OH-6.
It has been shown that UDP-5-thiogalactose (UDP-
5SGal)5 and UDP-N-acetyl-5-thiogalactosamine (UDP-
5SGalNAc)14 are substrates for galactosyltransferase,
giving 5SGalâ(1,4)GlcNAc and 5SGalNAcâ(1,4)GlcNAc
(5) Yuasa, H.; Hindsgaul, O.; Palcic, M. M. J . Am. Chem. Soc. 1992,
114, 5891.
(6) Hashimoto, H.; Kawanishi, M.; Yuasa, H. Chem.sEur. J . 1996,
2, 556.
(7) Mehta, S.; Andrews, J . S.; J ohnston, B. D.; Svensson, B.; Pinto,
B. M. J . Am. Chem. Soc. 1995, 117, 9783.
(8) Mehta, S.; Pinto, B. M. Tetrahedron Lett. 1992, 33, 7675.
(9) Hashimoto, H.; Izumi, M. Tetrahedron Lett. 1993, 34, 4949.
(10) Mehta, S.; J ordan, K. L.; Weimar, T.; Kreis, U. C.; Batchelor,
R. J .; Einstein, F. W. B.; Pinto, B. M. Tetrahedron: Asymmetry 1994,
5, 2367.
† Tokyo Institute of Technology.
‡ University of Alberta.
(11) Izumi, M.; Tsuruta, O.; Hashimoto, H.; Yazawa, S. Tetrahedron.
Lett. 1996, 37, 1809.
(12) Izumi, M.; Tsuruta, O.; Harayama, S.; Hashimoto, H. J . Org.
Chem. 1997, 62, 992.
(1) Giannis, A.; Ru¨bsam, F. Adv. Drug Res. 1997, 29, 1.
(2) Ferna´ndez, J . M. G.; Mellet, C. O.; Defaye, J . J . Org. Chem. 1998,
63, 3572 and references therein.
(3) Ravishankar, R.; Surolia, A.; Vijayan, M.; Lim, S.; Kishi, Y. J .
Am. Chem. Soc. 1998, 120, 11297 and references therein.
(4) Sulzenbacher, G.; Driguez, H.; Henrissat, B.; Schu¨lein, M.;
Davies, G. J . Biochemistry 1996, 35, 15280.
(13) Izumi, M.; Suhara, Y.; Ichikawa, Y. J . Org. Chem. 1998, 63,
4811.
(14) Tsuruta, O.; Shinohara, G.; Yuasa, H.; Hashimoto, H. Bioorg.
Med. Chem. Lett. 1997, 7, 2523.
10.1021/jo0300035 CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/17/2003
6400
J . Org. Chem. 2003, 68, 6400-6406

Synthesis of GDP-5-thiosugars
with complete regio- and stereoselectivity. This allows
the custom syntheses of 5S-glycosides with native se-
quences and anomeric configurations. In addition to
UDP-5S-sugars, if guanine-based 5S-sugar nucleotides
were donor substrates for glycosyltransferases, the en-
zymatic syntheses of many important 5S-sugar-based
oligosaccharide analogues would be possible (Figure 1).
We report the synthesis of two GDP-5S-sugar nucle-
otides [GDP-5-thiomannose (GDP-5SMan) and GDP-5-
thiofucose (GDP-5SFuc)] and their evaluation as sub-
strates for mannosyltransferase (ManT) and fucosyltrans-
ferase (FucT), respectively. The demonstration that these
sugar nucleotides are substrates for these glycosyltrans-
ferases implies that production of 5S-glycoside analogues
of oligomannose,15 H-type 2, and Lex 11 is feasible. All of
these have been chemically synthesized with considerable
effort. Of particular interest are analogues containing
5SFuc, since the ring sulfur atom tends to interact
preferably with receptors as exemplified by the strong
F IGURE 1. Construction of the 5-thiosugar-based oligosac-
charide mimetics library with glycosyltransferases.
SCHEME 1
J . Org. Chem, Vol. 68, No. 16, 2003 6401

Tsuruta et al.
SCHEME 2
inhibitory activity of 5SFuc against fucosidases16 and the
5SFuc-based H-type 2 mimetics against an anti-H-type
2 antibody.11
fucosyl bromide (6), though the R-phosphate was still a
major isomer (9r:9â ) 1.4:1). The stereoselectivity of the
phosphorylation might be under the control of competi-
tion between the kinetic anomeric effect favoring the
R-anomer formation and the neighboring-group partici-
pation that assists the â-anomer formation. If so, the
partial formation of the â-anomer with the benzoyl
derivative 6 is due to an enhanced participating ability
of the carbonyl oxygen of 2-benzoate by delocalization of
π-electrons from benzene.
