Substrate specificity of fucosyl transferase III: An efficient synthesis of sialyl Lewis(x) -, sialyl Lewis(a)- derivatives and mimetics thereof

Article abstract of DOI:10.1139/v00-081

Fucosyl transferase III (FucT III) has previously been characterized as the most general enzyme of the FucT family, as judged from its ability to catalyze the transfer of fucose to both Galβ(1-3)GlcNAc and Galβ(1-4)GlcNAc. In order to explore the synthetic potential of FucT III for the enzymatic synthesis of sialyl Lewis(x) and sialyl Lewis(a) derivatives, its substrate specificity has been probed using a number of natural substrate mimetics. A remarkable range of acceptor substrates was found when N-acetyl glucosamine was replaced by D-glucal, (R,R)-1,2-cyclohexanediol and (R,R)-butan-2,3-diol. Although the reaction rates were low compared to the reaction with the natural substrates, they proved to be sufficient for the synthesis of preparative amounts.

Full text of DOI:10.1139/v00-081

892
Substrate specificity of fucosyl transferase III: An
efficient synthesis of sialyl Lewisx-, sialyl Lewisa-
derivatives and mimetics thereof
Beat Ernst, Bea Wagner, Gabi Baisch, Andreas Katopodis, Tammo Winkler, and
Reinhold Öhrlein
Abstract: Fucosyl transferase III (FucT III) has previously been characterized as the most general enzyme of the FucT
family, as judged from its ability to catalyze the transfer of fucose to both Galβ(1–3)GlcNAc and Galβ(1–4)GlcNAc. In
order to explore the synthetic potential of FucT III for the enzymatic synthesis of sialyl Lewisx and sialyl Lewisa deriv-
atives, its substrate specificity has been probed using a number of natural substrate mimetics. A remarkable range of
acceptor substrates was found when N-acetyl glucosamine was replaced by D-glucal, (R,R)-1,2-cyclohexanediol and
(R,R)-butan-2,3-diol. Although the reaction rates were low compared to the reaction with the natural substrates, they
proved to be sufficient for the synthesis of preparative amounts.
Key words: fucosyl transferase III, sialyl Lewisa, sialyl Lewisx, carbohydrate mimetics.
Résumé : La fucosyle transférase III (FucT III) a déjà été caractérisée comme étant l’enzyme le plus général de la
famille des FucT, tel que le prouve sa capacité à catalyser le transfert du fucose aux deux Gal 1–3 GlcNAc et Gal 1–4
GlcVAc. Dans le but d’explorer le potentiel synthétique du FucT III pour la synthèse enzymatique des dérivés du sialyl
Lewisx et du sialyl Lewisa, on a mis en évidence la spécificité de son substrat en utilisant divers analogues de substrats
naturels. On trouvé un remarquable échantillonnage de substrats accepteurs lorsqu’on remplace la N-Acétyl
glucosamine par le D-glucal, le (R,R)-1,2-cyclohexanediol et le (R,R)-butan-2,3-diol. Bien que les vitesses de réaction
soient plus lentes que celles des produits naturels, elles se révèlent acceptables pour la synthèse à grande échelle.
Mots clés : fucosyle transférase III, sialyl Lewisx et du sialyl Lewisa., analogues d’hydrates de carbone.
[Traduit par la Rédaction] Ernst et al. 904
Introduction
ates this initial event (2). Since leukocyte rolling is a prereq-
uisite for later inflammatory events, inhibition of selectins is
a valuable strategy for preventing the deleterious conse-
quences of excessive leukocyte influx.
Numerous diseases and pathological situations are associ-
ated with excessive influx of leukocytes into tissues. Al-
though this influx normally represents an essential defense
mechanism against infection, excessive, or inappropriate leu-
kocyte accumulation results in injury to host tissues as ob-
served in ischemia-reperfusion injury, respiratory disease,
dermatitis, or gastro-intestinal inflammation (1).
Early in the inflammatory response leukocytes adhere to
and roll along endothelial cells on the inner surface of blood
vessels. It has been clearly shown that an inducible set of
calcium dependent adhesion molecules, the selectins, medi-
Studies of the carbohydrate specificity of E- and P-selectin
uncovered a recognition motif common to both receptors,
the sialylated and fucosylated tetrasaccharide antigens sialyl
Lewisx (1) (3) and sialyl Lewisa (2) (4). Later, L-selectin was
also shown to bind sLex and sLea containing oligosaccha-
rides (5). These two tetrasaccharides have therefore served
as lead structures in our search for selectin antagonists with
increased biological activity, simplified structure and im-
proved bioavailability (6). For the discovery of their struc-
ture-activity relationship, numerous derivatives of 1 and 2
have been synthesized. It is worth mentioning that these de-
rivatives have not only been obtained by chemical (7) but
also by enzymatic synthesis (Scheme 1); e.g., sialyl Lewisx
and sialyl Lewisa derivatives with a modified N-acyl group
of the D-glucosamine nitrogen (e.g., 3, 4) (8) or compounds
where fucose is replaced by other L-sugars (e.g., 5, 6) (9).
Although a number of studies showed that non-natural
substrates are accepted by glycosyl transferases (10), only a
limited number of structurally more distant compouds have
been probed to date (11). To further explore the synthetic
potential of the enzymatic approach for the synthesis of
sialyl Lewisx and sialyl Lewisa derivatives and to widen
the synthetic value of these biocatalysts, we decided to
Received September 17, 1999. Published on the NRC
Research Press Website on June 16, 2000.
This paper is dedicated to Professor Hanessian.
B. Ernst1 and B. Wagner. Institute of Molecular Pharmacy,
Pharmacenter, University of Basel, CH-4056 Basel,
Switzerland.
G. Baisch, A. Katopodis, and R. Öhrlein. Novartis Pharma
AG, CH-4002 Basel, Switzerland.
T. Winkler. Novartis Crop Protection AG, CH-4002 Basel,
Switzerland.
1Author to whom correspondence may be addressed
(e-mail: beat.ernst@unibas.ch).
Can. J. Chem. 78: 892–904 (2000)
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Ernst et al.
893
Scheme 1. sLex and sLea and enzymatically synthesized derivatives thereof (8, 9).
Scheme 2. (a) CMP-Neu-5-NAc, α(2–3)sialyl transferase (65%); (b) GDP-fucose, FucT III (83%).
investigate the substrate specificity of the enzymatic
fucosylation reaction. As judged from its ability to catalyze
the transfer of fucose to both α1,3 and α1,4 linkages FucT
III has previously been characterized as the most general en-
zyme of the FucT III family (12). We therefore concentrated
our investigation on the substrate specificity of FucT III.
advantages of using glycosyl transferases for the construc-
tion of glycosidic bonds include mild reaction conditions,
the lack of requirement for protection and deprotection steps
and the regio- and stereospecificity of the glycosylation.
In a first attempt, lactal 7, which can easily be obtained
from D-lactose (13) served as a mimic of N-acetyl-
lactosamine (Scheme 2). It was first incubated with recombi-
nant α(2–3)sialyl transferase (14) and subsequently with re-
combinant fucosyl transferase FucT III (15). As expected, α-
sialylation occurred at the 3-OH of galactose. Surprisingly,
the allylic OH-group of the glucal moiety was fucosylated
exclusively, generating the desired sLex derivative 9. A
Results and discussion
Since the chemical approach to sialyl Lewisx and sialyl
Lewisa analogs requires multi-step syntheses, the chemo-
enzymatic approach was considered as an alternative. The
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894
Can. J. Chem. Vol. 78, 2000
Scheme 3. (a) CMP-Neu-5-NAc, α(2–3)sialyl transferase (92%); (b) GDP-fucose, FucT III (82%).
Scheme 4. (a) (R,R)-cyclohexan-1,2-diol, TESOTf, CH3CN, 1 h (74%); (b) cat. NaOMe, MeOH, rt, 6 h (75%); (c) CMP-Neu-5-NAc,
α(2–3)sialyl transferase (64%); (d) GDP-fucose, FucT III (71%).
similar reaction sequence starting from 7 had previously
been carried out successfully by Wong et al. (16) using
FucT V instead of FucT III.
Galactosyl-β(1–3)glucal 10 (17), a type I congener of
lactal 7, could equally well be sialylated (→ 11) (Scheme 3).
Also in this case, FucT III accepted a glucal derivative as
substrate. Therefore, the N-acetyl group of the natural
glucosamine substrate is not a prerequisite for a successful
fucosylation.
pseudodisaccharide 15 was obtained in a two step procedure
by galactosylation of commercially available (R,R)-1,2-
cyclohexanediol using the trichloroacetimidate 13 (18) as
the galactosyl donor and TESOTf as the promotor (→ 14)
followed by removal of the benzoate protecting groups by
transesterification. In the first enzymatic step, 15 was incu-
bated with CMP-Neu-5-NAc and recombinant α (2–3)sialyl
transferase (14) to yield 16. As observed for 7 and 10, the
activity of the α(2–3)sialyl transferase was not affected by
the character of the aglycon at the reducing end of the galac-
tose moiety. In the subsequent fucosylation step, incubation
In a further step (see Scheme 4), the glucal ring system
was replaced by (R,R)-cyclohexan-1,2-diol. The precursor
© 2000 NRC Canada

Ernst et al.
895
Scheme 5. (a) (R,R)-cyclohexan-1,2-diol, DMTST (20), CH2Cl2, 4 Å molecular sieves, 3 h (59%); (b) cat. NaOMe, MeOH, rt, 3 h
n
(96%); (c) Bu2SnO, MeOH, rfl., 2 h; CsF, DMF, rt, 3 h, with benzyl (R)-3-cyclohexyl-2-trifluoromethansulfonyloxy-propionate 21a
(→ 22a, 76%), with benzyl (S)-3-cyclohexyl-2-trifluoromethansulfonyloxy-propionate 21b (→ 22b, 66%), with benzyl (R)-3-phenyl-2-
trifluoromethansulfonyloxy-proprionate 21c (→ 22c, 82%); (d) 20% Pd(OH)2, H2, dioxane–H2O–AcOH; Dowex 50 (Na+ form) (23a,
66%; 23b, 72%; 23c, 68%); (e) GDP-fucose, FucT III (24a, 54%; 24b, 61%; 24c, 96%).
4
of the trisaccharide 16 with GDP-fucose and recombinant
FucT III (15) gave 17, which can be regarded as mimic of
either sLex or a sLea. The connectivity pattern in 17 was
confirmed by ROESY.
groups of a D-glucose/D-glucosamine in the C1 conforma-
tion.
We further investigated (S,S)-cyclohexan-1,2-diol, which
represents
a
mimic of N-acetyl-L-glucosamine. The
Next, the enzymatic fucosylations of the pseudo-
trisaccharides 23a–c were examined. In these compounds
the D-glucosamine and the N-acetylneuraminic acid moiety
of the natural substrate are replaced by (R,R)-cyclohexan-
1,2-diol and R- and S-lactic acid derivatives, respectively.
