Synthesis of 5-thio-L-fucose-containing disaccharides, as sequence-specific inhibitors, and 2'-fucosyllactose, as a substrate of α-L-fucosidases

Article abstract of DOI:10.1021/jo961725h

Four 5-thio-L-fucose-containing disaccharides having α(1→6), α(1→3), α(1→4)GlcNAc, and α(1→2)Gal linkages (compounds 1-4, respectively) were synthesized as potential α-L-fucosidase inhibitors. The glycosylation reactions using 2,3,4-tri-O-acetyl-5-thio-L-fucopyranosyl trichloroacetimidate as a glycosyl donor and BF₃·OEt₂ as a catalyst gave mainly α-linked disaccharides. Only α(1→2)-linked disaccharide 4 showed inhibitory activity (K(i) = 0.21 mM) against Bacillus α-L-fucosidase which hydrolyzes the Fucα(1→2) linkage specifically. The results suggested that sequence specificity of an enzyme could be estimated from the inhibitory activities of the compounds 1-4. In contrast, every disaccharide showed inhibitory activity (K(i) = 30-91 μM) against bovine epididymis α-L-fucosidase.

Full text of DOI:10.1021/jo961725h

992
J . Org. Chem. 1997, 62, 992-998
Syn th esis of 5-Th io-L-fu cose-Con ta in in g Disa cch a r id es, a s
Sequ en ce-Sp ecific In h ibitor s, a n d 2′-F u cosylla ctose, a s a Su bstr a te
of r-L-F u cosid a ses†
Masayuki Izumi,‡ Osamu Tsuruta, Satoru Harayama, and Hironobu Hashimoto*
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku, Yokohama 226, J apan
Received September 9, 1996X
Four 5-thio-L-fucose-containing disaccharides having R(1f6), R(1f3), R(1f4)GlcNAc, and R(1f2)-
Gal linkages (compounds 1-4, respectively) were synthesized as potential R-L-fucosidase inhibitors.
The glycosylation reactions using 2,3,4-tri-O-acetyl-5-thio-L-fucopyranosyl trichloroacetimidate as
a glycosyl donor and BF3‚OEt2 as a catalyst gave mainly R-linked disaccharides. Only R(1f2)-
linked disaccharide 4 showed inhibitory activity (Ki ) 0.21 mM) against Bacillus R-L-fucosidase
which hydrolyzes the FucR(1f2) linkage specifically. The results suggested that sequence specificity
of an enzyme could be estimated from the inhibitory activities of the compounds 1-4. In contrast,
every disaccharide showed inhibitory activity (Ki ) 30-91 µM) against bovine epididymis R-L-
fucosidase.
The glycosidases cleave glycosidic bonds of oligosac-
charides in glycoconjugates. Glycosidase inhibitors have
been used for studies of N-linked oligosaccharide process-
ing in glycoproteins,1 metabolic disorders such as diabe-
tes,2 microbial infections,3 and metastasis.4 They are also
valuable tools for studying enzyme activity5 and purifica-
tion of enzymes by affinity chromatography.6 It has been
suggested that R-L-fucosidase (EC 3.2.1.15), which hy-
drolyzes the terminal R-L-fucopyranosyl linkage, partici-
pates in gamete recognition interacting with ascidian7
and mammal8 fucose-containing glycoconjugates. Previ-
ously, the author synthesized four R-L-fucopyranosyl
disaccharides with thioglycosidic linkages to create se-
quence specific inhibitors.9 However, their inhibitory
activities against two bovine R-L-fucosidases were low
and unsatisfactory. Furthermore, the fact that the
inhibition mode of three of these disaccharides was of
mixed-type suggested the additional binding of these
F igu r e 1.
inhibitors to sites different from the substrate binding
site of the enzyme. On the other hand, 5-thio-L-fucose,
which contains a sulfur atom in the pyranose ring instead
of oxygen, was a potent competitive inhibitor of bovine
R-L-fucosidase10 (Ki ) 42 µM), and a 5-thio-L-fucose-
containing disaccharide with an R(1f2)Gal linkage also
showed an excellent competitive inhibition11 (Ki ) 30 µM).
We report here the syntheses of four 5-thio-R-L-fucopy-
ranosyl disaccharides, the relationship between their
inhibitory activities against two R-L-fucosidases, and the
substrate specificity of these enzymes.
* Author for correspondence, Fax: 81-45-924-5805, e-mail:
hhashimo@bio.titech.ac.jp.
† Dedicated to Prof. Hans Paulsen on the occasion of his 75th
birthday.
‡ Present address: Dept. Pharmacology and Molecular Sciences, The
J ohns Hopkins Univ. Sch. Med., 725 North Wolfe St., Baltimore, MD,
21205.
X Abstract published in Advance ACS Abstracts, February 1, 1997.
(1) (a) Fuhrmann, U.; Bause, E.; Ploegh, H. Biochem. Biophys. Acta
1985, 825, 95. (b) Elbein, A. D. Annu. Rev. Biochem. 1987, 56, 497.
Elbein, A. D. FASEB J . 1991, 5, 3055.
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D.; Wingender, W. Angew. Chem., Int. Ed. Engl. 1981, 20, 744.
(3) (a) Schwarz, P. M.; Elbein, A. D. J . Biol. Chem., 1985, 260, 14452.
(b) Gruters, R. A.; Neefjes, J . J .; Tersmette, M.; De Goede, R. E. Y.;
Tulp, A.; Huisman, H. G.; Miedena, F.; Ploegh, H. L. Nature 1987,
330, 74. (c) Karpas, A.; Fleet, G. W. J .; Dwek, R. A.; Petursson, S.;
Namgoong, S. K.; Ramsden, N. G.; J acob, G. S.; Rademacher, T. W.
Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 9229.
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Cancer Res. 1986, 46, 5215.
(5) Defaye, J .; Gelas, J . In Studies in Natural Product Chemistry;
Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1991, Vol. 8, p 315.
(6) Orgeret, C.; Seillier, E.; Gautier, C.; Defaye, J .; Driguez, H.
Carbohydr. Res., 1992, 224, 29.
(7) (a) DeSantis, R.; Pinto, M. R. Gamete Interaction in Ascidians:
Sperm Binding and Penetration Through the Vitelline Coat. In
Mechanism of Fertilization: Plants to Humans; Dale, B., Ed.; Springer-
Verlag: Berlin, 1990; p 297. (b) Hoshi, M.; DeSantis, R.; Pinto, M. R.;
Cotelli, F.; Rosati, F. Zool. Sci., 1985, 2, 65.
(8) Huang, T. F., J r.; Ohzu, E.; Yanagimachi, R. Gamete Res., 1982,
5, 355.
Resu lts a n d Discu ssion
Syn th esis. Considering the frequency of L-fucosyl
linkages in glycoconjugates, disaccharides having R(1f6),
R(1f4), R(1f3)GlcNAc, and R(1f2)Gal linkages (1-4)
were selected as synthetic targets (Figure 1). As the
authors communicated previously,11 5-thio-R-L-fucopyra-
nosides could be synthesized using 2,3,4-tri-O-acetyl-5-
thio-L-fucopyranosyl trichloroacetimidate (6) as a glycosyl
12
donor and a catalytic amount of BF3‚OEt2 as an
activator. The trichloroacetimidate 6 was synthesized
from 5-thio-L-fucopyranose tetraacetate 513 in two steps
(10) Hashimoto, H.; Fujimori, T.; Yuasa, H. J . Carbohydr. Chem.,
1990, 9, 683.
(11) Hashimoto, H.; Izumi, M. Tetrahedron Lett., 1993, 31, 4949.
(12) Schmidt, R.; R. Kinzy, W. Adv. Carbohydr. Chem. Biochem.,
1994, 50, 21.
(9) Hashimoto, H.; Shimada, K.; Horito, S. Tetrahedron: Asymmetry
1994, 5, 2351.
S0022-3263(96)01725-2 CCC: $14.00 © 1997 American Chemical Society

Synthesis of 5-Thio-L-Fucose-Containing Disaccharides
J . Org. Chem., Vol. 62, No. 4, 1997 993
Sch em e 1
Sch em e 3
Sch em e 2
Sch em e 4
in 95% yield. The anomeric acetyl group of 5 was
removed by treatment with hydrazine acetate and the
resulting 1-OH derivative was treated with CCl3CN and
DBU14 to give 6 (Scheme 1).
To construct the (1f6) linkage, allyl 2-acetamido-3,4-
di-O-acetyl-2-deoxy-â-D-glucopyranoside15 was first ex-
amined as an acceptor, but migration of the acetyl group
from 4-OH to 6-OH was observed during the glycosylation
reaction and the desired disaccharide could not obtained
in a pure form. The glycosylation of allyl 2-acetamido-
2-deoxy-3,4-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
â-D-glucopyranoside (7)16 with the imidate 6 at room
temperature gave a mixture of R- and â-linked disaccha-
ride 8 in 70% yield after removal of the TIPDS group.
