New oligo-β-(1,3)-glucan derivatives as immunostimulating agents

Article abstract of DOI:10.1016/j.bmc.2009.10.053

Oligo-β-(1,3)-glucans were chemically modified in order to introduce a structural variation specifically on the reducing end of the oligomers. The impact of well defined structural modulations was further studied on cancer cells and murin models to evaluate their cytotoxicity and immunostimulating potential.

Full text of DOI:10.1016/j.bmc.2009.10.053

Bioorganic & Medicinal Chemistry 18 (2010) 348–357
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New oligo-b-(1,3)-glucan derivatives as immunostimulating agents
c,
Karine Descroix a,b, , Vaclav Vetvicka , Isabelle Laurent a,b, Frank Jamois d, Jean-Claude Yvin d,à
,
*
ˇ
ˇ
Vincent Ferrières a,b,
*
a Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, Avenue du Général Leclerc, CS 50837, 35708 Rennes Cedex 7, France
b Université européenne de Bretagne, France
c Department of Pathology, University of Louisville, 511 S. Floyd Street, MDR Building, Louisville, KY 40202, USA
d Laboratoire Goëmar, ZAC La Madeleine, Avenue du Général Patton, 35400 Saint Malo, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2009
Revised 26 October 2009
Accepted 27 October 2009
Available online 30 October 2009
Oligo-b-(1,3)-glucans were chemically modified in order to introduce a structural variation specifically on
the reducing end of the oligomers. The impact of well defined structural modulations was further studied
on cancer cells and murin models to evaluate their cytotoxicity and immunostimulating potential.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Carbohydrates
b-(1,3)-Glucans
Immunostimulation
1. Introduction
til now from polysaccharides that poor correlations can be estab-
lished with structural and conformational requirements,
It is now well established that natural b-(1,3)-glucans, which
are neither biosynthesized nor metabolized by mammals, are po-
tent immunological activators. Recent studies give evidence for re-
newed interest in these polysaccharides in the health domain,1–3
particularly with those related to clinical trials where they are used
as bioactive adjuvants in the treatment of patients receiving
chemotherapies.4,5 These studies notably highlight improvement
of the quality of life of cancer patients—a very important criterion
in oriental countries. During the last decade, the main receptors
able to interact specifically with b-(1,3)-glucans were identified.
Amongst them are the dectin-1,6,7 the complement receptor of
type 3 (CR3),8–10 a scavenger and a lactosylceramide. Since these
receptors are widely distributed on various immunocompetent
cells, b-(1,3)-glucans impact on both the innate and the adaptive
immunity. The related biological effects were recently reviewed
favorably.1,3 It was also shown that therapies based on antitumor
monoclonal antibodies are enhanced by administered b-glu-
cans.11–13 However, the structure–function relationships are still
objects of debate.2,14,15 It results from biological data obtained un-
molecular weight and degree of branching. In summary, glucans
have been extensively studied for their immunological and phar-
macological effects. More than 10,000 papers describing the bio-
logical activities of glucans exist.16 However, many of the
available data were obtained from extracted materials. As a result,
neither chemical structure nor purity of actual bioactive mole-
cule(s) can be rigorously specified. The absence of direct benefits
of polysaccharides on health therefore dampens the development
in western countries of such complementary and alternative med-
icine. It is important, therefore, to evaluate the possibility of using
synthetic oligosaccharides based on the structure of glucans.
In this context, and in order to increase our knowledge of lam-
inarine extracted from brown seaweeds,17 we have recently initi-
ated a program devoted to the chemical synthesis of well defined
and pure oligo-b-(1,3)-glucans.18 As a result, while they are not
able to adopt any helical arrangements, small linear oligo-b-
(1,3)-glucans possess biological activity comparable to that of the
native PhycarineÒ. Consequently, compared with polysaccharides,
minor chemical variations on small oligoglucans may present sig-
nificant changes in interactions with the targeted receptors. These
effects are expected to give a great deal of information on the
respective importance of hydroxyl groups. Upon this basis, we
now present a first chemical modulation that results in a new fam-
ily of oligo-b-(1,3)-glucans (Fig. 1) modified on the reducing end.
The designed glycosides are characterized by the presence of four
or five glucopyranose entities and a mannose residue at the reduc-
* Corresponding authors. Fax: +33 (0) 2 23 23 80 46 (V.F.).
E-mail address: vincent.ferrieres@ensc-rennes.fr (V. Ferrières).
Present address: School of Chemical Sciences and Pharmacy, Norwich NR4 7TJ,
UK.
à
Present address: AFI, 27 Avenue Franklin Roosevelt, BP 158, 35408 Saint Malo
Cedex, France.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.053

