Chemo-enzymatic synthesis of a divalent sialyl Lewisx ligand with restricted flexibility

Article abstract of DOI:10.1016/S0008-6215(03)00129-0

To study the influence of the entropic factor in cluster cooperative effects, a divalent sialyl Lewis^x ligand with restricted flexilbility was chemo-enzymatically synthesized. First, a cyclized precursor with both glucosamine residues bridged t

Full text of DOI:10.1016/S0008-6215(03)00129-0

Carbohydrate Research 338 (2003) 1163–1173
www.elsevier.com/locate/carres
Chemo-enzymatic synthesis of a divalent sialyl Lewisx ligand with
restricted flexibility
Fabrice Bintein, Claudine Auge´,* Andre´ Lubineau
Laboratoire de Chimie Organique Multifonctionnelle, UMR 8614, Uni6ersite´ de Paris-Sud, F-91405 Orsay, France
Received 23 December 2002; accepted 21 February 2003
Abstract
To study the influence of the entropic factor in cluster cooperative effects, a divalent sialyl Lewisx ligand with restricted
flexilbility was chemo-enzymatically synthesized. First, a cyclized precursor with both glucosamine residues bridged together by
a succinyl group was readily obtained in 42% yield by treatment of 2,2-bis(benzyloxymethyl)-1,3-bis(3,4,6-tri-O-acetyl-2-amino-2-
deoxy-b-D-glucopyranosyloxy)-propane with succinyl chloride. After deacetylation, this precursor was subjected to stepwise
enzymatic elongation utilizing successively, soluble galactosyltransferase, then recombinant sialyltransferase and fucosyltrans-
ferase; the latter enzymes immobilized on Ni2+-Agarose, to afford, after debenzylation, a divalent sialyl Lewisx ligand of restricted
flexibility, in 45% overall yield. Following the same enzymatic sequence, a totally flexible ligand, required as a reference compound
for evaluation of inhibitory activity toward selectins, was also prepared from 2,2(bis-benzyloxymethyl)-1,3-bis(2-acetamido-2-de-
oxy-b-D
-glucopyranosyloxy)-propane, as well as both related divalent Lewisx molecules lacking the sialic acids, the rigid one and
the flexible one. © 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Chemo-enzymatic synthesis; Divalent sialyl Lewisx; Selectin ligand; Cooperativity; Cluster effect
1. Introduction
E-selectin ligand so far, showing an inhibitory activity
at least 15-fold greater than that of the sialyl Lewisx
trisaccharide.2 In fact, physiological ligands on cell
surfaces like GlyCAM-1 and PSGL-1 have been shown
to bind with high affinity to individual selectins, proba-
bly because, as mucin-like molecules, they are highly
glycosylated and carry carbohydrates displayed in clus-
ters. The requirement of multivalency for strong bind-
ing has stimulated the synthesis of glyco-dendrimers,3
-polymers4 and liposomes,5 and also of several types of
glycoconjugates with multiple carbohydrate binding
sites.6–12 When these multivalent displays were tested as
inhibitors of selectins, the most powerful inhibitors
turned out to be five to six times more active than sialyl
Lewis x, only showing a moderate improvement in
binding as compared to the monovalent counterpart.1
Interestingly, remarkable IC50 values of 1 nanomolar
against L-selectin have been reported for branched
oligosaccharides containing four sialyl Lewisx epi-
topes.13 The strong binding of these ligands to their
receptor could be explained by the enhanced cluster
cooperative effects in these oligosaccharides, of limited
mobility, like in mucins.14
It is now well established that leukocyte adhesion to the
vascular endothelium is mediated in the early steps of
the inflammatory cascade, by selectins, a family of
calcium-dependent carbohydrate-binding cell adhesion
molecules. E-Selectin is induced on the vascular en-
dothelium after stimulation by cytokines released from
damaged tissues, P-selectin is immediately expressed on
the cell surface of platelets and endothelial cells in
response to inflammatory signals, while L-selectin is
constitutively expressed on the surface of leukocytes.
The tetrasaccharides sialyl Lewisx, sialyl Lewisa and
sulfated analogues thereof have been identified as mini-
mal carbohydrate epitopes recognized by the selectins.1
However, the affinity of selectins for these monovalent
epitopes is usually weak with dissociation constants
typically in the millimolar range, except for the sulfate
Lewisa pentasaccharide, the most potent monovalent
* Corresponding author
E-mail address: clauauge@icmo.u-psud.fr (C. Auge´).
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00129-0

1164
F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
These findings prompted us to consider the synthesis
drastic treatment of 3 with barium hydroxide17 afforded
the intermediate N-de-acetylated derivative which was
reprotected in situ with a tert-butyloxycarbonyl group
by treatment of the neutralized reaction mixture with
di-tert-butyl dicarbonate in the presence of triethy-
lamine,18 to give compound 4 in 82% overall yield.
Subsequent conventional acetylation led to compound
5 (Scheme 1). The inherent C2 symmetry of the
molecule was evidenced in the NMR spectrum of 5 in
deuterated dimethyl sulfoxide. The amine functions of
the di-glucosamine derivative 5 were deprotected by
treatment with trifluoroacetic acid affording compound
6 in 92% yield. Then, the acetylated derivative 6 was
reacted with succinyl chloride in dichloromethane in the
presence of an excess of triethylamine. Reaction condi-
tions were optimized to favour intramolecular reaction
leading to the expected cyclization product, rather than
the intermolecular reaction affording the di-substituted
derivative. Thus, a satisfactory yield of 42% for the
desired compound 7 was reached by high dilution con-
densation of compound 6 (1 mM) with a slight excess
of succinyl chloride (1.4 equiv). Yields could not be
increased by lowering the temperature to 0 °C or by
progressive addition of the reagent. However, a signifi-
cant proportion (32%) of the di-succinylated products,
isolated as the dimethyl ester 8 or the mixed mono-ester
and mono-acid 9, was also obtained, in addition to 15%
of the starting material 6 (Scheme 1). Deacetylation of
compound 7 afforded the precursor 10 that was elabo-
of selectin ligands with restricted conformational flexi-
bility. We herein wish to report on the chemo-enzy-
matic synthesis of the divalent sialyl Lewisx molecule 1
constructed on pentaerythritol with both amine func-
tions of the glucosamine residues bridged by a succinyl
group. For the synthesis of the sialyl Lewisx epitopes,
we turned to the well-known strategy based on the use
of glycosyltransferases, bringing thus new insights into
the specificity of these enzymes.
Both carbohydrate moieties of ligand 1 linked by two
amide bonds spaced by three single bonds have obvi-
ously limited mobility. To our knowledge, this is the
first synthesis of a divalent selectin ligand of low en-
tropy. Moreover, the divalent ligand 2 built on pen-
taerythritol but totally flexible was also prepared in
order to allow the direct comparison of the inhibitory
activity of both ligands.
2. Results and discussion
We chose the commercially available pentaerythritol as
the backbone for our divalent sialyl Lewis x cluster,
according to a research program initiated some years
ago.15 Two of the hydroxymethyl groups were substi-
tuted with benzyl groups and the other with glu-
cosamine units affording the symmetrical molecule 3,
the synthesis of which has been already described.16
A
Scheme 1. Reagents and conditions: (a) Ba(OH)2, 110 °C, 20 h; then M H2SO4“pH 2; (Boc)2O, water–dioxane, Et3N, 18 h, rt,
82%; (b) Ac2O, pyridine, 16 h, rt, 90%; (c) CF3COOH, CH2Cl2, 1 h, rt, 92%; (d) ClCOꢀ(CH2)2ꢀCOCl 1.4 equiv, CH2Cl2, Et3N,
13 h, rt, 42% for 7.