The R-linked disaccharide was formed predominantly in
a ratio of 81:19. The disaccharide mixture was acetylated
and separated several times on silica gel using CHCl3-
MeOH 75:1 as an eluate to give a pure R-linked disac-
charide 9. De-O-acetylation of the disaccharide 9 with
methanolic sodium methoxide gave R(1f6)-linked di-
saccharide 1 in 97% yield (Scheme 2).
The glycosylation of allyl 2-acetamido-2-deoxy-4,6-O-
isopropylidene-â-D-glucopyranoside (10)17 with the imi-
date 6 at 0 °C gave (1f3)-linked disaccharide 11 in 64%
yield. The ratio of R- and â-glycoside was 82:18. The
isopropylidene group was hydrolyzed by treatment with
1 M HCl in acetone17 and free hydroxyl groups were
acetylated to give pentaacetate 12 in 62% yield. The
R-linked disaccharide separated in this stage was de-O-
acetylated with methanolic sodium methoxide to give R-
(1f3)-linked disaccharide 2 (Scheme 3).
examined 1,6-anhydro-2-azido-2-deoxy-â-D-glucopyranose
derivative 1719 as a glycosyl acceptor. The compound 17
was synthesized in three steps from 1,6-anhydro-2-azido-
2-deoxy-â-D-glucopyranose (13) which could be conve-
niently prepared from D-glucal.20 Treatment of 13 with
1.2 mol equiv of tert-butyldimethylsilyl chloride (TBDM-
SCl) gave 4-O-TBDMS derivative 1421 and 3-O-TBDMS
derivative 15 in 89% yield in a 9:1 ratio. Compound 14
and 15 were separated by MPLC. The 4-O-TBDMS
derivative 14 was acetylated (94%), and subsequent
desilylation in hot aqueous acetic acid gave 17 in 73%
yield. Glycosylation of the acceptor 17 proceeded smoothly
at -20 °C to give only R-linked disaccharide 18 in 89%
yield. The 1,6-anhydro-ring of 18 was acetolyzed under
mild conditions, i.e. trifluoroacetic acid and acetic anhy-
dride, without a cleavage of the interglycosidic bond. The
azido group of 19 was converted to an acetamido group
by treatment with H2S in pyridine and water and
subsequent acetylation. O-Acetyl groups were removed
by treatment with methanolic ammonia to give R(1f4)-
linked disaccharide 3 in 67% yield.
The synthesis of (1f4)-linked disaccharide 3 required
many more steps for preparing the acceptor (Scheme 4).
Glycosylation of allyl 2-acetamido-3,6-di-O-benzoyl-2-
deoxy-â-D-glucopyranoside18 was unsuccessful due to a
lack of the reactivity of the 4-OH group. Next the authors
For the synthesis of R(1f2)-linked disaccharide, allyl
3,4,6-tri-O-acetyl-â-D-galactopyranoside (25) was used as
a glycosyl acceptor (Scheme 5). 1,3,4,6-tetra-O-acetyl-
(13) Izumi, M.; Tsuruta, O.; Hashimoto, H. Carbohydr. Res., 1996,
280, 287.
(14) Sugimoto, M.; Numata, M.; Koike, K.; Nakahara, Y.; Ogawa,
T. Carbohydr. Res., 1986, 156, c1.
(15) Roy, R.; J ennings, H. J . Carbohydr. Res., 1983, 112, 63.
(16) Kosma, P.; Bahnmu¨ller, R.; Schulz, G.; Brade, H. Carbohydr.
Res., 1990, 208, 37.
(17) Hasegawa, A.; Ozaki, M.; Kiso, M.; Azuma, I. J . Carbohydr.
Chem., 1984, 3, 331.
(19) Petitou, M.; Duchaussoy, P. Lederman, I.; Choay, J .; Sinay¨. P.;
J acquinet, J .-C.; Torri, G. Carbohydr. Res., 1986, 147, 221.
(20) Tailler, D.; J acquinet, J .-C.; Noirot, A.-M.; Beau, J .-M. J . Chem.
Soc., Perkin Trans. 1, 1992, 3163.
(21) van Boeckel, C. A. A.; van Aelst, S. F.; Beetz, T. Recl. Trav.
Chim. Pays-Bas, 1983, 102, 415.
(18) Nashed, N. M. Carbohydr. Res., 1979, 71, 299-304.

994 J . Org. Chem., Vol. 62, No. 4, 1997
Izumi et al.
Sch em e 5
Sch em e 6
Ta ble 1. Glycosyla tion Rea ction w ith th e
Tr ich lor oa cetim id a te 6
acceptor
(equiv)
entry
conditions
% yielda
R:â
1
2
3
4
7 (1.5) -20 °C, 3 h-rt, overnight
10 (3.0) 0 °C, 7 h
17 (3.0) -20 °C, 30 min
25 (1.6) -20 °C, 15 min
8, 70%
11, 76%
18, 85% >99:1
26, 86% 98:2
81:19
82:18
a
Isolated yield.
R-D-galactopyranose (21)22 was acylated with chloroacetyl
chloride and converted to the glycosyl bromide with
HBr-acetic acid. The glycosylation of allyl alcohol using
mercury(II) cyanide as an activator gave â-glycoside 24
in 81% yield. Dechloroacetylation with hydrazine acetate
gave the acceptor 25 in 82% yield. The glycosylation of
25 with the imidate 6 at -20 °C gave R-glycoside 26 in
86% yield. Deacetylation with methanolic sodium meth-
oxide gave target disaccharide 4 in 92% yield.
The results of glycosylation reactions with peracety-
lated 5-thio-L-fucopyranosyl trichloroacetimidate 6 are
summarized in Table 1. The reaction with reactive
acceptors (entries 3 and 4) proceeded smoothly at -20
°C with excellent stereoselectivity, and only the desired
R-linked disaccharides were obtained. On the other
hand, the reaction with less reactive acceptors (entries
1 and 2) required longer reaction times and higher
reaction temperatures. As a result of such reaction
conditions, the stereoselectivities were decreased to about
4:1. These results suggest that R-glycosides are kineti-
cally favored.
In addition to the above described four potential
inhibitors of R-L-fucosidase, 2′-fucosyllactose, which was
required for the enzyme assay as a substrate, was newly
synthesized in a convenient route as shown in Scheme
6. This synthetic method demonstrated the usefulness
of tri-O-isopropylidenelactose dimethylacetal 28 which
can be easily prepared by treatment of lactose with 2,2-
dimethoxypropane in the same manner as reported for
N-acetyllactosamine.23 Selective 6-O-acetylation followed
by glycosylation with per-O-p-methoxybenzylated 1-thio-
fucoside 27 in the presence of iodonium di(sym-collidine)
perchlorate (IDCP) gave R-linked trisaccharide derivative
Ta ble 2. Ki Va lu es of 5-Th io-L-fu cosyl Disa cch a r id es
a ga in st r-L-F u cosid a ses
Bacillus sp.
bovine epididymis,
inhibitor
K40Tm mM
µM
1f6GlcNAc (1)
1f3GlcNAc (2)
1f4GlcNAc (3)
1f2Gal (4)
>5
31
91
44
30
>5
>5
0.21
32 exclusively. Simultaneous deprotection of the O-p-
methoxylbenzyl and O-isopropylidene groups with CAN
followed by acetylation for purification and deacetylation
gave 2′-fucosyllactose 34 in 25% yield from 28.
En zym e In h ibition . The inhibitory activities of four
synthesized disaccharides having different glycosidic
linkages were examined against two R-L-fucosidases from
different biological sources. One is Bacillus sp. K40T R-L-
fucosidase which cleaves only R(1f2) linkage, and the
other is bovine epididymis R-L-fucosidase whose substrate
specificity is not clear. The inhibitory activities of the
disaccharides 1-4 against Bacillus sp. K40T R-L-fucosi-
dase were examined to investigate the relationship
between inhibitory activities and sequence specificity of
the enzyme. The assay of this enzyme was carried out
using 2′-fucosyllactose as a substrate. The Km value of
2′-fucosyllactose determined from an [S]/v vs [S] plot was
0.39 mM. The inhibition mode and the Ki value of these
compounds were determined from the Lineweaver-Burk
plot and are summarized in Table 2. R(1f2) Linked
disaccharide 4 was a potent competitive inhibitor (Ki )
0.21 mM), while the other three disaccharides 1-3 had
weak or no inhibitory activities up to 5 mM. These
results suggest that 5-thio-L-fucose-containing disaccha-
rides were recognized in the same way as natural
(22) Helferich, B.; Zirner, J . Chem. Ber., 1962, 95, 2604.
(23) Yoshino, T; Ishido, Y. 17th J apanese Carbohydrate Symposium,
J uly 18-20, 1995, Kyoto: Abstract p 170.

