The C-disaccharide α-C(1→3)-mannopyranoside of N-acetylgalactosamine is an inhibitor of glycohydrolases and of human α-1,3-fucosyltransferase VI. Its epimer α-(1→3)-mannopyranoside of N-acetyltalosamine is not

Article abstract of DOI:10.1021/jo991952u

The radical C-glycosidation of (-)-(1S,4R,5R,6R)-6-endo-chloro-3- methylidene-5-exo-(phenylseleno)-7-oxabicyclo[2.2.1]heptan-2-one ((-)-4) with 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl bromide gave (+)-(1S,3R,4R,5R,6R)- 6-endo-chloro-5-exo-(phenylseleno)-3-endo-(1',3',4',5',-tetra-O-acetyl-2',6'- anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-7-oxabicyclo[2.2.1]hept- 2-one ((+)-5) that was converted into (+)-(1R,2S,5R,6R)-5-acetamido-3-chloro- 2-hydroxy-6-(1',3',4',5'-tetra-O-acetyl)-2',6'-anhydro-7'-deoxy-D-glycero-D- manno-heptitol-7'-C-yl)cyclohex-3-en-1-yl acetate ((+)-10) and into (+)- (1R,2S,5R,6S)-5-bromo-3-chloro-2-hydroxy-6-(1,3,4,5 -tetra-O-acetyl- 2',6',anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)cyclohex-3-en-1-yl acetate ((+)-19). Ozonolysis of (+)-10 and further transformations provided 2-acetamido-2,3-dideoxy-3-C-(2',6'-anhydro-7'-deoxy-D-glycero-D-manno- heptitol-7'-C-yl)-D-galactose (α-C(1→3)-D-mannopyranoside of N- acetylgalactosamine (α-D-Manp- (1→3)CH₂-D-GalNAc): 1). Displacement of the bromide (+)-19 with NaN₃ in DMF provided the corresponding azide ((-)-20) following a S(N)2 mechanism. Ozonolysis of (-)-20 and further transformations led to 2-acetamido-2,3-dideoxy-3-C-(2',6'-anhydro-7'-deoxy-D-glycero-D-manno- heptitol-7'-C-yl)-D-talose (α-C(1→3)-D-mannopyranoside of N-acetyl D- talosamine (α-D-Manp-(1→3)CH₂-D-TalNAc): 2). The neutral C-disaccharide 1 inhibits several glycosidases (e.g., β-galactosidase from jack bean with K(i) = 7.5 μM, α-L-fucosidase from human placenta with K(i) = 28 μM, β- glucosidase from Caldocellum saccharolyticum with K(i) = 18 μM) and human α-1,3-fucosyltransferase VI (Fuc-TVI) with K(i) = 120 μM whereas it 2- epimer 2 does not. Double reciprocal analysis showed that the inhibition of Fuc-TVI by 1 displays a mixed pattern with respect to both the donor sugar GDP-fucose-and the acceptor LacNAc with K(i) of 123 and 128 μM, respectively.

Full text of DOI:10.1021/jo991952u

J . Org. Chem. 2000, 65, 4251-4260
4251
Th e C-Disa cch a r id e r-C(1f3)-Ma n n op yr a n osid e of
N-Acetylga la ctosa m in e Is a n In h ibitor of Glycoh yd r ola ses a n d of
Hu m a n r-1,3-F u cosyltr a n sfer a se VI. Its Ep im er
r-(1f3)-Ma n n op yr a n osid e of N-Acetylta losa m in e Is Not
Carla Pasquarello,† Sylviane Picasso,† Raynald Demange,† Martine Malissard,‡
Eric G. Berger,‡ and Pierre Vogel*,†
Section de Chimie, Universite´ de Lausanne, BCH, CH-1015 Lausanne-Dorigny, Switzerland,
and Physiologisches Institut der Universita¨t Zu¨rich, CH-8057 Zu¨rich-Irchel, Switzerland
Received December 21, 1999
The radical C-glycosidation of (-)-(1S,4R,5R,6R)-6-endo-chloro-3-methylidene-5-exo-(phenylseleno)-
7-oxabicyclo[2.2.1]heptan-2-one ((-)-4) with 2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyl bromide gave
(+)-(1S,3R,4R,5R,6R)-6-endo-chloro-5-exo-(phenylseleno)-3-endo-(1′,3′,4′,5′-tetra-O-acetyl-2′,6′-
anhydro-7′-deoxy-D-glycero-D-manno-heptitol-7′-C-yl)-7-oxabicyclo[2.2.1]hept-2-one ((+)-5) that was
converted into (+)-(1R,2S,5R,6R)-5-acetamido-3-chloro-2-hydroxy-6-(1′,3′,4′,5′-tetra-O-acetyl)-2′,6′-
anhydro-7′-deoxy-D-glycero-D-manno-heptitol-7′-C-yl)cyclohex-3-en-1-yl acetate ((+)-10) and into (+)-
(1R,2S,5R,6S)-5-bromo-3-chloro-2-hydroxy-6-(1′,3′,4′,5′-tetra-O-acetyl-2′,6′-anhydro-7′-deoxy-D-
glycero-D-manno-heptitol-7′-C-yl)cyclohex-3-en-1-yl acetate ((+)-19). Ozonolysis of (+)-10 and further
transformations provided 2-acetamido-2,3-dideoxy-3-C-(2′,6′-anhydro-7′-deoxy-D-glycero-D-manno-
heptitol-7′-C-yl)-D-galactose (R-C(1f3)-D-mannopyranoside of N-acetylgalactosamine (R-D-Manp-
(1f3)CH2-D-GalNAc): 1). Displacement of the bromide (+)-19 with NaN3 in DMF provided the
corresponding azide ((-)-20) following a SN2 mechanism. Ozonolysis of (-)-20 and further
transformations led to 2-acetamido-2,3-dideoxy-3-C-(2′,6′-anhydro-7′-deoxy-D-glycero-D-manno-
heptitol-7′-C-yl)-D-talose (R-C(1f3)-D-mannopyranoside of N-acetyl D-talosamine (R-D-Manp-(1f3)-
CH2-D-TalNAc): 2). The neutral C-disaccharide 1 inhibits several glycosidases (e.g., â-galactosidase
from jack bean with Ki ) 7.5 µM, R-L-fucosidase from human placenta with Ki ) 28 µM, â-glucosidase
from Caldocellum saccharolyticum with Ki ) 18 µM) and human R-1,3-fucosyltransferase VI (Fuc-
TVI) with Ki ) 120 µM whereas it 2-epimer 2 does not. Double reciprocal analysis showed that the
inhibition of Fuc-TVI by 1 displays a mixed pattern with respect to both the donor sugar GDP-
fucose and the acceptor LacNAc with Ki of 123 and 128 µM, respectively.
In tr od u ction
C-disaccharides5 are potential glycosidase and glycosyl-
transferase inhibitors and may represent nonhydrolyz-
The interaction between cell surface carbohydrates and
their protein receptors is implicated in several important
biological events.1 Carbohydrate mimetics are potentially
useful tools to study cellular interactions, the biosynthe-
sis of glycoproteins, the catabolism of glycoconjugates,2
and the mechanisms of digestion.3 Inhibitors of enzymes
involved in these processes such as the glycosidases and
the glycosyltransferases are potential anticancer, anti-
viral, and antidiabetic agents; they can also have insect
antifeedant effects.4 Disaccharide mimetics such as the
able epitopes.6 We present the syntheses of two new
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† Universite´ de Lausanne.
‡ Physiologisches Institut der Universita¨t Zu¨rich.
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Pergamon-Elsevier Science Ltd.: Oxford, 1995. Postema, M. H. D.
C-Glycoside Synthesis; CRC: Boca Raton, FL, 1995. Vogel, P.; Ferritto,
R.; Kraehenbuehl, K.; Baudat, A. Carbohydrate Mimics, Concept and
Methods; Chapleur, Y., Ed.; Wiley-VCH: Weinheim, 1997; Chapter 2,
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1996, 61, 1894. Streicher, H.; Geyer, A.; Schmidt, R. R. Chem. Eur.
1996, 2, 502. Witczak, Z. J .; Chhabra, R.; Chojnacki, J . Tetrahedron
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10.1021/jo991952u CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/10/2000

4252 J . Org. Chem., Vol. 65, No. 14, 2000
Pasquarello et al.
Sch em e 1
C-disaccharides, i.e.: R-D-Manp(1f3)CH2-D-GalNAc (1)7
and its epimer R-D-Manp(1f3)CH2-D-TalNAc (2) and
show that 1 inhibits several glycosidase and human
R-1,3-fucosyltransferase VI (Fuc-TVI) whereas 2 does not.
and C-glycosides of carbapentopyranoses19 and ana-
logues.20 Under modified conditions, the radical C-man-
nosidation between 2,3,4,6-tetra-O-acetyl-R-D-mannopy-
(6) See, for example, MacLean, G. D.; Bowen-Yacyshyn, M. B.;
Samuel, J .; Meikle, A.; Stuart, G.; Nation, J .; Poppema, S.; J erry, M.;
Koganty, R.; Wong, T.; Longenecker, B. M. J . Immunother. 1992, 11,
292. Rye, P. D.; Bovin, N. V.; Vlasova, E. V.; Walker, R. A. Glycobiology
1995, 5, 385.
(7) For a preliminary report on 1, see: Pasquarello, C.; Demange,
R.; Vogel, P. Bioorg. Med. Chem. Lett. 1999, 9, 793.
(8) Schenkel-Brunner, H. In Human Blood Groups; Springer-
Verlag: Vienna, 1995. Staudacher, E. Trends Glycosci. Glycotechnol.
1996, 8, 391.
(9) Costache, M.; Apoil, P. A.; Cailleau, A.; Elmgren, A.; Larson, G.;
Henry, S.; Blancher, A.; Iordachescu, D.; Oriol, R.; Mollicone, R. J . Biol.
Chem. 1997, 272, 29721.
(10) Mollicone, R.; Gibaud, A.; Francois, A.; Ratcliffe, M.; Oriol, R.
Eur. J . Biochem. 1990, 191, 169.
Fucosylated glycoconjugates play important roles in
physiological and pathological processes such as fertiliza-
tion, embryogenesis, lymphocyte trafficking, immune
response, and cancer metastasis.8 Among the eight Fuc-
Ts cloned to date,9 five of them catalyze the transfer from
GDP-Fucose to N-acetylglucosamine in an R-1,3 linkage.
These Fuc-Ts are designated Fuc-TIII to Fuc-TVII and
differ in substrate specificity, cation requirement, sen-
sitivity to inhibitors, and tissue distribution.10 Cell
surface R-1,3-fucosylated structures including the trisac-
charide Lewis x (Lex) and the tetrasaccharide sialyl-Lewis
x (sLex) are involved in a variety of cell-cell interactions
such as inflammation11 and tumor metastasis.12 Indeed,
R-1,3-fucosylated glycoconjugates have been identified as
ligands for E- and P-selectins, cell-adhesion molecules
involved in recruitment of leukocytes into the site of
inflammation.13 Furthermore, many studies have shown
that invasiveness of tumor cells is in correlation with an
increase of serum Fuc-T activity or in fucose incorpora-
tion into certain surface glycoproteins.14
(11) Lowe, J . B. Kidney Int. 1997, 51, 1418.
(12) Kim, Y. J .; Borsig, L.; Varki, N. M.; Varki, A. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 9325. Foxall, C.; Watson, S. R.; Dowbenko, D.;
Fennie, C.; Lasky, L. A.; Kiso, M.; Hasegawa, A.; Asa, D.; Brandley,
B. K. J . Cell Biol. 1992, 117, 895. Robinson, N. E.; De Vries, T.; Davis,
R. E.; Stults, C. L.; Watson, S. R.; Van den Eijnden, D. H.; Macher, B.
A. Glycobiology 1994, 4, 317.
(13) Walz, G.; Aruffo, A.; Kolanus, W.; Bevilacqua, M.; Seed, B.