Hydrogenolysis with 10% Pd/C as catalyst and alkaline
treatment of 7 and 9â gave the deprotected phosphates
10 and 11, respectively. When these compounds were
subjected to condensation with GMP-morpholidate (12)
in the usual manner,19 poor results were obtained, i.e.,
long reaction times of more than a week and low yields
(<10%), as was reported for the synthesis of GDP-Fuc.20
Recently, Wittmann and Wong reported that the addition
of 1H-tetrazole improved the GDP coupling reaction.21
However, attempts to use 1H-tetrazole in the reaction of
10 with 12 also gave low yields. Kadokura and co-workers
recently reported an efficient P-P coupling method.22
They showed that addition of a divalent metal chloride
Resu lts a n d Discu ssion
Synthetic schemes for GDP-5SMan and GDP-5SFuc
are shown in Scheme 1. The key step in these schemes
was the stereoselective formation of R- and â-glycosyl
phosphates required for GDP-5SMan and GDP-5SFuc,
respectively. The reported synthetic methods for R-man-
nosyl phosphate and â-fucosyl phosphate include the
coupling reaction between the per-O-acetylated glycosyl
bromide and a silver salt of dibenzyl phosphate.17 The
desired configuration predominated for each compound
due to anchimeric assistance from the 2-acetate. We used
the same methods for the syntheses of 5S-glycosyl
phosphates. Thus, R-5-thiomannosyl phosphate 7 was
stereoselectively obtained from per-O-acetyl-5-thioman-
nosyl bromide (4), as supported by optical rotation ([R]25
D
+57.3), which showed approximately the same value as
that of the R-mannosyl phosphate ([R]25 +58).18 In the
D
case of 5SFuc, however, only the undesired R-phosphate
8r was produced from per-O-acetyl-5-thiofucosyl bromide
(5). This result exemplifies the unusual axial glycoside
formation in 5S-glycosylation reactions7-13 despite the
presence of 2-acetate that would usually direct equatorial
glycoside formation. The desired â-phosphate was ob-
tained by the phosphorylation of per-O-benzoyl-5-thio-
(19) (a) Kochetkov, N. K.; Shibaev, V. N. Adv. Carbohydr. Chem.
Biochem. 1973, 28, 307. (b) Moffat, J . G. Methods Enzymol. 1966, 8,
136.
(20) (a) Prihar, H. S.; Behrman, E. J . Biochemistry 1973, 12, 997.
(b) Nunez, H. A.; O’Connor, J . V.; Rosevear, P. R.; Baker, R. Can. J .
Chem. 1981, 59, 2086. (c) Gokhale, U. B.; Hindsgaul, O.; Palcic, M. M.
Can. J . Chem. 1990, 68, 1063. (d) Schmidt, R. R.; Wegmann, B.; J ung,
K.-H. Liebigs Ann. Chem. 1991, 121. (e) Veeneman, G. H.; Broxterman,
H. J . G.; Marel, G. A.; van Boom, J . H. Tetrahedron Lett. 1991, 32,
6175.
(15) Yuasa, H.; Matsuura, S.; Hashimoto, H. Bioorg. Med. Chem.
Lett. 1998, 8, 1297.
(16) Hashimoto, H.; Fujimori, T.; Yuasa, H. J . Carbohydr. Chem.
1990, 9, 683.
(17) Putman, E. W. Methods Carbohydr. Chem. 1963, 2, 261.
(18) Colowick, S. P. J . Biol. Chem. 1938, 124, 557.
(21) Wittmann, V.; Wong, C.-H. J . Org. Chem. 1997, 62, 2144.
(22) Kadokura, M.; Wada, T.; Urashima, C.; Sekine, M. Tetrahedron
Lett. 1997, 38, 8359.
6402 J . Org. Chem., Vol. 68, No. 16, 2003

Synthesis of GDP-5-thiosugars
promoted the formation of P1-methyl-P3-(5′-guanosyl)-
triphosphate in the reaction of GDP with methyl phos-
phorimidazolidate, with a shorter reaction time.22 We
followed this method by the addition of 4 equiv of MgCl2
to the reaction of the deprotected phosphates 10 and 11
with GMP-imidazolidate (13) in DMF, to give GDP-
5SMan (14) and GDP-5SFuc (15) in acceptable to sig-
nificant yields (22% and 54%, respectively) within 36 h.
The reactions of 14 and 15 with tetramethylrhodamine
(TMR)-labeled glycosyl acceptors 16 and 1923 were carried
out in the presence of yeast R(1,2)ManT24 and milk R-
(1,3)FucT,25 respectively (Scheme 2). Analyses of these
reactions by capillary electrophoresis and MALDI-TOF
mass spectrometry revealed that the corresponding prod-
ucts were formed as a result of enzymatic 5S-saccharide
transfer. The relative rates of transfer of the thiodonors
compared to natural donor substrates were 0.1 % for 14
and 2.3% for 15. The relative rate of transfer for 14 was
considerably lower than that of 15. Reactions of acceptors
17 and 20 with 14 and 15, respectively, were carried out
to assign the structures of the saccharide products.