The glycosyl acceptors 23a–c were obtained by galactosy-
lation of (R,R)-cyclohexan-1,2-diol with ethyl 6-O-benzyl-
2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside 18 (19) us-
ing dimethylmethylthiosulfonium triflate (DMTST) (20) as
promoter (→ 19) followed by the removal of the benzoate
protecting groups by transesterification (→ 20). Finally,
alkylation of 20 with the lactic acid derivatives 21a,b (21)
and 21c (22), respectively, (→ 22a–c) and final deprotection
by hydrogenolysis gave the substrates 23a–c. As observed
for 16, the hydroxy group of the cyclohexane moiety was
fucosylated with good to excellent yield using FucT III. The
replacement of neuraminic acid by lactic acid derivatives
does not influence proper fucosylation. Furthermore, the
stereochemical orientation of the lactic acid substituents –
with S-configuration in 23a and R-configuration in 23b – did
not affect the outcome of the fucosylation.
pseudotrisaccharide 28 was obtained in analogy to 23a (see
Scheme 6): β-Galactosylation of (S,S)-cyclohexan-1,2-diol
gave the pseudodisaccharide 25 in moderate yield. After re-
moval of the benzoates, 26 was regioselectively alkylated
with benzyl (R)-3-cyclohexyl-2-trifluoromethansulfonyloxy-
propionate. Finally, the benzyl protective groups were re-
moved by hydrogenation to yield 28, which however did not
act as a substrate for FucT III as well as for FucT VI (23).
Finally, we examined (R,R)-butan-2,3-diol as a mimic of
the 3- and 4-hydroxy groups of a D-glucose/D-glucosamine.
For the synthesis of the corresponding substrate 33, the 3-
hydroxy group of ethyl 1-thio-β-D-galactopyranoside 29,
was regioselectively alkylated. For this purpose, the
stannylene acetal (24) of the two trans hydroxy groups (2-
and 3-position) of ethyl 1-thio-β-D-galactopyranoside 29
was formed in situ and treated with benzyl (R)-3-cyclohexyl-
2-trifluoromethansulfonyloxy-propionate 21a in DME at
room temperature in the presence of caesium fluoride. The
resulting thiogalactoside 30 was then perbenzoylated (BzCl,
pyridine, DMAP) to yield the glycosyl donor 31, which was
used for the galactosylation of (R,R)-2,3-butandiol with
DMTST (19) as promotor. After deprotecton by saponifi-
cation, the substrate for the enzymatic step was obtained in
excellent yield. The trisaccharide mimic 33 was then incu-
bated with FucT III and GDP-fucose. To our surprise, this
highly flexible compound proved to be an excellent substrate
D-Galactose carrying a substituent with the acceptor OH
group in the proper stereochemical arrangement at the re-
ducing end is therefore the only structural motif critical for
recognition by FucT III. (R,R)-cyclohexan-1,2-diol can be
considered as a perfect mimic for the 3- and 4-hydroxy
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896
Can. J. Chem. Vol. 78, 2000
Scheme 6. (a) (S,S)-cyclohexan-1,2-diol, DMTST (20), CH2Cl2, 4 Å molecular sieves, 3 h (59%); (b) cat. NaOMe, MeOH, rt, 3 h
n
(82%); (c) Bu2SnO, MeOH, rfl., 2 h; CsF, DMF, rt, 3 h, with benzyl (R)-3-cyclohexyl-2-trifuoromethansulfonyloxy-propionate 21a (→
26a, 69%); (d) 20% Pd(OH)2, H2, dioxane–H2O–AcOH; Dowex 50 (Na+ form) (78%); (e) GDP-fucose, FucT III or FucT VI: no reac-
tion.
n
Scheme 7. (a) Bu2SnO, MeOH, rfl., 2 h; benzyl (R)-3-cyclohexyl-2-trifuoromethansulfonyloxy-propionate (21a), CsF, DME, rt, 2 h
(67%); (b) benzoyl chloride, pyridine, DMAP (91%); (c) (R,R)-2,3-butandiol, CH2Cl2, 4 Å molecular sieves, DMTST (20), rt, 2 h
(70%); (d) LiOH, H2O–MeOH, 60°C, 5 h; Dowex 50 (Na+ form) (94%); (e) GDP-fucose, FucT III (47%).
© 2000 NRC Canada

Ernst et al.
897
of FucT III yielding the α-fucosylated product 34. This
fucose was donated by YAMASA–corp.2 CPM-Neu-5-NAc
was purchased from Forschungszentrum Jülich GmbH.3
1H NMR and 13C NMR spectra were recorded on a
BRUKER AC 400 and (or) a VARIAN Unity 500 spectrom-
eter. COSY, TOCSY, HMBC, and HSQC experiments were
performed using the manufacturer’s software. 1H NMR-
shifts and 13C NMR-shifts are referenced to the solvent line
(given in ppm) unless otherwise stated. Chemical shifts
marked with an asterisk (*) may be interchanged. ESI-MS
were recorded on VG Platform II (MicroMass, Manchester
U.K.) equipped with a HP 1050 and a CTC PAL
autosampler. Flow injection mass spectrometry was done
with 100 µL/min in H2O:CH3CN, 1:4 over a mass range of
(+) 100–1500 Da / 1.5 s, (–) 120–1500 Da / 1.5 s.
1
structure was confirmed based on the H and 13C chemical
shift of H-1 and C-1 of the fucose moiety (4.99 ppm (J =
3.3 Hz) and 96.4 ppm, respectively). In addition, the 13C sig-
nals of both CHO carbons of the butandiol moiety which are
shifted upfield by approximately 2 ppm proving the site of
the fucosylation.
In the examples shown, the FucT III was found to have a
remarkable flexibility in the recognition of acceptor sub-
strates. Further modifications are under current investigation.
However, in many cases the reaction rates were very low
compared to the reaction with the natural substrate
sialylα (2–3)lacNAc. Such rates, however, proved to be suffi-
cient for the synthesis of preparative amounts of
oligosaccharide derivatives and mimics.
5-Acetamido-3,5-dideoxy-D-glycero-α -D-galacto-2-nonulopy-
ranosylonic acid-(2–3)-O-β-D-galactopyranosyl-(1–4)-glucal
(8): In a buffered solution containing sodium cacodylate
buffer (75 µL, 0.5 M), MnCl2-solution (100 µL, 0.5 m) and
bidistilled water (2.1 mL), O-β-D-galactopyranosyl(1–4)-
1,5-anhydro-D-arabino-hex-1-enitol (D-lactal) 7 (20.0 mg,
64.9 µmol) (13), CMP-sialic acid (23.2 mg, 79.8 µmol), and
bovine serum albumine (1.5 mg) were dissolved. The clear
solution was incubated for 24 h with α(2–3)sialyl transferase
(20 µL, 160 µU) and calf intestine alkaline phosphatase
(2 µL, 30 U) at 37°C. A second portion of CMP-sialic acid
(24.9 mg, 85,6 µmol) α(2–3)sialyl transferase (20 µL, 160
mU) was added and the incubation continued for 2 days.
The reaction mixture was then lyophilized and passed over
sephadex G-10. The sugar containing fractions were com-
bined and chromatographed on silica (CH2Cl2:MeOH:H2O,
10:4:0.1 to 6:4:1) to yield 8 (24.5 mg, 65%): 1H NMR (D2O,
250.13 MHz) δ: 1.78 (1H, t, J = 12.8 Hz, H-3sia), 2.00
(3H, s, Ac), 2.73 (1H, dd, J = 4.2 Hz, 12.8 Hz, H-3sia), 3.51–
4.06 (17H, m), 4.10 (1H, dd, J = 2.8 Hz, 9.8 Hz, H-3gal),
4.59 (1H, d, J = 8.3 Hz, H-1gal), 6.41 (1H, dd, J = 1.4 Hz,
6.2 Hz, Hvinyl); 13C (D2O, 62.89 MHz) δ: 24.00, 41.57, 53.63,
61.54, 62.92, 64.51, 69.17, 69.37, 70.03, 70.34, 71.34,
73.73, 74.82, 77.05, 77.47, 78.74, 79.21, 101.79, 103.76,
104.57, 145.92, 175.88, 176.96; m/z = 598 [M+ – H]; Anal.
calcd. for C23H37NO17: C 46.1, H 6.2; found: C 45.6, H 6.0.
A severe drawback for the feasibility of the enzymatic
synthesis of oligosaccharides and derivatives thereof stems
from the availability of glycosyl transferases. Although an
increasing number of transferases are commercially avail-
able, they are still prohibitively expensive, particularly for
synthesis on a preparative scale. For this reason, we have
initiated programs to clone and overexpress our own trans-
ferases for mechanistic and synthetic studies.
The sugar nucleotides CMP-NeuNAc and GDP-Fuc are
commericially available (footnotes 2, 3) and numerous
chemical and chemo-enzymatic approaches for their synthe-
ses have been developed in recent years (25). This has deci-
sively improved the availability of activated sugars in
preparative amounts and therefore the feasibility of the enzy-
matic synthesis of oligosaccharides and derivatives thereof.
Experimental
General methods
Chemicals used were reagent grade and were used as sup-
plied except where noted. Solvents were either dried by stan-
dard procedures or used as purchased. Unless otherwise
stated, all reactions were performed under an argon atmo-
sphere. All chemical glycosylation reactions were performed
using molecular sieves, which were flame-dried under high
vacuum immediately before the reaction. Analytical thin-
layer-chromatography (TLC) was performed using silica gel
60 F254 glass plates (MERCK); compound spots were visual-
ized by UV light (254 nm) and by staining with a solution
containing p-anisaldehyde (25 mL), conc. sulfuric acid
(15 mL), glacial acidic acid (5 mL) in EtOH (900 mL).
Flash column chromatography was performed on silica gel
60 (0.040–0.063 mm, MERCK).