Science 1990, 250, 1132. Phillips, M. L.; Nudelman, E.; Gaeta, F. C.;
Perez, M.; Singhal, A. K.; Hakomori, S.; Paulson, J . C. Science 1990,
250, 1130.
(14) Hakomori, S. Adv. Cancer Res. 1989, 52, 257. Bolscher, J . G.;
Bruyneel, E. A.; Van Rooy, H.; Schallier, D. C.; Marcel, M. M.; Smet,
L. A. Clin. Exp. Metastasis 1989, 7, 557. Strout, G. B.; Anumula, K.
R.; Kline, T. F.; Caltabiano, M.; Stroup, M. Cancer Res. 1990, 50, 6787.
Wang, J . W.; Ambros, R. A.; Weber, P. B.; Rosano, T. G. Cancer Res.
1995, 55, 3654.
(15) Giese, B.; Witzel, T. Angew. Chem., Int. Ed. Engl. 1986, 25,
450.
(16) Bimwala, R. M.; Vogel, P. J . Org. Chem. 1992, 57, 2076.
(17) Ferritto, R.; Vogel, P. Tetrahedron: Asymmetry 1994, 5, 2077.
(18) Vieira, E.; Vogel, P. Helv. Chim. Acta 1983, 66, 1865. Black,
K. A.; Vogel, P. Ibid. 1984, 67, 1612. Reymond, J .-L.; Vogel, P.
Tetrahedron: Asymmetry 1990, 1, 729. Vogel, P.; Fattori, D.; Gasparini,
F.; Le Drian, C. Synlett 1990, 173. Vogel, P. Bull. Soc. Belg. 1990, 99,
295. Forster, A.; Kovac, T.; Mosimann, H.; Renaud, P.; Vogel, P.
Tetrahedron: Asymmetry 1999, 10, 567; see also: Saf, R.; Faber, K.;
Penn, G.; Griengl, H. Tetrahedron 1988, 44, 389. Ronan, B.; Kagan,
H. B. Tetrahedron: Asymmetry 1991, 2, 75. Corey, E. J .; Loh, T.-P.
Tetrahedron Lett. 1993, 34, 3979.
Syn th esis of th e C-Disa cch a r id es. Giese’s15 radical
C-glucosidation16 and C-galactosidation17 of enone (-)-4
derived from (+)-(1R,4R)-7-oxabicyclo[2.2.1]hept-5-en-2-
one ((+)-3: a “naked sugar” of the first generation)18 has
been shown to be highly diastereoselective, giving C-
glycosides that have been converted into C-disaccharides
(19) Cossy, J .; Ranaivosata, J .-L.; Bellosta, V.; Ancerewicz, J .;
Ferritto, R.; Vogel, P. J . Org. Chem. 1995, 60, 8351.

C-Disaccharide Inhibitor of Glycohydrolases
J . Org. Chem., Vol. 65, No. 14, 2000 4253
ranosyl bromide and (-)-4 is possible in benzene using
Ph3SnH as hydrogen donor, giving (+)-5 in 56% yield
(Scheme 1). The endo relative configuration of (+)-5 was
3
given by the coupling constant J (H-3exo,H-4) ) 5.6 Hz.21
No trace of the isomer exo C-mannoside could be detected
1
in the 400 MHz H NMR spectrum of the crude reaction
mixture.16,17 The R-configuration of the R-C-mannoside
3
(C-6′) was confirmed by the observation of J (H-2′,H-3′)
F igu r e 1. Average conformations of (+)-19 and (-)-20 as
given by the 3J (H,H) coupling constants.
3
) 2.7 Hz, J (H-5′,H-6′) ) 8.5 Hz, and of a NOE between
the signals of (+)-5 assigned to H-6′ (δH 3.98 ppm) and
H-1′ (δH 4.56 ppm).
moiety. Coupling constants between vicinal protons sug-
gest the half-chair conformation shown in Figure 1.
Product (+)-19 results probably from the cationic inter-
mediate 9 (Scheme 1) that is quenched by Br- onto its
less sterically hindered face. Alternatively, (+)-19 could
arise from an intramolecular transfer of Br- in the
zwitterion intermediate 18 (Scheme 2).25 Displacement
of the allylic bromide (+)-19 with NaN3/DMF (60 °C)
occurred with complete inversion at C-5 and without
allylic rearrangement giving (-)-20 in 98% yield. Ozo-
nolysis of the chloroalkene moiety of (-)-20, followed by
workup with Me2S in MeOH, furnished the methyl
taluronate (-)-21 (69% yield). This pyranoside exists
predominantly as the R-anomer as confirmed by its 13C
NMR spectrum showing δ(C-1) ) 92.8 ppm (1J (C,H) )
172 Hz).23 Silylation of (-)-21 with (i-Pr)3SiOSO2CF3 and
2,6-lutidine provided (-)-22 (100%), the â-pyranoside
structure being established by the 2D NOESY 1H NMR
spectrum of 25 (see below, Figure 3). The reduction of
azide (-)-22 (H2, PtO2) led to 23 that was acetylated into
(-)-24 (85%, 2 steps). Reduction of the uronic ester (-)-
24 with LiBH4 in THF at 0 °C generated 25 that was
acetylated to afford (-)-26 (76%, two steps). NOE’s
observed between signals assigned to H-1 (4.87 ppm), H-3
(2.15-2.02 ppm), and H-5 (3.83 ppm) in the 2D NOESY
1H NMR spectrum of (-)-26 confirmed the â-D-talopyra-
noside structure. Desilylation of (-)-26 gave (+)-27, the
R-D-pyranose configuration being given by δ(C-1) ) 93.4
ppm (1J (C,H) ) 171 Hz).23 Ammonolysis of (+)-27 in
MeOH delivered the unprotected C-disaccharide as a
mixture of 2 and furanoses 28 that was used as such in
the enzymatic assays (see below). With the hope of
increasing the overall yield in 2 + 28, we explored the
possibility to use the (tert-butyl)dimethylsilyl â-pyrano-
side (-)-29 (instead of (-)-22) that was obtained on
treating (-)-21 with (t-Bu)Me2SiOSO2CF3 and 2,6-luti-
dine (75% yield). The â-D-talo-pyranoside structure of (-)-
29 was given by NOE’s observed between the signals
assigned to H-1 (4.94 ppm) and H-5 (4.20-4.10 ppm) in
its 2D NOESY 1H NMR spectrum. Reduction of azide (-)-
29 (H2/PtO2 (cat.), EtOAc), followed by acetylation gave
(-)-30 (89%). Unfortunately, our attempts to convert the
uronic methyl ester into the corresponding talose deriva-
tive all failed, perhaps because of the lesser stability of
the (tert-butyl)dimethylsilyl â-pyranoside (-)-29 com-
pared with that of (-)-22.
As expected, reduction of the ketone (+)-5 with NaBH4
was exo face selective giving alcohol (-)-6 (79%). Oxida-
tive elimination of the benzeneselenyl group with m-
CPBA gave (+)-7, and subsequent acetylation provided
(+)-8 (86%). Acid-promoted (CF3SO3H) oxa-ring opening
of the 7-oxanorbornene (+)-8 in anhydrous acetonitrile
produced, after aqueous workup (NaHCO3), the amino-
conduritol derivative (+)-10 (84%). Ozonolysis of chloro-
alkene (+)-10 generated an acyl chloride-aldehyde in-
termediate that reacted with MeOH22 to produce a 1.4:1
mixture of the methyl uronates (+)-11 and (-)-12 sepa-
rated by flash chromatography on silica gel. The galacto
configuration of the pyranose moiety of (+)-11 was given
by the vicinal coupling constant 3J (H-2,H-3) ) 9.9 Hz;
3J (H-1,H-2) ) 3.0 Hz confirmed the R-anomer and δ(C-
1) ) 91.5 ppm the pyranose system (δ(C-1) ) 95.5 for
the furanose system).23 The high regioselectivity of the
acid-induced isomerization (+)-8 f (+)-10 can be inter-
preted in terms of the preferred cleavage of C(4)-O(7)
bond leading to the 1-chloroallyl cation intermediate 9.
Cleavage of the C(1)-O(7) bond would generate a 2-chlo-
roallyl cation intermediate less stable than 9. MeCN
reacts on the less sterically hindered face of 9 giving an
imminium ion intermediate that reacts with H2O to
afford (+)-10 (Ritter reaction24).
Silylation of (+)-11 with (i-Pr)3SiOSO2CF3 and 2,6-
lutidine gave (+)-13 (1:0.45 mixture of R- and â-pyrano-
side). Distinction between the R- (major) and â-pyrano-
side (minor) was based on the vicinal coupling constants
3J (H-1,H-2) ) 3.3 Hz (13r) and 7.8 Hz (13â). The â-D-
pyranoside structure of 13â was confirmed by the obser-
vation of NOE’s between signals assigned to H-1 (4.89
ppm), H-3 (2.43 ppm), H-5 (4.21 ppm). Uronic ester (+)-
13r was reduced with LiBH4 affording 14 that was
acetylated to give (+)-15 (46% based on (+)-11). Desily-
lation with Bu4NF of (+)-15 furnished (+)-16 (74%); then
ammonolysis in MeOH, followed by chromatographic
separation (Sep-Pak RP18, H2O), afforded 1 (mixture of
R- and â-pyranose) and 17 (mixture of R- and â-furanose)
that was tested as such for its inhibitory activities toward
glycosidases and glycosyltransferases (see below).
Treatment of the 7-oxanorbornene (+)-8 with BBr3 in
CH2Cl2 at 0 °C gave (+)-19 in 72% yield. Double irradia-
1
tions in the H NMR spectrum of (+)-19 demonstrated
that the hydroxy group resides at C-2 in the cyclohexenol
Con for m a tion of th e C(1f3)-Disa cch a r id es. Be-
cause the reducing sugar 1 and 2 are mixtures of R/â-
pyranoses and R/â-furanoses their conformational study
(20) Ferritto, R.; Vogel, P. Synlett 1996, 281.
(21) Gagnaire, D.; Payo-Subiza, E. Bull. Soc. Chim. Fr. 1963, 2627.
Nelson, W. L.; Allen, D. R. J . Heterocycl. Chem. 1972, 9, 561. Kienzle,
F. Helv. Chim. Acta 1975, 58, 1180.
(22) For related reactions, see, for example,Gasparini, F.; Vogel, P.
J . Org. Chem. 1990, 55, 2451. Pitzer, K.; Hudlicky, T. Synlett 1995,
803. Yan, F.; Nguyen, B. V.; York, C.; Hudlicky, T. Tetrahedron 1997,
53, 11541.
(23) Bock, K.; Pedersen, C. J . Chem. Soc., Perkin Trans. 2 1974,
293. J ones, C. In Advances in Carbohydrate Analysis; J AI Press:
Greenwich, CT, 1991; Vol. 1, p 145.
(25) For related reactions, see, for example, Koreeda, M.; J ung, K.-
Y.; Hirota, M. J . Am. Chem. Soc. 1990, 112, 7413. J ones, J . B.; Francis,
C. J . Can. J . Chem. 1984, 62, 2578. Guindon, Y.; Therien, M.; Girard,
Y.; Yoakim, C. J . Org. Chem. 1987, 52, 1680. Harwood: L. M.; J ackson,
B.; Prout, K.; Witt, F. J . Tetrahedron Lett. 1990, 31, 1885. Allemann,
S.; Vogel, P. Helv. Chim. Acta 1994, 77, 1. Mosimann, H.; Vogel, P.;
Pinkerton, A. A.; Kirschbaum, K. J . Org. Chem. 1997, 62, 3002.
J otterand, N.; Vogel, P.; Schenk, K. Helv. Chim. Acta 1999, 82, 821.
(24) Krimen, L. I.; Cota, D. J . Org. React. 1969, 17, 213.

4254 J . Org. Chem., Vol. 65, No. 14, 2000
Pasquarello et al.