Product isolation on a reversed-phase preparative MPLC
instrument afforded the 5-thiomannose-containing dis-
accharide 18 and the 5-thiofucose-containing trisaccha-
ride 21, whose structures were supported by their 1H
NMR spectra. The isolated yields of these reactions were
7.8% for 18 and 41% for 21, respectively.
The 2D ROESY spectrum for 18 (Figure 2) showed two
strong interglycosidic ROE signals for H-1/H-5′ and H-1′/
H-2, the latter being typical for the 1,2-glycosidic linkage.
Peters reported similar NOE signals, including that of
H-1/H-5′, for a natural ManR(1,2)Man disaccharide.26
These results suggest a similarity for 18 and its natural
counterpart with respect to their conformational popula-
tions on the basis of the flexibility of the glycosidic
linkages. Peters carried out HSEA calculations on the
conformation of the natural disaccharide with regard to
the glycosidic dihedral angles (φ ) H1′-C1′-O2-C2, ψ
) C1′-O2-C2-H2) and estimated the global minimum
A (φ ) -47°, ψ ) -20°) and local minimum B (φ ) 30°,
ψ ) 22°) conformations, the energy difference being 2.4
kcal/mol.26 Conformation A is consistent with the H-1/
H-5′ cross-peak. According to an MM2 (92) calculation
on the same disaccharide by Dowd and French, the global
minimum C (φ ) -25.5°, ψ ) 60.1°) is slightly (0.72 kcal/
mol) more stable than local minimum D (φ ) -42.4°, ψ
) -21.3°).27 Conformation D is identical to conformation
A, the global minimum from the HSEA calculation.
Conformations A ()D) and C have glyconic dihedral
angles (φ) in the range of the exo-anomeric effect and have
an aglyconic dihedral angle (ψ) of -20° or 60°, only the
former being consistent with the H-1/H-5′ cross-peak.
Since the sulfur atom is electronegative enough to induce
an exo-anomeric effect in 5-thioglycosides,10 the glyconic
dihedral angle of compound 18 would reside mainly
between -30° and -60°. Then, the aglyconic dihedral
F IGURE 2. Relevant section of the TROESY spectrum of 18
in D2O.
must be around -20° to generate the H-1/H-5′ cross-peak.
Overall, a large population of the dihedral angles for
compound 18 should reside near those forming conforma-
tion A (Figure 3). The 1H NMR spectrum of 21 shows
full accordance with that of the same trisaccharide
analogue synthesized by chemical methods.11 As has been
pointed out in a previous paper,28 the chemical shift for
H-5′′ (5SFuc) resides at a rather lower field (δ ca. 3.9
ppm) than does that of 5-thiofucose (δ 3.64 ppm). A
similar deshielding has been observed in the natural Lex
which is ascribed to the proximity of H-5′′ to the galactose
unit.29 The proximity effect observed for both the natural
and trisaccharide analogues suggests they have similar
conformations.
Accumulating data for 5S-glycosides, including this
study, suggest they are conformationally similar to their
natural counterparts.10,13 In addition, 5S-glycosides are
generally expected to be resistant to hydrolysis by
glycosidases. These characteristics of 5S-glycosides lend
themselves to the development of oligosaccharide-based
drugs, analogous to the amide bond isosteres of drugs
(23) (a) Zhao, J . Y.; Dovichi, N. J .; Hindsgaul, O.; Gosselin, S.; Palcic,
M. M. Glycobiology 1994, 4, 239. (b) Zhang, Y.; Le, X.; J . Dovichi, N.;
Compton, C. A.; Palcic, M. M.; Diedrich, P.; Hindsgaul, O. Anal.
Biochem. 1995, 227, 368.
(24) Wang, P.; Shen, G.-J .; Wang, Y.-F.; Ichikawa, Y.; Wong, C.-H.
J . Org. Chem. 1993, 58, 3935.
(25) Palcic, M. M. Methods Enzymol. 1994, 230, 300.
(26) Peters, T. Leibigs Ann. Chem. 1991, 135.
(28) Tsuruta, O.; Yuasa, H.; Hashimoto, H.; Kurono, S.; Yazawa, S.
Bioorg. Med. Chem. Lett. 1999, 9, 1019.
(29) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J .; Garcia-
J unceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.;
Paulson, J . C.; Wong, C.-H. J . Am. Chem. Soc. 1992, 114, 9298.
(27) Dowd, M. K.; French, A. D. J . Carbohydr. Chem. 1995, 14, 589.
