Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents

Article abstract of DOI:10.1016/j.carres.2006.03.044

The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-d-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.

Full text of DOI:10.1016/j.carres.2006.03.044

Carbohydrate Research 341 (2006) 2026–2036
Synthesis of globo- and isoglobotriosides bearing
a cinnamoylphenyl tag as novel electrophilic thiol-specific
carbohydrate reagents
Mohamed R. E. Aly,a Pascal Rochaix,b Mohamed Amessou,b
Ludger Johannesb and Jean-Claude Florenta,*
aLaboratoire de Chimie UMR 176 CNRS-Institut Curie, 26 rue d’Ulm, F-75248 Paris, France
bLaboratoire ‘Traffic and Signalisation’ UMR 144 CNRS-Institut Curie, 26 rue d’Ulm, 75248 Paris, France
Received 1 December 2005; received in revised form 7 March 2006; accepted 7 March 2006
Available online 13 June 2006
Abstract—The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled
with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globo-
trioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoyl-
phenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate
to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein
(StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Globotriose; Isoglobotriose; Chalcone; Thiol-specific reagents; Thiopropyl Sepharose 6B
1. Introduction
antigen responsible for immediate hyperacute rejection
upon xenotransplantation.6 Finally, isoGb3 was recently
identified as a key endogenous NKT cell antigen present
at the surface of antigen presenting cells D1.7 These tri-
saccharides, have been often used as probes in biological
applications. Thus, several globotriose-based artificial
STx ligands were investigated as blockers of the toxin-
host cell adhesion.8–11 Globotriose has been used as a
tool to study the retrograde transport (i.e., the directed
movement of membrane bound vesicles from recycling
endosomes back to the trans-Golgi network where they
are recycled for further rounds of transport) and in
immunotherapy.12 For its part, isoGb3, has been used
to form artificial isoglobotriosyl polymers as effective
antagonists of anti-Gal antibodies to prevent rejection
of xenografts.13 Furthermore the anti-Gal antibody,
mediated immunotherapy aimed at redirecting human
nature immune defence to kill viruses, bacteria and can-
cer cells has been investigated by Wang et al.6 Immobi-
lized isoglobotriose has been used for the separation of
toxins A and B.5
The Pk antigen globotriosylceramide (Gala1,4-Galb1,4-
Glcb1,1-OCer, Gb3), a cell surface glycosphingolipid
in various normal tissues,1 is also well known to be over-
expressed in several human tumour cells.2 The terminal
Gala(1!4)Gal or galabiosyl epitope of the trisaccharide
core, that is globotriose, or Gb3 is a ligand for Shiga
toxins3 excreted by E. coli strain O-157:H-7. This event
is responsible for gastroenteritis and the Haemolytic-
Uraemic Syndrome (HUS).4 A structurally related
glycosphingolipid, isoglobotriosyl ceramide (Gala1,3-
Galb1,4-Glcb1,1-OCer, isoGb3) characterized by the
terminal Gala(1!3)Gal moiety, the aGal epitope, of
the glycone isoglobotriose (isoGb3) is a known ligand
for two exotoxins, toxins A and B, produced by Clos-
tridium difficile, which are responsible for human intesti-
nal inflammation.5 IsoGb3 is also known as animal
*
Corresponding author. Fax: +33 1 42 34 66 31; e-mail: Jean-
Claude.Florent@curie.fr
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.044

M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
2027
Although chemical methods routinely used for the
synthesis of Gb3 and isoGb3 epitopes from different
galactosyl donors7,14–17 are well known, these methods
frequently lead to b-glycosides as sometimes unsepara-
ble byproducts. Our first objective was to investigate
the behaviour of 4,6-di-O-acetyl-2,3-di-O-benzyl-D-gal-
actopyranosyl trichloroacetimidate as galactosyl donor.
We hypothesized that conjugation of an expected
enhanced remote assistance of any of the two acetyl
groups, along with the well-established leaving group
ability of the trichloroacetimidate18 for the coupling
with reactive benzylated lactosides as galactosyl accep-
tors might lead to good stereocontrolled a-stereoselec-
tivity. The affinity of the cinnamoylphenyl group
(chalcone) to form stable thioether linkages with thi-
ols,19 prompted us to investigate this group conjugated
with these trisaccharides to prepare two novel electro-
philic thiol-specific carbohydrate reagents bearing a 6-
(p-cinnamoylphenoxy)hexyl tether 1 and 2 for neoglyco-
conjugate synthesis20 and biosensors preparation.21 As
an example, we realized the immobilization of 1 onto
Thiopropyl Sepharose 6B in order to isolate the Shiga
toxin StxB from periplasmic extract of E. coli strain
BH 101, and as an example of neoglycoconjugate syn-
thesis, the coupling of 2 to the protein StxB as thiol-
possessing protein (Fig. 1).
Scheme 1. Reagents and conditions: (a) EtSH, p-TsOH, CH2Cl2, 97%;
(b) Ac2O, pyridine, 94%; (c) TFA, CH2Cl2 2:1, 82%; (d) CCl3CN,
DBU, 93%.
silyl)ethyl group with 2:1 TFA in CH2Cl2 gave the 1-
O-unprotected derivative 6 in 82% yield. Further treat-
ment of 6 with trichloroacetonitrile in the presence of
1,8-diazo[5.4.0]bicycloundec-7-ene (DBU) furnished the
desired novel galactosyl trichloroacetimidate 7 (a/b
mixture 11:1, 93%).
2. Results and discussion
2.2. Synthesis of the globotriosyl chalcone conjugate 1
2.1. Synthesis of the galactosyl donor 7
As outlined in Scheme 2, compound 7 was coupled with
the glycosyl acceptor 814b,23 in 1:1 CH2Cl2–Et2O, for the
‘ether effect’,24 in the presence of TMSOTf (6 mol %) as
promotor at ꢀ25 ꢁC and under ‘inverse procedure’ con-
dition.25 This stereospecifically led to the a-glycoside 9,
which was obtained in 81% yield. It is noteworthy that
this result was obtained with an economical ratio of do-
nor to acceptor of 1.5:1. Assignment of the terminal gala-
ctopyranosyl moiety and hence the a-stereoselectivity
The benzylidene group of 2-(trimethylsilyl)ethyl 2,3-
di-O-benzyl-4,6-di-O-benzylidene-b-D-galactopyranoside
3
22 was smoothly removed by hydrolysis with a catalytic
amount of p-TsOH in the presence of an excess of EtSH
as scavenger of the released benzaldehyde, resulting in
the known diol 4 (97% yield)22 as depicted in Scheme
1. Acetylation of 4 with Ac2O–pyridine afforded 5 in
94% yield and subsequent removal of the 2-(trimethyl-
Figure 1.

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M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
Scheme 2. Reagents and conditions: (a) TMSOTf (6 mol %), Et2O–CH2Cl2, ꢀ25 ꢁC to rt, 81%; (b) NaOMe–MeOH, 98%; (c) 1: H2, Pd–C, MeOH; 2:
Ac2O, Pyridine, 89%; (d) N2H4ÆHOAc, DMF, 92%; (e) CCl3CN, DBU, 80%; (f) 6-chloro-1-hexanol, TMSOTf (6 mol %), CH2Cl2, ꢀ45 ꢁC to rt, 54%;
(g) NaOMe–MeOH, 96%; (h) p-cinnamoylphenol, K2CO3, DMF, 53%; (i) Ac2O, Pyridine, 88%.