Synthesis of 5-Thio-L-Fucose-Containing Disaccharides
J . Org. Chem., Vol. 62, No. 4, 1997 995
gradually warmed to rt and stirred overnight. The solution
was neutralized with Et3N (30 µL, 0.215 mmol), diluted with
CHCl3, and filtered through a pad of Celite. The filtrate was
concentrated in vacuo, and the residue was dissolved in THF
(49.5 mL). This solution was mixed with a solution of 1 M
Bu4NF in THF (0.5 mL). After being stirred for 45 min, the
solvent was evaporated, and the residue was dissolved in
CHCl3. The solution was washed twice with brine, and the
organic layer was dried (MgSO4) and concentrated in vacuo.
The residue was purified on a column of silica gel (8:1 CHCl3-
MeOH) to give 8 as a syrup (64.1 mg, 70%, R:â ) 81:19). The
compound 8 was dissolved in pyridine (1 mL) and acetic
anhydride (0.5 mL) and stirred overnight. Methanol was
added to the solution, and the solvents were evaporated. The
residue was chromatographed several times on a column of
silica gel (75:1 CHCl3-MeOH) to isolate R-linked disaccharide
9: mp 181-182 °C (from ether-petroleum ether); [R]20D -148
oligosaccharides by Bacillus R-L-fucosidase and the se-
quence specificity of the enzymes could be confirmed from
their inhibitory activities. Next, compounds 1-4 were
tested as inhibitors of the R-L-fucosidase from bovine
epididymis using p-nitrophenyl R-L-fucopyranoside as a
substrate. Each disaccharide was a competitive inhibitor,
which suggested that these disaccharides bind to the
catalytic site of the enzyme. Compounds 1, 3, and 4 have
Ki values that are similar to that of monosaccharide
5-thio-L-fucose. The Ki value of the disaccharide 2, which
carries R(1f3) linkage, was slightly larger than those of
the others. However, the ∆∆G between 2 with the largest
Ki value and 4 with the smallest Ki value was only 0.66
kcal/mol, which suggests that the difference is not
crucial.24 This result suggested that bovine epididymis
R-L-fucosidase has no sequence specificity.
1
(c 0.63, CHCl3); H NMR (270 MHz, CDCl3) δ 5.94-5.80 (m,
1H), 5.50 (bs, 1H), 5.46 (d, 1H, J ) 8.6 Hz, NH), 5.41-5.33
(m, 2H), 5.26 (dd, 1H, J ) 7.6, 9.9 Hz), 5.29-5.19 (m, 2H),
5.00 (t, 1H, J ) 9.9 Hz), 4.80 (d, 1H, J ) 2.0 Hz), 4.69 (d, 1H,
J ) 8.3 Hz), 4.34-4.27 (m, 1H), 4.12-4.04 (m, 1H), 3.89 (dd,
1H, J ) 2.3, 10.9 Hz), 3.83 (ddd, 1H, J ) 7.6, 8.3, 8.6 Hz),
3.65 (ddd, 1H, J ) 2.3, 5.6, 9.9 Hz), 3.52 (dd, 1H, J ) 5.6, 10.9
Hz), 3.48 (q, 1H, J ) 6.9 Hz), 2.18, 2.08, 2.04, 2.03, 1.98, 1.96
(each s, 3H × 6), 1.14 (d, 1H, J ) 6.9 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 170.9, 170.8, 170.8, 170.2, 169.9, 169.5, 133.5, 117.8,
99.5, 80.9, 73.4, 72.6, 72.4, 71.1, 69.6, 69.4, 69.0, 62.1, 54.7,
34.3, 20.9, 20.8, 20.7, 15.8. Anal. Calcd for C27H39NO14S: C,
51.18; H, 6.20; N, 2.21. Found: C, 50.98; H, 6.16; N, 2.14.
Allyl O-(5-Th io-r-L-fu cop yr a n osyl)-(1f6)-2-a ceta m id o-
2-d eoxy-â-D-glu cop yr a n osid e (1). To a solution of 9 (25.8
mg, 41 µmol) in MeOH (1.7 mL) was added a solution of 0.5
M NaOMe in MeOH (0.25 mL). After being stirred for 1 h,
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 chromatographed on a column of
Bio-Gel P-2 (water) and lyophilized to give 1 as a white powder
(16.7 mg, 97%): [R]23D -254 (c 0.21, H2O); 1H NMR (270 MHz,
D2O) δ 5.98-5.84 (m, 1H), 5.34-5.25 (m, 2H), 4.71 (d, 1H, J
) 3.0 Hz), 4.58 (d, 1H, J ) 8.2 Hz), 4.36-4.29 (m, 1H), 4.20-
4.13 (m, 2H), 4.03 (bs, 1H), 4.00 (dd, 1H, J ) 3.0, 10.2 Hz) ,
3.88 (dd, 1H, J ) 3.0, 10.2 Hz), 3.77-3.71 (m, 2H), 3.66 (m,
1H), 3.58-3.51 (m, 2H), 3.42 (q, 1H, J ) 7.3 Hz), 2.04 (s, 3H),
1.21 (d, 1H, J ) 7.3 Hz); 13C NMR (67.8 MHz, D2O) δ 175.4,
134.2, 119.0, 101.0, 84.1, 75.8, 75.0, 74.6, 71.9, 71.4, 71.3, 70.8,
68.3, 56.3, 37.0, 22.9, 16.4. Anal. Calcd for C17H29NO9S: C,
48.22; H, 6.90; N, 3.31. Found: C, 47.97; H, 6.50; N, 3.55.
Allyl O-(2,3,4-Tr i-O-a cetyl-5-th io-r/â-L-fu cop yr a n osyl)-
(1f3)-2-a ceta m id o-2-d eoxy-4,6-O-isop r op ylid en e-â-D-glu -
cop yr a n osid e (11). To a solution of 6 (80.5 mg, 0.179 mmol)
and allyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-â-D-glu-
copyranoside (10) (164 mg, 0.545 mmol) in CH2Cl2 (5.6 mL)
was added powdered MS4A (400 mg), and the mixture was
stirred for 1 h under Ar. After being cooled to 0 °C, a solution
of BF3‚OEt2 (7.2 µL, 58 µmol) in CH2Cl2 (1.5 mL) was added
dropwise. After being stirred for 7 h, the reaction mixture was
neutralized with Et3N and diluted with CHCl3. The solution
was filtered through a pad of Celite, and the filtrate was
concentrated in vacuo. The residue was purified on a column
of silica gel (1:1 hexane-EtOAc) to give 11 as a crystal (80.6
mg, 76%, R:â ) 82:18): mp 125-127 °C (from Et2O-petroleum
In summary, four 5-thio-L-fucose-containing disaccha-
ride analogs were synthesized using the trichloroace-
timidate method. A 5-thio-R-L-fucoside bond was formed
predominantly in each case. The fact that only an R-
(1f2)-linked disaccharide of these four disaccharides
showed inhibitory activity toward Bacillus R-1,2-L-fu-
cosidase suggested that these four disaccharides are
useful for estimating the sequence specificity of a fucosi-
dase.
Exp er im en ta l Section
Gen er a l. NMR spectra were recorded on 270 MHz or 400
MHz instrument. Melting points were uncorrected. Optical
rotations were determined with 0.5 dm cell. Column chroma-
tography was performed with Wakogel C-300 (200-300 mesh;
Wako Pure Chem.). Medium pressure liquid chromatography
(MPLC) was performed with Baker silica gel chromatography
packing (40 µm; J . T. Baker). TLC was carried out on plates
precoated with silica gel 60 F-254 (E. Merck). R-L-Fucosidase
(bovine epididymis), NADP, and L-fucose dehydrogenase
(Pseudomonas sp.) were purchased from Sigma Chemical Co.
R-L-Fucosidase (Bacillus sp. K40T) and p-nitrophenyl R-L-
fucopyranoside were purchased from Seikagaku Kogyo Co.
2,3,4-Tr i-O-a cet yl-5-t h io-L-fu cop yr a n osyl Tr ich lor o-
a cetim id a te (6). To a solution of 1,2,3,4-tetra-O-acetyl-5-thio-
L-fucopyranose (5) (99.8 mg, 287 µmol) in DMF (2.25 mL) was
added H2NNH2‚AcOH (45.0 mg, 489 µmol). After being stirred
at 50 °C for 2.5 h, the solution was cooled to rt, diluted with
EtOAc, and washed with brine (3 × 10 mL). The organic layer
was dried (MgSO4) and concentrated in vacuo. The residue
was dissolved in CH2Cl2 (0.33 mL) and CCl3CN (0.29 mL), and
the solution was cooled to 0 °C. To the stirred solution was
added DBU (4.2 µL). After being stirred overnight, the
reaction mixture was subjected to a column of silica gel and
eluted with 2:1 hexane-EtOAc to give 6 as a syrup (0.123 g,
95%, R:â ) 86:14): [R]25 -219 (c 0.67, CHCl3); 1H NMR (270
D
MHz, CDCl3) δ 8.64 (brs, 1H), 6.28 (d, 1H, J ) 3.0 Hz), 5.59
(brs, 1H, J ) 2.8 Hz), 5.52 (dd, 1H, J ) 3.0, 10.6 Hz), 5.42 (dd,
1H, J ) 2.8, 10.6 Hz), 3.68 (q, 1H, J ) 6.9 Hz), 2.20, 2.01, 2.00
(each s, 3H × 3), 1.18 (d, 3H, J ) 6.9 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 170.7, 170.1, 169.9, 160.9, 77.4, 72.2, 69.7, 69.3, 35.8,
20.7, 15.8. Anal. Calcd for C14H18Cl3NO7S: C, 37.30; H, 4.03;
N, 3.11; S, 7.11. Found: C, 37.32 ; H, 4.21; N, 3.00; S, 7.5.