F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
1165
Scheme 2. Reagents and conditions: (a) GalT, UDPGE, UDP-Glc 2 equiv, AP, 15 mM MnCl2, 25 mM sodium cacodylate buffer
pH 7.4, 37 °C, 4 days, 70% (for 11), 20% (for 12); (b) Ac2O, pyridine, 16 h, rt, 81%.
rated, using glycosyltransferases19–21 that catalyse the
regio- and stereospecific transfer of a monosaccharide
to oligosaccharide acceptors from a sugar-nucleotide
donor. Compound 10 was first incubated with commer-
cial b-(1“4)-galactosyltransferase (E.C.2.4.1.22), man-
ganese chloride, alkaline phosphatase22 together with
UDP-Glc and UDP-Glc epimerase that epimerize in
situ UDP-Glc into UDP-Gal. The glucosamine deriva-
tive with restricted flexibility could be galactosylated
very efficiently, a result in accordance with previous
reports on enzymatic galactosylation of non-natural
glucosamine acceptors.23 After 6 days of incubation, the
di-galactosylated compound 11 was isolated in 70%
yield, along with the mono-galactosylated compound
12 (20%) (Scheme 2). The structure of 11 was unam-
binant
CMP-Neu5Ac:b-
D
-Galp-(1“3/4)-b-
D-Glcp-
(EC 2.4.99.5,
NAc-(2“3%)-a-sialyltransferase
ST3Gal-III) adsorbed on Ni2+-Agarose and CMP-
Neu5Ac in 25 mM sodium cacodylate buffer, to afford
the disialylated derivative 14 in excellent yield (85%)
(Scheme 3). The downfield shift for both galactose H-3
1
at 4.11 ppm in the H NMR spectrum of 14 confirmed
the structure. The next step utilized recombinant GDP-
Fuc:b-
D
-Galp-(1“3/4)-b- -GlcpNAc-(2“3%)-a-fuco-
D
syl-transferase (EC 2.4.1.65, FucT-III), the only recom-
binant fucosyltransferase at our disposal,24 that is
known to exhibit a lower affinity for b-
D-Galp-(1“4)-
b- -GlcpNAc (Type 2) than for Galp-(1“3)-b-
D
D-Glcp-
NAc (Type 1) sequences. Nevertheless, provided that
high substrate concentration and a larger amount of
enzyme were used, fucosylation of compound 14 could
be achieved in good yield (80%), by incubation with
FucT-III expressed in insect cells and adsorbed on
Ni2+-Agarose, GDP-Fuc, manganese chloride and al-
kaline phosphatase. Finally, hydrogenolytic debenzyla-
tion led to the target di-sialyl Lewisx molecule 1 in 95%
yield. The flexible divalent ligand 2, required as a
reference compound for evaluation of its inhibitory
activity towards selectins, was obtained from the disia-
lylated compound 1616 through the same sequence,
fucosylation leading to compound 17 and debenzyla-
tion (Scheme 4). Intermediates lacking sialic acids, both
divalent Lewisx molecules, the rigid 19 and the
1
biguously assigned from the H NMR spectrum of the
peracetylated derivative 13, readily obtained by treat-
ment of 11 with acetic anhydride and pyridine, which
clearly showed a two-proton integrating signal at 5.34
ppm, with a small coupling constant, characteristic of
the most deshielded H-4 equatorial galactose protons.
The following two enzymatic steps were achieved with
recombinant mammalian glycosyltransferases produced
in baculovirus-infected insect cells. The recombinant
enzymes tagged with a six histidine tail were immobi-
lized on nickel-Agarose, a procedure that proved to be
of benefit for synthetic purposes.24 Mammalian biosyn-
thesis requires that sialylation precedes fucosylation.
Thus first, compound 11 was incubated with the recom-

1166
F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
flexible analog 22 were also prepared. Fucosylation was
achieved with another source of FucT-III: we used the
recombinant enzyme expressed in Chinese Hamster
Ovary (CHO) cells and immobilized on Ni2+
-
Agarose,24 an enzymatic preparation unsuitable for fu-
cosylation of sialylated oligosaccharides because it
contains residual sialidase activity. The fucosylated in-
termediates 18 and 21 were not obtained in as good
yields as compounds 15 and 17, probably because of
the lower enzyme affinity for these nonsialylated
oligosaccharide acceptors than for these sialylated ones.
Scheme 4. Reagents and conditions: (a) FucT-III immobilized
on Ni2+-NTA-Agarose, GDP-Fuc 2.8 equiv, AP, 15 mM
MnCl2, 25 mM sodium cacodylate buffer pH 6.6, 30 °C, 5
days, 82%; (b) 20% Pd(OH)2ꢀC, H2, MeOH–water, 16 h,
95%.
Again debenzylation led to the target molecules 19 and
22 (Scheme 5).
All of these divalent derivatives are now under bio-
logical evaluation towards E, L and P-selectins. It will
be most interesting to compare the inhibitory activity of
sialyl Lewisx ligands, 1 and 2. Compound 1, the first
divalent sialyl Lewisx with restricted flexibility is ex-
pected to have much better inhibition effect than diva-
lent sialyl Lewisx 2, in which the six single bonds
between both sialyl Lewisx epitopes allow for consider-
able conformational flexibility.
3. Experimental
3.1. General
Scheme 3. Reagents and conditions: (a) ST3Gal-III immobi-
lized on Ni2+-NTA-Agarose, CMP-Neu5Ac 3.2 equiv, 25
mM sodium cacodylate buffer pH 7.1, 37 °C, 4 days, 85%;
FucT-III immobilized on Ni2+-NTA-Agarose, GDP-Fuc 2.8
equiv, AP, 15 mM MnCl2, 25 mM sodium cacodylate buffer
pH 6.6, 30 °C, 5 days, 80%; (c) 20% Pd(OH)2ꢀC, H2, MeOH–
water, 16 h, 95%.
NMR spectra were recorded with Bruker AC-200 or
AC-250 spectrometers; the chemical shifts are given
relative to the signal of tetramethylsilane in CDCl3; for

F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
1167
1H and 13C NMR spectra in D2O, acetone (l 2.22 and
30.5 ppm) was used as an internal reference. Optical
rotations were measured with a JASCO digital microp-
olarimeter. Elemental analyses were performed at the
Service Central de Microanalyse, CNRS, Gif sur
Yvette, France. Mass spectra were performed on a
Finigan MATT 95 apparatus using ESI. Reactions were
monitored by TLC on Silica Gel 60F254 with detection
by charring with 10% H2SO4 in EtOH or 2% orcinol in
10% H2SO4. C18 Sep-Pak cartridges were from Mil-
lipore-Waters. CMP-Neu5Ac and GDP-Fuc were pur-
chased from Kyowa Hakko, UDP-Glc from Sigma, calf
intestine alkaline phosphatase from Roche Molecular
under Ag. The solution was acidified to pH 2 with M
sulfuric acid, filtered and the remaining solution evapo-
rated to dryness. The residue was dissolved in 1:1
water–dioxane (16 mL), triethylamine (0.60 mL, 4.14
mmol) and di-tert-butyl dicarbonate (1.8 g, 8.28 mmol)
were added and the mixture was stirred for 18 h at
room temperature (rt). Then solvents were evaporated
and the residue was purified by flash chromatography
on silica gel (95:5 EtOAc–MeOH) to afford compound
1
4 (1.42 g, 82%); [h]2D9 −13° (c 1, MeOH); H NMR
(CD3OD, 250 MHz): l 7.30–7.20 (m, 10 H, Ph), 6.4 (d,
2 H, JNH,2 6.4 Hz, NH), 4.44 (s, 4 H, 2 CH2Ph), 4.19 (d,
2 H, J1,2 7.8 Hz, 2 H-1), 3.95 (d, Jgem 9.8 Hz, 2 CH),
3.83 (d, 2 H, J6,6% 11.7 Hz, 2 H-6), 3.67 (dd, 2 H, J6%,5 5.3
Hz, 2 H-6%), 3.57–3.39 (m, 6 H, 2 H-3, 2 H-4, 2 CH),
3.37–3.16 (m, 8 H, 2 CH2, 2 H-5, 2 H-2) and 1.40 (s, 18
H, 2 (CH3)3C); 13C NMR (CD3OD, 62.9 MHz): l 158.3
(NHCO), 140.1, 129.3, 128.6, (CꢀAr), 104.3 (C-1), 80.2
((CH3)3C), 77.6, 75.8, 72.1 (C-3, C-4, C-5), 74.4
(CH2Ph), 69.9, 69.4, (CH2), 62.7 (C-6), 58.4 (C-2), 46.7
(Cq) and 29.0 (CH3); ES+MS: m/z 861.3 [M+Na]+.
Anal. Calcd for C41H62N2O16·0.5 H2O: C, 58.07; H,
7.49; N, 3.30; O, 31.13. Found: C, 58.23; H, 8.02; N,
3.08; O, 30.91.
Biochemicals, bovine milk -GlcNAc-b-(1“4)-galacto-
D
syltransferase and UDP-glucose 4-epimerase from
Calbiochem.