Sch em e 2
methano linker displayed one signal at 1.61 ppm that
couples with a large constant (11.2 Hz) with H-3 and a
small coupling constant with H-6′ (3.0 Hz), and the
second signal at 1.84 ppm that couples with a large
coupling constant with H-6′ (11.9 Hz) and a small
coupling constant with H-3 (3.1 Hz). Together with the
2D NOESY 1H NMR spectrum of 14 (NOE's between H-2
(4.18 ppm) and both protons H-7′ (1.84, 1.61 ppm),
between H-2 (3.59 ppm) and H-3 (2.19 ppm)), these data
are consistent with the conformation represented in
1
Figure 2. The 600 MHz H NMR spectrum 14 did not
change between -40 and +60 °C, suggesting that the
conformer of Figure 2 is significantly more stable than
other ones.
The coupling constants for the vicinal proton pairs (see
Experimental Section) in the 1H NMR spectrum (600
MHz, C5D5N) of 25 and NOE’s (2D NOESY 1H NMR, and
single irradiation experiments) confirmed that both the
R-C-mannopyranoside and the â-talopyranoside units
adopt chair conformations. The methano linker CH2
displayed one signal at δH 2.43 ppm assigned to proS
H-7′, that couples with H-3 (3J ) 4.3 Hz) and with H-6′
(3J ) 11.2 Hz), and a signal at δH 2.54 ppm assigned to
proR H-7′ that couples with H-3 (9.7 Hz) and with H-6′
(3.2 Hz). This pattern is very similar to that observed in
the 1H NMR spectrum of 14, suggesting therefore similar
conformations for 14 (Figure 2) and 25 (Figure 3). This
F igu r e 2. Preferred conformation of 14 (σ(C-5′,C-6′) and σ(C-
7′,C-3) are antiperiplanar; σ(C-6′,C-7′) and σ(C-3,C-2) are
antiperiplanar).
1
was confirmed by the 2D NOESY H NMR spectrum of
25 that showed cross-peaks for proton pairs H-1 (5.10
ppm) and H-3 (2.63-2.59 ppm), H-2 (4.70 ppm) and proS
H-7′ (2.43 ppm), H-3 (2.63-2.59 ppm) and H-6′ (4.85
ppm), proR H-7′ (2.54 ppm) and H-6′ (4.85 ppm), H-2′
(4.28 ppm) and proS H-7′ (2.43 ppm).
The 1H NMR spectrum of 25 (C5D5N, 600 MHz) did
not vary between 20 and 105 °C, suggesting that the
conformation shown in Figure 3 is more stable than
alternative ones. As for a large number of C-linked
disaccharides,17,26 the R-C-mannopyranosides 14 and 25
adopt conformations in which the exo-C-C glycosidic
bond σ(C-3,C-7′) is antiperiplanar with the σ(C-5′,C-6′)
bond, not with the σ(O-C-6′) bond. The preferred orien-
F igu r e 3. Preferred conformation of 25 (σ(C-5′,C-6′) and σ(C-
7′,C-3) are antiperiplanar; σ(C-6′,C-7′) and σ(C-3,C-2) are
antiperiplanar) with indication of the most significant NOE’s
1
observed in the 2D NOESY 600 MHz H NMR spectrum.
by 1H NMR could not be carried out. The coupling
constants for the vicinal proton pairs (see Experimental
Section) of 1H NMR spectrum (600 MHz, CD3OD) of the
tris(isopropyl)silyl R-galactopyranoside 14 and NOE’s (2D
NOESY 1H NMR and selective irradiation experiments)
confirmed that both the C-mannopyranoside and the
galactopyranoside units adopt chair conformations. The

C-Disaccharide Inhibitor of Glycohydrolases
J . Org. Chem., Vol. 65, No. 14, 2000 4255
Ta ble 1. In h ibition Ra te (in %) in th e P r esen ce of 1 m M
of 1 for th e Glycosid a se-Ca ta lyzed Hyd r olysis of th e
Cor r esp on d in g p-NO2-C6H4-glycosid e a t 37 °C, IC50 a n d Ki
in µM Va lu es
tation of the GalNAc and TalNAc moieties in which the
σ(C-2,C-3) and σ(C-6′,C-7′) bonds are antiperiplanar is
not the same than that found for R-D-Galp-(1f3)CH2-R-
D-Manp-OMe for which the rotamer with σ(C-3,C-4) and
σ(C-6′,C-7′) antiperiplanar is preferred.17
inhibn
enzyme
buffers
rate
IC50 Ki
Glycosid a se In h ibition . The C-disaccharides 1 and
2 were tested for their inhibitory activity toward two R-L-
fucosidases (from bovine epididymis, human placenta),
three R-galactosidases (from coffee bean, Aspergillus
niger, Escherichia coli), five â-galactosidases (bovine liver,
E. coli, A. niger, A. orizae, jack bean), three R-glucosidases
(maltases from yeast, from rice; isomaltase from baker
yeast), two amyloglucosidases (from A. niger, Rhizopus
mold), two â-glucosidases (from almond, Caldocellum
saccharolyticum), two R-mannosidases (from jack bean,
almond), one â-mannosidase (from Helix pomatia), one
â-xylosidase (from A. niger), one R-N-acetylgalactosamini-
dase (from chicken liver), three â-N-acetyl-glucosamini-
dases (from jack bean, bovine epididymis A and B). At 1
mM concentration of 1 and 2 and under optimal pH
conditions,27 no inhibition was observed for 2 whereas 1
inhibited some of the enzymes tested as shown in Table
1. N-Acetylgalactosamine did not inhibit the enzymes
tested. We verified that CH3CONH2, byproduct of the
ammonolysis, did not inhibit the enzymes tested as well.
r-L-fu cosid a se EC 3.2.1.51
bovine epididymis
human placenta
r-ga la ctosid a se EC 3.2.1.22
coffee beans
Aspergillus niger
Escherichia coli
â-ga la ctosid a se EC 3.2.1.23
Escherichia coli
bovine liver
Aspergillus niger
Aspergillus orizae
jack beans
r-glu cosid a se EC 3.2.1.20
yeast
rice
r-glu cosid a se EC 3.2.1.10
bakers’ yeasts
a m yloglu cosid a se EC 3.2.1.3
Aspergillus niger
Rhizopus mold
â-glu cosid a se EC 3.2.1.21
almonds
caldocellum saccharolyticum
R-mannosidase EC 3.2.1.24
jack beans
almonds
â-m a n n osid a se EC 3.2.1.25
Helix pomatia
â-xylosid a se EC 3.2.1.37
Aspergillus niger
r-N-a cetylga la ctosa m in id a se EC 3.2.1.49
chicken liver b) pH 4
a) pH 6
a) pH 6
95
94
19
70
25
28
a) pH 6
b) pH 4
a) pH 7
87
98
99
195
162
68
66
76
39
a) pH 7
a) pH 7
b) pH 4
b) pH 4
b) pH 4
86
34
-
-
92
500
-
-
-
-
-
-
-
9.4 7.5
a) pH 7
b) pH 4
97
-
38
-
40
-
a) pH 7
98
34
38
a) pH 5
a) pH 5
-
-
-
-
-
-
a) pH 5
a) pH 5
-
-
45
-
18
72c)
The fact that the two R-mannosidases tested ignore
both 1 and 2 indicates that these enzymes recognize
different disaccharides than R-D-Manp (1f3)-pyrano-
sides. The N-acetylgalactosamine moiety of 1 allows this
C-disaccharide to be recognized by the three R-galactosi-
dases and by one of the five â-galactosidases tested. The
highest inhibitory activity has been observed for the
â-galactosidase from jack bean (Ki ) 7.5 µM) for which
the Lineweaver and Burk plots showed a mixed type
(competitive, noncompetitive) of inhibition. It is probably
for the same reason that two out of the three â-N-
acetylglucosaminidases tested are inhibited moderately
by 1. The inhibition of the R-glucosidases from yeast and
from bakers’ yeasts are not explained readily. It appears
that these enzymes are not very restrictive in the
structural requirements of their sugar substrates. The
rather good inhibitory activity of 1 observed for the R-L-
fucosidases from bovine epididymis and human placenta
is a surprise. These enzymes are known to be inhibited
by 1-deoxymannonojirimycin with IC50 ) 22 µM28 and Ki
) 6.6 µM,29 respectively. This suggests that mimetics of
D-mannopyranose are recognized by them. It is thus
tempting to assign the inhibitory activity of 1 toward
these enzymes to its R-C-mannopyranoside moiety. If this
should be the explanation, we do not understand then
a) pH 5
a) pH 5
-
-
-
-
-
-
b) pH 4
a) pH 5
-
-
-
-
-
-
-
-
-
â-N-a cetylglu cosa m in id a se EC 3.2.1.30
jack bean
bovine epididymis A
bovine epididymis B
a) pH 5
b) pH 4
b) pH 4
-
99
99
-
130
55
-
135
102
a
b
0.1 M Na phosphate/citrate. 0.1 M Na citrate. c At 0.5 mM.
why 2, which possesses also a R-C-mannopyranoside unit,
is not an inhibitor of the same enzymes!
Glycosyltr a n sfer a se In h ibition Assa ys. Many gly-
cosidase inhibitors can behave as inhibitors of glycosyl-
transferases. This was shown for fucosyltransferases
which are inhibited moderately by some fucosidase
inhibitors such as 1-deoxy-L-fuconojirimycin and homo-
fuconojirimycin.30-32 Some inhibitors of glycosyltrans-
ferases based on acceptor substrates have been devel-
oped, but their potency is generally low with Ki in the
millimolar range.30,31 Fucosyltransferase inhibitors pre-
pared by J efferies and Bowen32 require GDP to be fully
active. They are composed of 1-deoxy-L-fucononojirimycin
linked to D-galactose, a disaccharide mimetic related to
the transition state of the reaction. The group of Hinds-
gaul33 has prepared a bisubstrate analogue composed of
a phenyl â-D-galactoside linked through a flexible ethyl-
ene bridge to GDP, an inhibitor of R-1,2-fucosyltrans-
ferase. Van Boom and co-workers34 have designed trisub-
(26) (a) Wang, Y.; Goekjian, P. G.; Ryckman, D. M.; Miller, W. H.;
Babirad, S. A.; Kishi, Y. J . Org. Chem. 1992, 57, 482. (b) O’Leary, D.
J .; Kishi, Y. Ibid. 1993, 58, 304. (c) Wei, A.; Kishi, Y. Ibid. 1994, 59,
88.
(27) Appropriate p-nitrophenyl glycoside substrates buffered to
optimum pH of the enzymes were used; for details, see: Picasso, S.;
Chen, Y.; Vogel, P. Carbohydr. Lett. 1994, 1, 1. Brandi, A.; Cicchi, S.;
Cordero, F. M.; Fignoli, B.; Goti, A.; Picasso, S.; Vogel, P. J . Org. Chem.
1995, 60, 6806.
(28) Evans, S. V.; Fellows, L. E.; Shing, T. K. M.; Fleet, G. W. J .
Phytochemistry 1985, 24, 1953. See also: Asano, N.; Oseki, K.; Kizu,
H.; Matsui, K. J . Med. Chem. 1994, 37, 3701. Zhou, P.; Salleh, H. M.;
Chan, P. C. M.; Lajoie, G.; Honek, J . F.; Chandra Nambiar, P. T.; Ward,
O. P. Carbohydr. Res. 1993, 239, 155.
(29) Legler, G.; Stu¨tz, A. E.; Immich, H. Carbohydr. Res. 1995, 272,
17. See also: Ekhart, G. W.; Fechter, M. H.; Hadwiger, P.; Mlaker, E.;
Stu¨tz, A. E.; Tauss, A.; Wrodnigg, T. M. In Iminosugars as Glycosidase
Inhibitors; Nojirimycin and Beyond; Stu¨tz, A. E., Ed., Wiley-VCH:
Weinheim, 1999; pp 254-390.
(30) 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, 9283.
(31) Qiao, L.; Murray, B. W.; Shimazaki, M.; Schultz, J .; Wong, C.
H. J . Am. Chem. Soc. 1996, 118, 7653.
(32) J efferies, I.; Bowen, B. R. Bioorg. Med. Chem. Lett. 1997, 7,
1171.