Allyl O-(2,3,4-Tr i-O-a cetyl-5-th io-r-L-fu cop yr a n osyl)-
(1f6)-2-a cet a m id o-3,4-d i-O-a cet yl-2-d eoxy-â-D-glu cop y-
r a n osid e (9). To a solution of 6 (74.9 mg, 0.166 mmol) and
allyl 2-acetamido-2-deoxy-3,4-O-(1,1,3,3-tetraisopropyldisilox-
ane-1,3-diyl)-â-D-glucopyranoside (7) (126 mg, 0.250 mmol) in
CH2Cl2 (5.6 mL) was added powdered MS4A (370 mg), and
the mixture was stirred for 1 h under Ar. To the solution
cooled to -20 °C was added dropwise a solution of BF3‚OEt2
(6.7 µL, 54 µmol) in CH2Cl2 (1.4 mL). The solution was
1
ether); H NMR (270 MHz, CDCl3) δ 5.85-5.79 (m, 1H), 5.65
(d, 1H, J ) 8.6 Hz), 5.48 (bs, 1H), 5.40 (dd, 1H, J ) 10.6, 3.0
Hz), 5.27 (dd, 1H, J ) 2.6, 10.6 Hz), 5.23-5.17 (m, 2H), 5.07
(d, 1H, J ) 2.6 Hz), 4.78 (d, 1H, J ) 8.2 Hz), 4.34-4.01 (m,
2H), 4.31 (bt, 1H), 3.93 (dd, 1H, J ) 5.3, 10.9 Hz), 3.83-3.61
(m, 4H), 3.35 (dt, 1H, J ) 5.3, 9.9 Hz), 2.17, 2.12, 1.98, 1.95
(each s, 3H × 4), 1.52, 1.37 (each s, 3H × 2), 1.10 (d, 1H, J )
6.9 Hz); 13C NMR (67.8 MHz, CDCl3) δ 171.2, 170.10, 170.09,
170.0, 133.7, 117.7, 99.9, 99.5, 86.1, 75.2, 73.3, 72.9, 71.7, 69.9,
68.8, 67.1, 62.2, 57.1, 34.2, 29.1, 23.4, 21.1, 20.8, 20.7, 19.1,
15.8. Anal. Calcd for C26H39NO12S: C, 52.96; H, 6.67; N, 2.38.
Found: C, 53.29; H, 6.93; N, 2.38.
Allyl O-(2,3,4-Tr i-O-a cetyl-5-th io-r-L-fu cop yr a n osyl)-
(1f3)-2-a cet a m id o-4,6-d i-O-a cet yl-2-d eoxy-â-D-glu cop y-
(24) Sphor, U.; Paszkiwicz-Hnatiw, E.; Morishima, N.; Lemieux, R.
U. Can J . Chem., 1992, 70, 254 and references therein.

996 J . Org. Chem., Vol. 62, No. 4, 1997
Izumi et al.
r a n osid e (12). To a solution of 11 (38.9 mg, 66.0 µmol) in
acetone (4.3 mL) was added 1 M HCl (0.29 mL). After being
boiled under reflux for 2 h, the solution was neutralized with
barium oxide, and the solid was filtered off. The filtrate was
concentrated in vacuo, and the residue was acetylated with
acetic anhydride and pyridine and a catalytic amount of
4-(dimethylamino)pyridine. The mixture was purified by silica
gel column chromatography (2:3 hexane-EtOAc) to give 12
as a syrup (25.8 mg, 62%, R:â ) 85:15). R-Linked disaccharide
was separated by silica-gel column chromatography (50:1
O-(2,3,4-Tr i-O-a cetyl-5-th io-r-L-fu cop yr a n osyl)-(1f4)-
3-O-a cet yl-1,6-a n h yd r o-2-a zid o-2-d eoxy-â-D-glu cop yr a -
n ose (18). To a solution of 6 (99.8 mg, 0.221 mmol) and 17
(155 mg, 0.675 mmol) in CH2Cl2 (6.8 mL) was added powdered
MS4A (500 mg), and the mixture was stirred for 1 h under
Ar. To the solution cooled to -20 °C was added dropwise a
solution of BF3‚OEt2 (8.9 µL, 71 µmol) in CH2Cl2 (1.8 mL). After
being stirred for 30 min, the solution was neutralized with
Et3N. The solution was diluted with CHCl3 and filtered
through a pad of Celite. The filtrate was concentrated in
vacuo, and the residue was purified by MPLC (1:1 hexane-
CHCl3-MeOH): [R]17 -153 (c 0.63, CHCl3); 1H NMR (270
D
EtOAc) to give 18 as a syrup (102 mg, 89%): [R]17 -122 (c
MHz, CDCl3) δ 5.88-5.82 (m, 1H), 5.66 (d, 1H, J ) 7.6 Hz),
5.46 (bs, 1H), 5.39 (m, 2H), 5.24-5.19 (m, 2H), 5.04 (t, 1H, J
) 8.9 Hz), 5.03 (d, 1H, J ) 2.3 Hz), 4.99 (d, 1H, J ) 7.9 Hz),
4.49 (t, 1H, J ) 8.9 Hz), 4.36-4.29 (m, 1H), 4.23 (dd, 1H, J )
5.3, 12.2 Hz), 4.12 (dd, 1H, J ) 3.0, 12.2 Hz), 4.10-4.03 (m,
1H), 3.70 (ddd, 1H, J ) 3.0, 5.3, 8.9 Hz), 3.43-3.32 (m, 2H),
2.16, 2.11 (each s, 3H × 2), 2.10 (s, 6H), 1.97, 1.95 (each s, 3H
× 2), 1.12 (d, 1H, J ) 7.3 Hz); 13C NMR (CDCl3, 67.8 MHz) δ
170.8, 170.7, 169.8, 169.5, 133.5, 118.0, 98.2, 80.6, 76.2, 72.6,
71.8, 71.4, 70.5, 70.1, 68.6, 62.6, 57.5, 34.8, 29.4, 23.5, 21.1,
20.8, 20.7, 20.6, 16.0. Anal. Calcd for C27H39NO14S: C, 51.18;
H, 6.20; N, 2.21. Found: C, 50.93; H, 6.47; N, 2.20.
D
0.85, CHCl3); 1H NMR (270 MHz, CDCl3) δ 5.55 (bs, 1H), 5.53
(s, 1H), 5.45 (dd, 1H, J ) 3.0, 10.6 Hz), 5.30 (dd, 1H, J ) 3.3,
10.6 Hz), 5.09 (d, 1H, J ) 3.0 Hz) 5.02 (s, 1H), 4.53 (bd, 1H, J
) 5.6 Hz), 3.97 (bd, 1H, J ) 7.3 Hz), 3.82 (dd, 1H, J ) 5.6, 7.3
Hz), 3.71 (s, 1H), 3.67 (q, 1H, J ) 6.9 Hz), 3.09 (s, 1H), 2.18,
2.12, 2.08, 1.99 (each s, 3H × 4), 1.17 (d, 1H, J ) 6.9 Hz); 13
C
NMR (67.8 MHz, CDCl3) δ 171.0, 170.1, 169.7, 169.4, 100.6,
78.1, 73.0, 72.5, 71.7, 71.2, 71.1, 69.1, 64.9, 58.4, 34.9, 21.0,
20.9, 20.7, 20.7, 15.9. Anal. Calcd for C20H27N3O11S: C, 46.42;
H, 5.26; N, 8.12. Found: C, 46.48; H, 5.35; N, 7.95.
O-(2,3,4-Tr i-O-a cetyl-5-th io-r-L-fu cop yr a n osyl)-(1f4)-
1,3,6-tr i-O-acetyl-2-azido-2-deoxy-r/â-D-glu copyr an ose (19).