3.2. 2,2-Bis-(benzyloxymethyl)-1,3-bis[2-tert-(butyloxy-
carbonyl-amino)-2-deoxy-b-D-glucopyranosyloxy]-
propane (4)
To a solution of 3 (1.496 g, 2.07 mmol) in degassed
water (14 mL) was added barium hydroxide (7.84 g,
24.84 mmol) and the mixture stirred at 110 °C for 20 h
3.3. 2,2-Bis-(benzyloxymethyl)-1,3-bis-[3,4,6-tri-O-ace-
tyl-2-(tert-butyloxycarbonyl)-amino-2-deoxy-b-D-gluco-
pyranosyloxy]-propane (5)
To a solution of 4 (0.70 g, 0.84 mmol) in pyridine (10
mL) cooled to 0 °C was added acetic anhydride (8 mL)
and the mixture was stirred at rt for 16 h. Then solvents
were evaporated, co-evaporated with toluene and the
residue was chromatographed on silica gel (2:1
petroleum ether–EtOAc) to give pure 5 (0.808 g, 90%);
1
[h]3D1 −26° (c 0.5, CH2Cl2); H NMR (Me2SO-d6, 250
MHz): l 7.35–7.24 (m, 10 H, Ph), 6.97 (d, JNH,2 10 Hz,
NH), 5.06 (t, 2 H, J4,5=J4,3 10 Hz, 2 H-4), 4.82 (t, 2 H,
J3,2 10 Hz, 2 H-3), 4.48–4.35 (m, 6 H, Jgem 12, J1,2 8.3
Hz, 2 CH2Ph, 2 H-1), 4.20 (dd, 2 H, J6%,6 12, J6,5 4.5 Hz,
2 H-6), 3.98 (dd, 2 H, J6%,5 2 Hz, 2 H-6%), 3.78 (d, 1 H,
Jgem 10 Hz, CH), 3.71 (m, 2 H, 2 H-2), 3.52–3.25 (m, 9
H, 2 H-5, 3 CH2, CH), 2.02 (2 s, 18 H, 6 OAc) and 1.45
(s, 18 H, 2 (CH3)3C); 13C NMR (Me2SO-d6, 50.3 MHz):
l 169.9, 169.7, 169.3 (CO), 155.1 (NHCO), 138.4,
128.1, 127.1 (CꢀAr), 101.7 (C-1), 72.5 (CH2Ph), 77.9,
72.4, 70.7 (C-3, C-4, C-5), 68.7, 68.3, 68.1 (CH2O), 61.8
(C-6), 54.7 (C-2), 44.9 (Cq), 28.0 (CH3)3) and 20.4
(COCH3); HRMS: Calcd for C53H74N2NaO22 [M+
Na]+ 1113.4630. Found: m/z 1113.4629.
3.4. 2,2-Bis-(benzyloxymethyl-1,3-bis(3,4,6-tri-O-acetyl-
2-amino-2-deoxy-b-D-glucopyranosyloxy)-propane (6)
Scheme 5. Reagents and conditions: (a) FucT-III immobilized
on Ni2+-NTA-Agarose, GDP-Fuc 2.8 equiv, AP, 15 mM
MnCl2, 25 mM sodium cacodylate buffer pH 6.6, 30 °C, 8
days, 74% (for 18), 50% (for 21); (b) 20% Pd(OH)2ꢀC, H2,
MeOH–water, 16 h, 95%.
To a solution of 5 (0.608 g, 0.56 mmol) in CH2Cl2 (2.8
mL) was added trifluoroacetic acid (2.7 mL) and the

1168
F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
solution was stirred for 1 h at rt. Then solvents were
evaporated and flash chromatography on silica gel of
the residue (97:3 CH2Cl2–MeOH) gave pure 6 (0.573 g,
170.7, 169.5 (CO), 138.8, 128.3, 127.6 (CꢀAr), 101.1
(C-1), 73.3 (CH2Ph), 72.2, 71.5, 69.4 (C-3, C-4, C-5),
68.8, 68.7 (CH2O), 62.1 (C-6), 55.1 (C-2), 45.0 (Cq),
31.6 (COCH2) and 20.7 (COCH3); HRMS: Calcd for
C47H60N2NaO20 [M+Na]+ 995.3637. Found: m/z
995.3636.
1
92%); [h]2D7 −3.5° (c 0.38, CH2Cl2) H NMR (CDCl3,
200 MHz): l 8.2–7.9 (m, 6 H, 2 NH3), 7.28 (s, 10 H,
Ph), 5.30 (t, 2 H, J4,3=J4,5 9.2 Hz, 2 H-4), 5.03 (t, 2 H,
J3,2 9.2 Hz, 2 H-3), 4.55 (d, 2 H, J1,2 8.3 Hz, 2 H-1), 4.5
(s, 4 H, 2 CH2Ph), 4.33 (dd, 2 H, J6%,6 11.7, J6,5 3 Hz, 2
H-6), 4.07 (d, 2 H, 2 H-6%), 3.92 (d, 2 H, Jgem 8.3 Hz, 2
CH), 3.78 (d, 2 H, 2 CH), 3.65 (m, 2 H, 2 H-5), 3.54 (2
d, 4 H, 4 CH), 3.31 (dd, 2 H, 2 H-2) and 2.05 (s, 18 H,
6 OAc); 13C NMR (CDCl3, 50.3 MHz): l 171.8, 170.1
(CO), 138.0, 128.2, 127.4 (CꢀAr), 98.5 (C-1), 73.5
(CH2Ph), 73.5, 71.6, 71.2 (C-3, C-4, C-5), 68.3 (CH2O),
61.7 (C-6), 54.8 (C-2), 44.5 (Cq), 20.6, 20.5 and 20.3
(COCH3); HRMS: Calcd for C43H59N2O18 [M+H]+
891.3763. Found: m/z 891.3765. Anal. Calcd for
C43H58N2O18: C, 57.90; H, 6.67; N, 3.14. Found: C,
58.02; H, 7.02; N, 3.21.
1
Compound 8: [h]2D6 −11° (c 1, CHCl3); H NMR
(Me2SO-d6, 250 MHz): l 7.99 (d, 2 H, JNH,2 8.8 Hz,
NH), 7.35–7.2 (m, 10 H, Bn), 5.07 (t, 2 H, J4,3=J4,5 9.8
Hz, 2 H-4), 4.82 (t, 2 H, J3,2 9.8 Hz, 2 H-3), 4.47 (d, 2
H, J1,2 8.3 Hz, 2 H-1), 4.43 (d, 2 H, Jgem 12.2 Hz, 2
CHPh), 4.37 (d, 2 H, 2 CHPh), 4.21 (dd, 2 H, J6,5 4.3,
J6,6% 12.2 Hz, 2 H-6), 3.96 (dd, 2 H, J6%,5 2 Hz, 2H-6%),
3.87–3.70 (m, 6 H, 2 H-5, 2 CH2), 3.53 (s, 6 H, 2
OCH3), 3.35–3.25 (m, 6 H, 2 H-2, 2 CH2), 2.46–2.12
(m, 8 H, 4 CH2), 1.97 (s, 6 H, 2 OAc), 1.95 (s, 6 H, 2
OAc) and 1.91 (s, 6 H, 2 OAc); 13C NMR (CDCl3, 50.3
MHz): l 173.7 (CO2Me), 171.3, 170.7, 169.4 (CO),
138.7, 128.4, 128.2, 127.7, 127.4 (CꢀAr), 101.4 (C-1),
73.4 (CH2Ph), 72.5, 71.7, 71.5 (C-3, C-4, C-5), 68.8,
68.7 (CH2O), 62.2 (C-6), 54.2 (C-2), 52.0 (CH3), 45.2
(Cq), 30.5 (CH2CO), 28.8 (CH2CO) and 20.6
(CH3CO); ES+MS: m/z 1141.3 [M+Na]+. Anal.
Calcd for C53H70N2O24: C, 56.88; H, 6.30. Found: C,
56.81; H, 6.56.
3.5. 2,2-Bis(benzyloxymethyl)-1,3-bis[3,4,6-tri-O-acetyl-
2-amino-2-deoxy-b-D-glucopyranosyloxy]-propane bu-
tane 1N,4N%-dioyl amide (7), 2,2-bis-(benzyloxy-
methyl)-1,3-bis-[3,4,6-tri-O-acetyl-2-deoxy-2-(3-methy-
loxycarbonyl-propanamido)-b-D-glucopyranosyloxy]-
propane (8) and 2,2-bis-(benzyloxymethyl)-1-[3,4,6-tri-
1
Compound 9: [h]2D4 −14° (c 1, CHCl3); H NMR
O-acetyl-2-deoxy-2-(3-methyloxycarbonyl-propanamido)-
(Me2SO-d6, 250 MHz): l 8.04 (d, 1 H, JNH,2 10.3 Hz,
NH), 8.0 (d, 1 H, JNH,2 10.3 Hz, NH), 7.35–7.2 (m, 10
H, Ph), 5.07 (t, 2 H, J4,5=J4,3 9.8 Hz, 2 H-4), 4.82 (t,
2 H, J3,4=J3,2 9.8 Hz, 2 H-3), 4.48 (d, 2 H, J1,2 8.3 Hz,
2 H-1), 4.43 (d, 2 H, Jgem 12.2 Hz, 2 CHPh), 4.37 (d, 2
H, 2 CHPh), 4.21 (dd, 2 H, J6,5 3, J6,6% 12.2 Hz, 2 H-6),
3.97 (d, 2 H, 2 H-6%), 3.87–3.69 (m, 6 H, 2 H-5, 2
CH2O), 3.54 (s, 3 H, OCH3), 3.34–3.18 (m, 6 H, 2 H-2,
2 CH2O), 2.46–2.12 (m, 8 H, 4 CH2), 1.97 (s, 6 H, 2
OAc), 1.95 (s, 6 H, 2 OAc) and 1.91 (s, 6 H, 2 OAc);
13C NMR (CDCl3, 50.3 MHz): l 172.3, 172.0 (CO2Me,
CO2H), 171.0, 169.6 (CO), 138.4, 128.1, 127.4 (CꢀAr),
101.2 (C-1), 73.3 (CH2Ph), 72.7, 72.4, 71.3 (C-3, C-4,
C-5), 68.6 (CH2O), 62.0 (C-6), 54.0, 53.8 (2 C-2), 51.6
(OCH3), 45.1 (Cq), 30.6, 30.3 (CH2CONH), 29.0, 28.7
(CH2COO) and 20.3 (CH3CO); ES+MS: m/z 575.1
[(M+2 Na)/2]+, 1127.4 [M+Na]+. Anal. Calcd for
C52H68N2O24: C, 56.52; H, 6.20; N, 2.53; O, 34.75.