K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
349
OR3
OR3
OH
OH
R3
O
R3O
O
O
O
O
HO
HO
O
O
O
R2
O
R3
OH
OH
H
H
OR3
OH
R3O
OH
R1
n
n
(a) Oligo-β-(1,3)-glucans
R1
R2
H, OH
R3
H
n
OH
2a
HO
HO
OH
O
4
4
4
5
5
5
a
b
O
3a
H, OBz
Br
Bz
Bz
H
HO
1a: n = 4
1b: n = 5
O
O
4a
OH
2b
H, OH
a
b
n
3b
Bz
Bz
H, OBz
Br
(b) Oligo-β-(1,3)-glucanyl-β-(1,3)-mannose
4b
Figure 1. Structure of oligo-b-(1,3)-glucan-mannose conjugates.
OBz
OBz
Bz
O
c
O
O
BzO
O
O
Bz
ing end. The impact of fine chemical variation on the phagocytic
activity and the secretion of interleukins was also studied in a mur-
ine model.
OBz
OBz
n
n
5a
5b
4
5
2. Results and discussion
Scheme 1. Synthesis of glycols 5. Reagents: (a) BzCl, pyridine (3a: 94%; 3b: 88%);
(b) HBr, HOAc (4a: 100%; 4b: 88%); (c) DBU (5a: 81%; 5b: 85%).
2.1. Synthesis of oligo-b-(1,3)-glucanyl-b-(1,3)-mannose
The introduction of a mannose entity on an oligoglucan chain
could be based on three different approaches: (1) a total synthesis
of the oligosaccharidic chain thanks to standard tools of glyco-
chemistry, (2) a coupling between an oligomer as donor and a suit-
ably protected mannopyranosyl acceptor, and (3) a controlled and
specific inversion of configuration at C-2 starting from already
formed oligo-b-(1,3)-glucans. In order to optimize time dedicated
to synthetic work, and because it is well known that reactivity of
glycosyl donors decreases by increasing the number of glycosyl
residues, we expected that the latter strategy would be the most
appropriate. This purpose required to distinguish the OH-2 group
on the reducing end from all the other OH-2 functions. Such an ap-
proach, first introduced by Lichtenthaler for the preparation of b-
mannopyranosides, involves the reduction of a 2-keto-glycoside
which is obtained by glycosidation of an ulosyl bromide, resulting
from bromination of a glucan derivative.19,20 Full experimental de-
tails and mechanistic analysis were described, included those re-
lated to the formation of by-products.21 Still limited to the
modification of mono- and disaccharides, we attempted to extend
this concept to laminaripenta- and hexaoses, 2a and 2b, respec-
tively (Scheme 1). Therefore, perbenzoylation of 2a and 2b, fol-
lowed by anomeric bromination of the reducing end, were
performed under standard conditions to afford glucanyl bromides
4a and 4b, respectively. The synthesis of glucans 5a and 5b re-
quired an elimination reaction with the assistance of 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU). Both enol ethers were thus
isolated in yields upper to 80%.
An additional step consisted of electrophilic bromination of the
ethylenic bond using N-bromosuccinimide (NBS) in situ followed
by oxidation of the C-2 position thanks to the action of ethanol
on the 2-benzoic ester (Scheme 2). However, under these initial
conditions, the non-expected dibromide derivative 621 was ob-
tained from the pentasaccharidic glycal in a low yield. At this stage,
we supposed competing reactivity between bromide ion, which
adds to bromonium intermediate, and ethanol, which acts on car-
bonyl group of the C-2 ester group to induce the formation of the
keto function. To corroborate this hypothesis, ethanol was first
substituted with ethanethiol in order to induce in situ production
of bromine thanks to an oxido-reduction reaction. As a result, 6
was obtained quantitatively. Its structure was established on the
basis of a new signal on the 13C NMR spectrum at 89.9 ppm corre-
sponding to C-2a, and on conformational changes, from a half-chair
conformation 4H for 5a to a boat 4C1 for 6. The value of the coupling
OBz
OBz
OBz
O
Bz
O
O
BzO
NBS,
HOEt or
HSEt
O
O
Bz
Bz
Br
OBz
Br
n
6
(n = 4)
5
OBz
OBz
O
NBS,
HOMe
Bz
O
O
BzO
O
O
OBz
Br
O
n
n
4
5
7a
7b
Scheme 2. Synthesis of ulosyl bromides 7. Reagents: NBS, HSEt (from 5a, n = 4,
100%); NBS, HOMe (7a: 100%; 7b: 100%).
constant between H-3a and H-4a of 9.3 Hz, as well as no possible
correlation with H-1a, corroborated the 13C NMR data. Subse-
quently, the same reaction was further performed in the presence
of anhydrous methanol. The desired ulosyl bromides 7a and 7b
were thus obtained in near quantitative yields.
With the required donors in hands, the protection of the ano-
meric center of the terminal reducing entity was studied under
various conditions (Scheme 3). In the presence of benzyl alcohol
and silver triflate as a Lewis acidic promoter, the desired glycosyl-
ation efficiently occurred but accompanied by an elimination reac-
tion at C-3, C-4. This reaction allowed us to isolate the enone 8 in
an excellent 75% yield. A similar result was observed under basic
conditions in the presence of silver carbonate. In order to limit
the elimination process, the introduction of benzyl alcohol was fur-
ther attempted under neutral conditions using triphenylphosphine
oxide as a promoter.22 Provided that reactions were carefully con-
trolled, the targeted 2-keto glycosides were thus obtained and iso-
lated in 87% and 85% in the corresponding hydrated form 9a and
9b, respectively. The coupling constant between C-1a and H-1a
of 171 Hz shows that the b-anomers were specifically synthesized.
This was quite surprising since initial studies based on this concept
favoured retention of C-1 configuration thanks to a double inver-
sion—the first one by triphenylphosphine oxide on starting bro-