(33) Palcic, M. M.; Heerze, L. D.; Srivastava, O. P.; Hindsgaul, O.
J . Biol. Chem. 1989, 264, 17174.

4256 J . Org. Chem., Vol. 65, No. 14, 2000
Pasquarello et al.
F igu r e 4. (A) Effect of LacNac concentration on Fuc-TVI inhibition at various concentrations of R-D-Manp(1f3)CH2-D-GalNAc
(1). Measurments were performed with 100 µM GDP-fuc at 37 °C, pH 6.2; (9) control without 1, (b) 83 µM of 1, (2) 166 µM of 1.
(B) Effect of GDP-fucose concentration on Fuc-TVI inhibition at various concentrations of 1. Measurements were performed with
20 mM LacNac at 37 °C, pH 6.2; (9) control without 1, (b) 77 µM of 1, (2) 154 µM of 1.
strate analogues containing D-glucose or D-N-acetyl-
glucosamine linked to a GDP-fucose derivative. The most
effective inhibitors of glycosyltransferases are often donor
substrate analogues such as fluorinated sugar nucle-
otides.35 These inhibitors are not expected to be specific
because they lack information of the substrate acceptor
(type of carbohydrate, position at which glycosylation
occurs). We have found that 1 inhibits human R-1,3-
fucosyltransferase VI but it is ignored by human R-2,6-
sialyltransferase and by â-1,4-galactosyltransferase from
human milk. We verified that acetamide, a possible
impurity contaminating 1, did not inhibit human R-1,3-
fucosyltransferase VI. Epimer 2 had no inhibitory activity
toward these enzymes at 2 mM concentration (see
Experimental Section).
With human R-1,3-fucosyltransferase VI (Fuc-TVI) 1
was found to be an effective inhibitor with IC50 of 71 µM.
Double reciprocal analysis showed that the inhibition
displayed a mixed pattern with respect to both the donor
sugar GDP-fucose and the acceptor substrate LacNAc
with Ki of 123 µM and 128 µM, respectively (see Figure
4).
TVI. As 1 is a mixture of R- and â-furanoses, we do not
know which form is selected by the enzyme. No effect on
the inhibition rate was detected on changing the prein-
cubation time. Among the several mechanisms respon-
sible of this inhibition, one is to invoke that 1 mimics
the R-L-Fuc-(1f3)-D-GlcNAc portion of the Lewis x
trisaccharide, the D-mannose replacing the fucose residue
on one hand, and D-GalNAc, the D-GlcNAc moiety, on the
other hand. The fact that GDP-mannose inhibits fuco-
syltransferases36 and that 1-deoxymannonojirimycin can
inhibit R-L-fucopyranoside28,37 suggests that the C-R-D-
mannopyranoside moiety of 1 can fit into the L-fucopy-
ranoside binding site of the enzyme. The D-GalNAc
moiety of 1 would thus take the place reserved for the
D-GlcNAc portion of Lewis x trisaccharide in the Fuc-
TVI active site, the enzyme permitting one “stereochem-
ical mistake” for that sugar (D-GalNAc is the 4-epimer
of D-GlcNAc). This hypothesis is consistent with the
observed mixed pattern inhibition. Compound 1 is the
first example of C-disaccharide to have glycosyltrans-
ferase inhibitory activity.
As the affinity of Fuc-TVI for its substrate LacNAc is
low (Km ) 32 mM), R-D-Manp-(1f3)CH2-D-GalNAc (1)
represents one of the most powerful inhibitors of Fuc-
TVI which is not an analogue of the donor substrate. As
reported for other fucosyl transferases,30 the product GDP
is a competitive inhibitor of the reaction with a Ki of 120
µM. A synergistic inhibition of fucosyltransferases can
be observed with iminosugar inhibitors in the presence
of GDP, and this synergy increases considerably the
power of the inhibition.30,36 With 1 we did not observe
such a synergistic inhibition of Fuc-TVI with GDP but,
on the contrary, preincubation of the enzyme with the
sugar nucleotide decreased the power of inhibition. This
suggests that the two inhibitors compete for binding to
the site of the enzyme.
Con clu sion
Efficient and stereoselective syntheses of two new
C-disaccharides, R-D-Manp-(1f3)CH2-D-GalNAc (1) and
R-D-Manp-(1f3)CH2-D-TalNAc (2), have been achieved
applying the “naked sugar” methodology16-18 and the
Giese’s radical C-glycosidation.15 Whereas 2 did not
inhibit any of the 25 glycosidases and 3 glycosyltrans-
ferases tested, 1 is a good inhibitor of â-galactosidase
from jack beans, (Ki ) 7.5 µM), of R-fucosidases from
bovine epididymis (Ki ) 25 µM) and from human placenta
(Ki ) 28 µM) and of human R-1,3-fucosyltransferase VI.38
Given the low affinity of Fuc-TVI for its substrate (Km
)
32 mM) and the fact that it does not require nucleotide
sugar GDP to inhibit significantly the enzyme, the
neutral C-disaccharide 1 represents a compound of great
No inhibition of Fuc-TVI was detected for N-acetylga-
lactosamine and for D-mannose at 5 mM concentrations.
This suggests that both sugar moieties of the C-disac-
charide 1 are necessary for its inhibitory activity of Fuc-
(37) Legler, G.; Stu¨tz, A. E.; Immich, H. Carbohydr. Res. 1995, 272,
17.
(38) Guanosine 5′-diphospho-2-deoxy-2-fluoro-â-1-fucose represents
the most potent inhibitor (Ki ) 4.2 µM) of R-1,3-fucosyltransferase V:
Murray, B. W.; Wittmann, V.; Burkhart, M. D.; Hung, S. C.; Wong, C.
H. Biochemistry 1997, 36, 823. Synthetic sulfated saccharides and the
triantennary sialylglycopeptide from fetuin are competitive inhibitors
of the transfer of L-fucose catalyzed by a partially purified R-1, 3/4-L-
fucosyltransferase preparation from Colo 205: Chandrasekaran, E. V.;
Rhodes, J . M.; J ain, R. K.; Bernacki, R. J .; Matta, K. L. Biochem.
Biophys. Res. Commun. 1994, 201, 78.
(34) Heskamp, B. M.; Veeneman, G. H.; Van der Marel, G. A.; Van
Broekel, C. A. A.; Van Boom, J . H. Tetrahedron 1995, 51, 8397.
(35) Murray, B. W.; Wittman, V.; Burkart, M. D.; Hung, S. C.; Wong,
C.-H. Biochemistry 1997, 36, 823.
(36) Wong, C.-H.; Dumas, D. P.; Ichikaea, Y.; Koseki, K.; Danishef-
sky, S. J .; Weston, B. W.; Lowe, J . B. J . Am. Chem. Soc. 1992, 114,
7321.

C-Disaccharide Inhibitor of Glycohydrolases
J . Org. Chem., Vol. 65, No. 14, 2000 4257
(+)-(1S,3R,4R,5R,6R)-6-en d o-Ch lor o-5-exo-(p h en ylse-
le n o)-3-en d o-(1′,3′,4′,5′-t e t r a -O-a ce t yl-2′,6′-a n h yd r o-7′-
d eoxy-D-glycer o-D-m a n n o-h ep titol-7′-C-yl)-7-oxa bicyclo-
[2.2.1]h ep ta n -2-on e ((+)-5). A mixture of (-)-416,17 (200 mg,
0.64 mmol), acetobromomannose (2,3,4,6-tetra-O-acetyl-R-D-
manno pyranosyl bromide 341 mg, 0.82 mmol), and AIBN (10.5
mg, 0.1 mmol) in anhydrous benzene (4 mL) was heated under
reflux for 20 min. A solution of Ph3SnH (225 mg, 0.64 mmol)
and AIBN (10.5 mg, 0.1 mmol) in anhydrous benzene (2 mL)
was added via a syringe in about 30 min. The mixture was
then heated under reflux for 60 min and allowed to cool to 20
°C. KF (270 mg) was added, and the mixture was stirred at
20 °C for 14 h. After filtration through Celite (EtOAc as eluent)
and solvent evaporation in vacuo, the residue was purified by
FC (EtOAc/light petroleum ether 2:3), yielding 231 mg (56%)
interest. Further work aimed at assaying 1 on recombi-
nant Fuc-TVII, an enzyme of crucial importance in the
biosynthesis of selective ligands involved in early inflam-
matory response,39 will be undertaken. Analogues of 1
and 2 deserve to be prepared and tested toward glycosi-
dases and glycosyltransferases.40
Exp er im en ta l Section
Gen er a l Rem a r k s. See ref 41. None of the procedures were
optimized. Flash column chromatography (FC) was performed
on Merck silica gel (230-400 mesh).42 Thin-layer chromatog-
raphy (TLC) was carried out on silica gel (Merck aluminum
foils). 1H NMR signals assignments were confirmed by double
irradiation experiments and, when required, by 2D NOESY
of (+)-5, white foam. [R]25 ) 5.8 (c ) 1.0, CHCl3). 1H NMR
D
1
and COSY spectra. J values are given in hertz. 600 MHz H
(400 MHz, CDCl3) δ 7.64-7.61 (m), 7.32-7.28 (m), 5.23 (dd,
NMR spectra were recorded on a Bruker AMX-600 FR spec-
trometer.
3
3
3J ) 4.8, 3.2), 4.95 (d, J ) 5.6), 4.91 (dd, J ) 8.5, 3.2), 4.85
3
2
3
3
(dd, J ) 4.8, 2.7), 4.56 (dd, J ) 12.2, J ) 9.1), 4.48 (d, J )
2-Aceta m id o-2,3-d id eoxy-3-C-(2′,6′-a n h yd r o-7′-d eoxy-D-
glycer o-D-m a n n o-h ep titol-7′-C-yl)-D-ga la ctose (r-C(1f3)-
D-m a n n op yr a n osid e of N-Acetyl D-ga la ctosa m in e: 1 +
17). A mixture of (+)-16 (29 mg, 0.05 mmol) and MeOH
saturated with gaseous NH3 (3 mL) was stirred at 20 °C for
9.5 h. The solvent was evaporated to dryness in vacuo and
the residue purified by column chromatography on Sep-Pak
RP18-2gr H2O as eluent. Yield: 18 mg (76%) of 1, yellowish
oil, contains a little of acetamide. IR (KBr) ν 3385, 1660, 1385,
1065. 1H NMR (400 MHz, CD3OD: mixture of R,â-D-pyranose
and R,â-D-furanose) δ 5.18 (d, 3J ) 4.4), 5.17 (d, 3J ) 2.1), 5.06
5.9), 4.32 (dd, 3J ) 5.9, 3.1), 4.05 (dd, 2J ) 12.2, 3J ) 3.9),
3
3
3
3.98 (ddd, J ) 10.3, 8.5, 2.0), 3.69 (d, J ) 3.1), 3.61 (ddd, J
) 9.1, 3.9, 2.7), 2.68 (ddd, 3J ) 10.5, 5.6, 5.4), 2.16-2.01 (4s +
2
3
m), 1.47 (ddd, J ) 14.8, J ) 10.5, 10.3).