J . Org. Chem, Vol. 68, No. 16, 2003 6403

Tsuruta et al.
commercially. R(1,3)Fucosyltransferase was isolated from hu-
man milk as has been described.25
2,3,4,6-Tetr a-O-a cetyl-5-th io-r-D-m an n opyr an osyl Diben -
zyl P h osp h a te (7). To a solution of 1,2,3,4,6-penta-O-acetyl-
5-thio-D-mannose (1)30 (49 mg, 121 µmol) in CH2Cl2 (1.0 mL)
was added dropwise 25% HBr-AcOH (0.5 mL) at 0 °C, and
the mixture was stirred for 2 h at room temperature. The
mixture was poured onto ice-water and extracted with CHCl3.
The extracts were washed with water and aqueous NaHCO3,
dried over MgSO4, and concentrated to give 2,3,4,6-tetra-O-
acetyl-5-thio-D-mannopyranosyl bromide (4). This compound
was used in the next step without further purification. Silver
dibenzyl phosphate (47 mg, 122 µmol) was added to a solution
of the bromide in benzene (2.0 mL). The mixture was refluxed
for 1 h in a round-bottom flask covered with aluminum foil to
shield it from light and then was filtered through Celite, and
the filtrate was concentrated. The residue was purified on a
column of silica gel (3:2 hexanes-EtOAc) to give 7 (49 mg,
1
63%) as a syrup: [R]25 +57.3 (c 2.44, CHCl3); H NMR (270
D
MHz, CDCl3) δ 7.39-7.34 (m, 10H), 5.44 (dd, 1H, J ) 3.3, 4.3
Hz), 5.43 (t, 1H, J ) 10.2 Hz), 5.41 (dd, 1H, J ) 4.3, 6.9 Hz),
5.25 (dd, 1H), 5.27-5.22 (m, 4H), 4.25 (dd, 1H, J ) 5.6, 12.2
Hz), 3.93 (dd, 1H, J ) 3.3, 12.2 Hz), 3.40 (ddd, 1H, J ) 3.3,
5.6, 12.2 Hz), 2.17, 2.04, 2.02, 2.00 (each s, 3H × 4); 13C NMR
(CDCl3) δ 170.40, 169.56, 169.47, 128.68, 128.61, 128.12,
127.91, 78.08, 77.99, 70.39, 70.24, 69.83, 69.76, 69.69, 69.54,
68.41, 61.33, 39.91, 20.85, 20.54; 31P NMR (CDCl3) δ -2.23;
HR-ESMS m/z calcd for C28H33O12PSNa (M + Na)+ 647.1328,
found 647.1330.
F IGURE 3. A presumed stable conformation of 18 with
glycosidic torsion angles of φ ) -47° and ψ ) -20°.
based on peptide mimetics. Developments of such car-
bohydrate drugs are now viable, since we can enzymati-
cally attach four different 5S-sugars, 5SGal, 5SGalNAc,
5SFuc, and 5SMan, to oligosaccharides. A further ad-
vantage expected for 5S-glycosides is their potentially
stronger affinity for receptors compared to that of their
natural counterparts. 5S-glycosides might find another
application in hydrolase-resistant vaccines, since a part
of the efforts to develop oligosacchride-based drugs has
been focused on the cancer vaccines with oligosaccharide
haptens.
Gu a n osin e 5′-Dip h osp h o-5-th io-r-D-m a n n ose Dia m m o-
n iu m Sa lt (14). To a solution of 4 (76 mg, 122 µmol) in MeOH
(2.0 mL) were added 10% Pd/C (100 mg) and tributylamine
(30 mg, 162 µmol). The mixture was hydrogenated under
hydrogen ballon with vigorous stirring for 24 h at room
temperature. The mixture was filtered through Celite, and the
filtrate was concentrated. The residue was allowed to stand
for 24 h at room temperature in MeOH-H2O-triethylamine
(7:3:1, 3.3 mL). The solution was concentrated to give 5-thio-
R-D-mannopyranosyl phosphate tributylammonium salt (10).
In the meantime, the following operations were performed. To
a solution of guanosine 5′-monophosphate (53 mg, 146 µmol)
and tributylamine (38 mL, 160 µmol) in DMF (1.5 mL) was
added N,N′-carbonyldiimidazole (26 mg, 160 µmol), and the
resulting mixture was stirred for 1 h at room temperature.
The reaction was quenched with a small amount of MeOH and
concentrated to give crude 13. The glycosyl phosphate 10 and
13 thus obtained were coevaporated twice with pyridine and
twice with DMF, respectively, and redissolved in DMF (2.0
mL). MgCl2 (46 mg, 483 µmol) was added to the mixture, which
gave rise to a clear homogeneous solution. It was stirred for
36 h at room temperature. After the mixture was concentrated,
the residue was applied to a column of anion-exchange resin
(Dowex 1-X-8, formate form) and fractionated by a gradient
elution of aqueous NH4HCO3 (0-1 M). The fractions containing
GDP-5-thiomannose were collected and concentrated. The
residue was further purified with a column of Sephadex G-15.