Bovine serum albumine (BSA) was obtained from
BOEHRINGER (no. 28031). Calf intestine alkaline phos-
phatase (CIAP) (E.C.3.1.3.1) was purchased from
BOEHRINGER (no. 108146, 7500 U / 498 µl). Recombi-
nant α (2,3)sialyl transferase (~100 U/l, from transfected in-
sect cells) (14) (EMBL accession no. M97754), recombinant
fucosyl transferase III (~10 U/l, baculovirus transfected in-
sect cells) (15) (EMBL accession no. X53578), and recombi-
nant fucosyl transferase VI (~16 U/l, from transfected CHO-
cells) (23) (EMBL accession no. LO1698) were used. GDP-
5-Acetamido-3,5-dideoxy-D-glycero-α -D-galacto-2-nonulopy-
ranosylonic acid-(2–3)-O-β-D-galactopyranosyl-(1–3)-O-[α-
L-fucopysanosyl]-(1–4)-glucal (9): GDP-fucose (25.1 mg,
39.5 µmol) and bovine serum albumine (1.6 mg) were added
to a mixture of sodium cacodylate buffer (450 µL, 250 mM,
pH 6.5), of MnCl2-solution (150 µL, 250 mM) and
bidistilled water (600 µl) 8 (20.0 mg, 33.4 µmol). The clear
solution was incubated at 37°C with a solution of recombi-
nant FucT III (83 µL, 498 µU) and calf intestine alkaline
phosphatase (2 µL, 30 U) for one day. After 2 h a second
portion of GDP-fucose (10.0 mg, 15.7 µmol) and transferase
(16.6 µL, 99.6 mU) were added and the incubation contin-
ued, until TLC control (EtOAc:iso-propanol:water, 2:2:1) in-
dicated the complete consumption of the substrate 8. For the
work-up, the reaction mixture was centrifugated, lyophilized
and chromatographed on silica (ethyl acetate:iso-propanol:
water, 2:2:1). The product containing fractions were
2 H. Kusakabe, YAMASA–corp., 1–23–8, Nihonbashi-Kakigaracho, Chou-ku, Tokyo 103, Japan. e-mail: kusakabe@yamasa.com.
3 U. Kragl and C. Wandrey, Forschungszentrum Jülich GmbH, P.O. Box 1913, D-5170 Jülich.
© 2000 NRC Canada

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Can. J. Chem. Vol. 78, 2000
combined, passed over a sephadex G-10 column and
lyophilized to yield 9 (20.6 mg, 83%): 1H NMR (D2O,
500.13 MHz) δ: 1.12 (3H, d, J = 6.5 Hz, H-6fuc), 1.82 (1H, t,
J = 13.0 Hz, H-3sia), 2.03 (3H, s, Ac), 2.74 (1H, dd, J =
4.8 Hz, 13.0 Hz, H-3sia), 3.53 (1H, dd, J = 7.0 Hz, 9.5 Hz,
H-2gal), 3.55–3.70 (7H, m, H-6gal, H-4,6,7,8, 9sia, H-6cycl),
3.78 (1H, dd, J = 3.6 Hz, 8.3 Hz, H-2fuc), 3.79 (1H, d, J =
cacodylate buffer (450 µL, 250 mM, pH 6.5), MnCl2-solu-
tion (150 µL, 250 mM) and bidistilled water (600 µL). The
clear mixture was incubated at 37°C with recombinant FucT
III (100 µL, 600 µU/mL) and calf intestine alkaline
phosphatase (2 µL, 30 U). After three days TLC control
(EtOAc:iso-propanol:water, 2:2:1) indicated that all the
starting material had been consumed. For the work-up, the
reaction mixture was centrifugated, lyophilized, and
chromatographed on silica. The product was finally passed
over a sephadex G-10 column and lyophilized to yield 12
3.0 Hz, H-4fuc), 3.82–3.92 (5H, m, H-6′gal, H-5,9′sia, H-3fuc
,
H-6′cycl), 3.93 – 4.00 (2H, m, H-5gal, H-5cycl), 4.09 (1H, dd, J
= 2.4 Hz, 10.0 Hz, H-3gal), 4.14–4.19 (2H, m, H-4gal, H-
1
4
cycl), 4.32 (1H, broad s, H-3cycl), 4.47 (1H, q, J = 6.5 Hz,
(18.1 mg, 82%). H NMR (D2O, 500.13 MHz) δ: 1.21 (3H,
H-5fuc), 4.62 (1H, d, J = 8.0 Hz, H-1gal), 4.98 (1H, dd, J =
d, J = 6.5 Hz, H-6fuc), 1.81 (1H, t, J = 13.0 Hz, H-3sia), 2.04
(3H, s, Ac), 2.79 (1H, dd, J = 4.8 Hz, 13.0 Hz, H-3sia), 3.58
(1H, dd, J = 7.0 Hz, 9.5 Hz, H-2gal), 3.60–3.76 (7H, m, H-
5,6gal, H-4,6,7,8, 9sia), 3.78 (1H, dd, J = 3.6 Hz, 8.3 Hz, H-
2fuc), 3.79 (1H, d, J = 3.0 Hz, H-4fuc), 3.83–3.93 (5H, m, H-
6′gal, H-5,9′sia, H-3fuc, H-6′cycl), 3.93–4.00 (2H, m, H-5gal, H-
5cycl), 4.05 (1H, broad t, J = 6.0 Hz, H-4cycl), 4.10 (1H, dd,
J = 2.4 Hz, 10.0 Hz, H-3gal), 4.15 (1H, m, H-5cycl), 4.46 (1H,
q, J = 6.5 Hz, H-5fuc), 4.53 (1H, broad t, J = 4 Hz, H-3cycl),
4.61 (1H, d, J = 8.0 Hz, H-1gal), 5.07 (1H, dd, J = 2.8 Hz,
6.0 Hz, H-2cycl), 5.10 (1H, d, J = 3.6 Hz, H-1fuc), 6.51 (1H,
d, J = 6.0 Hz, H-1cycl); 13C (D2O, 125 MHz) δ: 17.00, 23.57,
41.18, 53.23, 60.78, 62.77, 64.11, 68.57, 69.00, 69.53,
69.65, 69.96, 70.58, 70.82, 73.21, 73.33, 73.43, 73.70,
74.37, 76.35, 77.38, 78.96, 100.03, 100.59, 101.38, 101.72,
146.28, 175.55, 176.59; Structure confirmed by a ROESY
experiment as detailed for 9; m/z = 768 [M+ + Na]; Anal.
calcd. for C29H47NO21: C 46.7, H 6.3; found: C 46.9, H 6.4.
2.8 Hz, 6.0 Hz, H-2cycl), 5.04 (1H, d, = 3.6 Hz, H-1fuc),
J
6.26 (1H, d, J = 6.0 Hz, H-1cycl); 13C (D2O, 62.9 MHz) δ:
18.74, 25.52, 43.14, 55.17, 62.58, 64.68, 66.08, 70.26,
70.80, 71.25, 71.58, 71.85, 72.94 (2 × C), 73.41, 75.37,
75.87, 76.37, 78.44, 79.15, 80.91, 99.52, 102.34, 103.26,
105.33, 147.85, 177.41, 178.50. The connections of the car-
bohydrate residues were confirmed by a ROESY experi-
ment. In detail, sialylation of the 3-OH of the galactosyl
moiety is shown by a strong crosspeak between the axial H-
3
sia and H-3gal. Fucosylation at H-3cycl is similarily shown by
a strong crosspeak between H-1fuc and H-3cycl. A further
strong crosspeak between H-4cycl and H-1gal corroborates the
attachment of the galactose at O-C(4) of the glucal moiety.
The fucosyl residue occupies the usual preferred position be-
low the galactosyl residue as evidenced by a weak crosspeak
between H-5fuc and H-2gal; m/z = 768 [M+ + Na]; Anal.
calcd. for C29H47NO21: C 46.7, H 6.3; found: C 46.9, H 6.3.
(1R,2R) 2-Hydroxy-cyclohexyl β-D-galactopyranoside (15):
At room temperature the imidate 13 (640 mg, 1.0 mmol) and
(R,R)-cyclohexan-1,2-diol (174 mg, 1.50 mmol) were dis-
solved in CH3CN (5 mL) and treated with trimetylsilyl
triflate (20 µL). After 1 h the solvent was rotovaped off and
the residue chromatographed on silica (petrolether
40/60:EtOAc, 2:3) to yield (1R,2R) 2-hydroxy-cyclohexyl
2,3,4,6-tetra-O-benzoyl-β-D-galactopyranoside (14) (506 mg,
73%). 14 was dissolved in MeOH (5 mL) containing cata-
lytic amounts of NaOMe and stirred for 6 h. Then the reac-
tion mixture was neutalized with AcOH, evaporated and
chromatographed on silica (toluene:EtOH, 1:1) to give 15
5-Acetamido-3,5-dideoxy-D-glycero-α -D-galacto-2-nonulopy-
ranosylonic acid-(2–3)-O-β-D-galactopyranosyl-(1–3)-glucal
(11): D-galβ(1–3)glucal 10 (28.8 mg, 93.4 µmol) (16),
CMP-sialic acid (30.0 mg, 113.8 µmol) and bovine serum
albumine (2.1 mg) were dissolved in a buffered solution
containing sodium cacodylate buffer (75 µL, 0.5 M), MnCl2-
solution (100 µL, 0.5 m) and bidistilled water (2.1 mL). The
clear solution was then incubated at 37°C with α(2–3)sialyl
30 U transferase (200 µL, 700 µU) and calf intestine alkaline
phosphatase (2 µL, 30 U) for 3 days. A second portion of
CMP-sialic acid (45.0 mg, 167 µmol) and sialyl transferase
(100 µL, 350 µU) were added and the incubation continued.
After 3 days the reaction mixture was lyophilized and
chromatographed on silica (CH2Cl2:MeOH:H2O, 10:4:0.1 to
6:4:1). The product containing fractions were passed over
1
(152 mg, 75%): H NMR (CD3OD, 400.13 MHz) δ: 1.19
(4H, m), 1.62 (2H, m), 1.98 (2H, m), 3.35 (2H, m, 2 ×
CHOcycl), 3.44 (3H, m, H-2,3,5gal), 3.63 (1H, dd, J = 6.6 Hz,
9.8 Hz, H-6), 3.71 (1H, dd, J = 7.4 Hz, 11.5 Hz, H-6′),
3.76 (1H, broad d, J = 4.1 Hz, H-4), 4.28 (1H, d, J = 7.4 Hz,
H-1).
1
sephadex G-10 to yield 11 (51.2 mg, 92%): H NMR (D2O,
250.13 MHz) δ: 1.81 (1H, t, J = 12.8 Hz, H-3sia), 2.04
(3H, s, Ac), 2.78 (1H, dd, J = 4.2 Hz, 12.8 Hz, H-3sia), 3.51–
4.06 (17H, m), 4.14 (1H, dd, J = 2.8 Hz, 9.8 Hz, H-3gal),
4.41 (1H, m), 4.41 (1H, m), 4.99 (1H, d, J = 8.3 Hz, H-1gal),
6.49 (1H, dd, J = 1.4 Hz, 6.2 Hz, Hvinyl); 13C (D2O,
62.89 MHz) δ: 24.18, 41.73, 53.81, 62.15, 63.14, 64.71,
69.07 (2 × C), 69.61, 70.22, 70.49, 71.26, 73.89, 74.96,
77.20, 77.93, 78.83, 80.08, 101.85, 102.00, 103.29, 146.92,
175.99, 177.15; m/z = 598 [M+ – H].
(1R,2R) 2-Hydroxy-cyclohexyl 3-O-[2-O-(5-acetamido-3,5-
dideoxy-D-glycero-α-D-galacto-2-nonulopyranosonate)]-β-D-
galactopyranoside (16): The cyclohexane derivative 15
(23.5 mg, 84.4 µmol), CMP-sialic acid (68.5 mg,
104.0 µmol) and bovine serum albumine (1.8 mg) were dis-
solved in a buffered solution containing sodium cacodylate
buffer (2 mL, 0.05 M), MnCl2-solution (2 mL, 0.06 m) and
bidistilled water (1.3 mL). The clear solution was incubated
at 37°C with α(2–3)sialyl transferase (100 µL, 700 µU) and
calf intestine alkaline phosphatase (2 µL, 30 U) for 24 h. Af-
ter the addition of a second portion of CMP-sialic acid
(10 mg, 15.2 µmol) the incubation was continued for 5 h.
The reaction mixture was then lyophilized and
5-Acetamido-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopy-
ranosylonic acid-(2–3)-O-β-D-galactopyranosyl-(1–4)-O-[α-
L-fucopysanosyl]-(1–3)-glucal (12): Trisaccharide 11 (18.0 mg,
30.0 µmol), GDP-fucose (26.3 mg, 41.4 µmol) and bovine
serum albumine (1.6 mg) were added to a mixture of sodium
© 2000 NRC Canada

Ernst et al.