To a solution of 18 (42.3 mg, 81.7 µmol) in acetic anhydride
(0.5 mL) was added dropwise a solution of trifluoroacetic acid-
acetic anhydride (1:5, 0.6 mL) at 0 °C, and the mixture was
stirred overnight at rt. The solvents were evaporated in vacuo,
and the residue was purified on a column of silica gel (3:2
hexane-EtOAc) to give 19 as a syrup (45.4 mg, 90%, R:â )
3:1): 1H NMR (270 MHz, CDCl3) δ 6.26 (d, 0.75H, J ) 3.6 Hz),
5.56 (d, 0.25H, J ) 8.6 Hz), 5.54-5.46 (m, 2.75H), 5.30 (dd,
0.75H, J ) 3.0, 11.2 Hz), 5.23 (dd, 0.25H, J ) 3.0, 11.2 Hz),
5.15 (dd, 0.25H, J ) 8.6, 9.9 Hz), 4.71 (d, 0.75 H, J ) 2.6 Hz),
4.68 (d, 0.25 H, J ) 2.6 Hz), 4.51-4.42 (m, 1H), 4.35-4.25 (m,
1.25H), 4.07-4.02 (m, 0.75H), 3.81-3.74 (m, 1H), 3.55 (dd,
0.25H, J ) 8.6, 9.9 Hz), 3.54 (m, 1H), 3.81-3.43 (m, 1H), 3.42
(dd, 0.75H, J ) 3.6, 10.2 Hz), 2.24, 2.20, 2.19, 2.18, 2.17, 2.16,
2.08, 2.07, 2.06, 1.96, 1.95 (each s), 1.14 (d, 2.25H, J ) 6.9
Hz), 1.12 (d, 0.75H, J ) 6.9 Hz); 13C NMR (67.8 MHz, CDCl3)
δ 170.7, 170.4, 170.1, 170.0, 169.9, 168.7, 90.0, 83.5, 77.6, 72.6,
71.4, 71.1, 70.7, 68.6, 61.9, 60.9, 35.4, 21.6-20.6, 15.9. Anal.
Calcd for C24H33N3O14S: C, 46.52; H, 5.37; N, 6.78; S, 5.17.
Found: C, 46.78; H, 5.38; N, 6.41; S, 4.83.
O-(2,3,4-Tr i-O-a cetyl-5-th io-r-L-fu cop yr a n osyl)-(1f4)-
2-a cet a m id o-1,3,6-t r i-O-a cet yl-2-d eoxy-r/â-D-glu cop yr a -
n ose (20). A solution of 19 (21.6 mg, 34.9 mmol) in pyridine-
water (1:1, 1 mL) was saturated with H2S at rt. After being
stirred overnight, the reaction mixture was concentrated in
vacuo. The residue was dissolved in pyridine (1 mL) and acetic
anhydride (0.5 mL) and stirred overnight. To the solution was
added MeOH, and the solvents were evaporated. The residue
was purified by silica gel chromatography (1:4 hexane-EtOAc)
to give 20 as a syrup (21.2 mg, 100%): 1H NMR (270 MHz,
CDCl3) δ 6.09 (d, 0.75H, J ) 3.3 Hz), 5.68-5.65 (m, 0.5H),
5.58 (d, 0.75H, J ) 8.9 Hz), 5.51-5.44 (m, 2H), 5.31-5.24 (m,
1.75H), 5.12 (dd, 0.25H, J ) 7.6, 9.9 Hz), 4.79 (d, 1H, J ) 2.6
Hz), 4.55 (bd, 0.25H), 4.51 (bd, 0.75H), 4.38 (ddd, 0.75H, J )
3.3, 8.9, 10.2 Hz), 4.29-4.19 (m, 1.25H), 3.99-3.96 (m, 0.75
H), 3.89-3.80 (m, 1.25H), 3.50-3.45 (m, 1H), 2.23, 2.17, 2.13,
2.12, 2.10, 2.09, 2.08, 1.97, 1.94 (each s), 1.13 (d, 3H, J ) 6.9
Hz); 13C NMR (67.8 MHz, CDCl3) δ 171.8-168.8, 90.5, 82.8,
77.2, 72.5, 72.4, 71.1, 70.5, 68.7, 62.2, 51.4, 35.2, 23.1-20.6,
15.9. Anal. Calcd for C26H37NO15S: C, 49.13; H, 5.87; N, 2.20.
Found: C, 48.91; H, 6.15; N, 1.99.
Allyl O-(5-Th io-r-L-fu cop yr a n osyl)-(1f3)-2-a ceta m id o-
2-d eoxy-â-D-glu cop yr a n osid e (2). To a solution of 12 (28.1
mg, 44 µmol) in MeOH (1.7 mL) was added a solution of 0.5
M NaOMe in MeOH (0.25 mL). After being stirred for 2 h,
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 chromatographed on a column of
Bio-Gel P-2 (water) and lyophilized to give 2 as a white powder
(11.9 mg, 63%): [R]23D -213 (c 0.20, H2O); 1H NMR (270 MHz,
D2O) δ 5.97-5.83 (m, 1H), 5.33-5.24 (m, 2H), 4.83 (bs, 1H),
4.57 (d, 1H, J ) 7.9 Hz), 4.37-4.30 (m, 1 H), 4.19-4.12 (m, 1
H), 4.02 (bs, 1H), 3.98-3.84 (m, 4 H), 3.82 (dd, 1H, J ) 11.5,
2.6 Hz), 3.75 (dd, 1H, J ) 3.3, 11.9 Hz), 3.53 (q, 1H), 3.50-
3.49 (m, 2H), 2.01 (s, 3H), 1.16 (d, 1H, J ) 6.9 Hz); 13C NMR
(67.8 MHz, D2O) δ 175.2, 134.1, 119.0, 100.8, 84.2, 78.5, 76.6,
75.0, 71.6, 71.3, 69.3, 61.6, 56.0, 37.0, 23.0, 16.2. Anal. Calcd
for C17H29NO9S: C, 48.22; H, 6.90; N, 3.31. Found: C, 47.87;
H, 6.59; N, 3.44.
3-O-Acetyl-1,6-a n h yd r o-2-a zid o-4-O-(ter t-bu tyld im eth -
ylsilyl)-2-d eoxy-â-D-glu cop yr a n ose (16). To a solution of
1,6-anhydro-2-azido-2-deoxy-â-D-glucopyranose (13) (570 mg,
3.04 mmol) in DMF (3.7 mL) were added imidazole (470 mg,
6.90 mmol) and tert-butyldimethylsilyl chloride (600 mg, 3.98
mmol). After being stirred overnight, the solvent was evapo-
rated in vacuo, and the residue was dissolved in CHCl3. The
solution was washed subsequently with 1 M HCl and water.
The organic layer was dried (MgSO4) and concentrated in
vacuo. The residue was purified on a column of silica gel (3:1
hexane-EtOAc) to give 1,6-anhydro-2-azido-4-O-(tert-butyldim-
ethylsilyl)-2-deoxy-â-D-glucopyranose (14) and 1,6-anhydro-2-
azido-3-O-(tert-butyldimethylsilyl)-2-deoxy-â-D-glucopyra-
nose (15) as a syrup (813 mg, 89%, 14:15 ) 9:1). The
compound 14 was isolated by MPLC (3:1 hexane-EtOAc).
14: [R]25 +3.8 (c 1.1, CH2Cl2); lit.:20 [R]25 +5.7 (c 1, CH2Cl2).
D
D
The compound 14 (298 mg, 0.988 mmol) was acetylated with
pyridine (2 mL) and acetic anhydride (1 mL) to give crystalline
16 (320 mg, 94%): mp 68-70 °C (CHCl3); [R]20 +47 (c 1.0
D
CHCl3); 1H NMR (270 MHz, CDCl3) δ 5.50 (brs, 1H), 4.84 (m,
1H), 4.46 (brd, 1H, J ) 5.6 Hz), 4.00 (dd, 1H, J ) 1.0, 7.6 Hz),
3.76 (dd, 1H, J ) 5.6, 7.6 Hz), 3.59 (brs, 1H), 3.05 (s, 1H), 2.11
(s, 3H), 0.94 (s, 9H), 0.16 (s, 3H), 0.13 (s, 3H); 13C NMR (67.8
MHz, CDCl3) δ 169.4, 100.3, 76.9, 72.9, 69.5, 65.0, 58.8, 25.7,
21.0, 18.1, -4.9, -5.0. Anal. Calcd for C14H25N3O5Si: C,
48.96; H, 7.34; N, 12.23. Found: C, 48.87; H, 7.39; N, 12.20.
O-(5-Th io-r-L-fu cop yr a n osyl)-(1f4)-2-a ce t a m id o-2-
d eoxy-D-glu cop yr a n ose (3). A solution of 20 (32 mg, 52
µmol) in MeOH (2 mL) was saturated with NH3 and stirred
overnight. The reaction mixture was concentrated, and the
residue was purified on a column of silica gel (6:2:1 EtOAc-
MeOH-water) and Sephadex G-15 (water) and lyophilized to
3-O-Acetyl-1,6-a n h yd r o-2-a zid o-2-d eoxy-â-D-glu cop yr a -
n ose (17). A solution of 16 (0.317 g, 0.922 mmol) in 70%
aqueous AcOH (20 mL) was heated at 80 °C for 21 h. Most of
the solvents were evaporated in vacuo, and the remaining
solvents were coevaporated three times with toluene. The
residue was purified on a column of silica gel to give 17 as a
syrup (0.155 g, 73%): [R]27 -6.8 (c 1.9, CHCl3), lit.:18 [R]23
give 3 as a white powder (13.5 mg, 67%, R:â ) 3:2): [R]23
D
1
-237 (5 min) -241 (70 h) (c 0.50, H2O); H NMR (270 MHz,
D2O, at 60 °C) δ 5.20 (d, 0.6H, J ) 3.6 Hz), 4.79 (d, 0.6H, J )
3.3 Hz), 4.78 (d, 0.4H, J ) 3.3 Hz), 4.71 (d, 0.4H, J ) 7.6 Hz),
4.03-3.56 (m, 9H), 2.04 (s, 3H), 1.17 (d, 3H, J ) 7.3 Hz); 13C
D
D
-6.5 (c 1, CHCl3).