The appropriate fractions were collected and lyophilized to give
14 (18 mg, 22%) as a fluffy powder: [R]26D +39.5 (c 0.74, H2O);
1H NMR (400 MHz, D2O) δ 8.16 (s, 1H), 5.98 (d, 1H, J ) 6.4
Hz), 5.33 (dd, 1H, J ) 4.0, 9.2 Hz), 4.83 (dd, 1H, J ) 5.2, 6.4
Hz), 4.57 (dd, 1H, J ) 3.0, 5.2 Hz), 4.40 (m, 1H), 4.37 (dd, 1H,
J ) 2.3, 4.0 Hz), 4.28-4.25 (m, 2H), 3.94-3.78 (m, 4H), 3.32
(m, 1H); 31P NMR (D2O) δ -10.72 (J ) 20.6 Hz), -12.63 (J )
20.6 Hz); HR-ESMS m/z calcd for C16H25N5O15P2SNa (M +
Na)+ 644.0441, found 644.0436.
Con clu sion s
We demonstrate that GDP-5SMan and GDP-5SFuc are
donor substrates for R(1,2)ManT and R(1,3)FucT, afford-
ing a 5SMan-containing disaccharide and a 5SFuc-
containing Lex trisaccharide, respectively. The results of
this study and previous studies with UDP-5SGal and
UDP-5SGalNAc donors establish that 5-thiohexose nu-
cleotides are substrates for glycosyltransferases. In prin-
ciple, oligosaccharide analogues containing 5S-glycosides
of all structures containing these monosaccharides can
be enzymatically generated. 5S-glycosylation has the
potential of becoming a routine modification in the
development of oligosaccharide-mimetics-based drugs,
similar to thioamide isosteres in peptide mimetics and
phosphorothioate isosteres in antisense oligonucleotides.
Exp er im en ta l Section
Gen er a l P r oced u r es. Optical rotations were measured
using a 0.5 dm cell. 1H NMR spectra were obtained at 270
MHz with TMS as an internal standard in CDCl3 and at 400
and 600 MHz with HOD (δ 4.80) as an internal standard in
D2O. 13C NMR spectra were obtained at 67.8 MHz with TMS
as an internal standard. 31P NMR spectra were obtained at
109.25 MHz with 85% H3PO4 as an external standard. Column
chromatography was performed with Kieselgel 60 or Wakogel
C-300. TLC was carried out on plates precoated with Kieselgel
60 F254. R(1,2)Mannosyltransferase from Saccharomyces cer-
evisiae and calf intestine alkaline phosphatase were obtained
2,3,4-Tr i-O-a cetyl-5-th io-r-L-fu cop yr a n osyl Dib en zyl
P h osp h a te (8). To a solution of 1,2,3,4-tetra-O-acetyl-5-thio-
L-fucose (2)31 (70 mg, 201 µmol) in CH2Cl2 (2.0 mL) was added
(30) Yuasa, H.; Izukawa, Y.; Hashimoto, H. J . Carbohydr. Chem.
1989, 8, 753.
(31) Izumi, M.; Tsuruta, O.; Hashimoto, H. Carbohydr. Res. 1996,
280, 287.
6404 J . Org. Chem., Vol. 68, No. 16, 2003

Synthesis of GDP-5-thiosugars
dropwise 25% HBr-AcOH (1.0 mL) at 0 °C, and the mixture
was stirred for 2 h at room temperature. The mixture was
poured onto ice-water and extracted with CHCl3. The extracts
were washed with water and aqueous NaHCO3, dried over
MgSO4, and concentrated to give 2,3,4-tri-O-acetyl-5-thio-L-
fucopyranosyl bromide (5). This compound was used in the
next step without further purification. To a solution of the
bromide in benzene (1.0 mL) was added silver dibenzyl
phosphate (93 mg, 241 µmol). The mixture was refluxed for 1
h in a round-bottom flask covered with aluminum foil to shield
it from light and then was filtered through Celite, and the
filtrate was concentrated. The residue was purified on a
column of silica gel (3:2 hexanes-EtOAc) to give 8 (67 mg,
+ Na)+ 775.1743, found 775.1784. Anal. Calcd for C41H37O10
-
PS: C, 65.42; H, 4.95. Found: C, 65.48; H, 5.31.
Gu a n osin e 5′-Dip h osp h o-5-th io-â-L-fu cose Dia m m o-
n iu m Sa lt (15). To a solution of 9â (138 mg, 183 µmol) in
MeOH (15 mL) were added 10% Pd/C (50 mg) and triethyl-
amine (28 µL, 200 µmol). The mixture was hydrogenated under
hydrogen ballon with vigorous stirring for 12 h at room
temperature. The mixture was filtered through Celite, and the
filtrate was concentrated. The residue was allowed to stand
for 2 days in aqueous NH3 (28%)-pyridine (4:1, 15 mL) at room
temperature. The solution was concentrated, and the residue
was applied to a reversed-phase (C-18) column and eluted with
water. The elute was lyophilized to give 5-thio-â-fucopyranosyl
phosphate diammonium salt (53 mg, 98%) as a fluffy powder.