Found: C, 56.67; H, 6.46; N, 2.51; O, 34.48.
b- -glucopyranosyloxy]-3-[3,4,6-tri-O-acetyl-2-deoxy-2-
D
(3-carboxyl-propanamido)-b-D-glucopyranosyloxy]-
propane (9)
To a solution of 6 (150 mg, 0.134 mmol) in CH2Cl2
(130 mL) was added succinyl chloride (0.015 mL, 0.134
mmol) dissolved in CH2Cl2 (1 mL). After stirring for 5
min, triethylamine (0.113 mL, 0.67 mmol) was added
and the mixture was stirred for 10 h at rt. Succinyl
chloride (0.006 mL, 0.054 mmol) in CH2Cl2 (0.4 mL)
and triethylamine (0.023 mL, 0.134 mmol) were added
again and the solution was stirred for 3 h. The reaction
mixture was quenched with MeOH and solvents were
evaporated; the residue dissolved in CH2Cl2, washed
with water, concentrated to dryness, was purified by
flash chromatography on silica gel (97:3 CH2Cl2–
MeOH) to give in the order of elution: 8 (12 mg, 8%),
remaining starting material 6 (22 mg, 15%), 7 (55 mg,
42%), and 9 (35 mg, 24%).
Compound 7: [h]2D4 −5° (c 1, CH2Cl2); 1H NMR
(CDCl3, 200 MHz): l 7.40–7.20 (m, 10 H, Ph), 6.10 (d,
2 H, JNH,2 8.8 Hz, NH), 5.24 (t, 2 H, J4,3=J4,5 9.8 Hz,
2 H-4), 5.04 (t, 2 H, J3,2 9.8 Hz, 2 H-3), 4.57 (d, 2 H,
J1,2 8.8 Hz, 2 H-1), 4.46 (2 d, 4 H, Jgem 12.2 Hz, 2
CH2Ph), 4.26 (dd, 2 H, J6%,6 12.2, J6,5 4.4 Hz, 2 H-6),
4.04 (d, 2 H, 2 H-6%), 3.82–3.58 (m, 8 H, Jgem 9.3 Hz, 4
CH, 2 H-2, 2 H-5), 3.54 (d, 2 CH), 3.40 (d, 2 CH), 2.38
(2 d, 4 H, Jgem 13.2 Hz, 2 CH2) and 2.05–1.95 (m, 18
H, 6 OAc); 13C NMR (CDCl3, 50.3 MHz): l 172.1,
3.6. 2,2-Bis-(benzyloxymethyl-1,3-bis-[2-amino-2-deoxy-
b- -glucopyranosyloxy]-propane butane 1N,4N%-dioyl
D
amide (10)
Compound 7 (90 mg, 0.092 mmol) dissolved in 8:2:1
MeOH–triethylamine–water (5.5 mL) was stirred at rt
for 24 h, then concentrated to dryness. The residue was
chromatographed on silica gel (8:3:1 EtOAc–2-
propanol–water) to afford 10 (65 mg, 92%); [h]3D1 −3°
1
(c 1, MeOH); H NMR (D2O, 200 MHz): l 7.20 (s, 10

F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
1169
H, Ph), 4.37 (s, 4 H, CH2Ph), 4.22 (d, 2 H, J1,2 7.8 Hz,
2 H-1) and 2.5 (2 d, 4 H, Jgem 11.7 Hz, 2 CH2CO); 13C
NMR (D2O, 62.9 MHz): l 175.5 (NHCO), 138.9, 129.5,
129.1 (CꢀAr), 102.9 (C-1), 74.1 (CH2Ph), 76.6, 74.7,
70.8 (C-3, C-4, C-5), 69.8, 69.2 (CH2O), 61.7 (C-6), 56.7
(C-2), 45.6 (Cq) and 31.9 (COCH2); HRMS: Calcd for
C35H48N2O14Na [M+Na]+ 743.3003. Found: m/z
743.3003. Anal. Calcd for C35H48N2O14·1 H2O: C,
56.89; H, 6.83; N, 3.79; O, 32.59. Found: C, 56.62; H,
6.81; N, 3.69; O, 32.17.
70.9, 69.6 (C-2%, C-3%, C-4%, C-5%, 2 C-3, C-4, 2 C-5), 74.2
(CH2Ph), 69.8, 69.5 (CH2O), 62.0, 61.7, 61.1 (2 C-6,
C-6%), 56.7, 56.2 (2 C-2), 45.6 (Cq) and 31.9 (CH2CO);
ES+MS: m/z 905.2 [M+Na]+. Anal. Calcd for
C41H58N2O19·2 H2O: C, 53.58; H, 6.80; N, 3.05. Found:
C, 53.34; H, 6.64; N, 2.89.
Acetylation of compound 11: to 11 (22 mg, 0.021
mmol) dissolved in pyridine (4 mL) was added acetic
anhydride (2 mL); the mixture was stirred overnight at
rt, then evaporated to dryness, co-evaporated twice
with toluene and the residue was purified by flash
chromatography (9:1 EtOAc–petroleum ether) to af-
ford the peracetylated derivative 13 (26 mg, 81%); [h]D27
3.7. 2,2-Bis-(benzyloxymethyl)-1,3-bis-[O-b-
ranosyl-(1“4)-(2-amino-2-deoxy-b- -glucopyran-
osyloxy)]-propane butane-1N,4N%-dioyl amide (11) and
2,2-bis-benzyloxymethyl-1-(O-2-acetamido-2-deoxy-b-
glucopyranosyl-oxy)-3-[O-b- -galactopyranosyl-(1“4)-
(2-amino-2-deoxy-b- -glucopyranosyloxy)]-propane bu-
tane-1N,4N%-dioyl amide (12)
D-galactopy-
D
1
−12° (c 1, CH2Cl2); H NMR (CDCl3, 250 MHz): l
D-
7.38–7.27 (m, 10 H, Ph), 5.80 (d, 2 H, JNH,2 9.3 Hz, 2
NH), 5.34 (d, 2 H, J3%,4% 3.5 Hz, 2 H-4%), 5.11 (dd, 2 H,
J1%,2% 8.3, J2%,3% 10.3 Hz, 2 H-2%), 5.02–4.93 (m, 4 H, 2
H-3, 2 H-3%), 4.54–4.35 (m, 8 H, J1%,2% 8.3, Jgem 12, J6a,6b
12, J6a,5 2 Hz, 2 H-1%, 2 H-6a, 4 CHPh), 4.24 (d, 2 H,
J1,2 8.3 Hz, 2 H-1), 4.18–4.02 (m, 6 H, 2-H6b, 2 H-6%a,
2 H-6%b), 3.99–3.83 (m, 4 H, 2 H-2, 2 H-5%), 3.72–3.59
(m, 6 H, Jgem 9.3, J3,4 9.8, J4,5 9.3 Hz, 2 H-4, 4 CHO),
3.54–3.46 (m, 4 H, 2 H-5, 2 CHO), 3.37 (d, 2 H, 2
CHO), 2.49 (d, 2 H, Jgem 13 Hz, 2 CHCO), 2.20 (d, 2
H, 2 CHCO), 2.15 (s, 6 H, 2 OAc), 2.06 (s, 6 H, 2
OAc), 2.05 (s, 12 H, 4 OAc), 2.01 (s, 6 H, 2 OAc) and
1.97(s, 6 H, 2 OAc); 13C NMR (CDCl3, 50.3 MHz): l
172.5 (NHCO), 170.6, 170.4, 170.3, 170.1, 169.4 (CO),
138.5, 128.0, 127.1 (CꢀAr), 100.9 (C-1), 100.6 (C-1%),
76.3, 73.0, 72.6, 72.0, 70.8, 70.3, 68.9, 66.6 (C-2%, C-3%,
C-4%, C-5%, C-3, C-5, PhCH2, CH2O), 62.0 (C-6), 60.6
(C-6%), 53.7 (C-2), 44.5 (Cq), 31.0 (CH2CO), 20.4 and
20.2 (CH3CO); ES+MS: m/z 1571.5 [M+Na]+, 797.3
[(M+2 Na)/2]+.