899
chromatographed on silica (CH2Cl2:MeOH:H2O, 10:4:0.5)
to yield 16 (31.2 mg, 64%): 1H NMR (D2O, 250.13 MHz) δ:
1.30 (4H, m), 1.73 (2H, m), 1.85 (1H, t, J = 12.8 Hz, H-3sia),
2.08 (3H, s, Ac), 2.11 (2H, m), 2.80 (1H, dd, J = 4.2 Hz,
12.8 Hz, H-3sia), 3.51–3.96 (13H, m), 4.00 (1H, d, J =
2.8 Hz, H-4gal), 4.14 (1H, dd, J = 2.8 Hz, 8.3 Hz, H-3gal),
4.61 (1H, d, J = 8.3 Hz, H-1gal); 13C (D2O, 100.03 MHz) δ:
20.43, 21.76, 21.93, 28.15, 30.44, 37.94, 50.07, 59.48,
60.95, 65.97, 66.48, 66.80, 67.55, 70.14, 71.22, 71.30,
73.26, 74.19, 81.25, 98.27, 98.78, 172.34, 173.40.
400.13 MHz) δ: 1.22 (4H, m), 1.69 (3H, m), 2.08 (1H, m),
3.39 (1H, m), 3.50 (1H, m), 3.68 (2H, m, H-6,6′), 3.93
(1H, s, OH), 4.23 (1H, broad t, J = 4.4 Hz, H-5), 4.46 (1H,
d, J = 12.1 Hz, CH2Bn), 4.57 (1H, d, J = 12.1 Hz, CH2Bn),
4.87 (1H, d, J = 8.8 Hz, H-1), 5.59 (1H, dd, J = 7.7 Hz,
4.4 Hz, H-3), 5.76 (1H, dd, J = 8.8 Hz, 7.7 Hz, H-2), 5.97
(1H, dd, J = 4.4 Hz, 4.4 Hz, H-4), 7.24 (7H, m), 7.48
(6H, m), 7.64 (1H, m), 7.80 (2H, m), 7.98 (2H, m), 8.06
(2H, m); 13C (CDCl3, 100.03 MHz) δ: 23.65, 24.18, 30.75,
31.99, 67.62, 68.24, 70.00, 71.76, 72.87, 72.96, 73.64,
87.87, 101.90, 127.76–137.26 aromatics, 165.21, 165.51,
165.55; Anal. calcd. for C40H40O10: C 70.6, H 5.9; found: C
70.4, H 6.0.
2-O-{1-O-[(1R,2R) 2-O-(α-L-fucopyranosyl)cyclohexyl]β-D-
galactopyranos-3-yl} 5-acetamido-3,5-dideoxy-D-glycero-α-
D-galacto-2-nonulopyranosylonic acid (17): The sialic acid
derivative 16 (17.5 mg, 29.3 µmol), GDP-fucose (33.9 mg,
53.4 µmol) and bovine serum albumine (1.9 mg) were added
to a mixture of sodium cacodylate buffer (450 µL, 250 mM,
pH 6.5), MnCl2-solution (150 µL, 250 mM) and bidistilled
water (600 µL). The clear mixture was incubated at 37°C
with recombinant FucT III solution (83 µL, 498 µU/mL) and
calf intestine alkaline phosphatase (2 µL, 30 U) for one day,
until TLC control (CH2Cl2:MeOH, 6:4) of the turbid incuba-
tion mixture indicated that 16 was consumed. After
lyophilization the residue was chromatographed on silica
(CH2Cl2:MeOH, 6:4). The product containing fractions were
combined and finally passed over a sephadex G-10 column
and lyophilized to yield 17 (15.6 mg, 71%): [α]589 –17.8°
(1R,2R) 2-Hydroxy-cyclohexyl 6-O-benzyl-β-D-galactopy-
ranoside (20): The pseudodisaccharide 19 (2.0 g, 2.9 mmol)
was suspended in dry methanol (40 mL), a catalytic amount
of sodium methanolate was added and the reaction mixture
stirred at room temperature for 3 h until TLC control
(CH2Cl2:MeOH, 9:1) indicated complete consumption of the
starting material. Then the mixture was neutralized with
amberlyste 15 and filtered over celite. Evaporation leaves an
oil which is chromatographed on silica (CH2Cl2:MeOH,
19:1 to 9:1) to yield 20 (1.01 g, 94%): [α]589 –33.2° (c =
0.94, CH2Cl2); 1H NMR (CDCl3, 400.13 MHz) δ: 1.28
(4H, m), 1.73 (2H, m), 2.06 (2H, m), 3.40 (4H, m), 3.61
(1H, dd, J = 4.4 Hz, 8.8 Hz), 3.74 (4H, m), 4.01 (1H, t, J =
3.3 Hz), 4.34 (1H, d, J = 8.8 Hz, H-1), 4.60 (2H, q, 2 H, J =
12.1 Hz, CH2Bn), 7.43 (5H, m); 13C (CDCl3, 100.03 MHz)
δ: 23.84, 24.31, 30.98, 32.46, 69.00, 69.07, 71.34, 73.10,
73.48, 73.61, 73.66, 85.63, 102.43, 127.82, 128.46, 137.67;
Anal. calcd. for C19H28O7: C 61.9, H 7.7; found: C 61.7, H
7.7.
1
(c = 1.25, H2O); H NMR (D2O, 500.13 MHz) δ: 1.24 (3H,
d, J = 6.5 Hz, H-6fuc), 1.30 (4H, m), 1.76 (2H, m), 1.87 (1H,
t, J = 13.0 Hz, H-3sia), 2.09 (3H, s, Ac), 2.14 (1H, m), 2.19
(1H, m), 2.81 (1H, dd, J = 4.8 Hz, 13.0 Hz, H-3sia), 3.56
(1H, dd, J = 7.7 Hz, 9.5 Hz, H-2gal), 3.58 (1H, m, CHOcycl),
3.63–3.69 (4H, m, H-5gal, H-6,7,9sia), 3.70–3.79 (3H, m, H-
6gal, H-4,8sia), 3.80–3.86 (3H, m, H-2,4fuc, CHOcycl), 3.90
(1H, t, J = 10.0 Hz, H-5sia), 3.92–3.96 (2H, m, H-6gal, H-
8sia), 3.97 (1H, dd, J = 3.5 Hz, 10.0 Hz, H-3fuc), 3.98 (1H, d,
J = 3.2 Hz, H-4gal), 4.13 (1H, dd, J = 3.2 Hz, 9.5 Hz, H-
3gal), 4.63 (1H, d, J = 7.7 Hz, H-1gal), 4.68 (q, 1 H, J =
6.5 Hz, H-5fuc), 5.04 (1H, d, J = 5.5 Hz, H-1fuc); 13C (D2O,
100.03 MHz) δ: 15.55, 17.73, 22.36, 23.49, 29.54, 29.91,
39.90, 52.01, 61.75, 62.89, 66.78, 67.86, 68.25, 68.42,
68.74, 69.69, 69.87, 72.07, 72.34, 73.13, 74.92, 76.26,
77.45, 78.37, 95.77, 99.75, 100.17, 174.35, 175.34; Structure
confirmed by a ROESY experiment as detailed for 9; Anal.
calcd. for C29H49NO19: C 48.7, H 6.9; found: C 48.2, H 6.6.
Benzyl (2R) 3-cyclohexyl-2-O-{1-O-[(1R,2R) cyclohexyl]-6-
O-benzyl-β-D-galactopyranos-3-yl}propanoate (22a):
Glycoside 20 (0.98 g, 2.66 mmol) and dibutyltin oxide
(995 mg, 3.99 mmol) in MeOH (40 mL) were refluxed for
2.5 h to give a clear, colourless solution. After removal of
the solvent, the residue (white foam) was dissolved in dry
CH2Cl2 (29 mL), CsF (2.02 g,13.3 mmol) and the triflate
21a (5.25 g, 13.3 mmol) were added and the resulting solu-
tion stirred vigorously for 3 h. The reaction mixture was
then diluted with CH2Cl2 and washed with 10% KF–
KH2PO4 and brine. After drying the combined organic layers
with Na2SO4, the solvent was evaporated and the resulting
oil chromatographed on silica (hexane:EtOAc, 6:4) to yield
22a (1.25 g, 76%): [α]589 –40.7° (c = 0.59, CHCl3); 1H
NMR (CDCl3–D2O, 400.13 MHz) δ: 0.91 (2H, m), 1.28
(8H, m), 1.70 (9H, m), 2.04 (2H, m), 3.29 (1H, dd, J =
3.3 Hz, 8.8 Hz, H-2), 3.37 (1H, m, CHO-cyclohexanol), 3.44
(1H, m, CHO-cyclohexanol), 3.70 (3H, m, H-5, 6, 6′), 3.79
(1H, t, J = 3.3 Hz, H-4), 3.83 (dd, J = 3.3 Hz, 9.9 Hz, H-2),
4.28 (1H, dd, J = 2.3 Hz, 10.0 Hz), 4.31 (1H, d, J = 9.9 Hz,
H-1), 4.60 (2H, q, J = 12.1 Hz, CH2Bn), 5.19 (2H, q, J =
12.1 Hz, CH2Bn), 7.43 (10H, m); 13C (CDCl3, 100.03 MHz)
δ: 23.80, 24.34, 26.01, 26.21, 26.40, 31.21, 32.12, 32.20,
33.53, 33.92, 40.92, 66.83, 67.15, 68.95, 70.72, 73.05,
73.61, 73.66, 82.87, 86.78, 103.03, 127.72, 127.92, 128.40,
128.51, 128.62, 128.66, 135.12, 137.92, 174.72; Anal. calcd.
for C35H48O9: C 68.6, H 7.9; found: C 68.4, H 7.8.