Synthesis of 5-Thio-L-Fucose-Containing Disaccharides
J . Org. Chem., Vol. 62, No. 4, 1997 997
NMR (67.8 MHz, D2O) δ 175.3, 95.8, 91.5, 83.9, 76.22, 76.15,
75.8, 75.1, 73.4, 71.5, 71.3, 70.2, 60.8, 58.0, 55.3, 37.2, 23.0,
22.7, 16.3. Anal. Calcd for C14H25NO9S: C, 43.86; H, 6.57;
N, 3.65. Found: C, 43.60; H, 6.49; N, 3.92.
1,3,4,6-Tetr a -O-a cetyl-2-O-(2-ch lor oa cetyl)-r-D-ga la cto-
p yr a n ose (22). To a solution of 1,3,4,6-tetra-O-acetyl-R-D-
galactopyranose (21) (2.01 g, 5.77 mmol) in (CH2Cl)2 (17 mL)
at 0 °C was added a solution of chloroacetyl chloride (0.93 mL,
11.7 mmol) in (CH2Cl)2 (5.6 mL). After being stirred at 0 °C
for 1 h, ice was added to the reaction mixture and it was
diluted with CHCl3. The organic layer was washed subse-
quently with 1 M HCl, saturated NaHCO3, and water, dried
(MgSO4), and concentrated in vacuo. The residue was crystal-
for 15 min at -20 °C, the solution was neutralized with Et3N
(8 mL), diluted with EtOAc, and filtered through a pad of
Celite. The filtrate was evaporated, and the residue was
purified on a column of silica gel (3:2 f 8:7 f 1:1 hexane-
EtOAc) to give 26 as a syrup (24.6 mg, 86%). (R:â ) 98:2); R
anomer: mp 167-169 °C; [R]25D -191 (c 0.78, CHCl3); 1H NMR
(270 MHz, CDCl3) δ 6.01-5.87 (m, 1H), 5.45 (dd, 1H, J ) 1.3,
2.6 Hz), 5.37-5.24 (m, 2H), 5.35 (dd, 1H, J ) 2.6, 10.6 Hz),
5.32 (d, 1H, J ) 3.3 Hz), 5.23 (dd, 1H, J ) 2.6, 10.6 Hz), 5.09
(d, 1H, J ) 2.6 Hz), 5.08 (dd, 1H, J ) 3.3, 9.9 Hz), 4.53 (d, 1
H, J ) 7.6 Hz), 4.45-4.39 (m, 1H), 4.14-4.07 (m, 1H), 4.18
(dd, 1H, J ) 6.6, 10.9 Hz), 4.14 (dd, 1H, J ) 7.6, 9.9 Hz), 4.11
(dd, 1H, J ) 6.6, 10.9 Hz), 3.88 (d, 1H, J ) 6.6 Hz), 3.73 (dq,
1H, J ) 1.3, 7.3 Hz), 2.17, 2.14, 2.05, 2.00, 1.984, 1.981 (each
s, 3H × 6), 1.08 (d, 3H, J ) 7.3 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 170.8, 170.6, 170.4, 170.3, 170.1, 169.9, 133.1, 118.7,
100.8, 80.5, 73.6, 72.7, 72.0, 71.5, 70.7, 70.5, 68.7, 67.3, 61.2,
34.0, 20.7, 20.6, 20.5, 15.6. Anal. Calcd for C27H38O15S: C,
51.10; H, 6.04; S, 5.05. Found: C, 51.06; H, 5.90; S, 4.73.
Allyl O-(5-Th io-r-L-fu cop yr a n osyl)-(1f2)-â-D-ga la cto-
p yr a n osid e (4). To a solution of 26 (81.1 mg, 0.128 mmol)
in MeOH (3 mL) and CH2Cl2 (0.9 mL) was added a solution of
0.5 M NaOMe in MeOH (0.5 mL). After 75 min, 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 chromatographed on a column of Sephadex G-15
lized from EtOH-Et2O to give 22 (1.91 g, 78%): mp 115.5-
1
117 °C; [R]26 +89 (c 1.1, CHCl3); H NMR (270 MHz, CDCl3)
D
δ 6.40 (d, 1H, J ) 2.3 Hz), 5.52 (brs, 1H), 5.40-5.39 (m, 2H),
4.37 (brt, 1H, J ) 6.6 Hz), 4.12-4.09 (m, 2H), 4.01 (s, 2H),
2.17 (s, 6H), 2.05, 2.01 (each s, 3H × 2). Anal. Calcd for
C16H21ClO11: C, 45.24; H, 4.98. Found: C, 44.91: H, 5.03.
Allyl 3,4,6-Tr i-O-a cetyl-2-O-(2-ch lor oa cetyl)-â-D-ga la c-
top yr a n osid e (24). To a solution of 22 (0.559 g, 1.32 mmol)
in CH2Cl2 (7.5 mL) at 0 °C was added 25% HBr-AcOH (13.5
mL). After being stirred for 3 h at 0 °C, the solution was
diluted with CH2Cl2 and washed subsequently twice with ice-
water and saturated NaHCO3. The organic layer was dried
(MgSO4) and concentrated to give 3,4,6-tri-O-acetyl-2-O-(2-
chloroacetyl)-R-D-galactopyranosyl bromide (23) as a syrup
(0.570 g, 97%): 1H NMR (270 MHz, CDCl3) δ 6.70 (d, 1H, J )
4.0 Hz), 5.53 (dd, 1H, J ) 1.3, 3.3 Hz), 5.45 (dd, 1H, J ) 3.3,
10.6 Hz), 5.13 (dd, 1H, J ) 4.0, 10.6 Hz), 4.50 (dt, 1H, J ) 1.3,
6.6 Hz), 4.20 (dd, 1H, J ) 6.6, 11.4 Hz), 4.12 (dd, 1H, J ) 6.6,
11.4 Hz), 4.11 (s, 2H), 2.17, 2.07, 2.02 (each s, 3H × 3).
To a mixture of AllOH (1.8 mL, 26 mmol), Hg(CN)2 (1.33 g,
5.27 mmol), and powdered MS3A (0.855 g) in toluene-MeNO2
(1:1, 26 mL) was added dropwise a solution of 23 (0.570 g, 1.28
mmol) in toluene-MeNO2 (1:1, 3.9 mL), and the mixture was
stirred for 3 days and filtered through a pad of Celite. The
filtrate was diluted with CH2Cl2. The solution was washed
twice with water, dried (MgSO4), and concentrated in vacuo.
The residue was purified on a column of silica gel (3:2 hexane-
(water) and lyophilized to give 4 as a white powder (45.0 mg,
1
92%): [R]23 -269 (c 0.29, H2O); H NMR (400 MHz, D2O) δ
D
6.08-5.93 (m, 1H), 5.41-5.28 (m, 2H), 5.09 (d, 1 H, J ) 3.3
Hz), 4.53 (d, 1H, J ) 7.3 Hz), 4.44-4.38 (m, 1H), 4.25-4.18
(m, 1H), 4.02 (d, 1H, J ) 3.0 Hz), 4.00 (dd, 1 H, J ) 3.3, 10.2
Hz), 3.90-3.83 (m, 3H), 3.84 (dd, 1H, J ) 3.0, 10.2 Hz), 3.82-
3.72 (m, 2H), 3.68 (dd, 1H, J ) 4.6, 7.9 Hz), 3.52 (q, 1H, J )
7.3 Hz), 1.17 (d, 3H, J ) 7.3 Hz); 13C NMR (67.8 MHz, D2O) δ
134.1, 119.9, 101.8, 84.5 76.9, 75.8, 75.0, 74.6, 72.0, 71.4, 69.8,
61.7, 36.9, 16.4. Anal. Calcd for C15H26O9S : C, 47.11; H, 6.85.
Found: C, 46.89; H, 6.63.