(-)-(1S,2R,3S,5R,6R)-6-en d o-Ch lor o-5-exo-(p h en ylsele-
n o)-3-en do-(1′,3′,4′,5′-tetr a-O-acetyl-2′,6′-an h ydr o-7′-deoxy-
D-glycer o-D-m a n n o-h ep t it ol-7′-C-yl)-7-oxa b icyclo[2.2.1]-
h ep ta n -2-en d o-ol ((-)-6). NaBH4 (156 mg, 4.13 mmol) was
added to a solution of (+)-5 (940 mg, 1.45 mmol) in THF/MeOH
1:1 (40 mL) cooled to 0 °C. After stirring at 0 °C for 5 min, the
mixture was neutralized with 1 N HCl (if the pH is < 7,
saturated aqueous NaHCO3 solution is added until neutral
pH). Then a part of methanol was evaporated under vacuum,
and the aqueous phase was extracted with CH2Cl2. The organic
extract was washed with brine (twice) and dried (MgSO4), and
the solvent was evaporated in vacuo. Residue was purified by
FC (Et2O/light petroleum ether 9:1) yielding 742 mg (79%) of
3
3
(d, J ) 3.6), 4.57 (d, J ) 8.3, 4H-C(1)); 3.40-4.25 (m, 48H),
2.49-2.58 (m), 3.29-2.37 (m), 2.26-2.05 (m), 1.90-1.77 (m),
1.67-1.54 (m), 2.05-1.97 (5s, NHAc + NH2Ac). 13C NMR (101
MHz, CD3OD) δ 176.4, 173.8, 173.5, 172.8 (4s, 4CdO NHAc
+ CdO NH2Ac), 102.9, 98.9, 95.1, 92.2 (4d, 4C(1)), 83.9, 83.7,
79.9, 76.4, 76.3, 75.8, 75.7, 74.6, 74.5, 73.5, 73.1, 73.0 (*2), 72.8,
72.6, 72.3, 69.5, 69.3, 66.2, 65.6 (all d), 64.7, 63.3, 63.2 (*2),
63.0 (all t, 4C(1′) + 4 C(6)), 59.2, 53.1, 49.8 (*2), 43.1, 41.6,
37.6, 36.0 (all d), 32.4, 31.5, 26.3, 26.2 (all t, 4 C(7′)), 22.9,
22.8, 22.7, 22.6, 22.1 (q, 4CH3 + CH3CONH2). ESI + QIMS:
[M + 1]+) 382.3.
(-)-6, white foam. [R]25 ) -7.0 (c ) 1.0, CHCl3). 1H NMR
D
(400 MHz, CDCl3) δ 7.65-7.63 (m), 7.34-7.27 (m), 5.25 (dd,
3
3
3J ) 7.1, 3.3), 5.09 (dd, J ) 5.3, 3.3), 5.05 (dd, J ) 7.1, 5.9),
3
2
4.61 (d, J ) 5.5), 4.50-4.43 (m), 4.33-4.30 (m), 4.10 (dd, J
) 12.1, 3J ) 3.4), 4.00 (ddd, J ) 9.3, 5.3, 5.0), 3.77 (ddd, J )
3
3
2-Aceta m id o-2,3-d id eoxy-3-C-(2′,6′-a n h yd r o-7′-d eoxy-D-
glycer o-D-m a n n o-h ep titol-7′-C-yl)-D-ta lose (r-C(1f3)-D-
m a n n op yr a n osid e of N-Acetyl D-ta losa m in e: 2 + 28). A
mixture of (+)-27 (25 mg, 0.04 mmol) and MeOH saturated
with gaseous NH3 (3 mL) was stirred at 20 °C for 7.5 h. The
solvent was evaporated and the residue purified by column
chromatography on Sep-Pak RP18-2gr, with H2O as eluent,
yielding 17 mg (92%) of a mixture of pyranose 2 and furanose
28, contaminated with less than 20% of acetamide, yellowish
oil. IR (KBr) ν 3355, 1660, 1400, 1065. 1H NMR (400 MHz,
3
7.2, 5.9, 3.4), 3.65 (d, J ) 4.9), 2.45-2.36 (m), 2.10-2.00 (4s
+ m), 1.47 (ddd, J ) 14.4,3J ) 9.3 6.7).
2
(+)-(1S,2R,3S,4R)-6-Ch lor o-3-en d o-(1′,3′,4′,5′-t et r a -O-
acetyl-2′,6′-an h ydr o-7′-deoxy-D-glycer o-D-ma n n o-h eptitol-
7′-C-yl)-7-oxa bicyclo[2.2.1]h ep t-5-en -2-en d o-ol ((+)-7). A
solution of m-CPBA (70% 3-ClC6H4CO3H in 3-ClC6H4CO2H,
167 mg, 0.68 mmol) in anhydrous CH2Cl2 (3.3 mL) was added
dropwise to a stirred solution of (-)-6 (400 mg, 0.62 mmol) in
anhydrous CH2Cl2 (10.3 mL) cooled to -78 °C. The mixture
was stirred at -78 °C for 3 h, allowed to warm to 20 °C, and
stirred for 12 h. CH2Cl2 (20 mL) was added, and the solution
was washed with saturated aqueous solution of NaHCO3 (20
mL, twice). The aqueous phase was extracted with CH2Cl2 (20
mL, four times). The combined organic phases were washed
with brine (20 mL, twice) and dried (MgSO4). After solvent
evaporation in vacuo, the residue was purified by FC (Et2O/
3
CD3OD) δ 5.36 (d, J ) 4.6), 5.05 (br s), 5.01 (br s), 4.90-4.80
3
3
(br s, 4H-C(1) + H2O)), 4.57 (d, J ) 8.2), 4.47 (dd, J ) 9.1,
4.6), 4.36 (d, 3J ) 6.0), 4.20-4.05 (m), 3.94-3.56 (m), 3.50-
3.43 (m, 48H), 2.08, 2.05, 2.03, 2.01 (4s, NHAc), 1.98 (s, NH2-
Ac), 2.88-2.80 (m), 1.87-1.70 (m), 1.67-1.58 (m). 13C NMR
(101 MHz, CD3OD) δ 176.4, 173.8, 173.5, 172.7 (4s, 4CdO
NHAc + CdO NH2Ac), 101.8, 97.6, 97.1, 94.4 (4d, 4C(1)), 84.2,
83.9, 83.6, 80.6, 76.9, 76.6, 76.2, 76.0, 75.8, 75.3, 75.0, 74.2,
73.3, 72.9, 72.8, 72.6, 72.5, 72.3, 69.6, 69.5, 69.4, 67.2, 65.9
(all d), 65.9, 65.0, 64.5, 63.5, 63.2, 63.1, 62.9 (all t, 4C(1′) +
4C(6)), 60.0, 59.2, 55.4, 53.4, 52.8, 38.5, 36.8, 36.4, 32.7 (all
d), 29.4, 28.0, 27.9, 27.2 (all t, 4C(7′)), 23.3, 23.2, 22.5, 22.0 (q,
4CH3 + CH3CONH2). ESI + QIMS: [M + 1]+) 382.3.
light petroleum ether 9:1), yielding 314 mg (94%) of (+)-7,
1
white foam. [R]25 ) 5.4 (c ) 1.0, CHCl3). H NMR (400 MHz,
D
3
3
CDCl3) δ 6.34 (d, J ) 1.9), 5.25-5.15 (m), 4.90 (dd, J ) 4.4,
3
3
1.9), 4.73 (d, J ) 4.4), 4.63 (ddd, J ) 7.7, 6.0, 4.4), 4.42 (dd,
2J ) 12.1, 3J ) 6.7), 4.11-4.03 (m), 3.92 (ddd, 3J ) 7.1, 6.7,
2.8), 2.78 (d, 3J ) 6.0), 2.30-2.23 (m), 2.13-2.05 (4s), 1.83 (ddd,
2J ) 14.4, J ) 7.1, 6.9), 1.39 (ddd, J ) 14.4, J ) 7.7, 5.2).
(+)-(1S,2R,3S,4R)-6-Ch lor o-3-en d o-(1′,3′,4′,5′-t et r a -O-
acetyl-2′,6′-an h ydr o-7′-deoxy-D-glycer o-D-ma n n o-h eptitol-
7′-C-yl)-7-oxabicyclo[2.2.1]h ept-5-en -2-en do-yl Acetate ((+)-
8). A mixture of (+)-7 (500 mg, 1.02 mmol), anhydrous pyridine
(5.4 mL), acetic anhydride (482 µL), and a catalytic amount of
4-(dimethylamino)pyridine (8 mg) was stirred at 20 °C for 7
h. The solvent was evaporated in vacuo. Purification by FC
(EtOAc/light petroleum ether 3:2) gave 500 mg (92%) of (+)-
8, white foam. [R]25D ) 24 (c ) 1.0, CHCl3). 1H NMR (400 MHz,
CDCl3) δ 6.35 (d, 3J ) 2.0), 5.22 (dd, 3J ) 8.2, 4.4), 5.22 (dd, 3J
) 7.3, 3.2), 5.12 (dd, 3J ) 7.4, 7.3), 5.05 (dd, 3J ) 4.5, 3.2),
3
2
3
(39) Maly, P.; Thall, A. D.; Petryniak, B.; Rogers, G. E.; Smith, P.
L.; Marks, R. M.; Kelly, R. J .; Gersten, K. M.; Cheng, G.; Saunders:
T. L.; Camper, S. A.; Camphausen, R. T.; Sullivan, F. X.; Isogai, Y.;
Hindsgaul, O.; VonAndrian, U. H.; Lowe, J . B. Cell 1996, 86, 643.
(40) Decreased fucose incorporation of surface carbohydrates of
malignant cells is associated with inhibition of their invasive capacity;
see, for example, Bolscher, J . G.; Bruyneel, E. A.; Van Rooy, H.;
Schallier, D. C.; Mareel, M. M.; Smets, L. A. Clin. Exp. Metastasis 1989,
7, 557.
(41) Sevin, A. F.; Vogel, P. J . Org. Chem. 1994, 59, 5920. Kraehen-
buehl, K.; Picasso, S.; Vogel, P. Helv. Chim. Acta 1998, 81, 1439.
(42) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.

4258 J . Org. Chem., Vol. 65, No. 14, 2000
Pasquarello et al.
3
4
3
5.01 (ddd, J ) 4.3, 2.0, J ) 0.8), 4.99 (br d, J ) 4.4), 4.45
Da ta for (+)-r-13: white solid, mp 152-153 °C. [R]25
)
D
(dd, 2J ) 12.1, 3J ) 7.6), 4.14 (dd, 2J ) 12.1, 3J ) 3.0), 4.02
(ddd, J ) 10.5, 4.5, 3.1), 3.91 (ddd, J ) 7.6, 7.4, 3.0), 2.53-
2.46 (m), 2.11-2.04 (4s), 1.56-1.41 (2m).
43 (c ) 0.5, CHCl3). H NMR (400 MHz, CDCl3) δ 5.55 (br s),
1
3
3
3
3
3
5.52 (d, J ) 10.0), 5.35 (d, J ) 3.3), 5.25 (dd, J ) 5.9, 3.1),
5.02 (dd, 3J ) 5.9, 4.3), 4.92 (dd, 3J ) 6.9, 3.1), 4.66 (br s),
2
3
3
3
(+)-(1R,2S,5R,6R)-5-Acet a m id o-3-ch lor o-2-h yd r oxy-6-
(1′,3′,4′,5′-tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-
D-m a n n o-h ep titol-7′-C-yl)cycloh ex-3-en -1-yl Aceta te ((+)-
10). CF3SO3H (90 µL, 1.03 mmol) was added to a solution of
(+)-8 (100 mg, 0.19 mmol) in dry MeCN (4.4 mL) cooled to 0
°C. After stirring at 0 °C for 5 min, the reaction was allowed
to warm to 20 °C and stirred for an additional 40 min. The
reaction was quenched with ice-cold saturated solution of
NaHCO3 and extracted with CH2Cl2 (20 mL, four times). The
combined organic extracts were dried (MgSO4), and after
solvent evaporation in vacuo the residue was purified by FC
(EtOAc/light petroleum ether 4:1) gaving 94 mg (84%) of (+)-
4.61 (dd, J ) 12.1, J ) 7.7), 4.34 (ddd, J ) 12.0, J ) 10.0,
2 3 3
3.3), 4.18 (dd, J ) 12.1, J ) 4.4), 4.10 (ddd, J ) 10.8, 6.9,
2.4), 3.98 (ddd, 3J ) 7.7, 4.4, 4.3), 3.72 (s), 2.32 (dddd, 3J )
2
3
12.0, 11.4, 3.0, 2.9), 2.11-1.99 (6s), 1.65 (ddd, J ) 14.2, J )
2
3
10.8, 3.0), 1.45 (ddd, J ) 14.2, J ) 11.4, 2.4), 1.17-1.06 (m).