(1R,2R)-2-Hydroxy-cyclohexyl 6-O-benzyl-2,3,4-tri-O-benzoyl-
β-D-galactopyranoside (19): Under argon 23.0 g of finely
ground 4 Å molecular sieves were added to a mixture of
ethyl 6-O-benzyl-2,3,4-tri-O-benzoyl-thio-β-D-galactopyranoside
18 (11.41 g, 18.0 mmol) (19) and (R,R)-trans-1,2-cyclo-
hexandiol (1.76 g, 15.2 mmol) in 100 mL dry CH2Cl2. After
stirring for 2 h at room temperature, dimethylmethylthio-
sulfonium triflate (DMTST) (7.83 g, 30.0 mmol) (20) dis-
solved in dry CH2Cl2 (70 mL) was added portionwise over
1 h. The slurry was stirred for 3 h and then diluted with
CH2Cl2. After filtration over celite, the organic phase was
washed with 10% NaHCO3 and brine, dried over NaSO4 and
concentrated. The residue was chromatographed on silica
(toluene:EtOAc, 95:1 to 85:15) to yield 19 (6.85 g, 67%):
[α]589 +105.2° (c = 0.79, CHCl3); 1H NMR (CDCl3,
© 2000 NRC Canada

900
Can. J. Chem. Vol. 78, 2000
Benzyl (2S) 3-cyclohexyl-2-O-{1-O-[(1R,2R) cyclohexyl]-6-
O-benzyl-β-D-galactopyranos-3-yl}propanoate (22b): Ac-
cording the protocol for the preparation of 22a, 22b (270 mg,
46%) was obtained starting from 20 (350 mg, 0.95 mmol)
and the triflate 21b (922 mg, 2.375 mmol): [α]589 –24.4°
Sodium (2S) 3-phenyl-2-O-{1-O-[(1R,2R) cyclohexyl]β-D-
galactopyranos-3-yl}propanoate (23b): Following the proto-
col for 23a, the pseudotrisaccharide 23b (235 mg, 72%) was
obtained from 22b (438 mg, 0.71 mmol) as a white solid:
1
[α]589 +21.6° (c = 2.1, H2O); H NMR (D2O, 400.13 MHz)
1
(c = 2.0, CHCl3); H NMR (CDCl3–D2O, 400.13 MHz) δ:
δ: 1.01 (2H, m), 1.29 (7H, m), 1.69 (10H, m), 2.08 (2H, m),
3.46 (1H, dd, J = 3.5 Hz, 12.9 Hz, H-3), 3.55 (4H, m, H-2,
5, 2 × CHO-cyclohexanol), 3.77 (1H, dd, J = 4.0 Hz,
12.1 Hz, H-6), 3.83 (1H, dd, J = 8.7 Hz, 12.1 Hz, H-6′), 4.07
(broad d, J = 3.1 Hz, H-4), 4.11 (1H, dd, J = 4.3 Hz,
7.8 Hz), 4.55 (1H, d, J = 7.8 Hz, H-1); 13C (D2O,
100.03 MHz) δ: 25.54; 25.72, 27.89, 28.08, 28.34, 32.00,
33.83, 34.20, 35.82, 35.84, 42.74, 63.36, 68.25, 71.66,
75.14, 77.50, 80.34, 83.31, 85.16, 103.03, 184.16; m/z =
431.2 [M+ – Na].
0.94 (2H, m), 1.26 (8H, m), 1.72 (9H, m), 2.06 (2H, m),
3.35 (2H, m), 3.48 (1H, m, CHO-cyclohexanol), 3.78
(4H, m), 3.83 (dd, J = 3.3 Hz, 9.9 Hz, H-2), 4.28 (1H, dd,
J = 2.3 Hz, 10.0 Hz), 4.31 (1H, d, J = 9.9 Hz, H-1), 4.60
(2H, q, J = 12.1 Hz, CH2Bn), 5.18 (2H, q, J = 12.1 Hz,
CH2Bn), 7.43 (10H, m); 13C (CDCl3, 100.03 MHz) δ: 23.79,
24.27, 25.98, 26.25, 26.36, 30.84, 32.18, 32.25, 33.83,
33.98, 40.13, 67.11, 68.55, 69.81, 71.29, 73.16, 73.67,
73.76, 82.32, 87.35, 100.31, 127.86, 127.91, 128.41, 128.52,
128.58, 128.66, 134.98, 137.44, 169.06.
Sodium (2R) 3-phenyl-2-O-{1-O-[(1R,2R) cyclohexyl]β-D-
galactopyranos-3-yl}propanoate (23c): Following the proto-
col for 23a, the pseudotrisaccharide 23c was obtained
(155 mg, 50%) from 22c (420 mg, 0.69 mmol) as a white
solid: [α]589 –34.9° (c = 2.1, H2O); 1H NMR (D2O,
400.13 MHz) δ: 1.23 (4H, m), 1.69 (2H, m), 2.00 (2H, m),
2.99 (1H, dd, J = 12.0 Hz, 13.2 Hz, CH2Ph), 3.13 (1H, dd, J
= 4.4 Hz, 13.2 Hz, CH2Ph), 3.30 (1H, dd, J = 4.4 Hz,
7.7 Hz, H-3), 3.51 (3H, m, H-2, 2 × CHO-cyclohexanol),
3.51 (1H, m, H-5), 3.70 (1H, dd, J = 8.7 Hz, 12.1 Hz, H-6′),
4.88 (1H, dd, J = 9.9 Hz, 12.1 Hz, H-6), 3.92 (broad d, J =
3.1 Hz, H-4), 4.14 (1H, dd, J = 4.4 Hz, 12.0 Hz, CHBn),
4.39 (1H, d, J = 8.8 Hz, H-1), 7.32 (5H, m, phenyl); 13C
(D2O, 100.03 MHz) δ: 23.67, 23.83, 30.10, 32.34, 39.62,
61.48, 66.42, 70.18, 73.23, 74.86, 82.40, 82.89, 83.41,
100.82, 127.00, 128.87, 129.81, 138.41, 180.90; m/z = 425
[M+ – Na].
Benzyl (2R) 3-phenyl-2-O-{1-O-[(1R,2R) cyclohexyl]-6-O-
benzyl-β-D-galactopyranos-3-yl}propanoate (22c): Accord-
ing the protocol for the preparation of 22a, 22c (437 mg,
82%) was obtained starting from 20 (320 mg, 0.87 mmol)
and R-phenyl lactic acid dervivative 21c (1.68 g,
4.343 mmol): [α]589 –26.4° (c = 2.0, CHCl3); 1H NMR
(CDCl3, 400.13 MHz) δ: 1.28 (4H, m), 1.72 (3H, m), 2.00
(2H, m), 2.99 (1H, dd, J = 9.5 Hz, 14.4 Hz, CH2Ph), 3.11
(1H, dd, J = 3.2 Hz, 9.5 Hz, H-3), 3.20 (1H, dd, J = 3.2 Hz,
14.4 Hz, CH2Ph), 3.29 (2H, m, H-5, CHO-cyclohexanol),
3.41 (1H, m, CHO-cyclohexanol), 3.58 (1H, dd, J = 5.7 Hz,
6.4 Hz, H-6), 3.68 (3H, m, H-2, 6′, OH), 3.76 (1H, m, H-4),
4.06 (1H, broad s, OH), 4.20 (1H, d, J = 8.0 Hz, H-1), 4.32
(dd, J = 3.2 Hz, 9.5 Hz, BnCHO), 4.51 (1H, d, J = 11.1 Hz,
OCH2Ph), 4.61 (1H, d, J = 11.1 Hz, OCH2Ph), 5.16 (1H, d,
J = 11.1 Hz, OCH2Ph), 5.22 (1H, d, J = 11.1 Hz, OCH2Ph),
7.43 (15H, m); 13C (CDCl3, 100.03 MHz) δ: 23.80, 24.32,
31.05, 32.24, 39.54, 66.54, 67.49, 68.82, 70.34, 73.00,
73.40, 73.65, 80.22, 84.19, 86.52, 105.48, 127.19, 127.69,
127.88, 128.37, 128.63, 128.70, 128.76, 129.20, 134.83,
136.66, 137.91, 173.17; m/z = 624 [M+ + NH4].
(2R) 3-cyclohexyl-2-O-{1-O-[(1R,2R) 2-O-(α-L-fucopyrano-
syl)cyclohexyl]β-D-galactopyranos-3-yl}propionic acid (24a):
The pseudotrisaccharide 23a (42.5 mg, 93.5 µmol), GDP-
fucose (79.1 mg, 124.5 µmol) and bovine serum albumine
(1.5 mg) were added to a mixture of sodium cacodylate
buffer (450 µL, 250 mM, pH 6.5), MnCl2-solution (150 µL,
250 mM) and bidistilled water (600 µL). The clear mixture
was incubated at 37°C with recombinant FucT III (100 µL,
200 mU) and calf intestine alkaline phosphatase (3 µL, 45
U) for two days. The turbid incubation mixture was ab-
sorbed on a C-18 reversed phase column. After washing
with H2O, the product was eluted with MeOH. MeOH was
then evaported and the residue chromatographed on silica
(CH2Cl2:MeOH:H2O, 10:3:0.3). The product containing
fractions were combined and finally passed over a sephadex
G-10 column to yield 24a (30.2 mg, 54%). An analogous in-
cubation with recombinant FucT VI gave no detectable
amounts of 24a: [α]589 –42.4° (c = 0.4, H2O); 1H NMR
(CD3OD, 400.13 MHz) δ: 0.77–0.95 (2H, m); 1.12 (3H, d,
J = 6.6 Hz, H-6fuc); 1.12–1.38 (7H, m); 1.45–1.74 (9H, m);
1.84 (1H, broad d, J = 12.5 Hz,); 1.94–2.05 (2H, m); 3.13
(1H, dd, J = 2.3, 7.7 Hz, H-3gal); 3.41 (1H, broad t, J =
4.4 Hz, H-5gal); 3.43–3.52 (2H, m, H-2gal, CHOcycl); 3.57–
3.74 (5H, m, H-6,6′gal, H-2,3fuc, CHOcycl); 3.59–3.86 (3H, m,
H-4gal, H-4fuc, CH(O)O); 4.25 (1H, d, J = 8.6 Hz, H-1gal);
4.50 (1H, q, J = 6.6 Hz, H-5fuc); fucose H-1 beyond water
(DHO) at 4.80 ppm; 13C (CD3OD, 100.03 MHz, shifts in
Sodium (2R) 3-cyclohexyl-2-O-{1-O-[(1R,2R) cyclohexyl]β-
D-galactopyranos-3-yl}propanoate (23a): The benzyl pro-
tected 22a (287 mg, 0.46 mmol) was dissolved in a mixture
of dioxane (9 mL), water (4.5 mL) and acetic acid (2.3 mL).
After addition of 300 mg (20%) of Pd (OH)2, the resulting
slurry was vigorously stirred for 22 h at room temperature in
an atmosphere of hydrogen. The reaction mixture was then
filtered and evaporated three times with water. The residue
was dissolved in H2O–MeOH and treated with Dowex 50
(Na+ form). Filtration and lyophilization gave a white pow-
der which was chromatographed on RP-18 silica (H2O–
MeOH, 7:3) to yield 23a (141 mg, 66%): [α]589 –43.2° (c =
0.21, CHCl3); 1H NMR (D2O, 400.13 MHz) δ: 0.92 (2H, m),
1.23 (7H, m), 1.62 (10H, m), 2.04 (2H, m), 3.41 (1H, dd, J =
3.7 Hz, 12.8 Hz, H-3), 3.55 (3H, m, H-2, 2xCHO-
cyclohexanol), 3.66 (1H, ddd, J = 0.9 Hz, 4.6 Hz, 8.2 Hz, H-
5), 3.74 (1H, q, J = 11.9 Hz, H-6), 3.76 (q, J = 11.9 Hz, H-
6′), 3.92 (1H, dd, J = 3.5 Hz, 3.7 Hz, H-4), 4.49 (1H, d, J =
8.3 Hz, H-1); 13C (D2O, 100.03 MHz) δ: 22.12; 22.29,
24.67, 24.93, 28.60, 30.62, 30.80, 32.03, 32.38, 39.97,
59.96, 65.06, 68.68, 71.70, 73.30, 78.18, 81.59, 81.97,
99.62, 181.41; m/z = 432 [M+ – Na].
© 2000 NRC Canada

Ernst et al.