1
was determined unambiguously by H NMR data of
H-1c (d = 5.14, J1,2 3.1 Hz) as well as by 13C NMR data
C-1c (d = 100.25). This high selectivity can be attributed
to the participation of any of the remote acetyl groups as
well as the reaction conditions used, and thus support
our starting hypothesis.
debenzylated in the presence of a catalytic amount of
Pd/C (10%) in MeOH at room temperature followed
by a treatment with Ac2O–pyridine, 1115d was obtained
in 89% yield. Besides the ease of debenzylation of 10,
deacetylation of 9 removed any traces of unreacted
acceptor from the coupling step and was taken into
account in estimating the yields of both 9 and 10. Ano-
meric deacetylation of 11 was performed by treatment
with hydrazinium acetate in DMF to afford 1215d in
92% yield. Subsequent treatment with trichloroacetonit-
rile in the presence of DBU furnished the known a-tri-
chloroacetimidate 1315d in 80% yield. Reaction of
trisaccharide donor 13 with 6-chloro-1-hexanol (under
inverse procedure condition)25 with TMSOTf
(6 mol %) as catalyst in MeCN at low temperature,
afforded the desired b-linked 6-chlorohexyl glycoside
Further important step was the replacement of the
benzyl groups by their acetyl counterparts in order to
control the b configuration of the spacer. In our first
attempt for reductive debenzylation, compound 9 was
treated directly with Pd/C (10%, 1:1 w/w) in 3:1
MeOH–AcOH, then acetylated with Ac2O/pyridine. In
these conditions, an array of products was obtained fur-
nishing only a low yield of 11 (data not shown). Fortu-
´
nately, when 9 was deacetylated first under Zemplen
conditions,26 affording 10 (98%) and the latter product

M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
2029
14 in a moderate (54%) yield. The b-anomeric configura-
tion was determined from the neighbour 2-H proton d
4.88 (J1,2 8.1 Hz) because of overlapping of signals in
the H-1a region. The modest yield of this coupling reac-
tion could originate from the low donor to acceptor
ratio (1:6) and the relatively high acidic conditions used
in order to avoid orthoester formation.15d Under these
conditions, hydrolysis of the donor could be a competi-
tive reaction. It is noteworthy that no glycoside forma-
tion was observed using the softer Lewis acid
Et2OÆBF3. Attempts to obtain 14 directly by reacting
11 with 6-chloro-1-hexanol in the presence of Et2OÆBF3
led only to 12 (data not shown). Deacetylation of 14
tion proceeded smoothly and could be monitored easily
by virtue of both deacetylation and the fluorescent
nature of the linker. For detailed confirmation of the
structure of 1, it was transformed into 16 for easier
1
assignment by H NMR spectroscopy.
2.3. Synthesis of the isoglobotriose chalcone conjugate 2
The synthesis of the target compound 2 followed exactly
the same sequence as described for the synthesis of 1
(Scheme 3). Thus, treatment of 7 with 1727 afforded 18
in higher yield as expected (87%), due to the higher reac-
tivity of the equatorial 3-OH group. The a-stereoselec-
1
´
under Zemplen conditions yielded 15 in 96% yield,
tivity was unambiguously determined by the H NMR
then treatment with p-cinnamoylphenol and K2CO3 in
DMF afforded the first target 1 in 53% yield. The reac-
data of H-1c (d 5.16, J1,2 2.9 Hz) as well from the 13C
NMR value for C-1c (d 95.50). Subsequent deacetylation
Scheme 3. Reagents and conditions: (a) TMSOTf (6 mol %), Et2O–CH2Cl2, ꢀ20 ꢁC to rt, 87%; (b) NaOMe–MeOH, 76%; (c) 1: H2, Pd–C, MeOH; 2:
Ac2O, Pyridine, 72%; (d) N2H4ÆHOAc, DMF, 70%; (e) CCl3CN, DBU, 82%; (f) 6-chloro-1-hexanol, TMSOTf (6 mol %), CH2Cl2, ꢀ45 ꢁC to rt, 44%;
(g) NaOMe–MeOH, quant.; (h) p-cinnamoylphenol, K2CO3, DMF, 77%; (i) Ac2O, Pyridine, 91%.

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M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
followed by debenzylation and acetylation afforded 20
in good overall yield. Deacetylation of 18 removed easily
traces of unreacted acceptor from the coupling step and
was taken into account in estimating the yields of both
18 and 19. Anomeric deacetylation followed by trichloro-
acetimidate activation gave the trisaccharide donor
22,15d which was coupled with 6-chloro-1-hexanol to
afford the b-linked conjugate 23 (44%). The b-anomeric
configuration of 23 was unambiguously determined
from the signal of the neighbouring H-2 proton at d
4.86 (J1,2 8.0 Hz) because of overlapping of signals in
the H-1a region as in 14. Deacetylation and condensa-
tion with p-cinnamoylphenol afforded the second target
compound 2 in 77% yield. Compound 2 was similarly
transformed into the acetylated derivative 25, the struc-
disulfide-bond formation with the cysteine residue of the
protein.21a Periplasmic extracts from bacteria expressing
StxB were then passed over the column, and after wash-
ing, proteins were eluted with a linear guanidinium
chloride gradient. The elution profile showed one large
peak at 4.5 M gradient concentration of guanidinium
chloride (Fig. 2A). The peak fractions were pooled,
and the StxB analyzed by Western Blot with specific
antibody (Fig. 2B, left) and by Coomassie blue staining
of the gels (Fig. 2B, right). Identical amounts of total
protein were loaded in lanes 2–8. The Western Blot
allowed identifying the StxB containing fractions. Note-
worthy, the column flow through contained only very
little amount of StxB (lane 4), indicating that the column
retained most of Stx. As shown by Coomassie blue
staining, pools 5–7 all contained StxB with purity
greater than 99%. For comparison, lane 8 contained
the eluted protein from a MonoQ cation exchange col-
umn over which the crude periplasmic extract was
passed once. The procedure allowed the purification of
at least 0.75 mg of StxB from 1 mL of globotriose func-
tionalized column material.
1
ture of which has been completely assigned by both H
and 13C NMR spectroscopy.