A solution of this ammonium salt (18 mg, 61 µmol) in a small
volume of water was passed through a column of cation-
exchange resin (Dowex 50W-X-8, pyridinium form) with water,
and the elute was concentrated. To a solution of the residue
in pyridine (1.0 mL) was added tributylamine (16 µL, 67 µmol).
The mixture was concentrated and coevaporated twice with
pyridine. A solution of this residue in a small volume of water
was lyophilized to give 5-thio-L-fucopyranosyl phosphate tri-
butylammonium salt (11). In the meantime, the following
operations were performed. To a solution of guanosine 5′-
monophosphate (44 mg, 121 µmol) and tributylamine (32 mL,
134 µmol) in DMF (1 mL) was added N,N′-carbonyldiimidazole
(24 mg, 148 µmol), and the resulting mixture was stirred for
2 h at room temperature. The reaction was quenched with a
small amount of MeOH and concentrated to give crude 13. This
imidazolide 13 was coevaporated twice with pyridine and twice
with DMF and dissolved in DMF (1.0 mL) together with the
lyophilized phosphate 11. To the mixture was added MgCl2
(23 mg, 244 µmol), and the mixture was stirred for 36 h at
room temperature. The mixture was purified in the same
manner as described for the preparation of 14 to give 15 (21
59%) as a syrup: [R]26 -160.5 (c 0.91, CHCl3); 1H NMR (270
D
MHz, CDCl3) δ 7.36-7.26 (m, 10H), 5.68 (br d, 1H, J ) 8.6
Hz), 5.46 (br s, 1H), 5.37-5.29 (m, 2H), 5.16-5.03 (m, 4H),
3.44 (br q, 1H, J ) 6.9 Hz), 2.17, 1.99, 1.88 (each s, 3H × 3),
1.08 (d, 3H, J ) 6.9 Hz); 13C NMR (CDCl3) δ 170.55, 170.04,
169.74, 135.40, 128.60, 128.57, 128.54, 127.95, 127.83, 78.03,
77.92, 72.19, 70.01, 69.92, 69.58, 69.49, 69.40, 68.43, 35.20,
20.63, 20.56, 20.50, 15.55; 31P NMR (CDCl3) δ -1.31; HR-
ESMS m/z calcd for C26H32O10PS (M + H)+ 567.1454, found
567.1453.
2,3,4-Tr i-O-ben zoyl-5-th io-â-L-fu cop yr a n osyl Diben zyl
P h osp h a te (9â). To a solution of 2 (100 mg, 287 µmol) in
MeOH (3 mL) was added NaOMe (catalytic). After being
stirred overnight, the solution was neutralized with Dowex
50W-X8 (H+), and the resin was filtered off. The filtrate was
concentrated in vacuo, and the residue was dissolved in
pyridine (3 mL). Benzoyl chloride (0.2 mL, 1.72 mmol) was
added dropwise at 0 °C, and the mixture was stirred overnight
at room temperature. The mixture was diluted with CHCl3,
washed with 1 M HCl and water, dried over MgSO4, and
concentrated. The residue was purified on a column of silica
gel (10:1 hexanes-EtOAc) to give 1,2,3,4-tetra-O-benzoyl-5-
thio-L-fucopyranose (3) concomitant with a small amount of
reagent. This mixture was used in the next step without
further purification. To a solution of this crude residue in CH2-
Cl2 (5 mL) was added dropwise 25% HBr-AcOH (1.5 mL) at
0 °C, and the mixture was stirred for 2 h at room temperature.
The mixture was poured onto ice-water and extracted with
CHCl3. The extracts were washed with water and aqueous
NaHCO3, dried over MgSO4, and concentrated to give 2,3,4-
tri-O-benzoyl-5-thio-L-fucopyranosyl bromide (6). This com-
pound was used in the next step without further purification.
To a solution of the bromide in benzene (11 mL) was added
silver dibenzyl phosphate (150 mg, 390 µmol). The mixture
was refluxed for 1 h in a round-bottom flask wrapped with
aluminum foil to shield the light and filtered through Celite,
and the filtrate was concentrated. The residue was purified
on a column of silica gel (3:1 hexanes-EtOAc) to give 9r (112
mg, 52%) and 9â (78 mg, 36%) each as a syrup. Data for 9r:
mg, 54%) as a fluffy powder: [R]26 -29.9 (c 0.64, H2O); 1H
D
NMR (400 MHz, D2O) δ 8.17 (s, 1H), 5.98 (d, 1H, J ) 6.3 Hz),
5.15 (t, 1H, J ) 8.9 Hz), 4.86 (dd, 1H, J ) 5.3, 6.3 Hz), 4.60
(dd, 1H, J ) 3.3, 5.3 Hz), 4.40 (t, 1H, J ) 3.3 Hz), 4.28 (m,
2H), 3.95 (br s, 1H), 3.84 (dd, 1H, J ) 8.9, 9.6 Hz), 3.52 (dd,
1H, J ) 3.1, 9.6 Hz), 3.26 (br q, 1H, J ) 7.0 Hz), 1.21 (d, 3H,
J ) 7.0 Hz); 31P NMR (D2O) δ -10.42 (J ) 20.6 Hz), -11.81
(J ) 20.6 Hz); HR-ESMS m/z calcd for C16H26N5O14P2S (M +
H)+ 606.0672, found 606.0680.