901
ppm relative to solvent) δ: 16.56; 24.49; 27.26; 27.56;
27.83; 29.91; 30.95; 33.49; 34.74; 35.50; 43.32; 63.15;
67.43; 67.72; 69.89; 71.45; 71.68; 73.78; 75.87; 77.04;
79.24; 80.76; 85.53; 96.84; 102.60; 183.40; m/z = 623 [M+ +
Na]; Anal. calcd. for C27H46O13: C 56.0, H 8.0; found: C
55.8, H 7.8.
0.7, 3.7 Hz, H-4), 7.19–8.09 (20H, m, aromatics); 13C
(CDCl3, 100.03 MHz) δ: 25.02, 25.30, 32.42, 32.97, 68.13,
68.54, 71.05, 71.51, 72.89, 74.52, 74.98, 85.11, 101.00,
127.76–135.61 aromatics, 165.21, 165.51, 165.55.
(1S,2S) 2-Hydroxy-cyclohexyl 6-O-benzyl-β-D-galactopyr-
anoside (26): According the protocol for the preparation of
compound 15, 26 (115 mg, 82%) was obtained from
glycoside 25 (262 mg, 0.39 mmol): [α]589 +5.6° (c = 1.5,
(2S) 3-cyclohexyl-2-O-{1-O-[(1R,2R) 2-O-(α-L-fucopyrano-
syl)cyclohexyl]β-D-galactopyranos-3-yl}propionic acid (24b):
Following the protocol for 24a, 24b (34.1 mg, 61%) was ob-
tained from the pseudotrisaccharide 23b (42.5 mg,
93.5 µmol) and GDP-fucose (81.8 mg, 128.7 µmol): 1H
NMR (CD3OD, 400.13 MHz) δ: 0.78–0.92 (2H, m); 1.09 (d,
3-H, J = 6.6 Hz, H-6fuc); 1.10–1.37 (7H, m); 1.48–1.70
(9H, m); 1.80 (1H, broad d, J = 12.6 Hz); 1.91–2.00
(2H, m); 3.23 (1H, dd, J = 2.6 Hz, 7.8 Hz, H-3gal); 3.32 (1H,
broad t, J 6.0 Hz, H-5gal); 3.42–3.70 (7H, m, H-2,6,6′gal, H-
2,4fuc, 2 × CHOcycl); 3.80 (1H, dd, J = 4.2 Hz, 10.8 Hz, H-
3fuc); 3.88 (1H, d, J = 4.8 Hz, H-4gal); 3.98 (1H, m,
CH(O)O); 4.23 (1H, d, J = 7.2 Hz, H-1gal); 4.47 (1H, q, J =
6.6 Hz, H-5fuc); fucose H-1 beyond water (DHO) at
4.80 ppm; 13C (CD3OD, 100.03 MHz) δ: 16.58; 24.37;
27.28; 27.46; 27.76; 29.85; 30.87; 33.64; 35.17; 35.35;
43.22; 62.79; 67.39; 67.59; 70.00; 70.19; 70.55; 73.81;
76.36; 76.93; 79.29; 82.45; 85.53; 96.85; 102.91; C=O not
resolved; m/z = 623 [M+ + Na]; Anal. calcd. for C27H46O13:
C 56.0, H 8.0; found: C 55.6, H 7.9.
1
CHCl3); H NMR (CD3OD, 400.13 MHz) δ: 1.21 (4H, m),
1.61 (2H, m), 1.87 (1H, m), 2.12 (1H, m), 3.29 (1H, m,
CHO-cyclohexanol), 3.38 (1H, m, CHO-cyclohexanol), 3.40
(1H, dd, J = 3.7 Hz, 10.3 Hz, H-3), 3.48 (1H, dd, J = 7.8 Hz,
10.3 Hz, H-2), 3.52 (3H, m, H-5, 6, 6′), 3.73 (1H, d, J =
3.5 Hz, H-4), 4.34 (1H, d, J = 7.8 Hz, H-1), 4.50 (2H, m,
CH2Bn), 7.23 (5H, m); 13C (CD3OD, 100.03 MHz) δ: 24.28,
24.37, 32.49, 32.93, 69.61, 69.78, 72.14, 73.19, 73.87,
73.97, 74.06, 86.38, 105.24, 127.60, 127.86, 137.67; m/z =
391 [M+ + Na].
Benzyl (2R) 3-cyclohexyl-2-O-{1-O-[(1S,2S) cyclohexyl]-6-
O-benzyl-β-D-galactopyranos-3-yl}propanoate (27): Accord-
ing the protocol for the preparation of compound 22a, 27
(94 mg, 59%) was obtained from 26 (96 mg, 0.26 mmol)
and the R-cyclohexyl lactic acid derivative 21a (514 mg,
1
1.30 mmol): [α]589 –4.5° (c = 1.5, chloroform); H NMR
(CDCl3, 400.13 MHz) δ: 0.92 (2H, m), 1.22 (6H, m), 1.46
(2H, m), 1.69 (9H, m), 2.00, (1H, m), 2.11 (1H, m), 3.25
(2R) 3-phenyl-2-O-{1-O-[(1R,2R) 2-O-(α-L-fucopyranosyl)cyclo-
hexyl]β-D-galactopyranos-3-yl}propionic acid (24c): Fol-
lowing the protocol for 24a, 24c (16.0 mg, 96%) was ob-
tained from the pseudotrisaccharide 23c (12.5 mg,
29.3 µmol) and GDP-fucose (28.1 mg, 44.2 µmol): [α]589
(1H, dd, J = 3.7 Hz, 9.2 Hz, H-3), 3.38 (2H, m, CHOcycl
,
OH), 3.51 (1H, m, CHOcycl), 3.59 (1H, t, J = 6.1 Hz, H-5),
3.75 (3H, m, H-4, 6, 6′), 3.89 (dd, J = 7.9 Hz, 9.2 Hz, H-2),
4.16 (1H, dd, J = 4.3 Hz, 9.2 Hz, CHC(O)O), 4.49 (1H, d, J
= 7.9 Hz, H-1), 4.57 (1H, d, J = 11.9 Hz, CH2Bn), 4.60 (1H,
d, J = 11.9 Hz, CH2Bn), 5.14 (1H, d, J = 12.2 Hz, CH2Bn),
5.19 (1H, d, J = 12.2 Hz, CH2Bn), 7.35 (10H, m); 13C
(CDCl3, 100.03 MHz) δ: 25.67, 24.47, 25.95, 26.12, 26.32,
32.03, 32.21, 32.43, 33.61, 33.75, 40.84, 66.63, 67.17,
69.39, 71.09, 73.48, 73.58, 74.33, 77.34, 83.91, 86.92,
104.39, 127.61, 127.67, 128.34, 128.48, 128.62, 135.05,
138.19, 174.77; m/z = 657 [M+ + HCOO].
1
–84.4° (c = 0.8, H2O); H NMR (CD3OD, 400.13 MHz) δ:
1.18 (3H, d, J = 6.6 Hz, H-6fuc); 1.18–1.29 (4H, m); 1.62–
1.74 (2H, m); 2.00–2.11 (2H, m); 2.97 (1H, dd, J = 8.4 Hz,
12.0 Hz, CH2Ph); 3.14 (1H, dd, J = 5.4, 12.6 Hz, CH2Ph);
3.28 (1H, dd, J = 3.0, 9.6 Hz, H-3gal); 3.44–3.56 (3H, m, H-
2,5gal, CHOcycl); 3.65–3.80 (5H, m, H-6,6′gal, H-2,3fuc
,
CHOcycl); 3.88–3.93 (2H, m, H-4gal, H-4fuc); 4.17 (1H, dd,
J = 5.4 Hz, 9.6 Hz, CH(O)O); 4.28 (1H, d, J = 7.2 Hz, H-
1gal); 4.60 (1H, q, J = 6.6 Hz, H-5fuc); 4.96 (1H, d, J =
3.2 Hz, H-1fuc); 7.31 (1H, m, Ph); 7.39 (4H, m, Ph);13C
(CD3OD, 100.03 MHz) δ: 16.54; 24.43; 24.48; 29.91; 30.87;
41.44; 47.92; 63.05; 67.40; 69.91; 71.46; 71.60; 73.79; 75.89;
77.00; 79.13; 83.94; 85.94; 85.58; 96.85; 102.13; 127.23;
129.23 (2 × C); 130.62 (2 × C); 140.61; 181.49; m/z = 617
[M+ + Na]; Anal. calcd. for C27H40O13: C 56.6, H 7.0;
found: C 56.2, H 6.8.
Sodium (2R) 3-cyclohexyl-2-O-{1-O-[(1S,2S) cyclohexyl]β-
D-galactopyranos-3-yl}propanoate (28): According the pro-
tocol for the preparation of compound 23a, 28 (40 mg, 58%)
is obtained from 27 (93 mg, 0.15 mmol): [α]589 –3.1° (c =
1
1.5, H2O); H NMR (D2O, 400.13 MHz) δ: 0.92 (2H, m),
1.22 (7H, m), 1.62 (9H, m), 1.79 (1H, m), 1.97 (1H, m),
2.12 (1H, m), 3.41 (1H, dd, J = 3.9 Hz, 9.8 Hz, H-3), 3.55
(2H, m, 2 × CHOcycl), 3.61 (1H, dd, J = 8.2 Hz, 9.8 Hz, H-
2), 3.66 (1H, broad dd, J = 3.1, 5.5 Hz, H-5), 3.74 (2H, m,
H-6, 6′), 3.91 (1H, d, J = 3.1 Hz, H-4), 3.96 (1H, m,
CHC(O)O), 4.56 (1H, d, J = 8.2 Hz, H-1); 13C (D2O,
100.03 MHz) δ: 21.45, 21.71, 23.79, 24.06, 24.24, 29.73,
29.96, 30.25, 31.36, 31.70, 39.32, 59.01, 64.23, 68.56,
71.70, 72.56, 77.49, 80.79, 83.52, 101.58, 180.76; m/z = 432
[M+].