2.4. Purification of StxB using globotriose functionalized
Thiopropyl Sepharose 6B
For immobilization of 1 onto Thiopropyl Sepharose 6B,
commercially available as the 2-dithiopyridyl gel, thio-
pyridyl protecting groups were removed before loading
into a FPLC column by thorough washing with 2-
mercaptoethanol. Then, compound 1 was coupled to
Sepharose in two steps. First, Sepharose was treated
with 1 (17 mg) dissolved in absolute MeOH in the pres-
ence of InCl3 (10 mol %), catalyst accelerator of thiol
addition into chalcone derivatives.19b Second, to ensure
maximum saturation of the column, the recovered com-
pound 1 (10 mg) obtained by evaporation of the flow
through, poorly soluble in water, was dissolved in
DMF, before being diluted with phosphate buffer
(1:1), to avoid precipitation. It was then incubated again
with the same gel equilibrated with DMF–phosphate
buffer (1:1) at pH 7.5. Unreacted thiol groups of Sephar-
ose were then trapped by N-methylmaleimide to prevent
2.5. Coupling of conjugate 2 to the protein StxB/Cys
To demonstrate the ability of compound 2 to fix sulfhy-
dryl containing proteins, we used a variant of StxB to
which a Cys was added such as to avoid the formation
of inter- and intra-chain disulfide bonds.28 The coupling
reaction was performed with different molar excess of
compound 2 over StxB/Cys for 16 h in borate buffer at
pH 9 and dialysis against deionized water, then analyzed
by MALDI-TOFMS (Fig. 3). It can clearly be seen that,
with increasing molar ratios, the coupling product be-
comes the major species. The ease of synthesis and puri-
fication of 2 besides the high coupling efficiency to
protein may make this linker an addition to the already
available thiol-reactive reagents. Thus, this method
Figure 2. Purification of StxB. (A) Elution profile from globotriose functionalized Sepharose 6B loaded with periplasmic extracts from bacteria
expressing StxB. The A280 line measures total protein, and the A254 line measures tyrosine absorbance (and reflects ribosomal RNA contamination).
Fraction 3–5, 6–8 and 9–11 were sampled in pools 5, 6 and 7, respectively. (B) Western blotting and (C) Coomassie blue analysis of the following
samples: 1 and 9: markers; 2: crude extract; 3: crude extract after ultracentrifugation at 100,000g/30 min; 4: column flow through; 5–7: fraction pools;
8: MonoQ fraction. Arrows indicate StxB.

M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
2031
layer thickness 0.2 mm). The detection was achieved
by treatment with a soln of 20 g ammonium molybdate
and 0.4 g cerium(IV) sulfate in 400 mL 10% H2SO4 or
with 15% H2SO4, and heating at 150 ꢁC. Flash chromato-
graphy was carried out on silica gel (Baker, 30–60 lm)
or Lichroprep Si 60 (E. Merck; / 15–25 lm), type-B
silica column. Optical rotation was determined at
20 ꢁC with a Perkin–Elmer 241/MC polarimeter (1 dm
cell). NMR spectra were recorded with a Bruker AC
300 DRX instrument with Me4Si as internal standard;
J values are given in Hertz. The assignment of 1H
NMR (300 MHz) spectra was based on chemical shift
correlation (DQFCOSY) while, the assignment of 13C
NMR (75 MHz) spectra were based on Carbon-Proton
Shift-Correlation Heteronuclear Multiple Quantum
Coherence (HMQC). Mass spectra were recorded with
a MALDI-Kompakt (Kratos) equipment using a-cyano-
3,5-dihydroxycinamic acid (CHCA), while FAB spectra
were recorded with a Jeol MS700 apparatus at the Ecole
´
Normale Superieure (Paris). Microanalysis was per-
formed in the Microanalysis Unit of CNRS at Lyon.
Thiopropyl Separose 6B was provided from General
Electric Amersham Bioscience and the column was
equipped with AKTA Purifier 10-General Electric
Amersham Bioscience.
Figure 3. MALDI-TOF analysis of compound 2 coupling to StxB/
Cys. The molar excess of compound 2 over StxB B-chain monomer is
noted to the right. In the absence of compound 2, StxB B-chain
monomer is the only species at 7793 (theoretical mass: 7791). With
increasing excess of compound 2, the coupling product at 8604
becomes the major product. B-chain dimer was frequently detected at
15,582 as byproduct.
3.2. 2-(Trimethylsilyl)ethyl 4,6-di-O-acetyl-2,3-di-O-benz-
yl-b-D-galactopyranoside (5)
A mixture of 3 (6.62 g, 12.0 mmol), EtSH (6.5 mL,
87.7 mmol) and p-TsOH (0.13 g, 0.70 mmol) in dry
CH2Cl2 (10 mL) was stirred at rt. After 48 h, the mixture
was neutralized with Et3N, evaporated under dimin-
ished pressure and the residue was purified by flash
chromatography (3.5:1 toluene–acetone) to yield 4
(5.42 g, 97%) as a colourless powder. Rf 0.22 in 3.5:1 tol-
uene–acetone. The product from the last step was stirred
with pyridine (80 mL) and Ac2O (40 mL) for 24 h at rt,
then co-evaporated with toluene under diminished pres-
sure. A purification by flash chromatography (5:1 petro-
leum ether–EtOAc) furnished 5 (6.13 g, 94%) as a
colourless oil; [a]D +19.6 (c 0.3, CHCl3); Rf 0.23 (5:1
petroleum ether–EtOAc); 1H NMR (CDCl3): d 7.37–
7.26 (m, 10H, 2Ph), 5.49 (d, 1H, J3,4 = J4,5 1.8 Hz, H-
4), 4.90, 4.73 (2d, 2-H, Jgem 10.9 Hz, CH2Ph), 4.75,
4.53 (2d, 2-H, Jgem 11.4 Hz, CH2Ph), 4.40 (d, 1-H, J1,2
7.3 Hz, H-1), 4.22–4.11 (m, 2-H, H-60, H-6), 4.06–3.97
(m, 1-H, –OCHH), 3.78–3.74 (m, 1-H, H-5), 3.66–3.54
(m, 3-H, –OCHH, H-2, H-3), 2.14, 2.07 (2s, 6-H,
2CH3CO), 1.11–1.01 [m, 2-H, –CH2Si(CH3)3], 0.03 [s,
9-H, –Si(CH3)3]; 13C NMR (CDCl3): d 170.52, 170.45
(2C@O), 138.64–127.57 (2Ph), 103.26 (C-1), 79.11,
78.89 (C-2, C-3), 76.57, 72.18, (2CH2Ph), 70.56 (C-5),
67.81 (–OCH2–), 66.50 (C-4), 62.00 (C-6), 20.89, 20.74
(2CH3CO), 18.48 [CH2Si(CH3)3], ꢀ1.48 [Si(CH3)3];
FABMS m/z 567.3 [M+Na]+. Anal. Calcd for
avoids the use of cross-coupling reagents and amin-
oalkyl glycosides for ligation of glycosides into cys-
teine-containing proteins29 or thiol-coated surfaces
used for preparation of artificial biosensors.21a
2.6. Conclusion
We described here a novel and convenient synthesis of
globo- and isoglobotrioses, as well as their grafting to
a thiol-reactive chalcone. It is noteworthy that the new
glycosyl donor afforded stereospecifically globo- and
isoglobotrioses in good yields. Moreover, ligation of
the novel conjugates to Thiopropyl Sepharose as well
as to a thiol-possessing model-protein was efficiently
performed with the new chalcone based spacer. This
constitute a valuable addition to methods, which
already exist in this context for diverse bio-applications.
3. Experimental
3.1. General methods
Solvents were purified in the usual way. TLC was per-
formed on plastic plates Silica Gel 60 F254 (E. Merck,

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M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
C29H40O8Si (544.78): C, 63:93; H, 7.41. Found: C, 64.10;
H, 7.43.