Da ta for â-13: colorless, viscous oil. 1H NMR (400 MHz,
3
3
CDCl3) δ 5.49 (br s), 5.47 (d, J ) 9.0), 5.20 (dd, J ) 7.5, 3.4),
3
3
3
5.09 (dd, J ) 7.5, 6.7), 5.01 (dd, J ) 4.1, 3.4), 4.89 (d, J )
2
3
3
7.8), 4.46 (dd, J ) 12.1, J ) 6.6), 4.21 (d, J ) 1.2), 4.16 (dd,
2J ) 12.1, J ) 4.0), 4.11 (ddd, J ) 12.3, 4.1, 3.0), 3.89 (ddd,
3
3
3J ) 6.7, 6.6, 4.0), 3.70 (s), 3.64 (ddd, J ) 12.1, 9.0, 7.8), 2.43
3
3
2
(dddd, J ) 12.1, 11.3, 2.6, 2.5), 2.13-1.99 (6s), 1.85 (ddd, J
) 14.5, 3J ) 12.3, 2.6), 1.36 (ddd, 2J ) 14.5, 3J ) 11.3, 3.0),
1.29-1.07 (m).
10, white foam. [R]25 ) 16 (c ) 1.0, CHCl3). 1H NMR (400
D
3
3
MHz, CDCl3) δ 5.93 (d, J ) 9.5), 5.91 (d, J ) 2.5), 5.23 (dd,
3J ) 7.4, 3.1), 5.20 (dd, 3J ) 3.1, 2.2), 5.07-5.03 (m), 4.56 (ddd,
Tr is(isop r op yl)silyl 2-Aceta m id o-2,3-d id eoxy-3-C-(2′,6′-
a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-h ep titol-7′-C-yl)-r-
D-ga la ctop yr a n osid e (14). A 2 M solution of LiBH4 in
anhydrous THF (135 µL, 0.27 mmol) was added slowly to a
stirred solution of (+)-R-13 (28 mg, 36 µmol) in anhydrous THF
(3.8 mL) cooled to 0 °C. After stirring at 0 °C for 5 min, the
mixture was allowed to warm to 20 °C and stirred for 23 h.
H2O (3 mL) was added under vigorous stirring. The solvent
was evaporated in vacuo, and the residue was purified by
chromatography on a short column (h ) 3 cm, L ) 1 cm) of
Dowex 50WX8 (100-200 mesh, 3.7% aq HCl, then H2O, then
MeOH). Solvent evaporation to dryness yielded 14 mg (72%)
3J ) 11.0, 9.5, 2.5), 4.53 (dd, J ) 12.0, J ) 7.8), 4.10-4.05
(m), 4.02-3.98 (m), 3.50-3.40 (br d), 2.12-2.04 (5s + m), 1.82
(ddd, 2J ) 14.6, 3J ) 11.4, 2.9), 1.52 (ddd, 2J ) 14.6, 3J ) 10.6,
3.0).
2
3
Me t h yl
2-Ace t a m id o-4-O-a ce t yl-2,3-d id e oxy-3-C-
(1′,3′,4′,5′-tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-
D-m a n n o-h ep titol-7′-C-yl)-r-D-ga la ctop yr a n u r on a te ((+)-
11) a n d Mixtu r e of Meth yl 2-Aceta m id o-5-O-a cetyl-2,3-
d id e oxy-3-C-(1′,3′,4′,5′-t e t r a -O-a ce t yl-2′,6′-a n h yd r o-7′-
d eoxy-D-glycer o-D-m a n n o-h ep t it ol-7′-C-yl)-r-D- a n d â-D-
Ga la ctofu r a n u r on a te ((-)-12). NaHCO3 (66 mg, 0.78 mmol)
was added to a solution of (+)-10 (80 mg, 0.14 mmol) in MeOH/
CH2Cl2 1:1 (4.8 mL). The solution was cooled to -78 °C, and
ozone was bubbled through it until starting material has
disappeared by TLC (10-20 min). A stream of N2 was bubbled
for 10 min, and a few drops of Me2S were added. After 10 min
the mixture was allowed to warm to 20 °C and was stirred for
6 h. H2O (10 mL) was added, and the aqueous phase was
extracted with CH2Cl2 (15 mL, four times). The combined
organic extracts were dried (MgSO4), and after solvent evapo-
ration in vacuo a 1.4:1 mixture of (+)-11 and (-)-12 was
obtained. They were separated by FC (MeOH/AcOEt/CH2Cl2:
30:485:485), yielding 20 mg (23%) of (-)-12 (a 1.7:1 mixture
of anomers) and 27 mg (31%) of (+)-11.
1
of 14 as a viscous oil. H NMR (600 MHz, CD3OD) δ 5.28 (d,
3
3J ) 3.3, H-C(1)), 4.18 (dd, J ) 12.2, 3.3, H-C(2)), 4.09 (ddd,
3J (HproS-C(7′)-H-C(6′)) ) 11.9, 3J (HproR-C(7′)-H-C(6′)) ) 3.0,
3J (H-C(5′)-H-C(6′))) 2.8, H-C(6′)), 3.84 (dd, 2J ) 11.5, 3J (H-
C(1′)-H-C(2′)) ) 2.9, H-C(1′)), 4.06-4.03 (m, 2H), 3.81-3.76
(m, 2H), 3.74-3.71 (m, 2H), 3.69-3.64 (m, 2H, H-C(1′), H-C(3′),
H-C(4′), H-C(5′), H-C(4), H-C(5), 2H-C(6)), 3.59 (ddd, 3J ) 7.9,
6.7, 2.9, H-C(2′)), 2.19 (dddd, 3J (H-C(2)-H-C(3)) ) 12.2,
3
3J (HproR-C(7′)-H-C(3)) ) 11.2, J (HproS-C(7′)-H-C(3)) ) 3.1,
3J (H-C(4)-H-C(3)) ) 2.8, H-C(3)), 2.03 (s, NHAc), 1.84 (ddd,
3
3
2J ) 14.5, J (HproS-C(7′)-H-C(6′)) ) 11.9, J (HproS-C(7′)-H-
C(3)) ) 3.1, HproS-C(7′)), 1.61 (ddd, 2J ) 14.5, 3J (HproR-C(7′)-
H-C(3)) ) 11.2, 3J (HproR-C(7′)-H-C(6′)) ) 3.0, HproR-C(7′)),
1.24-1.15 (m, 21H, TIPS). 13C NMR (101 MHz, CD3OD) δ
Da ta for (+)-11: white foam. [R]25 ) 50 (c ) 0.5, CHCl3).
D
1
1
1H NMR (400 MHz, CDCl3) δ 5.81 (d, 3J ) 9.8), 5.57 (br s),
173.3 (s, CdO), 97.6 (d, J (C,H) ) 168, C(1)), 76.2 (d, J (C,H)
1 1
3
3
3
5.36 (d, J ) 3.0), 5.22 (dd, J ) 7.2, 3.3), 5.04 (dd, J ) 7.2,
) 139), 74.1 (d, J (C,H) ) 148), 73.2 (d, J (C,H) ) 134), 72.9
1 1 1
3
2
3
7.1), 5.00 (dd, J ) 4.9, 3.3), 4.75 (br s), 4.48 (dd, J ) 12.0, J
(d, J (C,H) ) 142), 72.3 (d, J (C,H) ) 145), 69.5 (d, J (C,H) )
1 1
3
3
) 8.3), 4.32 (ddd, J ) 9.9, 9.8, 3.0), 4.17 (dd, J ) 12.0, 4.9),
4.14-4.10 (m), 4.01-3.96 (m), 3.74 (s), 3.70-3.60 (br s), 2.37-
145), 65.8 (d, J (C,H) ) 146), 51.0 (d, J (C,H) ) 141), 35.5 (d,
1J (C,H) ) 128), C(2), C(3), C(4), C(5), C(2′), C(3′), C(4′), C(5′),
C(6′), 62.9 (t, 1J (C,H) ) 141), 62.4 (t, 1J (C,H) ) 142), C(1′),
C(6), 26.9 (t, 1J (C,H) ) 126, C(7′)), 22.9 (q, 1J (C,H) ) 128,
NHAc), 18.4, 18.3 (q, 1J (C,H) ) 120, TIPS), 13.5 (d, 1J (C,H) )
118, TIPS).
3
2.29 (dd, J ) 9.9, 9.7), 2.14-2.04 (6s), 1.80-1.60 (m), 1.40-
1.25 (m).
Da ta for (-)-12: white foam. [R]25D ) -30 (c ) 0.5, CHCl3).
1H NMR (400 MHz, CDCl3) δ (1.7:1 mixture of anomers) 6.16,
6.07 (2s), 5.89 (d, 3J ) 7.1), 5.85 (d, 3J ) 7.8), 5.25-5.04 (2m),
4.35-4.03 (m), 3.84 (ddd, 3J ) 7.5, 7.4, 3.0), 3.74-3.69 (m),
3.48 (s), 3.44 (s), 2.75-2.67 (m), 2.20-2.05 (m), 1.89 (ddd, J
) 14.8, J ) 14.7, 4.3), 1.82-1.60 (m).
Tr is(isop r op yl)silyl 2-Acet a m id o-4,6-d i-O-a cet yl-2,3-
d id e oxy-3-C-(1′,3′,4′,5′-t e t r a -O-a ce t yl-2′,6′-a n h yd r o-7′-
d eoxy-D-glycer o-D-m a n n o-h ep t it ol-7′-C-yl)-r-D-ga la ct o-
p yr a n osid e ((+)-15). A mixture of 14 (14 mg, 26 µmol),
anhydrous pyridine (3.5 mL), Ac2O (1 mL), and 4-(dimethy-
lamino)pyridine (1 mg) was stirred at 20 °C for 12 h. The
solvent was evaporated in vacuo. The residue was purified by
2
3
Me t h yl
2-Ace t a m id o-4-O-a ce t yl-2,3-d id e oxy-3-C-
(1′,3′,4′,5′-tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-
D-ma n n o-h eptitol-7′-C-yl)-1-O-tr is(isopr opyl)silyl-r-D- an d
â-D-Ga la ctop yr a n u r on a te ((+)-13, r/â 1:0.45). 2.6-Lutidine
(38 µL, 0.33 mmol) and TIPSOTf (59 µL, 0.22 mmol) were
added to a solution of (+)-11 (68 mg, 0.11 mmol) in CH2Cl2
(5.4 mL) cooled at 0 °C under N2. After stirring at 0 °C for 5
min, the reaction was allowed to warm to 20 °C and stirred
for 1.5 h; by TLC there was still starting material so the
reaction was cooled again to 0 °C, and 2,6-lutidine (38 µL, 0.33
mmol) and TIPSOTf (59 µL, 0.22 mmol) were added. The
reaction was allowed to warm to 20 °C and stirred for 2 h more.
Brine (15 mL) was added, and the aqueous phases were
extracted with CH2Cl2 (20 mL, four times). After drying
(MgSO4) and solvent evaporation in vacuo, the residue was
purified by FC (EtOAc/light petroleum ether 3:2), yielding 16
mg of â-13 and 40 mg (59%) of R-13. Pure â-13 was obtained
by FC (Et2O).
FC (EtOAc/light petroleum ether 3:2), yielding 21 mg (96%)
1
of (+)-15, colorless oil. [R]25 ) 43 (c ) 0.5, CHCl3). H NMR
D
3
(400 MHz, CDCl3) δ 5.52 (d, J ) 10.0), 5.34 (br s), 5.28-5.24
3
3
(m), 5.02 (dd, J ) 6.9, 4.5), 4.91 (dd, J ) 7.0, 3.1), 4.65 (dd,
2J ) 12.1, J ) 7.6), 4.30-4.20 (m), 4.20-4.10 (m), 4.00-3.90
3
(m), 2.26 (dddd, 3J ) 11.6, 11.5, 2.8, 2.7), 2.12-1.99 (7s), 1.65-
2
3
1.57 (m), 1.40 (dddd, J ) 11.7, J ) 11.6, 2.4, 2.3), 1.20-1.07
(m).