D
D
Compound 10 (60 mg, 0.083 mmol), UDP-Glc (51 mg,
0.083 mmol), bovine milk -GlcNAc b-(1“4)-galacto-
D
syltransferase (0.3U), UDP-glucose-4-epimerase (1.5
U), were incubated at 37 °C in 25 mM sodium cacody-
late buffer pH 7.4 (6 mL) containing 15 mM MnCl2.
The reaction was monitored by TLC on silica gel (3:3:1
EtOAc–2-propanol–water). After 48 h UDP-Glc (51
mg, 0.083 mmol), bovine milk
D-GlcNAc b-(1“4)-
galactosyltransferase (0.2 U), UDP-glucose-4-epimerase
(1 U) and MnCl2 (10 mg, 0.05 mmol) were added again
together with alkaline phosphatase (2 U) and the incu-
bation was continued for 48 h. The reaction mixture
divided into five portions was applied to Sep-Pak C18
cartridges; the combined MeOH eluates were evapo-
rated to dryness and the residue was purified by flash
chromatography on silica gel (6:4:1 EtOAc–2-
propanol–water) to give the di-galactosylated com-
pound 11 (60 mg, 70%) and the mono-galactosylated
compound 12 (15 mg, 20%).
3.8. 2,2-Bis-(benzyloxymethyl)-1,3-bis[O-(5-acetamido-
3,5-dideoxy-a-
osylonate)-(2“3)-O-b-
(2-amino-2-deoxy-b- -glucopyranosyloxy)]-propane bu-
D-glycero-
D-galacto-2-nonulopyran-
D-galactopyranosyl-(1“4)-
Compound 11: [h]2D7 −4° (c 1, water); 1H NMR
(D2O, 200 MHz): l 7.4 (s, 10 H, Ph), 4.60–4.43 (m, 6
H, J1%,2% 8.8, Jgem 12.2 Hz, 2 H-1%, 2 CH2Ph), 4.36 (d, 2
H, J1,2 7.8 Hz, 2 H-1) and 2.62 (2 d, 4 H, Jgem 12.7 Hz,
2 CH2CO); 13C NMR (D2O, 50.3 MHz): l 175.5
(NHCO), 138.7, 129.6, 129.2 (CꢀAr), 103.9 (C-1%), 102.9
(C-1), 79.4 (C-4), 76.3, 75.6, 74.2, 73.5, 73.2, 71.9, 69.6
(C-2%, C-3%, C-4%, C-5%, C-3, C-5, PhCH2), 69.5, 69.3
(CH2O), 62.0, 61.0 (C-6, C-6%), 56.2 (C-2), 45.5 (Cq)
and 31.9 (CH2CO); HRMS: Calcd for C47H68N2O24Na
[M+Na]+ 1067.4076. Found: m/z 1067.4060.
D
tane-1N,4N%-dioyl amide (14)
Compound 11 (40 mg, 0.038 mmol), CMP-NeuAc (25
mg, 0.038 mmol) and ST3Gal-III adsorbed on Ni2+
-
Agarose (50 mU) were incubated in 50 mM sodium
cacodylate buffer pH 7.1 (12 mL) at 30 °C for 4 days
with gentle stirring. Additional CMP-NeuAc (27 mg,
0.041 mmol) was added twice, after 24 and 48 h. At the
end of incubation, the gel was filtered off, washed with
10 mM sodium cacodylate buffer pH 7.1 and filtrate
and washings were combined, divided into four por-
tions and applied to Sep-Pak C18 cartridges; the com-
bined methanol eluates were evaporated to dryness;
then the residue taken up in water was purified on
Bio-Gel P2 and passed through a small column of AG
50W-X8 ion-exchange resin (Na+ form) to give 14 as
1
Compound 12: [h]2D7 +1° (c 1, MeOH); H NMR
(D2O, 250 MHz): l 7.4–7.3 (m, 10 H, Bn), 4.55–4.41
(m, 5 H, J1%,2% 8.8 Hz, H-1%, 2 CH2Ph), 4.31 (d, 2 H, J1,2
7.8 Hz, 2 H-1) and 2.60 (2 d, 4 H, Jgem 11.7 Hz, 2
CH2CO); 13C NMR (D2O, 50.3 MHz): l 175.4, 175.3
(NHCO), 138.8, 129.6, 129.1 (CꢀAr), 103.9 (C-1%), 102.9
(C-1), 79.4 (C-4), 76.7, 76.4, 75.6, 74.7, 73.6, 73.4, 72.0,
1
its sodium salt (54 mg, 85%); [h]2D7 −4° (c 1, water); H

1170
F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
NMR (D2O, 250 MHz): l 7.5–7.3 (m, 10 H, Ph),
4.59–4.51 (m, 4 H, J1%,2% 7.3, Jgem 12.2 Hz, 2 H-1%, 2
CHPh), 4.48 (d, 2 H, 2 CHPh), 4.36 (d, 2 H, J1,2 7.3 Hz,
2 H-1), 4.11 (dd, 2 H, J3%,4% 3, J3%,2% 9.8 Hz, 2 H-3%), 3.39
(d, 2 H, Jgem 9.7 Hz, 2 CH), 2.8–2.67 (m, 4 H, J3¦e,3¦a
12.5, J3¦a,4¦ 4.5, Jgem 12.7 Hz, 2 H 3-¦e, 2 CHCO), 2.5
(d, 2 H, 2 CHCO), 2.02 (s, 6 H, 2 OAc) and 1.79 (t, 2
H, J3¦a,4¦ 12.5 Hz, 2 H-3¦a); 13C NMR (D2O, 50.3
MHz): l 176.4, 175.9, 175.3 (NHCO, CO2Na), 139.1,
130.1, 129.7 (CꢀAr), 104.0 (C-1%), 103.3 (C-1), 101.2
(C-2¦), 74.7 (CH2Ph), 79.6 (C-4), 76.9, 76.6, 76.0 (C-5,
C-5%, C-3%), 74.3 (C-6¦), 73.6 (C-3), 73.2 (C-8¦), 71.0,
70.8, 70.3, 69.5, 68.9 (CH2O, C-2%, C-4%, C-4¦, C-7¦),
64.0 (C-9¦), 62.5, 61.4 (C-6, C-6%), 56.6 (C-2), 53.1
(C-5¦), 45.9 (Cq), 41.0 (C-3¦), 32.3 (CH2CO) and 23.4
(CH3CO); HRMS (negative mode): Calcd for
C69H100N4O40 [(M−2 Na)/2]− 812.2957. Found: m/z
812.2959.
C-4, C-4%, C-4¦, C-7¦, C-2§, C-3§, C-4§, C-5§), 64.0
(C-9¦), 63.0 (C-6), 60.0 (C-6%), 57.4 (C-2), 53.1 (C-5¦),
45.9 (Cq), 41.2 (C-3¦), 32.2 (CH2CO), 23.4 (CH3CO)
and 16.7 (CH3); HRMS (negative mode): Calcd for
C81H120N4O48 [(M−2 Na)/2]− 958.3536. Found: m/z
958.3531.