(CDCl3): d 170.22–170.13 (2CH3C@O), 139.03–137.45
(m, 9-ArC), 128.82–127.27 (m, 45-ArCH), 102.80,
102.38 (C-1a, C-1b), 100.25 (C-1c), 82.47–66.60 (m,
30-C), 20.80, 20.65 (2-CH3CO); FAB+HRMS: Calcd
for C85H90O18, [M+Na]+ 1421.6006. Found: m/z
1421.6025.
3.3. O-(4,6-Di-O-acetyl-2,3-di-O-benzyl-a/b-D-galacto-
pyranosyl) trichloroacetimidate (7)
To a soln of 5 (6.07 g, 11.1 mmol) in anhyd CH2Cl2
(30 mL) was added dropwise TFA (70 mL) at rt with
stirring. After 5 h, the mixture was co-evaporated with
toluene under diminished pressure and dried. The resi-
due was purified by flash chromatography (1.5:1 petro-
leum ether–EtOAc) to yield 6 (4.06 g, 82%) as a yellow
oil. Rf 0.25 (1.5:1 petroleum ether–EtOAc). Compound
6 (0.89 g, 2.0 mmol) was stirred with CCl3CN (1.5 mL,
15.0 mmol) and DBU (0.05 mL, 0.3 mmol) in dry
CH2Cl2 (3.0 mL) at rt. After 5 h, the mixture was co-
evaporated with EtOAc under diminished pressure and
the residue was purified by flash chromatography (1:1
petroleum ether–EtOAc + 1% Et3N) to yield 7 (1.11 g,
93%) as a yellow syrup a/b ꢁ 11:1. Rf 0.79 (7a), 0.63
(7b) (1:1 petroleum ether–EtOAc + 1% Et3N); 1H
NMR (300 MHz, CDCl3): d 8.68 (s, 0.08-H, NHb),
8.61 (s, 0.92-H, NHa), 7.30–7.26 (m, 10-H, 2Ph), 6.52
(d, 0.92-H, J1,2 <1.0 Hz, H-1a), 5.78 (d, 0.02-H, J1,2
8.1 Hz, H-1b), 5.63 (d, 0.92-H, J3,4 = J4,5 <1.0 Hz, H-
4a), 5.55 (d, 0.08-H, J3,4 = J4,5 <1.0 Hz, H-4b), 4.85–
4.74 (m, 3-H, 1.5CH2Ph), 4.58 (d, 1-H, Jgem 11.6 Hz,
CHHPh), 4.33 (m, 1-H, H-5), 4.15 (m, 1-H, H-60),
4.11–3.96 (m, 3-H, H-6, H-3, H-2), 2.17, 2.07 (2s, 0.48-
H, 2CH3CO), 2.13, 2.03 (2s, 5.52-H, 2CH3COa).
3.5. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-(1!4)-
2,3,6-tri-O-acetyl-b-D-galactopyranosyl-(1!4)-1,2,3,6-
tetra-O-acetyl-D-glucopyranose (11)
A mixture of 9 (1.52 g, 1.1 mmol) and NaOMe (0.57 g,
10.5 mmol) in dry MeOH (150 mL) was stirred at room
temperature for 48 h, then neutralized with Amberlite
IR-resin (H+ form), filtered and evaporated under
diminished pressure. The residue was purified by flash
chromatography (1.5:1 petroleum ether–EtOAc) to
afford 10 (1.41 g, 98%) as a colourless syrup. Rf 0.23
(1.5:1 petroleum ether–EtOAc). This was dissolved in
dry MeOH (60 mL) and stirred with 10% Pd–C (0.4 g)
under H2 at rt for 48 h then filtered through Celite,
washed with (1:1 MeOH–water), evaporated under
diminished pressure and dried well. The residue was stir-
red with pyridine (50 mL) and Ac2O (25 mL) at rt for
48 h, then co-evaporated with toluene under diminished
pressure. The residue was purified by flash chromatogra-
phy (3:1 toluene–acetone) to afford 11 (1.03 g, 89%) as
colourless foam. Rf 0.42 (2:1 toluene–acetone); 1H
NMR (CDCl3): d 6.21 (d, 0.75-H, J1,2 3.6 Hz, H-1aa),
5.65 (d, 0.25-H, J1,2 8.2 Hz, H-1ab), 5.55 (d, 1-H, J
<1.0 Hz, H-4c), 5.44 (dd, 0.75-H, J2,3 10.1, J3,4 9.2 Hz,
H-3aa), 5.37 (m, 1-H, H-3c), 5.18–4.98 (m, 4.25-H, H-
3ab, H-2c, H-2b, H-2aa, H-2ab, H-1b), 4.97 (d, 1-H, J1,2
3.4 Hz, H-1c), 4.71 (m, 1-H, H-3b), 4.51–4.39 (m, 3-H,
H-60ab, H-60 ; H-60 , H-5c), 4.15–4.06 (m, 4-H, H-6ab,
3.4. Benzyl 4,6-di-O-acetyl-2,3-di-O-benzyl-a-D-galacto-
pyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-galactopyranos-
yl-(1!4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (9)
aa
b
Compound 8 (1.4 g, 1.4 mmol) in dry 1:1 CH2Cl2–Et2O
(6 mL) was stirred with 0.02 M TMSOTf in CH2Cl2
(6.5 mL) under argon at ꢀ25 ꢁC while compound 7
(1.5 equiv, 1.25 g, 2.16 mmol) in anhyd CH2Cl2
(4.0 mL) was added dropwise. After 1.5 h, the mixture
was allowed to warm up to rt over 30 min, then diluted
with CH2Cl2 (100 mL), washed with satd NaHCO3
(2 · 25 mL), dried over MgSO4 and evaporated under
diminished pressure. The residue was purified by flash
chromatography (3.5:1 petroleum ether–EtOAc) to yield
9 (1.63 g, 81%) as a yellow syrup. Traces of unreacted
acceptor, easily removed after deacetylation of 9 (next
step), were taken into account for the calculation of
the yield. [a]D +42.9 (c 0.55, CHCl3); Rf 0.22 (3.5:1
petroleum ether–EtOAc); 1H NMR (CDCl3): d 7.40–
7.18 (m, 45-H, 9Ph), 5.57 (d, 1-H, J 1.6 Hz, H-4c),
5.14 (d, 1-H, J1,2 3.1 Hz, H-1c), 5.10 (d, 1-H, Jgem
11.1 Hz, CHHPh), 5.00 (d, 1-H, Jgem 12.1 Hz, CHHPh),
4.95–4.63 (m, 14-H, 7CH2Ph), 4.60–4.52 (m, 3-H, H-5c,
H-1a, H-1b), 4.44 (d, 1-H, Jgem 12.1 Hz, CHHPh), 4.32–
3.34 (m, 17-H), 2.09, 2.06 (2s, 6-H, 2CH3CO); 13C NMR
H-6aa, H-60 , H-6c, H-6b), 3.40 (m, 0.75-H, H-5aa), 3.99
c
(d, 1-H, J <1.0 Hz, H-4b), 3.82–3.72 (m, 2.25-H, H-5b,
H-4aa, H-5ab, H-4ab), 2.14–1.95 (sev. s, 33-H,
11CH3CO); 13C NMR (CDCl3): d 170.57–168.74 (11-
CH3CO), 101.09 (C-1ba), 100.85 (C-1bb), 99.52 (C-1c),
91.46 (C-1a), 88.91 (C-1aa), 76.82 (C-4b), 73.37–71.80
(C-3b, C-4a, C-5b, C-5ab), 70.57–67.00 (C-5aa, C-3c,
C-2c, C-2b, C-3a, C-2a, C-4c, C-5c), 61.81–60.17 (C-6b,
C-6c, C-6a), 21.34–20.41 (11-CH3CO); FAB+HRMS:
Calcd for C43H50O25, [M+Na]+ 989.2750. Found: m/z
989.2735.