2-Aceta m id o-4,6-d i-O-a cetyl-2,3-d id eoxy-3-C-(1′,3′,4′,5′-
tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-
h ep titol-7′-C-yl)-r-D-ga la ctop yr a n ose ((+)-16). A mixture
of (+)-15 (58 mg, 0.07 mmol), THF (3.5 mL), and tetrabuty-
lammonium fluoride (0.11 mL, 0.11 mmol) was stirred at 0 °C
under N2 atmosphere for 5 min. H2O (6 mL) was added, and
the aqueous layer was extracted with Et2O (6 mL) and then

C-Disaccharide Inhibitor of Glycohydrolases
J . Org. Chem., Vol. 65, No. 14, 2000 4259
with CH2Cl2 (6 mL, four times). The combined organic extracts
were dried (MgSO4), and the solvent was evaporated. The
residue was purified by FC (CH2Cl2/MeOH 96:4), yielding 34
was incomplete (control by TLC), and the mixture was stirred
at 20 °C for 4 more days. Brine (10 mL) was added, and the
aqueous phase was extracted with CH2Cl2 (10 mL, 4 times).
The combined organic extracts were dried (MgSO4), and the
solvent was evaporated. The residue was purified by FC
mg (74%) of (+)-16, white foam. [R]25 ) 37 (c ) 0.5, CHCl3).
D
1H NMR (400 MHz, CDCl3) δ 5.82 (d, 3J ) 9.8), 5.29 (br s),
5.24-5.19 (m), 5.02 (dd, 3J ) 6.3, 6.2), 4.98 (dd, 3J ) 5.2, 3.3),
4.48 (dd, 2J ) 12.0, 3J ) 8.4), 4.32 (dd, 3J ) 6.5, 6.4), 4.24
(EtOAc/light petroleum ether 2:3), yielding 95 mg (100%) of
1
(-)-22, yellowish oil. [R]25 ) -51 (c ) 1.0, CHCl3). H NMR
D
3
(ddd, J ) 12.7, 9.8, 3.5), 4.19-4.08 (m), 4.02-3.93 (m), 2.24
(400 MHz, CDCl3) δ 5.37 (br t, 3J = 1.5), 5.24 (dd, 3J ) 6.5,
3.3), 5.10 (dd, 3J ) 6.6, 6.5), 5.01 (dd, 3J ) 5.0, 3.3), 4.98 (d, 3J
) 1.4), 4.49 (dd, 2J ) 12.2, 3J ) 6.5), 4.18-4.12 (m), 3.92 (ddd,
3J ) 6.6, 6.5, 3.4), 3.41 (br d, 3J = 2.0), 2.16-2.08 (m + 5s),
1.76-1.70 (m), 1.21-1.09 (m).
(dddd, 2J ) 12.7, 3J ) 12.0, 2.8, 2.2), 2.14-2.05 (7s), 1.70 (ddd,
2
2J ) 14.0, 3J ) 12.0, 2.2), 1.33 (ddd, J ) 14.0, 3J ) 10.7, 2.2).
(+)-(1R ,2S ,5R ,6S )-5-B r o m o -3-c h lo r o -2-h y d r o x y -6-
(1′,3′,4′,5′-tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-
D-m a n n o-h ep titol-7′-C-yl)cycloh ex-3-en -1-yl Aceta te ((+)-
19). A 1 M solution of BBr3 in anhydrous CH2Cl2 (1.14 mL,
1.14 mmol) was added to a solution of (+)-8 (402 mg, 0.76
mmol) in anhydrous CH2Cl2 (20 mL) cooled to 0 °C under N2
atmosphere. After stirring at 0 °C for 15 min, a saturated
aqueous solution of NaHCO3 (25 mL) was added and the
mixture was extracted with CH2Cl2 (25 mL, four times). The
combined organic extracts were dried (MgSO4), and the solvent
was evaporated in vacuo. The residue was purified by FC
(Et2O/light petroleum ether 7:3) yielding 338 mg (72%) of (+)-
Met h yl
2-Acet a m id o-4-O-a cet yl-2,3-d id eoxy-3-C--
(1′,3′,4′,5′-tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-
D-m a n n o-h ep t it ol-7′-C-yl]-1-O-(t r is(isop r op yl)silyl)-â-D-
ta lop yr a n u r on a te ((-)-24). A mixture of (-)-22 (38 mg, 0.05
mmol), EtOAc (3.3 mL), and PtO2 (80 mg) was degassed and
pressurized with H2 (1 atm.). After shaking for 1 h at 20 °C,
the catalyst was filtered off through Celite and the solvent
evaporated in vacuo. The residue (compound 23) was taken
up in Ac2O (1.6 mL) and pyridine (42 µL). After stirring at 20
°C for 2 h, the solvent was evaporated in vacuo. The residue
was purified by FC (EtOAc/light petroleum ether 1:1), yielding
19, white foam. [R]25 ) 36 (c ) 1.0, CHCl3). 1H NMR (400
D
MHz, CDCl3) δ 6.24 (d, 3J ) 2.8), 5.24 (dd, 3J ) 7.9, 3.3), 5.24-
33 mg (85%) of (-)-24, white foam. [R]25 ) -0.6 (c ) 1.0,
D
3
3
1
3
5.22 (m), 5.16 (dd, J ) 7.9, 7.5), 5.13 (dd, J ) 3.4, 3.3), 4.56
(dd, 3J ) 8.3, 2.8), 4.44 (dd, 2J ) 12.2, 3J ) 7.2), 4.13-4.07
(m), 4.08 (dd, 2J ) 12.2, 3J ) 2.4), 3.96 (ddd, 3J ) 7.5, 7.2,
2.4), 2.95-2.80 (br s), 2.58 (dddd, 3J ) 10.9, 8.3, 2.9, 2.9), 2.32-
CHCl3). H NMR (400 MHz, CDCl3) δ 5.95 (d, J ) 10.4), 5.43
3 3
(br s), 5.20-5.10 (br d), 5.05 (dd, J ) 2.7, 2.6), 4.89 (d, J )
1.7), 4.41 (dd, 2J ) 12.2, 3J ) 6.0), 4.28-4.22 (br d, 3J = 10.0),
4.19 (d, 3J ) 1.3), 4.10 (dd, 2J ) 12.2, 3J ) 3.1), 4.14-4.08 (m),
3.90-3.85 (m), 3.71 (s), 2.25-2.17 (m), 2.09-2.00 (6s), 1.80
(ddd, 2J ) 11.1, 3J ) 11.2, 3.1), 1.51 (ddd, 2J ) 11.1, 3J ) 11.0,
2.9), 1.10-1.00 (m).
2
3
2.24 (m), 2.13-2.05 (5s), 1.51 (ddd, J ) 14.4, J ) 10.9, 2.8).
(1R,2S,5S,6R)-5-Azid o-3-ch lor o-2-h yd r oxy-6-(1′,3′,4′,5′-
tetr a -O-a cetyl-2,6-a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-
h eptitol-7′-C-yl)cycloh ex-3-en -1-yl Aceta te ((-)-20). A mix-
ture of (+)-19 (340 mg, 0.55 mmol), DMF (18.3 mL), and NaN3
(70 mg, 1.1 mmol) was heated in the dark to 60 °C for 15 min.
After cooling to 20 °C, H2O (20 mL) was added. The aqueous
phase was extracted with Et2O (20 mL, 4 times). The combined
organic extracts were washed with H2O (20 mL, twice) and
dried (MgSO4). After solvent evaporation, the residue was
purified by FC (Et2O/light petroleum ether 7:3), yielding 310
mg (98%) of (-)-20, white foam. [R]25D ) -173 (c ) 1.0, CHCl3).
Tr is(isop r op yl)silyl 2-Aceta m id o-2,3-d id eoxy-3-C-(2′,6′-
a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-h ep titol-7′-C-yl)-â-
D-ta lop yr a n osid e (25). A 2 M solution of LiBH4 in anhydrous
THF (0.12 mL, 0.24 mmol) was added to a stirred solution of
(-)-24 (24 mg, 0.03 mmol) in anhydrous THF (3.4 mL) cooled
to 0 °C. After stirring at 0 °C for 2 h, the mixture was stirred
at 20 °C for 22 h. H2O (2 mL) was added, and the solvent was
evaporated in vacuo. The residue was purified by filtration
through a column (L ) 1 cm, h ) 3 cm) of Dowex 50WX8 and
rinsing with MeOH. (The Dowex was prewashed with 3.7%
aqueous HCl first and then with H2O and finally with MeOH).
Solvent evaporation yielded 13 mg (78%) of 25, yellowish oil.
3
3
1H NMR (400 MHz, CDCl3) δ 6.19 (d, J ) 5.3), 5.26 (dd, J )
3
3
7.9, 3.3), 5.17 (dd, J ) 2.2, 2.1), 5.14 (dd, J ) 7.9, 7.0), 5.09
(dd, 3J ) 4.0, 3.3), 4.50 (dd, 2J ) 12.2, 3J ) 6.8), 4.15-4.08
(m), 4.04 (dd, 2J ) 12.2, 3J ) 2.6), 3.97 (ddd, 3J ) 7.0, 6.8,
2.6), 3.90 (dd, 3J ) 5.3, 5.2), 2.44-2.39 (m), 2.17-2.07 (5s),
2.05-1.94 (m), 1.69-1.63 (m).
3
1H NMR (600 MHz, C5D5N) δ 7.34 (d, J ) 10.0, NH), 5.10 (d,
3J ) 1.6, H-C(1)), 4.85 (ddd, 3J (HproS-C(7′)-H-C(6′)) ) 11.2,
3J (HproR-C(7′)-H-C(6′)) ) 3.2, 3J (H-C(5′)-H-C(6′)) ) 3.1, H-C(6′)),
3
Meth yl (4-O-Acetyl-2-a zid o-2,3-d id eoxy-3-C-(1′,3′,4′,5′-
tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-
h ep titol-7′-C-yl)-r-D-ta lop yr a n u r on a te ((-)-21). NaHCO3
(11 mg, 0.13 mmol) was added to a solution of (-)-20 (120 mg,
0.21 mmol) in MeOH/CH2Cl2 1:1 (8 mL). The solution was
cooled to -78 °C, and ozone was bubbled through it for 3 h. A
stream of N2 was bubbled for 10 min, and a few drops of Me2S
were added. The mixture was allowed to warm to 20 °C and
stirred for 12 h. H2O (10 mL) was added, and the aqueous
phase was extracted with CH2Cl2 (10 mL, four times). The
combined organic extracts were dried (MgSO4), and after
solvent evaporation in vacuo the residue was purified by FC
4.70 (br d, J = 10.0, H-C(2)), 4.58-4.55 (m, H-C(4), H-C(1′)),
3
2
3
4.49 (dd, J ) 7.9, 3.3, H-C(4′)), 4.46 (dd, J ) 11.0, J ) 3.3,
2
H-C(1′)), 4.39-4.37 (m, H-C(3′), H-C(5′)), 4.35 (dd, J ) 10.8,
3J ) 6.7, H-C(6)), 4.28 (ddd, J ) 7.5, 7.5, 3.3, H-C(2′)), 4.23
3
(dd, 2J ) 10.8, 3J ) 5.4, H-C(6)), 3.85 (br t, 3J = 10.0, H-C(5)),
2.63-2.59 (m, H-C(3)), 2.54 (ddd, 2J ) 14.4, 3J (HproR-C(7′)-
H-C(3)) ) 9.7, 3J (HproR-C(7′)-H-C(6′)) ) 3.2, HproR-C(7′)),
2.43 (ddd, 2J ) 14.4, 3J (HproS-C(7′)-H-C(6′)) ) 11.2, 3J (HproS-
C(7′)-H-C(3)) ) 4.3, HproS-C(7′)), 1.93 (s, NHAc), 1.20-1.00
(m, 21H, TIPS).