3.10. 2,2-Bis-[O-(5-acetamido-3,5-dideoxy-a-
-galacto-2-nonulopyranosylonate)-(2“3)-O-b-
galactopyranosyl-(1“4)-(2-amino-2-deoxy-3-O-a-
fucopyranosyl-b- -glucopyranosyloxy)]-propane butane-
1N,4N%-dioyl amide]-1,3-diol (1)
D-glycero-
D
D-
L-
D
A solution of 15 (8 mg, 0.004 mmol) in 1:1 MeOH–wa-
ter (0.5 mL) was stirred under hydrogen with 20%
Pd(OH)2 on charcoal (10 mg) for 16 h at rt. The
solution was then filtered through Celite; the filtrate
was purified on Bio-Gel P2 and passed through a small
column of AG 50W-X8 ion-exchange resin (Na+ form)
to afford 7 mg of 1 (95%); [h]2D8 −17° (c 0.83, water);
1H NMR (D2O, 400 MHz): l 5.18 (d, 2 H, J1§,2§ 4 Hz,
2 H-1§), 4.88 (m, 2 H, J5§,6§ 6.5 Hz, 2 H-5§), 4.59 (d, 2
H, J1%,2% 7.8 Hz, 2 H-1%), 4.53 (d, 2 H, J1,2 7.8 Hz, 2 H-1),
4.07 (dd, 2 H, J3%,2% 9.3, J3%,4% 3 Hz, 2 H-3%), 4.08 (d, 2 H,
Jgem 11.5 Hz, 2 CHO), 3.53 (d, 2 H, 2 CHO), 2.87–2.76
(m, 4 H, J3¦a,3§e 12 Hz, J3¦e,4¦ 4.5 Hz, Jgem 12.5 Hz, 2
H-3¦e, 2 CHCO), 2.46 (d, 2 H, 2 CHCO), 2.04 (s, 6 H,
2 OAc), 1.87 (t, 2 H, J3¦a,4 12 Hz, 2 H-3¦a) and 1.24 (d,
6 H, J6§,5¨ 6.5 Hz, 2 CH3); 13C NMR (D2O, 62.9 MHz):
l 176, 175.3, 174.9 (NHCO, CO2Na), 102.6 (C-1%, C-1),
100.6 (C-2¦), 99.6 (C-1§), 76.6, 76.1, 75.9, 74.3, 73.9; 73,
72.9, 70.6, 70.5, 70.2, 69.6, 69.5, 69.3, 69.1, 68.7, 68.3,
67.7 (C-5, C-5%, C-3%, C-3, C-6¦, C-8¦, C-2%, CH2O, C-4,
C-4%, C-4¦, C-7¦, C-2§, C-3§, C-4§, C-5§), 63.6 (C-9¦),
62.5, 61.2, 60.6 (CH2OH, C-6%, C-6), 57.0 (C-2), 52.7
(C-5¦), 46.1 (Cq), 40.8 (C-3¦), 31.8 (CH2CO), 23
(CH3CO) and 16.3 (CH3); ESMS (negative mode): m/z
868.3 [(M−2 Na)/2]− C67H108N4O48Na2.
3.9. 2,2-Bis-(benzyloxymethyl)-1,3-bis-[O-(5-acetamido-
3,5-dideoxy-a-
osylonate)-(2“3)-O-b-
(2-amino-2-deoxy-3-O-a-
D
-glycero-
D
-galacto-2-nonulopyran-
-galactopyranosyl-(1“4)-
-fucopyranosyl-b- -glucopy-
D
L
D
ranosyloxy)]-propane butane-1N,4N% dioyl amide (15)
Compound 14 (8 mg, 0.0048 mmol), GDP-Fuc (3 mg,
0.0048 mmol), MnCl2 (12 mg, 0.060 mmol) and FucT-
III adsorbed on Ni2+-Agarose (190 mU) were incu-
bated in 25 mM sodium cacodylate buffer pH 6.6 (4
mL) at 30 °C for 5 days with gentle stirring. Additional
GDP-Fuc was added twice, after 2 days (3 mg, 0.0048
mmol) and after 4 days (2.4 mg, 0.0038 mmol) together
with alkaline phosphatase (2 U) and MnCl2 (4 mg). At
the end of incubation the gel was filtered off, washed
with 10 mM sodium cacodylate buffer pH 6.6 and
filtrate and washings were combined and applied to
Sep-Pak C18 cartridges; the methanol eluate was evapo-
rated to dryness; then the residue dissolved in water
was purified on Bio-Gel P2 and passed through a small
column of AG 50W-X8 ion-exchange resin (Na+ form)
affording 7.5 mg of 15 (80%); [h]2D5 −32° (c 0.75,
3.11. 2,2-Bis-(benzyloxymethyl)-1,3-bis-[O-(5-acetamido-
3,5-dideoxy-a-
osylonate)-(2“3)-O-b-
(2-acetamido-2-deoxy-3-O-a-
glucopyranosyloxy)]-propane (17)
D
-glycero-
-galactopyranosyl-(1“4)-
-fucopyranosyl-b-
D-galacto-2-nonulopyran-
D
1
water); H NMR (D2O, 200 MHz): l 7.44–7.32 (m, 10
H, 2 Ph), 5.08 (d, 2 H, J1§,2§ 4 Hz, 2 H-1§), 4.83 (m, 2
H, 2 H-5§), 4.60–4.44 (m, 6 H, Jgem 11.7, J1%,2% 7.3 Hz,
2 H-1%, 2 CH2Ph), 4.35 (d, 2 H, J1,2 7.8 Hz, 2 H-1), 4.07
(dd, 2 H, J3%,2% 9.3, J3%,4% 3 Hz, 2 H-3%), 3.38 (d, 2 H, Jgem
9.7 Hz, 2 CH), 2.82–2.64 (m, 4 H, J3¦e,3¦a 12.5, J3¦e,4¦
4.5, Jgem 12.7 Hz, 2 H-3¦e, 2 CHCO), 2.46 (d, 2 H, 2
CHCO), 2.02 (s, 6 H, 2 OAc), 1.75 (t, 2 H, J3¦a,4¦ 12.5
Hz, 2 H-3¦a) and 1.16 (d, 6 H, J6§,5§ 6.3 Hz, 2 CH3);
13C NMR (D2O, 50.3 MHz): l 176.5, 175.7, 175.4
(NHCO, CO2Na), 139.1, 130.1, 129.7 (CꢀAr), 102.9
(C-1%, C-1), 101.1 (C-2¦), 100.0 (C-1§), 74.6 (CH2Ph),
77.1, 76.4, 74.5, 74.3, 73.3, 70.6, 70.1, 69.7, 69.5, 69.1,
68.7, 68.1 (C-5, C-5%, C-3%, C-3, C-6¦, C-8¦, C-2%, CH2O,
L
D-
Compound 16 (7 mg, 0.0042 mmol), GDP-Fuc (3 mg,
0.0048 mmol), MnCl2 (12 mg, 0.060 mmol) and FucT-
III adsorbed on Ni2+-Agarose (50 mU) were incubated
in 25 mM sodium cacodylate buffer pH 6.6 (4 mL) at
30 °C for 5 days with gentle stirring. Additional GDP-
Fuc was added twice, after 2 days (2.7 mg, 0.0042
mmol) and after 4 days (2.1 mg, 0.0034 mmol) together
with alkaline phosphatase (3 U) and MnCl2 (4 mg). At
the end of incubation the gel was filtered off, washed
with 10 mM sodium cacodylate buffer pH 6.6 and

F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
1171
filtrate and washings were combined and applied to
3.13. 2,2-Bis-(benzyloxymethyl)-1,3-bis-[O-b-
galactopyranosyl-(1“4)-(2-amino-2-deoxy-3-O-a-
fucopyranosyl-b- -glucopyranosyloxy)]-propane butane
1N,4N%-dioyl amide (18)
D-
Sep-Pak C18 cartridges; the methanol eluate was evapo-
rated to dryness; then the residue dissolved in water,
purified on Bio-Gel P2 and passed through a small
column of AG 50W-X8 ion-exchange resin (Na+ form)
affording 7 mg of 17 (82%); [h]2D4 −33°(c 0.75, water);
1H NMR (D2O, 200 MHz): l 7.44–7.29 (m, 10 H, 2
Ph), 5.04 (d, 2 H, J1§2§ 4 Hz, 2 H-1§), 4.75 (m, 2 H, 2
H-5§), 4.51–4.43 (m, 6 H, J1%,2% 7.8 Hz, 2 H-1%, 2
CH2Ph), 4.34 (d, 2 H, J1,2 7.8 Hz, 2 H-1), 4.05 (dd, 2 H,
J3%,2% 9.3, J3%,4% 2.5 Hz, 2 H-3%), 2.72 (dd, 2 H, J3¦e,3¦a 12.5,
J3¦e,4¦ 4.5 Hz, 2 H-3¦e), 1.99 (s, 6 H, 2 OAc), 1.89 (s, 6
H, 2 OAc), 1.76 (t, 2 H, J3¦a,4¦ 12.5 Hz, 2 H3-¦a) and
1.14 (d, 6 H, J6§,5§ 6.3 Hz, 2 CH3); 13C NMR (D2O,
50.3 MHz): l 176.0, 174.9, 174.7 (NHCO, CO2Na),
138.6, 129.7, 129.1 (CꢀAr), 102.6 (C-1%, C-1), 100.7
(C-2¦), 99.5 (C-1§), 74.4 (CH2Ph), 76.6, 76.2, 75.8, 75;7,
74.3, 73.9, 72.8, 70.2, 69.8, 69.7, 69.3, 69.1, 68.7, 68.3,
67.6 (C-5, C-5%, C-3%, C-3, C-6¦, C-8¦, C-2%, CH2O, C-4,
C-4%, C-4¦, C-7¦, C-2§, C-3§, C-4§, C-5§), 63.6 (C-9¦),
62.4 (C-6), 60.7 (C-6%), 56.8 (C-2), 52.7 (C-5¦), 45.7
(Cq), 40.8 (C-3¦), 23.3, 23.0 (CH3CO) and 16.3 (CH3);
ESMS (negative mode): m/z 959.3 [(M−2 Na)/2]−
C81H122N4O48Na2.