3.6. O-[2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-
(1!4)-2,3,6-tri-O-acetyl-b-D-galactopyranosyl-(1!4)-
2,3,6-tetra-O-acetyl-a-D-glucopyranosyl] trichloroacet-
imidate (13)
Compound 11 (0.93 g, 0.96 mmol) was treated with
hydrazinium acetate (0.12 g, 1.3 mmol) in anhyd DMF
(3 mL), then co-evaporated with toluene and water
under diminished pressure. The residue was purified by

M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
2033
flash chromatography (2.5:1 toluene–acetone) to yield
12 (0.82 g, 92%) as a colourless foam. Rf 0.13 (2.5:1 tol-
uene–acetone). A mixture of 12 (0.29 g, 0.31 mmol) and
CCl3CN (1.0 mL, 10.0 mmol) in dry CH2Cl2 (1 mL) was
stirred at rt in the presence of DBU (0.05 mL, 0.3 mmol)
for 5 h, then co-evaporated with EtOAc under dimin-
ished pressure. The residue was purified by flash chro-
matography (10:1 EtOAc–toluene + 1% Et3N) to yield
13 (0.27 g, 80%) as a yellow foam. Rf 0.64 (10:1
3.8. 6-(4-Cinnamoylphenoxy)-hexyl a-D-galactopyran-
osyl-(1!4)-b-D-galactopyranosyl-(1!4)-b-D-glucopyran-
oside (1)
A mixture of 14 (0.13 g, 0.12 mmol) and NaOMe
(5.38 M, 50 lL) in dry MeOH (10 mL) was stirred at
rt overnight, then neutralized with Amberlite IR 120
resin (H+-form) and concentrated under diminished pres-
sure to yield 15 (74 mg, 96%) as an amorphous mass.
Rf 0.29 (6:3:1 EtOAc–iso-PrOH–H2O). Compound 15
(0.13 g, 0.2 mmol) was stirred with p-cinnamoylphenol
(1.0 g, 0.42 mmol) and K2CO3 (60 mg, 0.43 mmol) in
dry DMF (1.0 mL) at 95–100 ꢁC. After 24 h, the mixture
was diluted with MeOH, acidified with glacial AcOH
(0.2 mL) and co-evaporated with toluene under dimin-
ished pressure. The residue was purified by flash chro-
matography type-B silica (6:3:1 EtOAc–i-PrOH–water)
to yield 1 (90 mg, 53%) as a yellowish white solid. [a]D
+30.9 (c 0.11, CHCl3); Rf 0.23 (6:3:1 EtOAc–i-PrOH–
1
EtOAc–toluene + 1% Et3N); H NMR (CDCl3): d 8.64
(s, 1-H, NH), 6.48 (d, 1-H, J1,2 3.8 Hz, H-1a), 5.59
(dd, 1-H, J3,4 3.1, J4,5 3.5 Hz, H-4c), 3.56 (dd, 1-H, J2,3
9.7, J3,4 9.3 Hz, H-3a), 5.40 (dd, 1-H, J2,3 11.0, J3,4
3.1 Hz, H-3c), 5.20 (dd, 1-H, J1,2 3.5, J2,3 11.0 Hz, H-
2c), 5.13–5.05 (m, 2-H, H-2b, H-2a), 4.99 (d, 1-H, J1,2
7.8 Hz, H-1b), 4.50–4.42 (m, 3-H, H-60 , H-5c, H-60 ),
a
b
4.21–4.11 (m, 5-H, H-5a, H-60 , H-6c, H-6a, H-6b), 4.02
c
(dd, 1-H, J3,4 ꢁ J4,5 2.3 Hz, H-4b), 3.87 (dd, 1-H, J3,4
9.7, J4,5 9.3 Hz, H-4a), 3.77 (m, 1-H, H-5b), 2.17–1.98
(sev. s, 33-H, 11CH3CO).
1
water); H NMR (CDCl3): d 8.11–6.76 (m, 11-H, 9Ar,
ACH@CHA), 4.94 (d, 1-H, J1,2 3.4 Hz, H-1c), 4.41 (d,
1-H, J1,2 7.3 Hz, H-1), 4.28 (d, 1-H, J1,2 7.8 Hz, H-1),
4.25–3.20 (m, 22-H), 1.83–1.50 (m, 8-H, 4-CH2–);
(MALDI+MS) Calcd for C39H54O18 (810.3310). Found:
m/z 833.7470 [M+Na]+, 849.7293 [M+K]+.
3.7. 6-Chloro-n-hexyl 2,3,4,6-tetra-O-acetyl-a-D-galacto-
pyranosyl-(1!4)-2,3,6-tri-O-acetyl-b-D-galactopyranos-
yl-(1!4)-2,3,6-tri-O-acetyl-b-D-glucopyranoside (14)
Compound 13 (0.26 g, 0.24 mmol) in dry CH3CN
(1.5 mL) was added dropwise, under Ar, to 6-chloro-1-
hexanol (0.2 g, 1.5 mmol) in CH3CN (1 mL) and
TMSOTf (0.02 M in CH3CN, 0.76 mL) at ꢀ25 ꢁC under
stirring, then allowed to reach rt (2.5 h). The mixture
was diluted with EtOAc (50 mL), washed with satd
NaHCO3 (25 mL), dried over MgSO4 and evaporated
under diminished pressure. The residue was purified by
flash chromatography (4:1 toluene–acetone) to yield 14
(0.14 g, 54%) as a yellow oil. [a]D +33.3 (c 0.3, CHCl3);
Rf 0.15 (4:1 toluene–acetone); 1H NMR (CDCl3): d 5.58
(d, 1-H, J3,4 = J4,5 2.2 Hz, H-4c), 5.38 (dd, 1-H, J2,3 10.9,
J3,4 3.2 Hz, H-3c), 5.23–5.15 (m, 2-H, H-3a, H-2c), 5.10
(dd, 1-H, J1,2 7.7, J2,3 10.9 Hz, H-2b), 4.98 (d, 1-H, J1,2
3.4 Hz, H-1c), 4.88 (dd, 1-H, J1,2 8.1, J2,3 9.1 Hz, H-
2a), 4.72 (dd, 1-H, J2,3 10.9, J3,4 1.8 Hz, H-3b), 4.52–
4.40 (m, 5-H, H-1b, H-5c, H-1a, H-60 ; H-60 ), 4.19–4.06
3.9. 6-(4-Cinnamoylphenoxy)-hexyl 2,3,4,6-tetra-O-
acetyl-a-D-galactopyranosyl-(1!4)-2,3,6-tri-O-acetyl-
b-D-galactopyranosyl-(1!4)-2,3,6-tri-O-acetyl-b-D-
glucopyranoside (16)
A mixture of 1 (9 mg, 11 lmol), pyridine (6 mL) and
Ac2O (3 mL) was stirred at rt for 48 h, then co-evapo-
rated with toluene under diminished pressure. The resi-
due was purified by flash chromatography (3:1 toluene–
acetone) to yield 16 (12.0 mg, 88%) as a colourless syrup.