r(1,2)Ma n T Ca p illa r y Electr op h or esis Assa y. 14 (10
nmol) was incubated with R-Mn-TMR (16; 5 nmol) in the
presence of R(1,2)ManT (745 µU) in a buffer (100 mM MOPS,
20 mM MnCl2, 0.1% Triton X-100, 1 mg/mL BSA, pH 7.7, total
volume 5 µL) at 37 °C for 2 h. As a reference, a reaction
mixture consisting of GDP-D-mannose (10 nmol), 16 (5 nmol),
and R(1,2)ManT (5.7 µU) in the same buffer was incubated at
37 °C for 2 h. An aliquot (0.5 µL) of each reaction mixture was
diluted with water (50 µL) and then loaded onto a C-18 Sep-
Pak cartridge preequilibrated with MeOH (10 mL) and water
(10 mL). The cartridge was washed with water (10 mL), 30%
MeOH (8 mL), and 50% MeOH (4 mL). The substrates and
products bearing TMR were eluted with MeOH (4 mL) and
concentrated under vacuum. The analysis of the relative rate
by capillary electrophoresis with laser-induced fluorescence
detection was performed as described before.32
En zym a tic Rea ction w ith Don or 14. The mannosyl
acceptor 17 (1.00 mg, 2.85 µmol) was incubated with 14 (2.4.
mg, 3.71 µmol) in the presence of R(1,2)ManT (280 mU, 175
µL) and alkaline phosphatase (1 U/µL, 1 µL) in buffer (100
mM MOPS, 5 mM MnCl2, 0.1% Triton X-100, 1 mg/mL BSA,
175 µL) at room temperature. Reaction was continued for 44
days. During the reaction, 1.2 mg of 14 at the 12th and 33rd
days, 1.72 µL of 1 M MnCl2 at the 33rd day, and 172 µL of the
buffer at the 22nd day were added to the reaction mixture.
[R]26 -254.3 (c 1.26, CHCl3); 1H NMR (270 MHz, CDCl3) δ
D
8.14-7.18 (m, 25H), 5.98 (dd, 1H, J ) 2.0, 7.9 Hz), 5.93-5.91
(m, 3H), 5.09-5.00 (m, 4H), 3.69 (br q, 1H, J ) 6.9 Hz), 1.20
(d, 3H, J ) 6.9 Hz); 13C NMR (CDCl3) δ 165.88, 165.73, 165.50,
135.49, 135.29, 133.53, 133.28, 133.15, 129.96, 129.70, 129.63,
129.27, 129.04, 128.95, 128.66, 128.52, 128.32, 128.25, 127.82,
127.75, 78.53, 78.42, 73.17, 71.39, 71.30, 69.54, 69.36, 35.99,
15.80; 31P NMR (CDCl3) δ -1.45; HR-ESMS m/z calcd for
C
41H37O10PSNa (M + Na)+ 775.1743, found 775.1744. Data for
9â: [R]25 -147.7 (c 1.38, CHCl3); 1H NMR (270 MHz, CDCl3)
D
δ 8.30-6.94 (m, 25H), 6.12 (dd, 1H, J ) 9.2, 9.6 Hz), 5.92 (dd,
1H, J ) 1.7, 2.6 Hz), 5.86 (dd, 1H, J ) 8.6, 9.2 Hz), 5.43 (dd,
1H, J ) 2.6, 9.6 Hz), 5.09, 4.79 (each dd, 1H · 2, J ) 7.3, 11.6
Hz), 5.02, 4.68 (each dd, 1H × 2, J ) 7.3, 11.6 Hz), 3.59 (dq,
1H, J ) 1.7, 6.9 Hz), 1.35 (d, 3H, J ) 6.9 Hz); 13C NMR (CDCl3)
δ 165.77, 165.46, 165.34, 135.35, 135.24, 133.50, 133.24,
129.99, 129.74, 129.69, 129.22, 128.95, 128.70, 128.60, 128.48,
128.34, 128.30, 128.23, 127.85, 127.46, 76.43, 72.76, 72.13,
72.02, 71.90, 69.76, 69.67, 69.50, 69.44, 38.20, 16.03; 31P NMR
(CDCl3) δ -1.64; HR-ESMS m/z calcd for C41H37O10PSNa (M
(32) Sujino, K.; Uchiyama, T.; Hindsgaul, O.; Seto, N. O. L.;
Wakarchuk, W. W.; Palcic, M. M. J . Am. Chem. Soc. 2000, 122, 1261.