(1S,2S)-2-Hydroxy-cyclohexyl 6-O-Benzyl-2,3,4-tri-O-benzoyl-
β-D-galactopyranoside (25): According the protocol for the
preparation of compound 14, 25 (265 mg, 59%) is obtained
from ethyl 6-O-benzyl-2,3,4-tri-O-benzoyl-1-thio-β-D-galacto-
pyranoside (500 mg, 0.80 mmol) and (S,S) cyclohexan-1,2-
1
diol (77 mg, 0.66 mmol): H NMR (CDCl3, 400.13 MHz) δ:
1.24 (3H, m), 1.46 (1H, m), 1.70 (2H, m), 1.97 (1H, m),
2.14 (1H, m), 3.45 (2H, m, 2 × CHOcycl), 3.71 (2H, m, H-6,
6′), 4.19 (dt, 1 H, J = 0.7, 5.9 Hz, H-5), 4.46 (1H, d, J =
12.1 Hz, CH2Bn), 4.54 (1H, d, J = 12.1 Hz, CH2Bn), 4.98
(1H, d, J = 7.3 Hz, H-1), 5.60 (1H, dd, J = 3.7 Hz, 11.4 Hz,
H-3), 5.76 (1H, dd, J = 7.3,11.4 Hz, H-2), 5.94 (1H, dd, J =
Benzyl (2R) 3-cyclohexyl-2-O-[1-O-ethyl-β-D-thio-galactopy-
ranos-3-yl]propanoate (30): Thioglycoside 29 (5.0 g,
22.29 mmol) and di-nbutyl tinoxide (6.7 g, 26.8 mmol) were
refluxed in methanol (150 mL) for 2 h. The methanol was
then evaporated and the residue thoroughly dried. The
© 2000 NRC Canada

902
Can. J. Chem. Vol. 78, 2000
resulting foam was dissolved in dimethoxyethane (40 mL)
and treated with triflate 21a (13.2 g, 33.4 mmol) (20) and
cesium fluoride (4.1 g, 26.8 mmol) in dimethoxyethane
(20 mL) at room temperature for 90 min. The turbid mixture
was diluted with EtOAc and then washed with 2N potassium
dihydrogenphosphate containing 15 g KF (200 mL), 10%
potassium fluoride solution and brine. The organic phase
was dried with sodium sulfate and evaporated. The residue
was purified on silica (acetone:hexane, 1:4 to 1:2) yielding
OCH2Ph), 5.19 (1H, d, J = 17.6 Hz, OCH2Ph), 5.62 (1H, t, J
= 12.0 Hz, H-2), 5.92 (1H, d, J = 3.3 Hz, H-4), 7.58
(14H, m, aromatics), 8.15 (6H, m, aromatics); 13C (CDCl3,
100.03 MHz) δ: 16.71, 18.23, 25.30, 25.56, 25.87, 32.35,
33.14, 33.21, 40.17, 62.53, 66.46, 69.52, 70.34, 71.88,
72.54, 78.02, 84.29, 101.29, 128.10, 128.20, 128.23, 128.32,
128.35, 128.38, 129.31, 129.40, 129.70, 129.91, 132.88,
132.98, 133.13, 135.25, 164.70, 165.53, 165.95, 172.20; m/z
= 831 [M+ + Na].
1
30 (7.0 g, 67%): H NMR (CDCI3, 400. 13 MHz) δ: 0.92
Sodium (2R) 3-cyclohexyl-2-O-{2-O-[(2R,3R)butyl]β-D-thio-
galactopyranos-3-yl}propanoate (33): The protected glyco-
side 32 (500 mg, 0.62 mmol) and 10 eq. of LiOH × H2O
(0.26 g) in a mixture of water (15 mL) and methanol (5 mL)
were dissolved and stirred for 5 h at 60°C. After evaporation
of the solvent the residue was chromatographed on a P-2 gel,
passed over Dowex 50 (Na+ form) and finally
chromatographed on RP-18 silica (H2O:MeOH, 9.5:0.5 to
1:1) to yield 33 (250 mg, 94%) as a white solid: [α]589
(2H, m, cycl), 1.23 (4H, m, cycl), 1.32 (3H, t, J = 8.8 Hz,
CH3), 1.68 (7H, m), 2.29 (broad, OH), 2.41 (broad, OH),
2.78 (2H, m, CH2S), 3.30 (1H, dd, J = 2.2 Hz, 9.9 Hz, H-3),
3.41 (broad, OH), 3.49 (1H, m), 3.73 (1H, m), 3.86 (2H, m),
3.93 (1H, m), 4.30 (2H, m), 5. 8 (1H, d, J = 13.2 Hz,
CH2O), 5.12 (1H, d, J = 13,2 Hz, CH2O), 7.38 (5H, m, aro-
matic).
Benzyl (2R) 3-cyclohexyl-2-O-[1-O-ethyl-2,4,6-tri-O-benzoyl-
β-D-thio-galactopyranos-3-yl]propanoate (31): 30 (7.0 g,
14.9 mmol) was dissolved in pyridine (75 mL), cooled to
0°C, and treated with benzoyl chloride (18.88 g,134.4 mmol)
and a catalytic amount of DMAP (0.5 g). The mixture was
stirred for 6 h at room temperature and then evaporated. The
resulting residue was dissolved in EtOAc and subsequently
washed with 0.1 N HCl, sat. NaHCO3, and finally with
brine. The combined organic layers were dried with Na2SO4
and evaporated. The residue was chromatographed on silica
1
–35.1° (c = 1.9, water); H NMR (D2O, 400.13 MHz) δ:
0.96 (2H, m), 1.19 (8H, m), 1.64 (7H, m), 1.81 (1H, m),
3.41 (1H, dd, J = 2.3, 9.4 Hz, H-3), 3.58 (1H, t, J = 9.4 Hz,
H-2), 3.66 (1H, m, H-5), 3.74 (4H, m, H-6,6′, 2 × CHCH3),
3.91 (1H, broad t, J = 2.3 Hz, H-4), 3.97 (1H, m,
OCHC(O)ONa), 4.45 (1H, d, J = 9.4 Hz, H-1); 13C (D2O,
100.03 MHz) δ: 17.00, 19.16, 27.45, 27.71, 27.90, 33.59,
35.01, 35.36, 42.96, 62.93, 68.03, 71.72, 72.24, 76.22,
81.17, 81.56, 102.52, 184.46; MS found: 405.3 [M+ – Na].
1
(EtOAc:hexane, 1:6) to give 31 (10.7 g, 91%): H NMR
(CDCI3, 400. 13 MHz) δ: 0.50 (2H, m, cycl), 0.71 (1H, m,
cycl) 0.84 (2H, m, cycl), 1.07 (1H, m, cycl), 1.29 (6H, m,
cycl, 3H, CH3), 2.78 (2H, m, CH2S), 3.91 (1H, dd, J = 2.2,
9.9 Hz, H-3), 4.01 (broad t, 1H; J = 6.6 Hz, H-5), 4.20
(1H, m, CHO), 4.43 (1H, dd, J = 6.6, 12.1 Hz, H-6), 4.49
(dd,1 H, J = 8.8, 12.1 Hz, H-6′), 4.62 (1H, d, J = 9.9 Hz, H
1), 5.08 (1H, d, J = 13.2 Hz, CH2O), 5.21 (1H, d, J =
13.2 Hz, CH2O), 5.68 (1H, t, J = 9.9 Hz, H-2), 6.00 (1H, d,
J = 2.2 Hz, H-4), 7.33 (5H, m, aromatic), 7.47 (6H, m, aro-
matic), 7.59 (3H, m, aromatic), 8.10 (6H, m, aromatic).
(2R) 3-cyclohexyl-2-O-{2-O-[(2R,3R) 3-O-(α-L-fucopyano-
syl)butyl]β-D-thio-galactopyranos-3-yl}propionic acid (34):
The pseudotrisaccharide 33 (20.9 mg, 51.5 µmol), GDP-
fucose (47.0 mg, 74.0 µmol) and bovine serum albumine
(2.8 mg) was treated with sodium cacodylate buffer (450 µL,
250 mM, pH 6.5), MnCl2-solution (150 µL, 250 mM) and
bidistilled water (600 µL). The clear mixture was incubated
at 37°C with recombinant FucT III solution (210 µL, 410
mU) and calf intestine alkaline phosphatase (3 µL, 45 U) for
two days until TLC control (CH2Cl2:MeOH, 6:4) indicated
that the starting material was completely consumed. After
lyophilization the residue was chromatographed on silica
(CH2Cl2:MeOH, 6:4). The product containing fractions were
combined, passed over a sephdex G-10 column and
lyophilized to yield 34 (13.3 mg, 47%):[α]589 –88.1° (c =
Benzyl (2R) 3-cyclohexyl-2-O-{2-O-[(2R,3R)butyl]2,4,6-tri-
O-benzoyl-β-D-thio-galactopyranos-3-yl}propanoate (32):
The thioglycoside 31 (500 mg, 0.64 mmol) and (R,R)-2,3-
butandiol (0.29 g, 3.20 mmol) were dissolved in CH2Cl2
(5 mL). After the addition of molecular sieves 4 Å (0.25 g),
the mixture was stirred for 1 h at room temperaure. Within
1 h, three portions of DMTST (0.5 eq each) (19) were
added. After strring for an additional hour, the suspension
was filtered on celite, diluted with CH2Cl2 and washed with
sat. NaHCO3, brine, and water. The residue obtained after
evaporation of the organic phase was chromatographed on
silica (EtOAc:hexane, 1:4 to 1:2) yielding 32 (367 mg,
1
1.19, water); H NMR (D2O, 400.13 MHz) δ: 0.91 (2H, m),
1.19 (12H, m), 1.59 (7H, m), 1.78 (1H, m), 3.40 (1H, dd, J =
3.3 Hz, 9.9 Hz, H-3gal), 3.51–3.81 (6H, m, 1 × CHCH3, H-
2gal, H-6,6′gal, H-2,3,4fuc), 3.89, (2H, m, H-4,5gal), 3.98
(2H, m, 1 × CHCH3, CHC(O)O), 4.35 (1H, q, J = 6.5 Hz, H-
5fuc), 4.46 (1H, d, J = 9.8 Hz, H-1gal), 4.99 (1H, d, J =
3.3 Hz, H-1fuc); 13C (D2O, 100.03 MHz) δ: 14.26, 16.10,
16.52, 26.26, 26.35, 26.52, 32.17, 33.48, 34.91, 41.74,
61.30, 65.22, 67.39, 68.28, 69.91, 70.00, 72.35, 74.84,
75.22, 77.74, 79.57, 82.97, 96.35, 101.30, C=O not resolved;
Anal. calcd. for C25H44O13: C 54.3, H 8.0; found: C 54.8, H
7.8.
1
70%): [α]589 +20.1° (c = 2.1, CHCl3); H NMR (CDCl3,
400.13 MHz) δ: 0.53 (2H, m), 0.71 (1H, m), 0.89 (2H, m),
0.95 (3H, d, J = 8.8 Hz, CH3), 1.09 (3H, d, J = 8.8 Hz,
CH3), 1.34 (8H, m), 3.41 (1H, broad s, OH), 3.43 (1H, dq,
J = 2.2 Hz, 8.8 Hz, CHCH3), 3.60 (1H, dq, J = 2.2 Hz,
8.8 Hz, CHCH3) 3.91 (1H, dd, J = 3.3 Hz, 9.9 Hz, H-3),
3.99 (1H, broad t, J = 8.8 Hz, H-5), 4.19 (1H, dd, J =
3.3 Hz, 8.9 Hz, OCHC(O)OBn), 4.40 (1H, dd, J = 12.1,
14.8 Hz, H-6), 4.52 (1H, dd, J = 7.6, 14.8 Hz, H-6′), 4.59
(1H, d, J = 12.0 Hz, H-1), 5.06 (1H, d, J = 17.6 Hz,
Acknowledgement
We thank Dr. Hitoshi Kusakabe, YAMASA Corp., Tokyo,
Japan for the generous gift of GDP-fucose.
© 2000 NRC Canada

Ernst et al.
903
Eur. J. 3, 1571 (1997); H.C. Kolb and B. Ernst. Pure Appl.
Chem. 69, 1879 (1997).
References
8. G. Baisch, R. Öhrlein, A. Katopodis, and B. Ernst. Bioorg.
Med. Chem. Lett. 6, 759 (1996); R. Öhrlein, G. Baisch, A.
Katopodis, M. Streiff, and F. Kolbinger. J. Mol. Catal. B:
Enzym. 5, 125 (1998); G. Baisch, R. Öhrlein, and M. Streiff.