Rf 0.32 (3:1 toluene–acetone); 1H NMR (CDCl3): d 8.04–
6.95 (m, 11-H, 9Ar, ACH@CHA), 5.58 (d, 1-H,
J3,4 = J4,5 3.2 Hz, H-4c), 5.38 (dd, 1-H, J2,3 11.1,
J3,4 = 3.3 Hz, H-3c), 5.36–5.15 (m, 2-H, H-3a, H-2c),
5.10 (dd, 1-H, J1,2 7.7, J2,3 10.9 Hz, H-2b), 4.97 (d, 1-H,
J1,2 3.4 Hz, H-1c), 4.88 (dd, 1-H, J1,2 7.8, J2,3 9.4 Hz, H-
2a), 4.72 (dd, 1-H, J2,3 10.9, J3,4 2.4 Hz, H-3b), 4.52–
4.40 (m, 5-H, H-1b, H-5c, H-1a, H-60 ; H-60 ), 4.19–3.97
b
a
(m, 4-H, H-60 , H-6c, H-6a, H-6b), 4.00 (d, 1-H,
c
J3,4 = J4,5 <1.0 Hz, H-4b), 3.87–3.73 (m, 3-H, –OCHH,
H-4a, H-5b), 3.62 (m, 1-H, H-5a), 3.56–3.42 (m, 3-H,
1.5 –CH2–), 2.14–1.69 (m, 30-H, 10CH3CO), 1.75–1.13
[2m, 8-H, –(CH2)4–]; 13C NMR (CDCl3): d 168.87–
170.68 (10C@O), 100.53 (C-1a), 101.11 (C-1b), 99.63
(C-1c), 76.91, 76.48, 73.11, 72.79, 72.51, 71.79, 69.86,
(C-2a, C-4a, C-5b, C-5a, C-3b, C-3a, C-4b, –OCH2–),
69.84, 68.98, 68.83, 67.89, 67.06, (C-5c, C-3c, C-4c, C-
2b, C-2c), 62.21, 61.32, 60.36 (C-6c, C-6a, C-6b), 44.91
(–CH2Cl), 32.44, 29.21, 26.47, 25.10 [–(CH2)4–], 20.90,
b
a
(m, 7-H, H-4b, H-60 , H-6c, H-6a, H-6b, –OCH2), 3.87–
c
3.73 (m, 3-H, H-4a, H-5b, –OCHH–), 3.63 (m, 1-H, H-
5a), 3.62 (m, 1-H, H-5a), 3.47 (m, 1-H, –OCHH–), 2.17–
1.98 (m, 30-H, 10CH3CO), 1.61–1.06 [m, 8-H, –(CH2)4–].
3.10. Benzyl 4,6-di-O-acetyl-2,3-di-O-benzyl-a-D-galacto-
pyranosyl-(1!3)-2,4,6-tri-O-benzyl-b-D-galactopyrano-
syl-(1!4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside
(18)
20.48
(10CH3CO);
FAB+HRMS:
Calcd
for
C44H63ClO26, [M+Na]+ 1065.3194 and 1067.3197.
Compound 17 (1.42 g, 1.4 mol) was treated with 7 (1.2 g,
Found: m/z 1065.3132 and 1067.3148.
2.0 mmol) in the presence of TMSOTf (0.02 M in

2034
M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
CH2Cl2, 6.6 mL), then worked up exactly as described
for 9. The residue was purified by flash chromatography
(3:1 petroleum ether–EtOAc) to yield 18 (1.78 g, 87%) as
a light yellow oil. Traces of unreacted acceptor, easily
removed after deacetylation of 18 (next step), were taken
into account for the calculation of the yield. [a]D
+55.2 (c 0.7, CHCl3); Rf 0.15 (3:1 petroleum ether–
EtOAc); 1H NMR (CDCl3): d 7.32–7.07 (m, 45-H,
9Ph), 5.16 (d, 1-H, J1,2 2.9 Hz, H-1c), 5.14 (d, 1-H,
J3,4 = J4,5 <1.0 Hz, H-4c), 5.04 (d, 1-H, Jgem 10.6 Hz,
0.5CH2Ph), 5.01 (d, 1-H, Jgem 10.0 Hz, 0.5CH2Ph),
4.93 (d, 1-H, Jgem 12.0 Hz, 0.5CH2Ph), 4.90 (d, 1-H, Jgem
10.7 Hz, 0.5CH2Ph), 4.83 (d, 1-H, Jgem 11.4 Hz,
0.5CH2Ph), 4.79–4.59 (m, 8-H, 4CH2Ph), 4.51–4.34
(m, 4-H, H-1a, H-1b, CH2Ph), 4.40–4.23 (m, 4-H, H-
5c, 1.5CH2Ph), 4.05–3.75, 3.67–3.32 (m, 13-H), 2.09,
1.91 (2s, 6-H, 2CH3CO); 13C NMR (CDCl3): d 170.38
(C@O), 139.19–127.06 (9Ph), 102.94, 102.48 (C-1a,
C-1b), 95.49 (C-1c), 82.94–62.23 (24C), 20.94, 20.92
(2CH3CO); FAB+HRMS: Calcd for C85H90O18,
[M+ Na]+1421.6025. Found: m/z 1421.6060.
NH), 6.19 (d, 1-H, J1,2 3.9 Hz, H-1a), 5.56 (dd, 1-H,
J2,3 10.0, J3,4 9.5 Hz, H-3a), 5.44 (d, 1-H, J3,4 2.2, J4,5
<1.0 Hz, H-4c), 5.33 (d, 1-H, J3,4 = J4,5 2.8 Hz, H-4b),
5.28–5.24 (m, 2-H, H-2c, H-1c), 5.18 (dd, 1-H, J1,2 7.8,
J2,3 10.1 Hz, H-2b), 5.09 (dd, 1-H, J2,3 10.4, J3,4
2.2 Hz, H-3c), 4.93 (dd, 1-H, J1,2 3.9, J2,3 10.0 Hz, H-
2a), 4.48–4.43 (m, 2-H, H-60 , H-1b), 4.26–4.02 (m, 7-H,
a
H-5c, H-6c, H-60 , H-6a, H-6b, H-60 , H-5b), 3.85–3.79
c
b
(m, 3-H, H-3b, H-4a, H-5a), 2.16–1.94 (sev. s, 30-H,
10CH3CO); 13C NMR (CDCl3):
170.41–168.63
d
(10CH3CO), 100.75 (C-1b), 93.43 (C-1c), 89.92 (C-1a),
74.98–61.20 (18C), 20.83–20.63 (10CH3CO).