Met h yl 2,3,4-Tr i-O-(p -m et h oxyb en zyl)-1-t h io-â-L-fu -
cop yr a n osid e (27). To a solution of methyl 2,3,4-tri-O-acetyl-
1-thio-â-L-fucopyranoside (1.564 g, 4.88 mmol) in MeOH (19
mL) was added a catalytic amount of 0.5 M NaOMe, and the
mixture was stirred for 1 h at rt. The solution was neutralized
with Dowex 50W-X8 (H+), and the resin was filtered off. The
filtrate was concentrated, and the residual syrup was dried
in high vacuum. To a solution of the residue in DMF (11.5
mL) was added NaH (1.02 g, 55%) at 0 °C, and the mixture
was stirred for 30 min. To the solution was added MpmCl
(3.2 mL) and stirred overnight at rt. Methanol was added to
the solution, and the mixture was diluted with EtOAc. The
solution was washed four times with brine, and the organic
layer was dried and concentrated. The residue was purified
on a column of silica gel (3:1 f 12:5 hexane-EtOAc) to give
EtOAc) to give 24 as a syrup (0.438 g, 81%): [R]25 -1.6 (c
D
1.3, CHCl3); 1H NMR (270 MHz, CDCl3) δ 5.93-5.79 (m, 1H),
5.41 (dd, 1H, J ) 1.0 Hz), 5.29 (dd, 1H, J ) 7.9, 10.6 Hz), 5.32-
5.19 (m, 2H), 5.07 (dd, 1H, J ) 3.3, 10.6 Hz), 4.57 (d, 1H, J )
7.9 Hz), 4.40-4.32 (m, 1H), 4.15-4.07 (m, 1H), 4.20 (dd, 1H,
J ) 6.6, 11.2 Hz), 4.13 (dd, 1H, J ) 6.6, 11.2 Hz), 4.04 (s, 2H),
3.92 (dt, 1H, J ) 1.0, 6.6 Hz), 2.16, 2.06, 1.99 (each s, 3H × 3);
13C NMR (67.8 Hz, CDCl3) δ 170.4, 170.2, 170.1, 166.0, 133.1,
118.0, 99.6, 70.7, 70.7, 70.6, 70.2, 67.0, 61.2, 40.5, 20.6, 20.5.
Anal. Calcd for C17H23ClO10: C, 48.29; H, 5.48. Found: C,
48.16 ; H, 5.46.
Allyl 3,4,6-Tr i-O-a cetyl-â-D-ga la ctop yr a n osid e (25). To
a solution of 24 (0.438 g, 1.04 mmol) in EtOAc-MeOH (1:1,
16 mL) was added H2NNH2‚AcOH (0.292 g, 3.17 mmol). After
being stirred for 5 h, the solvents were evaporated and the
residue was dissolved in EtOAc. The solution was washed
with brine, and the organic layer was dried (MgSO4) and
concentrated in vacuo. The residue was purified on a column
of silica gel (1:1 f 1:2 hexane-EtOAc) to give 25 as a syrup
(0.295 g, 82%): [R]25D +2.6 (c 1.8, CHCl3); 1H NMR (270 MHz,
CDCl3) δ 6.03-5.88 (m, 1H), 5.39 (dd, 1H, J ) 1.0, 3.6 Hz),
5.37-5.23 (m, 2H), 4.94 (dd, 1H, J ) 3.6, 10.2 Hz), 4.45-4.38
(m, 1H), 4.20-4.13 (m, 1H), 4.42 (d, 1H, J ) 7.6 Hz), 4.19 (dd,
1H, J ) 6.6, 11.2 Hz), 4.12 (dd, 1H, J ) 6.6, 11.2 Hz), 3.90 (dt,
1H, J ) 1.0, 6.6 Hz), 3.84 (dd, 1H, J ) 7.6, 10.2 Hz), 2.49 (brs,
1H), 2.13 (s, 3H), 2.05 (s, 6H); 13C NMR (67.8 Hz, CDCl3) δ
170.4, 170.3, 170.2, 133.3, 118.5, 102.0, 72.5, 70.7, 70.5, 69.1,
67.1, 61.3, 20.7, 20.6, 20.6. Anal. Calcd for C15H22O9: C, 52.02;
H, 6.40. Found: C, 51.81; H, 6.26.
27 as a crystal (1.965 g, 73%): [R]26 -8.1 (c 1.2, CHCl3); mp
D
1
73-75 °C; H NMR (270 MHz, CDCl3) δ 7.34-7.26 (m, 6H),
6.90-6.83 (m, 6H), 4.92-4.59 (m, 6H), 4.26 (d, 1H, J ) 9.6
Hz), 3.816 (s, 3H), 3.804 (s, 3H), 3.796 (s, 3H), 3.78 (dd, 1H, J
) 9.2, 9.6 Hz), 3.56 (d, 1H, J ) 3.0 Hz), 3.52 (dd, 1H, J ) 3.0,
9.2 Hz), 3.46 (q, 1H, J ) 6.3 Hz), 2.19 (s, 3H), 1.16 (d, 3H, J )
6.3 Hz). Anal. Calcd for C31H38O7S: C, 67.12; H, 6.90.
Found: C, 67.03; H, 7.35.
O-(3,4-O-Isop r op ylid en e-â-D-ga la ctop yr a n osyl)-(1f4)-
2,3;5,6-d i-O-isop r op ylid en e-D-glu cose Dim et h yl Acet a l
(28). To a solution of CSA (13 mg) in Me2C(OMe)2 (6.16 mL)
was added lactose (1.00 g, 2.78 mmol), and the mixture was
stirred at 80 °C until it became clear. The solution was cooled
to rt and neutralized with Et3N. The solution was concen-
trated in vacuo, and the residue was purified by MPLC (2:3
f 1:3 hexane-EtOAc) to give 28 (0.594 g, 42%) and its 6′-O-
(2-methoxy-2-propyl) isomer 29 (0.500 g, 31%). The structure
Allyl O-(2,3,4-Tr i-O-acetyl-5-th io-L-fu copyr an osyl)-(1f2)-
3,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osid e (26). To a solu-
tion of 6 (20.2 mg, 44.8 µmol) and 25 (25.0 mg, 72.2 µmol) in
(CH2Cl)2 (1.04 mL) was added powdered MS4A (152 mg), and
the mixture was stirred for 1 h under Ar. To the solution
cooled to -20 °C was added dropwise a solution of BF3‚OEt2
(1.9 µL, 15 µmol) in (CH2Cl)2 (0.40 mL). After being stirred
of 28 was determined as its diacetate 30. 28: [R]23 +35.7 (c
D
1
2.0, CHCl3); H NMR (270 MHz, CDCl3) δ 4.60 (dd, 1H, J )
6.9, 7.9 Hz), 4.43 (d, 1H, J ) 8.3 Hz), 4.37 (d, 1H, J ) 6.9 Hz),
3.50 (s, 3H), 3.49 (s, 3H), 1.50 (s, 6H), 1.39 (s, 6H), 1.32 (s,
3H), 1.31 (s, 3H); 13C NMR (67.8 MHz, CDCl3) δ 110.0, 109.4,
107.8, 106.6, 102.9, 78.9, 77.7, 77.1, 75.3, 74.9, 74.2, 73.6, 73.1,
64.0, 61.9, 57.1, 53.9 27.6-23.4. Anal. Calcd for C23H40O12
:

998 J . Org. Chem., Vol. 62, No. 4, 1997
Izumi et al.
C, 54.32; H, 7.93. Found: C, 54.62; H, 8.17. 29: 1H NMR
(270 MHz, CDCl3) δ 3.44 (s, 3H), 3.43 (s, 3H), 3.21 (s, 3H),
1.52 (s, 3H), 1.50 (s, 3H), 1.38 (s, 6H), 1.34 (s, 9H), 1.33 (s,
3H). 30: 1H NMR (270 MHz, CDCl3) δ 5.02 (dd, 1H, J ) 6.9,
7.9 Hz), 4.79 (d, 1H, J ) 7.9 Hz), 4.46 (t, 1H, J ) 6.3 Hz), 4.39
(dd, 1H, J ) 5.0, 11.9 Hz), 4.36 (d, 1H, J ) 6.3 Hz), 4.31 (dt,
1H, J ) 2.3, 6.3 Hz), 4.24 (dd, 1H, J ) 7.3, 11.9 Hz), 4.16-
4.10 (m, 2H), 4.08 (dd, 1H, J ) 1.7, 2.3 Hz), 3.98-3.93 (m,
4H), 3.42 (s, 6H), 2.13 (s, 3H), 2.09 (s, 3H), 1.56 (s, 3H), 1.48
(s, 3H), 1.37 (s, 3H), 1.33 (s, 3H), 1.32 (s, 3H); 13C NMR (67.8
MHz, CDCl3) δ 170.8, 169.6, 110.9, 110.7, 108.0, 105.0, 100.2,
78.2, 78.1, 77.3, 75.1, 73.8, 73.7, 72.6, 70.9, 64.6, 63.4, 55.5,
53.0, 27.7, 27.6, 26.4, 26.3, 26.1, 24.7, 20.9, 20.8.
75.0, 74.3, 74.2, 73.6, 72.8, 72.3, 70.8, 66.4, 65.2, 63.3, 55.9,
55.2, 53.1, 27.8, 27.3, 26.84, 26.76, 26.5, 25.2, 20.8, 16.8. Anal.
Calcd for C55H76O20: C, 54.54; H, 7.69. Found: C, 54.71; H,
7.95.