Tr is(isop r op yl)silyl 2-Acet a m id o-4,6-d i-O-a cet yl-2,3-
d id e oxy-3-C-(1′,3′,4′,5′-t e t r a -O-a ce t yl-2′,6′-a n h yd r o-7′-
d eoxy-D-glycer o-D-m a n n o-h ep titol-7′-C-yl)-â-D-ta lop yr a -
n osid e ((-)-26). A mixture of 25 (13 mg, 24 µmol), pyridine
(2.8 mL), Ac2O (0.9 mL), and 4-(dimethylamino)pyridine (2 mg)
was stirred at 20 °C for 12 h. The solvent was evaporated in
vacuo and the residue purified by FC (EtOAc/light petroleum
(EtOAc/light petroleum ether 3:2), yielding 88 mg (69%) of (-)-
1
21, white foam. [R]25 ) -13 (c ) 1.0, CHCl3). H NMR (400
D
MHz, CDCl3) δ 5.62 (d, 3J ) 3.1), 5.49 (br s), 5.26 (dd, 3J )
3
3
7.0, 3.2), 5.10 (dd, J ) 7.1, 7.0), 5.05 (dd, J ) 4.6, 3.2), 4.82
(d, 3J ) 1.5), 4.49 (dd, 2J ) 12.1, 3J ) 7.2), 4.19 (ddd, 3J )
2
3
3
12.1, 4.6, 4.5), 4.12 (dd, J ) 12.1, J ) 2.9), 3.93 (ddd, J )
7.2, 7.1, 2.9), 3.74 (s), 3.42 (br d, 3J = 2.9), 2.60-2.52 (m), 2.12-
2.08 (5s), 1.80-1.66 (m).
ether 1:1), yielding 18 mg (93%) of (-)-26, yellowish oil. [R]25
D
1
) -5.0 (c ) 0.25, CHCl3). H NMR (400 MHz, CDCl3) δ 5.87
3
3
(d, J ) 10.4), 5.20-5.10 (m), 5.06 (dd, J ) 2.8, 2.7), 4.87 (d,
2
3
3
Meth yl 4-O-Acetyl-2-a zid o-2,3-d id eoxy-3-C-(1′,3′,4′,5′-
t e t r a -O -a c e t y l-2′,6′-a n h y d r o -7′-d e o x y -D -g l yc er o-D -
m a n n o-h e p t it ol-7′-C-yl)-1-O-(t r is(isop r op yl)silyl)-â-D-
ta lop yr a n u r on a te ((-)-22). A mixture of (-)-21 (71 mg, 0.12
mmol), CH2Cl2 (6 mL), 2,6-lutidine (41 µL, 0.36 mmol), and
triisopropylsilyl trifluoromethanesulfonate (TIPSOTf, 65 µL,
0.24 mmol) was stirred at 0 °C under N2 atmosphere for 5 min
and then at 20 °C for 5 h. More 2,6-lutidine (41 µL, 0.36 mmol)
and TIPSOTf (65 µL, 0.24 mmol) were added if the reaction
3J ) 1.7), 4.42 (dd, J ) 12.2, J ) 5.9), 4.26-4.22 (br d, J =
9.0), 4.15-4.00 (m), 3.90-3.86 (m), 3.83 (br t, 3J = 6.8), 2.15-
2
3
2.02 (7s + m), 1.80 (ddd, J ) 14.4, J ) 14.5, 3.2), 1.50 (ddd,
3
2J ) 14.4, J ) 14.3, 2.8), 1.12-1.04 (m).
2-Aceta m id o-4,6-d i-O-a cetyl-2,3-d id eoxy-3-C-(1′,3′,4′,5′-
tetr a -O-a cetyl-2′,6′-a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-
h ep titol-7′-C-yl)-r-D-ta lop yr a n ose ((+)-27). A mixture of
(-)-26 (70 mg, 0.09 mmol), THF (4.2 mL), and tetrabutylam-
monium fluoride (130 µL, 0.13 mmol) was stirred at 0 °C under

4260 J . Org. Chem., Vol. 65, No. 14, 2000
Pasquarello et al.
Glycosid a se In h ibition Assa ys. See ref 27.
N2 atmosphere for 5 min. After filtration through a column of
Florisil (L ) 2 cm, h ) 3 cm) and rinsing with EtOAc (50 mL),
the solvent was evaporated in vacuo. The residue was purified
by FC on Florisil (EtOAc), yielding 42 mg (74%) of (+)-27,
Glycosyltr a n sfer a se In h ibition Assa ys. The â-1,4-ga-
lactosyltransferase from human milk was assayed following
the protocol of Berger43 using the following conditions: 10 mM
MnCl2, 10 mM GlcNAc, 400 µM UDP-Gal, 0.15 µM 14C-UDP,
and enzyme in Tris-HCl (100 mM, pH 7.4), final volume of 50
µL. The R-2,6-sialtransferase, a soluble form of which that had
been expressed in Pichia pastoris has been tested as described
previously44 using the following conditions: 10 mM LacNAc,
500 µM CMP-Nana (N-acetylneuraminic acid), 0.035 µmol 14C-
CPM-Nana, and enzyme cocodylate buffer (100 µM, pH 6.8),
final volume of 50 µL. The R-1,3-fucosyltransferase VI (Fuc-
TVI), a soluble form of which that has been expressed in Pichia
pastoris, was assayed as described previously44 with the
following modifications:45 5 mM ATP, 10 mM MnCl2, 100 µM
GDP-fucose, 5 mL LacNAc (unless specified), 1.6 µM 14C-GDP-
fucose, and enzyme in cacodylate buffer (1 M, pH 6.2), final
volume of 50 µL. The C-disaccharides 1 and 2, and test
compounds such as D-mannose, N-acetylgalactosamine, and
CH3CONH2, were soluble in H2O. For inhibition measurements
the enzymes were preincubated for 5 min at 20 °C with the
inhibitor of various concentrations. The reactions were initi-
ated on adding the substrate and were run for 20 min at 37
°C for Fuc-TVI46,47 and galactosyltransferase, and for 2 h at
37 °C for sialyltransferase.
yellow oil. [R]25 ) 14 (c ) 1.0, CHCl3). 1H NMR (400 MHz,
D
3
CDCl3) δ 6.29 (d, J ) 9.9), 5.24 (br s), 5.21-5.11 (m), 5.09 (d,
3J ) 3.7), 5.07 (dd, 3J ) 3.3, 3.2), 4.43-4.36 (m), 4.15-3.98
(m), 3.85 (ddd, 3J ) 7.2, 7.1, 3.0), 2.60 (ddd, 3J ) 10.2, 6.4,
2
3
3.4), 2.17-2.02 (7s), 1.68 (ddd, J ) 14.5, J ) 12.0, 3.4), 1.44
2
3
(ddd, J ) 14.5, J ) 10.7, 3.0).
Meth yl (4-O-Acetyl-2-a zid o-1-O-(ter t-bu tyld im eth ylsi-
lyl)-2,3-dideoxy-3-C-(1′,3′,4′,5′-tetr a-O-acetyl-2′,6′-an h ydr o-
7′-d eoxy-D-glycer o-D-m a n n o-h ep titol-7′-C-yl)-â-D-ta lop y-
r an u r on ate ((-)-29). (-)-21 (631 mg, 1.04 mmol) was dissolved
in CH2Cl2 (19 mL) at 0 °C under N2, and then 2,6-lutidine (334
mg, 3.12 mmol) and (tert-butyl)dimethylsilyl trifluoromethane-
sulfonate (TBDMSOTf, 550 mg, 2.08 mmol) were added. After
stirring at 0 °C for 20 min, the mixture was allowed to warm
to 20 °C and stirred for 2.5 h; by TLC there was still unreacted
(-)-21. The mixture was thus cooled again to 0 °C, and 2,6-
lutidine (167 mg, 1.56 mmol) and TBDMSOTf (275 mg, 1.04
mmol) were added. The mixture was allowed to warm to 20
°C and stirred for 1.5 h. Brine (20 mL) was added, and the
aqueous phase was extracted with CH2Cl2 and (20 mL, four
times). The combined extracts were dried (MgSO4). After
solvent evaporation in vacuo, the residue was purified by FC
(EtOAc/light petroleum ether 2:3), yielding 560 mg (75%) of
(-)-29 contaminated by 10-20% of the R-pyranoside. White
foam. [R]25D ) -56 (c ) 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
Ack n ow led gm en t . We thank the Swiss National
Science Foundation, the Fonds Herbette (Lausanne), for
support and the European Programm COSTD13/0001/
99 for encouragement. We thank also Mr. Martial Rey
and Francisco Sepulveda for their technical help.
3
3
3
δ 5.38 (dd, J ) 3.0, 2.0), 5.25 (dd, J ) 7.4, 3.2), 5.12 (dd, J
) 7.4, 6.5), 5.03 (dd, 3J ) 5.0, 3.2), 4.94 (d, 3J ) 1.7), 4.49 (dd,
2J ) 12.2, 3J ) 6.8), 4.20-4.10 (m), 3.95 (ddd, 3J ) 6.8, 6.5,
3.3), 3.74 (s), 3.44 (dd, 3J ) 3.6, 1.7), 2.20-2.00 (5s + m), 1.83-
1.67 (m), 0.96 (s), 0.24 (s), 0.17 (s).
1
Meth yl (2-Aceta m id o-4-O-a cetyl-1-O-(ter t-bu tyld im e-
t h ylsilyl)-2,3-d id eoxy-3-C-(1′,3′,4′,5′-t et r a -O-a cet yl-2′,6′-
a n h yd r o-7′-d eoxy-D-glycer o-D-m a n n o-h ep titol-7′-C-yl)-â-
D-ta lop yr a n u r on a te ((-)-30). After degassing, a mixture of
(-)-29 (508 mg, 0.71 mmol), EtOAc (47 mL) and PtO2 (250
mg) was pressurized with H2 (1 atm) and shaken at 20 °C for
18 h. The catalyst was filtered off and the solvent evaporated.
The residue was taken up in Ac2O (14 mL). After the addition
of pyridine (0.35 mL), the mixture was stirred at 20 °C for 2
h. The solvent was evaporated in vacuo and the residue
purified by FC (EtOAc/light petroleum ether 7:3), yielding 465
mg (89%) of (-)-30, white foam. [R]25D ) -4.3 (c ) 1.0, CHCl3).
Su p p or tin g In for m a tion Ava ila ble: Detailed H and 13C
NMR spectra and signal assignments, further optical rotation
data, and UV, IR, and MS spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
J O991952U
(43) Malissard, M.; Borsig, L.; DiMarco, S.; Gru¨tter, M. G.; Kragl,
U.; Wandrey, C.; Berger, E. G. Eur. J . Biochem. 1996, 239, 340.
(44) Malissard, M.; Zeng, S.; Berger, E. G. Glycoconjugate J . 1999,
16, 125-129.
(45) Sarnesto, A.; Ko¨hlin, T.; Hidsgaul, O.; Vogele, K.; Blaszczyk-
Thurin, M.; Thurin, J . J . Biol. Chem. 1992, 267, 2745.
(46) The so-called plasma type of fucosyltransferases are expressed
in liver47 and endothelial cells.48
3
1H NMR (400 MHz, CDCl3) δ 5.94 (d, J ) 10.4), 5.43 (br s),
3
3
5.19-5.17 (m), 5.06 (dd, J ) 3.0, 2.9), 4.84 (d, J ) 1.8), 4.42
(47) Brinkman Van der Linden, E. C. M.; Mollicone, R.; Oriol, R.;
Larson, G.; Van den Eijnden, D. H.; Van Dijk, W. J . Biol. Chem. 1996,
271, 14492.
2
3
(dd, J ) 12.2, J ) 6.0), 4.23-4.18 (m), 4.15-4.09 (m), 3.91-
2
3
3.85 (m), 3.73 (s), 2.30-2.03 (5s + m), 1.80 (ddd, J ) 14.5, J
) 12.0, 3.1), 1.52 (ddd, 2J ) 14.5, 3J ) 10.7, 3.1), 0.90 (s), 0.17
(s), 0.11 (s).
(48) Majuri, M. L.; Pinola, M.; Niemela, R.; Tiisala, S.; Natunen,
J .; Renkonen, O.; Renkonen, R. Eur. J . Immunol. 1994, 24, 3205.