3.13. 6-Chlorohexyl 2,3,4,6-tetra-O-acetyl-a-D-galacto-
pyranosyl-(1!3)-2,3,6-tri-O-acetyl-b-D-galactopyran-
osyl-(1!4)-2,3,6-tri-O-acetyl-b-D-glucopyranoside (23)
To compound 22 (0.43 g, 0.4 mmol) in dry CH3CN
(3 mL) were added 6-chloro-1-hexanol (0.4 g, 3 mmol)
and TMSOTf (0.02 M in CH3CN, 1.3 mL) at ꢀ25 ꢁC.
The soln was then stirred and worked up as described
for the synthesis of 14. The residue was purified by flash
chromatography on Type-B Silica Gel E. Merck (6:1
then 5:1 toluene–acetone) to yield 23 (0.18 g, 44%) as a
colourless foam. [a]D +40.0 (c 0.1, CHCl3); Rf 0.15
3.11. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-
(1!3)-2,4,6-tri-O-acetyl-b-O-galactopyranosyl-(1!4)-
1,2,3,6-tetra-O-acetyl-D-glucopyranose (20)
1
(5:1 toluene–acetone); H NMR (CDCl3): d 5.43 (dd,
Compound 18 (1.77 g, 1.3 mmol) was treated with
NaOMe and worked up as described for the synthesis
of 10. Flash chromatography (1:1 petroleum ether–
EtOAc) yielded 19 (1.26 g, 76%). Treatment of 19
(1.2 g, 0.9 mmol) with Pd–C, followed by acetylation
as described for the synthesis of 11 and purification by
flash chromatography (2.5:1 toluene–acetone) gave 20
(0.64 g, 72%) as a colourless foam. Rf 0.29 (2.5:1 tolu-
1-H, J3,4 = J4,5 2.5 Hz, H-4c), 5.30 (dd, 1-H, J3,4 = J4,5
2.2 Hz, H-4b), 5.26–5.11 (m, 4-H, H-3a, H-2b, H-2c, H-
1c), 5.07 (dd, 1-H, J2,3 10.2, J3,4 2.5 Hz, H-3c), 4.86
(dd, 1-H, J1,2 8.0, J2,3 9.5 Hz, H-2a), 4.48–4.38 (m, 3-
H, H-60 , H-1a, H-1b), 4.20–4.00 (m, 6-H, H-6b, H-60 ,
a
b
H-5c, H-6a, H-6c, 60 -H), 3.85–3.73 (m, 4-H, H-5b, H-4a,
c
H-3b, –CHH–) 3.63–3.41 (m, 4-H, H-5a, 1.5 –CH2–),
2.15–1.93 (sev. s, 30-H, 10CH3CO), 1.76–1.24 (m, 8-
H, 4-CH2–); 13C NMR (CDCl3); d 170.63–168.71
(10CH3CO), 101.10 (C-1b), 100.51 (C-1a), 93.40 (C-1c),
67.10–60.97 (16C), 44.92 (CH2Cl), 32.44, 29.20, 26.46,
25.09 (4-CH2–), 20.79–20.41 (10CH3CO); FAB+
HRMS: Calcd for C44H63ClO26, [M+Na]+ 1065.3194
and 1067.3197. Found: m/z 1065.3132 and 1067.3179.
1
ene–acetone); H NMR (CDCl3): d 6.22 (d, 0.5-H, J1,2
3.6 Hz, H-1aa), 5.65 (d, 0.5-H, J1,2 8.2 Hz, H-1ab),
5.47–3.74 (m, 20-H), 2.17–1.92 (sev. s, 33-H, 11CH3CO).
1
These H NMR data are in good agreement with the
published data.15d
3.12. (2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-(1!3)-
2,4,6-tri-O-acetyl-b-O-galactopyranosyl-(1!4)-2,3,6-tri-
O-acetyl-a-D-glucopyranosyl) trichloroacetimidate (22)
3.14. 6-(4-Cinnamoylphenoxy)-hexyl a-D-galactopyran-
osyl-(1!3)-b-D-galactopyranosyl-(1!4)-b-D-glucopyran-
oside (2)
Compound 20 (0.17 g, 0.17 mmol) was treated with
hydrazinium acetate, then worked up as described for
the synthesis of 12. The residue was purified by flash
chromatography (2.5:1 toluene–acetone) to yield 21
(0.11 g, 70%) as a colourless foam Rf 0.31 (2:1 tolu-
ene–acetone), which was next stirred with CCl3CN
(0.8 mL, 8.0 mmol) in dry CH2Cl2 (1 mL) at rt for 4 h,
then co-evaporated with EtOAc under diminished pres-
sure. The residue was purified by flash chromatography
(10:1 EtOAc–toluene + 1% Et3N) to yield 22 (0.11 g,
82%) as a yellow foam. Rf 0.64 (10:1 + 1% Et3N
EtOAc–toluene); 1H NMR (CDCl3): d 8.65 (s, 1-H,
To a soln of compound 23 (0.17 g, 0.16 mmol) in MeOH
(10 mL) was added NaOMe ( 5.38 M, 60 lL). The
mixture was stirred at rt overnight. After neutralization
with IR 120Amberlite resin (H+ form), 24 (0.1 g, quant)
was obtained as an amorphous yellowish residue.
(MALDI+MS) Calcd for C24H43ClO16 (623.12). Found:
m/z 645.1946, 647.1907 [M+Na]+; 661.2392, 663.1588
[M+K]+. The residue from the last step was treated with
4-cinnamoylphenol then worked up and purified exactly
under the same condition used for synthesis of 1 to yield
2 (0.1 g, 77%) as amorphous colourless mass. [a]D +42.4

M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
2035
(c 0.62, CHCl3); Rf 0.31 (6:3:1 EtOAc–isoPrOH–H2O);
1H NMR (CD3OD): d 8.03–7.03 (m, 11-H, Ph, Ar,
ACH@CHA), 5.03 (d, 1-H, J1,2 1.0 Hz, H-1c), 4.41 (d,
1-H, J1,2 7.3 Hz, H-1), 4.29 (d, 1-H, J1,2 7.8 Hz, H-1),
4.22 (t, 1-H, J5,6, 6.1 Hz), 4.09 (t, 2-H, J 6.3 Hz, –
OCH2–), 4.03 (dd, 1-H, J3,4 ꢁ J4,5 <1.0 Hz, H-4), 3.92–
3.49 (m, 16-H), 3.39 (m, 1-H, H-5), 3.24 (dd, 1-H, J1,2
7.8, J2,3 8.4 Hz, H-2), 1.83 (t, 2-H, J 6.7 Hz, –CH2–),
1.67 (t, 2-H, J 6.5 Hz, –CH2–), 1.5 (m, 4-H, 2(–CH2–));
13C NMR (CD3OD): d 190.75 (C@O), 164.97–115.55
(Ar, ACH@CHA), 10511, 104.24 (C-1a, C-1b), 97.75
(C-1c), 81.00–58.33 (17C), 30.69, 30.18, 26.90, 26.83
(4-CH2–); MALDI+MS: m/z 833.2652 [M+Na]+,
849.2392 [M+K]+.