O-(2,3,4-Tr i-O-a cetyl-r-L-fu cop yr a n osyl)-(1f2)-O-(3,4,6-
tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-1,2,3,6-tetr a -O-
a cetyl-r/â-D-glu cop yr a n ose (33). A solution of 32 (0.219 g,
0.206 mmol) and CAN (0.68 g, 1.24 mmol) in 9:1 (v/v) MeCN-
H2O (3.1 mL) was stirred at rt. After TLC indicated comple-
tion of the reaction, pyridine (22 mL) and Ac2O (11 mL) and a
catalytic amount of DMAP was added and stirred overnight.
To the reaction mixture was added MeOH. The mixture was
then diluted with CHCl3. The solution was washed subse-
quently with water and saturated aqueous NaHCO3. The
organic layer was dried and concentrated in vacuo. The
residue was purified on a column of silica gel (3:2 hexane-
EtOAc) to give 33 as a syrup (0.128 g, 72%): 1H NMR (400
MHz, CDCl3) δ 6.30 (d, 0.25H, J ) 4.0 Hz), 5.69 (d, 0.75H, J
) 8.3 Hz), 5.37 (d, 1H, J ) 3.6 Hz), 4.41 (d, 1H, J ) 7.6 Hz),
1.22 (d, 3H, J ) 7.6 Hz); 13C NMR for R anomer (67.8 MHz,
CDCl3) δ 170.6-168.6, 100.0, 95.6, 88.8, 73.5, 73.2, 71.6, 71.0,
70.7, 70.6, 69.02, 68.95, 67.9, 67.4, 66.9, 64.7, 60.9, 60.7, 20.9-
O-(6-O-Acetyl-3,4-O-isop r op ylid en e-â-D-ga la ctop yr a n o-
syl)-(1f4)-2,3:5,6-d i-O-isop r op ylid en e-D-glu cose Dim eth -
yl Aceta l (31). To a solution of 28 (3.356 g, 6.59 mmol) in
CH2Cl2 (33.7 mL) was added Ac2O (1.18 mL) and Et3N (2.67
mL). After being stirred for 18 h at rt, MeOH was added and
the solution was concentrated in vacuo. The residue was
purified by MPLC (3:2 f 1:1 hexane-EtOAc) to give 31 (2.434
g, 67%) and diacetate 30 (0.188 g, 5%) and recovered 28 (0.963
g, 28%): [R]25 +32.1 (c 2.4, CHCl3); 1H NMR (400 MHz,
D
CDCl3) δ 4.45 (dd, 1H, J ) 6.2, 7.6 Hz), 4.42 (d, 1H, J ) 8.2
Hz), 4.37 (d, 1H, J ) 6.2 Hz), 4.35 (dd, 1H, J ) 4.7, 11.6 Hz),
4.30 (dd, 1H, J ) 7.2, 11.6 Hz), 4.28 (dt, 1H, J ) 6.5, 2.6 Hz),
4.16 (dd, 1H, J ) 6.5, 8.7 Hz), 4.11 (dd, 1H, J ) 2.3, 5.7 Hz),
4.09 (d, 1H, J ) 1.5, 2.6 Hz), 4.07 (t, 1H, J ) 5.7, 7.6 Hz), 4.02
(dd, 1H, J ) 2.6, 8.7 Hz), 3.95 (ddd, 1H, J ) 2.3, 4.7, 7.2 Hz),
3.90 (dd, 1H, J ) 1.5, 7.6 Hz), 3.56 (dd, 1H, J ) 7.6, 8.2 Hz),
3.47 (bs, 1H), 3.44 (s, 3H), 3.44 (s, 3H); 13C NMR (67.8 MHz,
CDCl3) δ 170.7, 110.4, 110.4, 108.3, 105.1, 103.7, 79.0, 78.0,
77.8, 76.4, 75.0, 74.1, 73.3, 71.4, 64.6, 63.5, 56.1, 53.2, 28.1,
20.4, 15.3. Anal. Calcd for C38H52O25
Found: C, 50.53; H, 5.98.
: C, 50.22; H, 5.77.
O-r-L-F u cop yr a n osyl-(1f2)-O-â-D-ga la ct op yr a n osyl-
(1f4)-D-glu cop yr a n ose (34). To a solution of 33 (0.102 g,
0.118 mmol) in MeOH (16.8 mL) was added a catalytic amount
of NaOMe, and the mixture was stirred overnight at rt. The
solution was neutralized with Dowex 50W-X8 (H+), and the
resin was filtered off. The filtrate was evaporated, and the
residue was chromatographed on a column of Sephadex G-15
(water) and lyophilized to give 34 as a white solid (47 mg,
81%): 1H NMR (400 MHz, D2O) δ 5.37 (d, J ) 2.7 Hz), 5.28
(d, J ) 3.7 Hz), 4.70 (d, J ) 8.1 Hz), 4.58 (d, J ) 7.8 Hz), 4.31
(q, J ) 6.5 Hz), 4.28 (q, J ) 6.5 Hz), 1.28 (d, J ) 6.5 Hz).
En zym e In h ibition Assa ys. A reaction solution which
contained R-L-fucosidase (Bacillus sp. K40T) and 2′-fucosyl-
lactose (0.37, 0.53, 1.0, 1.7 mM) and inhibitors (three different
concentrations in the range 0.14-0.56 mM and 0 mM) and
bovine serum albumin in 56 mM phosphate buffer (pH 6.5,
150 µL) was incubated at 37 °C for 20 or 30 min. The enzyme
reaction was quenched at 100 °C for 5 min and the released
L-fucose was analyzed using fucose dehydrogenase-NADP
system.25,26
27.2, 26.3, 26.2, 25.6, 23.8, 20.8. Anal. Calcd for C25H42O13
C, 54.54; H, 7.69. Found: C, 54.71; H, 7.95.
:
O-(2,3,4-Tr i-O-(p-m eth oxyben zyl)-r-L-fu cop yr a n osyl)-
(1f2)-O-(6-O-a cetyl-3,4-O-isop r op ylid en e-â-D-ga la ctop y-
r a n osyl)-(1f4)-2,3:5,6-d i-O-isop r op ylid en e-D-glu cose Di-
m eth yl Aceta l (32). A mixture of 31 (1.172 g, 2.12 mmol)
and IDCP (0.719 g, 1.52 mmol) in (CH2Cl)2-Et2O (1:5) (12.3
mL) and MS4A (1.03 g) was stirred for 1 h at 0 °C under Ar
atmosphere. To the stirred solution was added 27 (0.567 g,
1.02 mmol), and the mixture was stirred for 1 h at rt. The
solution was diluted with CHCl3, and the solid was filtered
off. The filtrate was washed with 2 M aqueous Na2S2O3, and
the organic layer was dried and concentrated in vacuo. The
A reaction solution containing p-nitrophenyl R-L-fucopyra-
noside (0.067, 0.10, 0.17, 0.33 mM), bovine epididymis R-L-
fucosidase, and inhibitors (three different concentrations in
the range of 0.018-0.21 mM) in 20 mM sodium citrate buffer
(pH 5.8, 300 µL) was incubated at 25 °C for 30 or 40 min. The
reaction was quenched with cooling in an ice bath and by
adding glycine buffer (pH 10.0, 500 µL), and the amount of
p-nitrophenol was measured by the absorbance at 400 nm.
residue was purified by MPLC (3:2 hexane-EtOAc) to give
1
32 as a syrup (0.701 g, 65%): [R]25 -34.0 (c 1.7, CHCl3); H
D
NMR (400 MHz, CDCl3) δ 7.34-7.23 (m, 6H), 6.90-6.82 (m,
6H), 5.53 (d, 1H, J ) 3.6 Hz), 4.90-4.63 (m, 6H), 4.61 (d, 1H,
J ) 6.6 Hz), 4.49 (dd, 1H, J ) 5.9, 6.3 Hz), 4.36 (d, 1H, J )
6.3 Hz), 4.33 (dd, 1H, J ) 4.0, 11.5 Hz), 4.25-4.21 (m, 2H),
4.18 (t, 1H, J ) 5.9 Hz), 4.08-3.87 (m, 9H), 3.81 (s, 3H), 3.802
(s, 3H), 3.797 (s, 3H), 3.69 (t, 1H, J ) 6.9 Hz), 3.42 (s, 6H),
2.09 (s, 3H), 1.46 (s, 3H), 1.43 (s, 3H), 1.38 (s, 3H), 1.35 (s,
3H), 1.32 (s, 3H), 1.29 (s, 3H), 1.05 (d, 3H, J ) 6.3 Hz); 13C
NMR (67.8 MHz, CDCl3) δ 170.7, 159.1, 158.9, 131.5, 131.2,
131.0, 129.9, 129.2, 128.9, 113.7, 113.52, 113.47, 110.2, 110.0,
108.6, 105.2, 101.2, 95.2, 80.1, 79.0, 77.6, 77.5, 77.4, 76.1, 75.2,
J O961725H
(25) Tsay, G. C.; Daeson, G. Anal. Biochem., 1977, 78, 423.
(26) Compound 1 showed 7% inhibition at 5 mM.