L-
D
Compound 11 (8.5 mg, 0.008 mmol), GDP-Fuc (5.4
mg, 0.008 mmol), MnCl2 (12 mg, 0.060 mmol) and
FucT-III adsorbed on Ni2+-Agarose (50 mU) were
incubated in 25 mM sodium cacodylate buffer pH 6.6
(4 mL) at 30 °C for 7 days with gentle stirring. Addi-
tional GDP-Fuc was added three times, after 2 days
(5.4 mg, 0.008 mmol) and after 4 days (2.1 mg, 0.0032
mmol), after 6 days ((2.1 mg, 0.0032 mmol) together
with alkaline phosphatase (2 U) and MnCl2 (4 mg). At
the end of incubation the gel was filtered off, washed
with 10 mM sodium cacodylate buffer pH 6.6 and
filtrate and washings were combined and applied to
Sep-Pak C18 cartridges; the MeOH eluate was evapo-
rated to dryness; then the residue taken up in water was
purified on Bio-Gel P2 to give pure 18 (8 mg, 75%) and
a mixture of 18 and the monofucosylated product (1.5
1
mg); [h]2D7 −43° (c 0.67, water); H NMR (D2O, 400
MHz): l 7.44–7.33 (m, 10 H, 2 Ph), 5.1 (d, 2 H, J1¦,2¦
4 Hz, 2 H-1¦), 4.84 (m, 2 H, 2 H-5¦), 4.54 (d, 2 H, Jgem
12 Hz, 2 CHPh), 4.48 (d, 2 H, 2 CHPh), 4.44 (d, 2 H,
J1%,2% 7.8 Hz, 2 H-1%), 4.36 (d, 2 H, J1,2 8.3 Hz, 2 H-1),
3.38 (d, 2 H, Jgem 9.8 Hz, 2 CH), 2.7 (d, 2 H, Jgem 13
Hz, 2 CHCO), 2.48 (d, 2 H, 2 CHCO) and 1.16 (d, 6 H,
J6¦,5¦ 6.5 Hz, 2 CH3); 13C NMR (D2O, 62.9 MHz): l
175.2 (NHCO), 138.8, 129.6, 129.2 (Ph), 102.8, 102.6
(C-1%, C-1), 99.7 (C-1¦), 74.2 (PhCH2), 76.1, 76, 73.5,
73.1, 73, 70.3, 69.7, 69.4, 69.2, 68.8, 67.7, 62.6, 62.1,
60.8 (C-3, C-4, C-5, C-2%, C-3%, C-4%, C-5%, C-2¦, C-3¦,
C-4¦, C-5¦, C-6, C-6%, CH2O), 57.0 (C-2), 45.5 (Cq),
32.0 (CH2CO) and 16.4 (CH3); HRMS: Calcd for
C59H88N2O32Na2 [(M+Na)/2]+ 691.2558. Found: m/z
691.2555.
3.12. 2,2-Bis-[O-(5-acetamido-3,5-dideoxy-a-
-galacto-2-nonulopyranosylonate)-(2“3)-O-b-
galactopyranosyl)-(1“4)-(2-acetamido-2-deoxy-3-O-
a- -fucopyranosyl-b- -glucopyranosyloxy)]-propane-
1,3-diol (2)
D-glycero-
D
D-
L
D
A solution of 17 (9 mg, 0.0047 mmol) in 1:1 MeOH–
water (0.6 mL) was stirred under hydrogen with 20%
Pd(OH)2 on charcoal (12 mg) for 16 h at rt. The
solution was then filtered through Celite; the filtrate
was purified on Bio-Gel P2 and passed through a small
column of AG 50W-X8 ion-exchange resin (Na+ form)
to afford 8 mg of 2 (95%); [h]2D8 −28°(c 0.75, water);
1H NMR (D2O, 400 MHz): l 5.02 (d, 2 H, J1§,2§ 4 Hz,
2 H-1§), 4.73 (m, 2 H, 2 H-5§), 4.43 (d, 2 H, J1%,2% 7.8
Hz, 2 H-1%), 4.34 (d, 2 H, J1,2 7.8 Hz, 2 H-1), 3.99 (dd,
2 H, J3%,2% 10.2, J3%,4% 3 Hz, 2 H-3%), 4.00 (d, 2 H, Jgem 9.8
Hz, 2 CH), 2.74 (dd, 2 H, J3¦e,3¦a 12.5, J3¦e,4¦ 4.5 Hz, 2
H-3¦e), 2.05 (s, 6 H, 2 OAc), 2.03 (s, 6 H, 2 OAc), 1.78
(t, 2 H, J3¦a,4§ 2.5 Hz, 2 H-3¦a) and 1.16 (d, 6 H%, J6§,5§
6.5 Hz, 2 CH3); 13C NMR (D2O, 62.9 MHz): l 176.0,
175.1, 174.9 (NHCO, CO2Na), 102.7, 102.6 (C-1%, C-1),
100.7 (C-2¦), 99.6 (C-1§), 76.7, 76.2, 75.9, 75.7, 74.3,
73.9, 72.9, 70.2, 70.0, 69.3, 69.1, 68.8, 68.3, 67.7 (C-5,
C-5%, C-3%, C-3, C-6¦, C-8¦, C-2%, CH2O, C-4, C-4%, C-4¦,
C-7¦, C-2§, C-3§, C-4§, C-5§), 63.6 (C-9¦), 62.5 (C-6),
61.6, 60.6 (CH2OH, C-6%), 56.9 (C-2), 52.7 (C-5¦), 46.1
(Cq), 40.8 (C-3¦), 23.2, 23.0 (CH3CO) and 16.3 (CH3);
ESMS (negative mode): m/z 869.3 [(M−2Na)/2]−
C67H110N4O48Na2.
3.14. 2,2-Bis-[O-b-
D
-galactopyranosyl-(1“4)-(2-amino-
2-deoxy-3-O-a- -fucopyranosyl-b-
L
D-glucopyranosyloxy)-
propane butane-1N,4N%-dioyl amide]-1,3-diol (19)
A solution of 18 (9 mg, 0.0063 mmol) in 1:1 MeOH–
water (0.9 mL) was stirred under hydrogen with 20%
Pd(OH)2 on charcoal (18 mg) for 16 h at rt. The
solution was then filtered through Celite; the filtrate
was purified on Bio-Gel P2 to afford 8 mg of 19
1
(quantitative yield); [h]3D1 0° (c 0.25, water); H NMR
(D2O, 250 MHz): l 5.14 (d, 2 H, J1¦,2¦ 4.5 Hz, 2 H-1¦),
4.8 (m, 2 H, 2 H-5¦), 4.35 (d, 4 H, J1%,2% 7.8, J1,2 7.8 Hz,
2 H-1, 2 H-1, 2 H-1%), 3.99 (d, 2 H, Jgem 12 Hz, 2 CH),
3.44 (d, 2 H, Jgem 12 Hz, 2 CH), 2.74 (d, 2 H, Jgem 13
Hz, 2 CHCO), 2.51 (d, 2 H, 2 CHCO) and 1.17 (d, 2 H,
J6¦,5¦ 6.5 Hz, 2 CH3); 13C NMR (D2O, 50.3 MHz): l
175.3 (NHCO), 102.8, 102.6 (C-1, C-1%), 99.6 (C-1¦),
76.1, 76.0, 75.9, 74.3, 73.5, 72.9, 72.0, 70.2, 69.6, 69.3,

1172
F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
68.7, 67.7 (C-3, C-4, C-5, C-2%, C-3%, C-4%, C-5%, C-2¦,
C-3¦, C-4¦, C-5¦, CH2O), 62.5 (C-6), 61.2, 60.7 (C-6%,
CH2OH), 57.0 (C-2), 46.1 (Cq), 31.8 (CH2CO) and 16.3
(CH3); HRMS: Calcd for C45H76N2O32 [(M+2 Na)/
2]+ 601.2088. Found: m/z 601.2087.