350
K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
diploid control fibroblastic cells which weakly divided. This con-
firmed no aspecific toxicity induced by the molecules.
Then, the specific effects of the molecules onto cells involved
into the immune response were monitored. The effects of various
glucans on macrophages are well established (for a review, see No-
OBz
O
OBz
BnOH
O
BzO
AgOTf or Ag2CO3
O
O
Bz
OBn
OBz
7
O
n
8 (n = 4)
vak and Vetvicka16). However, in order to demonstrate that syn-
thetic oligosaccharides built up with linear short chains of
glucans unable to adopt helical arrangements, really exhibit immu-
nomodulatory characteristics, an evaluation of some of the im-
mune reactions is necessary. Moreover, the effects observed with
glucan-mannose conjugates 1a, b were also compared with those
induced by pure laminaripentaose and laminariheptaose (Fig. 1a,
n = 4 and 6, respectively). We first showed that intra-peritoneal
injection of individual samples resulted in an increase in the per-
centage of granulocytes in peripheral blood. This was accompanied
by a decrease of percentage of lymphocytes (Fig. 2). Similarly, all
four samples caused an influx of peritoneal macrophages (Fig. 3).
Glucans are well known to stimulate phagocytosis.23,24 There-
fore, in the next step we measured the effects of oligosaccharides
on phagocytosis of synthetic HEMA microspheres in peripheral
blood (Fig. 4) and peritoneal cavity (Fig. 5). Our results showed that
all tested samples significantly increased phagocytic activity of
ˇ
ˇ
OBz
O
OBz
BnOH, Ph3PO
Bz
O
OBn
BzO
O
O
O
Bz
OBz
O
n
OBz
OH
OBz
Bz
O
O
O
BzO
O
O
OBn
Bz
OH
OBz
n
n
4
5
9a
9b
Scheme 3. Glycosylation of benzyl alcohol from ulosyl bromides. Reagents: BnOH,
AgOTf (starting from 7a, n = 4, 75%); BnOH, Ph3PO (9a: 87%; 9b: 85%).
100
80
mide and the second one by the acceptor on the intermediate
phosphonium. A possible explanation to justify the observed oppo-
site selectivity could be connected with the use of 2 equiv of the
neutral promoter. This possibly resulted in a shift of the a,b-equi-
60
librium for the 2-keto phosphonium intermediate towards the
a-
form, and hence to the synthesis of the b-glycoside.
40
20
0
Further reduction of 9a and 9b was efficiently performed with
L-Selectride and afforded derivatives 10a and 10b, respectively,
in near quantitative yields and with total manno selectivity
(Scheme 4). The target oligo-b-(1,3)-glucanyl-b-(1,3)-mannose
were finally obtained after Zemplén transacylation and palla-
dium-catalyzed hydrogenolysis in high degree of purity.
Monocytes
Control
Granulocytes Lymphocytes
Laminariheptaose
Laminaripentaose
1a
1b
2.2. Immunostimulating effects of glucan-mannose conjugates
1a and 1b
Figure 2. Effect of intra-peritoneal injection of 100
peripheral blood cells. The results represent the mean of three independent
experiments SD.
l
g of samples on percentages of
We first verified that our set of new molecules did not induce
potential aspecific toxicity onto cultured cells. Assays were per-
formed by monitoring cell viability and growth activity of five dif-
ferent tumor cell lines representative of the most frequent solid
tumors in human (liver, colon, prostate, breast, and lung) and
one resting normal diploid skin fibroblastic cell line, exposed to
80
*
increasing concentrations of the molecules (from 0.1 to 25 lM)
for 1 and 2 days. None of the tested molecules were able to induce
changes in the growth kinetics of actively dividing cells as well as
*
60
*
*
*
*
40
20
0
OR
RO
RO
OH
O
O
a
RO
9
O
O
OBn
R
OR
n
n
R
10a
4
4
5
5
Bz
b
b
11a
10b
11b
H
Macrophages Lymphocytes
Mast cells
Bz
H
Laminariheptaose
Laminaripentaose
Control
1a
1b
c
1a,b
Figure 3. Effect of intra-peritoneal injection of 100 lg of samples on percentages of
Scheme 4. Synthesis of mannose derivatives and final deprotection steps.
Reagents: (a) L-Selectride (10a: 100%; 10b: 98%); (b) NaOMe, HOMe (11a: 91%;
11b: 96%); (c) H2, Pd(OAc)2 (1a: 100%; 1b: 100%).
peritoneal cells. The results represent the mean of three independent experi-
ments SD. *represents significant differences between samples and control at P
<0.05 level.