Bioorg. Med. Chem. Lett. 8, 161 (1998).
1. S.R. Sharar, R.K. Winn, and J.M. Harlan. Springer Semin.
Immunopathol. 16, 359 (1995); Physiology and pathophysi-
ology of leukocyte adhesion. Edited by D.N. Granger and
G.W. Schmid-Schönbein. New York, Oxford University Press.
1995.
2. J. Bischoff. Trends Glycosci. Glycotechnol. 6, 351 (1994);
R.P. McEver. Curr. Opin. Immunol. 6, 75 (1994); L.A. Lasky.
Annu. Rev. Biochem. 64, 113 (1995); S.A. Mousa and D.A.
Cheresh. TDD (Trends in Drug Design) 2, 187 (1997); G.S.
Kansas. Blood, 88, 3259 (1996).
3. L. Phillips, E. Nudelman, F.C.A. Gaeta, M. Perez, A.K.
Singhal, S.-I. Hakomori, and J.C. Paulson. Science (Washing-
ton, D.C.), 250, 1130 (1990); G. Walz, A. Aruffo, W. Kolanus,
M. Bevilacqua, and B. Seed. Science (Washington, D.C.), 250,
1132 (1990); M.J. Polley, M.L. Phillips, E. Wayner, E.
Nudelman, A.K. Singhal, S.-I. Hakomori, and J.C. Paulson.
Proc. Natl. Acad. Sci. U.S.A. 88, 6224 (1991).
4. J.L. Magnani. Science (Washington, D.C.), 250,1132 (1990);
E.L. Berg, M.K. Robinson, O. Mansson, E.C. Butcher, and
J.L. Magnani. J. Biol. Chem. 266,14869 (1991).
9. R. Öhrlein, G. Baisch, A. Katopodis, M. Streiff, and F.
Kolbinger. Bioorg. Med. Chem. Lett. 7, 2447 (1997); R.
Öhrlein, G. Baisch, and A. Katopodis. Bioorg. Med. Chem.
Lett. 7, 2431 (1997); G. Baisch, R. Öhrlein, M. Streiff, and F.
Kolbinger. Bioorg. Med. Chem. Lett. 8, 755 (1998).
10. B. Ernst and R. Öhrlein. Glycoconjugate J. 16, 161 (1999),
and refs. therein; R. Öhrlein. Top. Curr. Chem. 200, 227
(1999), and refs. therein; S. Ogawa, N. Matsunaga, H. Li, and
M.M. Palcic. Eur. J. Org. Chem. 631 (1999); P.-P. Lu, O.
Hindsgaul, H. Li, and M.M. Palcic. Can. J. Chem. 75, 790
(1997); J.A.L.M. van Dorst, J.P. Kamerling, and J.F.G.
Vliegenthart. Pure Appl. Chem. 69, 537 (1997); M.M. Palcic
and O. Hindsgaul. Trends Glycosci. Glycotechnol. 8, 37 (1996),
and refs therein; Y. Nishida, T. Wiemann, and J. Thiem. Tetra-
hedron Lett. 34, 2905 (1993); C.-H. Wong, Y. Ichikawa, T.
Krach, C. Gautheron-Le Narvor, D.P. Dumas, and G.C. Look.
J. Am. Chem. Soc. 113, 8137 (1991).
5. E.L. Berg, J.L. Magnani, R.A. Warnock, M.K. Robinson, and
E.C. Butcher. Biochem. Biophys. Res. Commun. 184, 1048 (1992).
6. Determination of the bioactive conformation: K. Scheffler, B.
Ernst, A. Katopodis, J.L. Magnani, W.T. Wang, R. Weisemann,
and T. Peters. Angew. Chem. Int. Ed. Engl. 34, 1841 (1995); K.
Scheffler, J.-R. Brisson, R. Weisemann, J.L. Magnani, B. Ernst,
and T. Peters. J. Biomol. NMR, 9, 423 (1997); D. Henrichsen,
B. Ernst, J.L. Magnani, W.-T. Wang, B. Meyer, and T. Peters.
Angew. Chem. Int. Ed. Engl. 38, 98 (1999); R. Harris, G.R.
Kiddle, R.A. Field, B. Ernst, J.L. Magnani, and S. Homans. J.
Am. Chem. Soc. 121, 2546 (1999); W. Jahnke, H.C. Kolb,
M.J.J. Blommers, J.L. Magnani, and B. Ernst. Angew. Chem.
Int. Ed. Engl. 36, 2603 (1997); Design of sialyl Lewisx mimics:
H.C. Kolb and B. Ernst. Pure Appl. Chem. 69, 1879 (1997);
H.C. Kolb and B. Ernst. Chem. Eur. J. 3, 1571 (1997); S.
Hanessian, G.V. Reddy, H.K. Huynh, J. Pan, S. Pedatella, H.C.
Kolb, and B. Ernst. Bioorg. Med. Chem. Lett. 7, 2729 (1997);
R. Bänteli and B. Ernst. Tetrahedron Lett. 38, 4059 (1997); D.
Roche, R. Bänteli, T. Winkler, F. Casset, and B. Ernst. Tetrahe-
dron Lett. 39, 2545 (1998); S. Hanessian, H.K. Huynh, G.V.
Reddy, G. McNaughton-Smith, B. Ernst, H.C. Kolb, J.L.
Magnani, and C. Sweeley. Bioorg. Med. Chem. Lett. 8, 2803
(1998); C.-Y. Tsai, W.K.C. Park, G. Weitz-Schmidt, B. Ernst,
and C.-H. Wong. Bioorg. Med. Chem. Lett. 8, 2333 (1998); Bi-
ological evaluation of selectin antagonists: K.E. Norman, G.P.
Anderson, H.C. Kolb, K. Ley, and B. Ernst. Blood, 91, 475 (1998).
7. The pharmacophores of sialyl Lewisx have been determined
by: M. Yoshida, A. Uchimura, M. Kiso, and A. Hasegawa.
Glycoconjugate J. 10, 3 (1993); B.K. Brandley, M. Kiso, S.
Abbas, P. Nikrad, O. Srivastava, C. Foxall, Y. Oda, and A.
Hasegawa. Glycobiology, 3, 633 (1993); R.M. Nelson, S.
Dolich, A. Aruffo, O. Cecconi, and M.P. Bevilacqua. J. Clin.
Invest. 91, 1157 (1993); W. Stahl, U. Sprengard, G.
Kretzschmar, and H. Kunz. Angew. Chem. 106, 2186 (1994);
A. Hasegawa, T. Ando, M. Kato, H. Ishida, and M. Kiso.
Carbohydr. Res. 257, 67 (1994); J.Y. Ramphal, Z.-I. Zheng, C.
Perez, L.E. Walker, S.A. Defrees, and F.C.A. Gaeta. J. Med.
Chem. 37, 3459 (1994); S. Komba, H. Ishida, M. Kiso, and A.
Hasegawa. Glycoconjugate J. 13, 241 (1996); G. Kuznik, B.
Hörsch, G. Kretzschmar, and C. Unverzagt. Bioorg. Med.
Chem. Lett. 7, 577 (1997); H.C. Kolb and B. Ernst. Chem.
11. X. Qian, O. Hindsgaul, H. Li, and M.M. Palcic. J. Am. Chem.
Soc. 120, 2184 (1998); S. Ogawa, T. Furuya, H. Tsunoda, O.
Hindsgaul, K. Stangier, and M.M. Palcic. Carbohydr. Res. 271,
197 (1995); C. Hällgren and O. Hindsgaul. J. Carbohydr.
Chem. 14, 453 (1995).
12. D.J. Legault, R.J. Kelly, Y. Natsuka, and J.B. Lowe. J. Biol.
Chem. 270, 20987 (1995); T. de Vries, C.A. Srnka, M.M.
Palcic, S.J. Swiedler, D.H. van den Eijnden, and B.A. Macher.
J. Biol. Chem. 270, 8712 (1995); O. Zöllner and D. Vestweber.
J. Biol. Chem. 271, 33002 (1996).
13. P.W. Kent and S.D. Dimitrijevich. J. Fluorine Chem. 10, 455
(1977).
14. Cloning and expression of α(2–3)sialyl transferase: D.X. Wen,
B.D. Livingston, K.F. Medziharaszky, S. Kelm, A.L.
Burlingame, and J.C. Paulson. J. Biol. Chem. 267, 21011 (1992).
15. Cloning and expression of FucT III: K. Sasaki, K. Kurata, K.
Funayama, M. Nagata, E. Watanabe, S. Ohta, N. Hanai, and T.
Nishi. J. Biol. Chem. 269, 14730 (1994).
16. D.P. Dumas, Y. Ichikawa, C.-H. Wong, J.B. Lowe, and R.P.
Nair. Bioorg. Med. Chem. Lett. 1, 425 (1991); S.A. DeFrees,
F.C.A. Gaeta, Y.-C. Lin, Y. Ichikawa, and C.-H. Wong. J. Am.
Chem. Soc. 115, 7549 (1993).
17. G.C. Look, Y. Ichikawa, G.-J. Shen, P.-W. Cheng, and C.-H.
Wong. J. Org. Chem. 58, 4326 (1993).
18. R.R. Schmidt, J. Michel, and M. Roos. Liebigs Ann. Chem.
86, 1343 (1984).
19. 18 was obtained in quantitative yield by benzoylation of ethyl
6-O-benzyl-1-thio-β-D-galactopyranoside; S. Sato, Y. Ito, and
T.A. Ogawa. Carbohydr. Res. 155, C1 (1986).
20. H. Meerwein, K.F. Zenner, and R. Gipp. Liebigs Ann. Chem.
67, 688 (1965).
21. M.N. Qabar, J.P. Meara, C. Lum, H.-K. Kim, and M. Kahn.
Tetrahedron Lett. 39, 5895 (1998).
22. F. Degerbeck, B. Fransson, L. Grehn, and U. Ragnarsson. J.
Chem. Soc. Perkin Trans 1, 11 (1993).
23. Cloning and expression of FucT VI: K.L. Koszdin and B.R.
Bowen. Biochem. Biophys. Res. Commun. 187, 152 (1992).
24. D.J. Jenkins and B.V.L. Potter. Carbohydr. Res. 265, 145
(1994); H. Qin and T.B. Grindley. J. Carbohydr. Chem. 15, 95
(1996).
© 2000 NRC Canada

904
Can. J. Chem. Vol. 78, 2000
25. CMP-Neu-5-NAc: M.D. Chappell and R.L. Halcomb. Tetrahe-
dron, 53, 109 (1997); Y. Kajihara, T. Ebata, K. Koseki, H.
Kodama, H. Matsushita, and H. Hashimoto. J. Org. Chem. 60,
5732 (1995); M. Kittelmann, T. Klein, U. Kragl, C. Wandrey,
and O. Ghisalba. Appl. Microbiol. Biotechnol. 44, 59 (1995);
GDP-Fuc: K. Adelhorst and G.M. Whitesides. Carbohydr.
Res. 242, 69 (1993); V. Wittmann and C.-H. Wong. J. Org.
Chem. 62, 2144 (1997); G. Baisch and R. Öhrlein. Bioorg.
Med. Chem. 5, 383 (1997).
© 2000 NRC Canada