102.8, 102.7 (C-1, C-1%), 99.6 (C-1¦), 76.3, 75.9, 75.7,
74.3, 73.4, 72.9, 72.0, 70.2, 70.1, 69.3, 68.7, 67.7 (C-3,
C-4, C-5, C-2%, C-3%, C-4%, C-5%, C-2¦, C-3¦, C-4¦, C-5¦,
CH2O), 62.5 (C-6), 61.6, 60.7 (C-6%, CH2OH), 56.8
(C-2), 46.1 (Cq), 23.2 (CH3CO) and 16.2 (CH3);
HRMS: Calcd for C45H78N2O32 [(M+2 Na)/2]+
602.2166. Found: m/z 602.2167.
3.15. 2,2-Bis-(benzyloxymethyl)-1,3-bis-[O-b-
topyranosyl-(1“4)-(2-acetamido-2-deoxy-3-O-a-
fucopyranosyl-b- -glucopyranosyloxy)]-propane (21)
D-galac-
L-
D
Acknowledgements
Compound 20 (8 mg, 0.0076 mmol), GDP-Fuc (5.1 mg,
0.0076 mmol), MnCl2 (12 mg, 0.060 mmol) and FucT-
III adsorbed on Ni2+-Agarose (60 mU) were incubated
in 25 mM sodium cacodylate buffer pH 6.6 (4 mL) at
30 °C for 7 days with gentle stirring. Additional GDP-
Fuc was added three times, after 2 days (5.1 mg, 0.0076
mmol), after 4 days (2.1 mg, 0.0032 mmol), after 6 days
(2.1 mg, 0.0032 mmol) together with alkaline phos-
phatase (2 U) and MnCl2 (4 mg). At the end of
incubation the gel was filtered off, washed with 10 mM
sodium cacodylate buffer pH 6.6 and filtrate and wash-
ings were combined and applied to Sep-Pak C18 car-
tridges; the methanol eluate was evaporated to dryness;
then the residue taken up in water was purified on
Bio-Gel P2 to afford pure 21 (5 mg, 50%), and a
mixture of 21 and the monofucosylated product (30%);
We thank CNRS and the University of Paris-Sud for
financial support.
References
1. Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.;
Wong, C.-H. Chem. Re6. 1998, 98, 833–862.
2. Yuen, C.-T.; Bezouska, K.; O’Brien, J.; Stoll, M.;
Lemoine, R.; Lubineau, A.; Kiso, M.; Hasegawa, A.;
Bockovich, N. J.; Nicolaou, K. C.; Feizi, T. J. Biol.
Chem. 1994, 269, 1595–1598.
3. Palcic, M. M.; Li, H.; Zanini, D.; Bhella, R. S.; Roy, R.
Carbohydr. Res. 1997, 305, 433–442.
4. Miyauchi, H.; Tanaka, M.; Koike, H.; Kawamura, N.;
Hayashi, M. Bioorg. Med. Chem. Lett. 1997, 7, 985–988.
5. Spevak, W.; Foxall, C.; Charych, D. H.; Dasgupta, F.;
Nagy, J. O. J. Med. Chem. 1996, 39, 1018–1020.
6. DeFrees, S. A.; Kosch, W.; Way, W.; Paulson, J. C.;
Sabesan, S.; Halcomb, R. L.; Huang, D.-H.; Ichikawa,
Y.; Wong, C.-H. J. Am. Chem. Soc. 1995, 117, 66–79.
7. Wittmann, V.; Takayama, S.; Gong, K. W.; Weitz-
Schmidt, G.; Wong, C.-H. J. Org. Chem. 1998, 63, 5137–
5143.
1
[h]2D5 −31° (c 0.67, water); H NMR (D2O, 250 MHz):
l 7.43–7.29 (m, 10 H, 2 Ph), 5.04 (d, 2 H, J1¦2¦ 4 Hz, 2
H-1¦), 4.77 (m, 2 H, 2 H-5¦), 4.45 (s, 4 H, 2 CH2Ph),
4.39 (d, 2 H, J1%,2% 7.8 Hz, 2 H-1%), 4.33 (d, 2 H, J1,2 7.8
Hz, 2 H-1), 1.88 (s, 6 H, 2 OAc) and 1.12 (d, 6 H, J6¦,5¦
6.5 Hz, 2 CH3); 13C NMR (D2O, 62.9 MHz): l 174.8
(NHCO), 138.6, 129.7, 129.2, (CꢀAr), 102.8, 102.5 (C-
1%, C-1), 99.6 (C-1¦), 74.4 (CH2Ph), 76.2, 75.9, 74.3,
73.5, 72.9, 72.0, 70.2, 69.9, 69.6, 69.4, 68.7, 67.7 (C-3,
C-4, C-5, C-2%, C-3%, C-4%, C-5%, C-2¦, C-3¦, C-4¦, C-5¦,
CH2O), 62.5, 60.7 (C-6, C-6%), 56.9 (C-2), 45.6 (Cq),
23.3 (CH3CO) and 16.3 (CH3); HRMS: Calcd for
C59H90N2O32Na2 [(M+Na)/2]+ 692.2636. Found: m/z
692.2615.
8. Lin, C.-H.; Shimazaki, M.; Wong, C.-H.; Koketsu, M.;
Luneja, L. R.; Kim, M. Biorg. Med. Chem. 1995, 3,
1625–1630.
8
9. Baisch, G.; Ohrlein, R. Angew. Chem., Int. Ed. Engl.
1996, 35, 1812–1815.
10. Gege, C.; Schmidt, R. R. Carbohydr. Res. 2002, 337,
1089–1094.
11. Miyauchi, H.; Yuri, M.; Tanaka, M.; Kawamura, N.;
Hayashi, M. Bioorg. Med. Chem. Lett. 1997, 7, 989–992.
12. Sprengard, U.; Schudock, G.; Schmidt, W.; Kretzschmar,
G.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1996, 35,
321–324.
13. Renkonen, O.; Toppipila, S.; Penttila, L.; Saminen, H.;
Maaheimo, H.; Costello, C. E.; Turunen, J. P.;
Renkonen, R. Glycobiology 1997, 7, 453–461.
14. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew.
Chem., Int. Ed. Engl. 1998, 37, 2754–2794.
3.16. 2,2-Bis-[O-b-
amido-2-deoxy-3-O-a-
osyloxy)]-propane-1,3-diol (22)
D
-galactopyranosyl-(1“4)-(2-acet-
L
-fucopyranosyl-b- -glucopyran-
D
A solution of 21 (4.5 mg, 0.0034 mmol) in 1:1 MeOH–
water (0.6 mL) was stirred under hydrogen with 20%
Pd(OH)2 on charcoal (8 mg) for 16 h at rt. The solution
was then filtered through Celite; the filtrate was purified
on Bio-Gel P2 to afford 4 mg of 22 (quant. yield); [h]D30
15. Dagron, F. Ph.D. Thesis, 1999, University of Paris-Sud,
Orsay, France.
16. Ivannikova, T.; Bintein, F.; Malleron, A.; Juliant, S.;
Cerutti, M.; Harduin-Lepez, A.; Delannoy, P.; Auge´, C.;
Lubineau, A. Carbohydr. Res. 2003, 338, see following
communication doi:10.1016/S0008-6215(03)00130-7.
17. Auzanneau, F.-I.; Mondange, M.; Charon, D.; Szabo, L.
Carbohydr. Res. 1992, 228, 37–45.
18. Suhara, Y.; Hildreth, J. E. K.; Ichikawa, Y. Tetrahedron
Lett. 1996, 37, 1575–1578.
19. Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J.;
Garcia-Junceda, E.; Williams, M. A.; Bayer, R.;
1
−76° (c 0.25, water); H NMR (D2O, 200 MHz): l
5.12 (d, 2 H, J1¦,2¦ 4.5 Hz, 2 H-1¦), 4.90 (m, 2 H, 2
H-5§), 4.45 (d, 4 H, J1%,2% 7.8, J1,2 7.8 Hz, 2 H-1, 2 H-1%),
3.98 (d, 2 H, Jgem 12 Hz, 2 CH), 3.42 (d, 2 H, 2 CH),
2.04 (s, 6 H, 2 OAc) and 1.17 (d, 6 H, J6¦,5¦ 6.5 Hz, 2
CH3); 13C NMR (D2O, 50.3 MHz): l 175.1, (NHCO),

F. Bintein et al. / Carbohydrate Research 338 (2003) 1163–1173
1173
Ketcham, C.; Walker, L. E.; Paulson, J. C.; Wong, C.-H.
J. Am. Chem. Soc. 1992, 114, 9283–9298.
20. Chuen Liu, Y.; Li, H.; Otter, A.; Kamath, V. P.;
Streiff, M. B.; Palcic, M. M. Can. J. Chem. 2002, 80,
540–545.
21. Barstro¨m, M.; Bengtsson, M.; Blixt, O.; Norberg, T.
Carbohydr. Res. 2000, 328, 525–531.
22. Unverzagt, C.; Kunz, H.; Paulson, J. C. J. Am. Chem.
Soc. 1990, 112, 9308–9309.
8
23. Schwardt, O.; Baisch, G.; Ohrlein, R. Biorg. Med. Chem.
2001, 9, 1857–1869.
24. Auge´, C.; Malleron, A.; Tahrat, H.; Marc, A.; Goergen,
J.-L.; Cerutti, M.; Steelant, W. F. A.; Delannoy, P.;
Lubineau, A. Chem. Commun. 2000, 2017–2018.