J . Org. Chem, Vol. 68, No. 16, 2003 6405

Tsuruta et al.
The enzyme retained 75% and 25% of its original activity after
12 and 33 days, respectively. The yield of this reaction was
estimated from TLC char to be 20% based on acceptor 17. The
reaction mixture was loaded onto a C18 Sep-Pak cartridge
preequilibrated with MeOH (10 mL) and water (10 mL). The
cartridge was washed with water (50 mL), and then the
product was eluted with MeOH (40 mL). The MeOH eluate
was concentrated, and the resulting residue was loaded onto
a column of Iatrobeads (4.0 g) which was eluted with 10:1
CH2Cl2-MeOH (25 mL), 4:1 CH2Cl2-MeOH (24 mL), and 80:
20:1 CH2Cl2-MeOH-H2O (101 mL). The fractions containing
the product were collected and concentrated. The residue was
again loaded onto a preequilibrated C-18 Sep-Pak cartridge,
and the cartridge was washed with water (30 mL) and then
MeOH (10 mL). The MeOH eluate was concentrated, and the
product was dissolved in water (10 mL). This solution was
(m, 12H, -(CH)2-); HR-ESMS m/z calcd for C22H40O12NaS (M
+ Na)+ 551.2138, found 551.2138.
E n zym a t ic R ea ct ion w it h Don or 15. The N-acetyl-
lactosaminyl acceptor 20 (0.5 mg, 0.9 µmol) was incubated with
15 (0.6 mg, 0.9 µmol) in the presence of milk R(1,3)FucT (33.75
mU, in 450 µL of 25 mM sodium cacodylate buffer, pH 6.5,
containing 5 mM MnCl2 and 25% glycerol) and 50 µL of
concentrated buffer (200 mM Hepes, pH 7.0, containing 200
mM MnCl2 and 2% BSA) at 37 °C. Additional 12 (0.6 mg, 0.9
µmol) was added 8, 24, and 48 h after the initiation of the
reaction. The reaction was terminated after 7 days. The yield
of this reaction was estimated from TLC char to be 75% based
on acceptor 20. The reaction mixture was purified in the same
manner as described for the reaction of 14 with R(1,2)ManT
to give the trisaccharide 21 containing 25% starting compound
1
20 as a fluffy powder (0.5 mg, 41%). The H NMR spectrum of
passed through
a filter (0.22 µm), and the filtrate was
21 shows a full accordance with that of the same trisaccharide
analogue synthesized by chemical methods.10 MALDI-TOF MS
m/z 716.3 (M + H)+, 738.3 (M + Na)+.
lyophilized to give 18 as a fluffy white powder (0.117 mg,
7.8%): 1H NMR δ 5.11 (d, 1H, J 1,2 ) 1.5 Hz, H-1), 4.87 (d, 1H,
J 1′,2′ ) 3.7 Hz, H-1′), 4.35 (dd, 1H, J 2′,3′ ) 2.6 Hz, H-2′), 4.04
(dd, 1H J 2,3 ) 3.3 Hz, H-2), 3.94 (dd, 1H, J 6′a,5′ ) 3.1, J 6′a,6′b
11.9 Hz, H-6′a), 3.93 (dd, 1H, J 3,4 ) 9.8 Hz, H-3), 3.89 (dd,
1H, J 6a,5 ) 2.3, J 6a,6b ) 11.6 Hz, H-6a), 3.82 (dd, 1H, J 6′b,5′
)
Ack n ow led gm en t. We are indebted to Professor Ole
Hindsgaul, University of Alberta, for providing com-
pounds 16, 17, 19, and 20. This work was funded in part
by a grant from the Natural Sciences and Engineering
Research Council of Canada (to M.M.P.).
)
6.9 Hz, H-6′b), 3.80 (t, 1H, J 4′,3′ ) J 4′,5′ ) 9.8 Hz, H-4′), 3.79-
3.76 (m, 1H, H-3′), 3.78 (dd, 1H, H-6b), 3.75-3.72 (m, 1H,
-CH2O-), 3.70 (t, 1H, J 4,3 ) J 4,5 ) 9.8 Hz, H-4), 3.69 (s, 3H,
OMe), 3.65-3.62 (m, 1H, H-5), 3.60-3.56 (m, 1H, -CH2O-),
3.09 (m, 1H, H-5′), 1.67-1.53 (m, 2H, -CH2(O)-), 1.39-1.28
J O0300035
6406 J . Org. Chem., Vol. 68, No. 16, 2003