2.1 Hz, H-4b), 5.12–5.27 (m, 4-H, H-2b, H-3a, H-2c, H-
1c), 5.08 (dd, 1-H, J2,3 10.5, J3,4 2.9 Hz, H-3c), 4.87 (dd,
1-H, J1,2 8.0, J2,3 9.4 Hz, H-2a), 4.46–4.39 (m, 3-H,
H-60 , H-1a, H-1b), 4.21–4.00 (m, 8-H, H-5c, H-6c, H-60 ,
a
c
H-6a, H-6b, H-60 , –OCH2–), 3.88–3.73 (m, 4-H, H-4a,
b
H-3b, H-5b, –OCHH–), 3.60 (m, 1-H, H-5a), 3.46 (m, 1-
H, –OCHH–), 2.15–1.93 (several s, 30-H, 10CH3CO),
1.79–124 (m, 8-H, 4-CH2–); 13C NMR (CDCl3): d
188.64 (–COACH@CHA), 170.35–168.0 (10CH3CO),
162.94–142.87 (–HC@CH–), 135.04–114.23 (Ph, Ar),
101.08 (C-1b), 100.50 (C-1a), 93.27 (C-1c) 76.58–60.93
(20C), 30.89–25.55 (4-CH2–), 20.78–20.45 (10CH3CO).
3.17. Coupling of 6-(p-cinnamoylphenoxy)hexyl a-D-
galactopyranosyl-(1!4)-b-D-galactopyranosyl(1!4)-b-
D-glucopyranoside 1 to thiopropyl Sepharose 6B
3.15. 6-(4-Cinnamoylphenoxy)-hexyl a-D-galactopyran-
osyl-(1!3)-b-D-galactopyranosyl-(1!4)-b-D-glucopyran-
oside (2)
Thiopropyl Sepharose 6B (1 g) was suspended in water
(200 mL), filtered over sintered glass, then suspended
again in a soln containing 2-mercaptoethanol (0.5 M),
NaHCO3 (0.3 M, pH 8.3) and disodium EDTA (1 mM),
and mixed gently for 40 min at rt. The gel was filtered
on a sintered glass then washed thoroughly with AcOH
(0.1 M, 400 mL) containing NaCl (0.5 M) and disodium
EDTA (1 mM) to remove 2-mercaptopyridine. The gel
(1 mL) was transferred into a C10/10 column, washed
with water (20 mL), MeOH (20 mL) then gently mixed
for 4 h at rt with a soln of 1 (17 mg) in MeOH (2 mL) con-
taining InCl3 (10 mol %). The gel (1 mL) was eluted and
the eluent was evaporated under diminished pressure to
recover the non-coupled 1 (10 mg), which was taken up
in DMF (1 mL) and diluted with phosphate buffer
(pH 7.4, 1 mL). The column was equilibrated by washing
with (20 mL) of (1:1 DMF–phosphate buffer), then incu-
bated with the above-mentioned soln of 1 overnight at
45 ꢁC then drained. The unreacted thiol groups on the
gel were capped by gentle mixing with N-methylmalei-
mide (1%) in a phosphate buffer at pH 6.8 for 2 h at rt.
The column was drained and equilibrated with PBS at
pH 7.5 and a periplasmic extract of E. coli strain BH
101 (10 mL) was run through it at a rate of 0.1 mL/min
then washed with a phosphate buffer containing NaCl
(0.5 M, 5 mL). The attached protein was eluted at a rate
of 0.1 mL/min with a linear gradient ranging from 0 to
6 M of guanidinium chloride and the protein in the eluted
fractions (250 lL/fraction) was monitored by absorbance
at 280 and 254 nm. Fractions corresponding to the peaks
in the elution profile were dialyzed against PBS and ana-
lyzed on a PAGE gel and by WB.
Compound 23 (0.17 g, 0.16 mmol) was treated with
NaOMe, then worked up as described for the synthesis
of 15 to yield 24 (0.1 g) as an amorphous yellowish res-
idue. (MALDI+MS) Calcd for C24H43ClO16 (623.12).
Found: m/z 645.1946, 647.1907 [M+Na]+; 661.2392,
663.1588 [M+K]+. The residue from the last step was
treated with 4-cinnamoylphenol, then worked up and
purified exactly under the same conditions as that used
for the synthesis of 1 to yield 2 (0.1 g, 77%) as an amor-
phous colourless mass. [a]D +42.4 (c 0.62, CHCl3); Rf
1
0.31 (6:3:1 EtOAc–isoPrOH–H2O); H NMR (CD3OD):
d 8.03–7.03 (m, 11-H, Ph, Ar, ACH@CHA), 5.03 (d, 1-
H, J1,2 1.0 Hz, H-1c), 4.41 (d, 1-H, J1,2 7.3 Hz, H-1), 4.29
(d, 1-H, J1,2 7.8 Hz, H-1), 4.22 (t, 1-H, J5,6, 6.1 Hz), 4.09
(t, 2-H, J 6.3 Hz, –OCH2–), 4.03 (dd, 1-H, J3,4 ꢁ J4,5
<1.0 Hz, H-4), 3.92–3.49 (m, 16-H), 3.39 (m, 1-H, H-
5), 3.24 (dd, 1-H, J1,2 7.8, J2,3 8.4 Hz, H-2), 1.83 (t, 2-
H, J 6.7 Hz, –CH2–), 1.67 (t, 2-H, J 6.5 Hz, –CH2–),
1.5 (m, 4-H, 2(–CH2–)); 13C NMR (CD3OD): d 190.75
(C@O), 164.97–115.55 (Ar, ACH@CHA), 10511,
104.24 (C-1a, C-1b), 97.75 (C-1c), 81.00–58.33 (17C),
30.69, 30.18, 26.90, 26.83 (4-CH2–); (MALDI+MS)
Calcd for C39H54O18 (810.3310). Found: m/z 833.2652
[M+Na]+, 849.2392 [M+K]+.
3.16. 6-(4-Cinnamoylphenoxy)-hexyl 2,3,4,6-tetra-O-acet-
yl-a-D-galactopyranosyl-(1!3)-2,4,6-tri-O-acetyl-b-D-
galactopyranosyl-(1!4)-2,3,6-tri-O-acetyl-b-D-glucopyr-
anoside (25)
Compound 2 (24 mg, 2.9 mmol) was acetylated and
worked up as described for the synthesis of 16. The resi-
due was purified by flash chromatography (3:1 toluene–
acetone) to yield 25 (33 mg, 91%) as a yellowish foam.
Rf 0.5 (3:1 toluene–acetone); 1H NMR (CDCl3): d
8.03–6.94 (m, 11-H, Ph, Ar, ACH@CHA), 5.43 (dd, 1-
H, J3,4 2.9, J4,5 2.1 Hz, H-4c), 5.31 (dd, 1-H, J3,4 = J4,5
3.18. Coupling of StxB-cys with 2
StxB-cys (23 lg) in borate buffer pH 9 (20 mM, 9 lL)
including NaCl (150 mM) was incubated for 16 h at rt
at four different concentrations (2.34, 7.10, 21.32 and
63.96 mg/mL) of compound 2 suspended in Me2SO

2036
M. R. E. Aly et al. / Carbohydrate Research 341 (2006) 2026–2036
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Acknowledgements
Our work was supported by grants from Institut Curie
(PIC Vectorization), Fondation de France, and Associ-
ation pour la Recherche sur le Cancer (no. 3105 and
´
5177). We are particularly grateful to Lucien Cabanie
and Wolfgang Faigle for technical help.
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