K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
351
800
*
*
60
40
20
0
*
*
*
600
*
*
400
200
0
Monocytes
Granulocytes
Control
Laminariheptaose
Laminaripentaose
1a
1b
Laminariheptaose
Laminaripentaose
1a
1b
Control
Figure 6. Effect of intra-peritoneal injection of 100 lg of samples on in vitro
Figure 4. Potentiation of phagocytosis of synthetic microspheres (HEMA particles)
by different oligosaccharides injected intra-peritoneally 24 h before test. Monocytes
and granulocytes with three and more HEMA particles were considered positive.
Each value represents the mean SD. All differences were significant at P <0.05
level except when marked as *.
secretion of IL-2 by spleen cells. Control cells were isolated from mice injected with
PBS. The results represent the mean of three independent experiments SD.
*represents significant differences between samples and control at P <0.05 level.
modulation involved ulosyl bromides as keys intermediates. Intro-
duction of benzyl alcohol on the reducing anomeric center re-
quired neutral but neither acidic nor basic activation, otherwise
elimination occurred. Further selective reduction by L-Selectride
followed by standard deprotection steps gave the desired glucan-
mannose conjugates in 60.3% and 52.6% overall yields for eight
steps for 1a and 1b, respectively. Although they are not able to
form helices, these oligosaccharides were however efficient to
stimulate immune responses in vivo after intra-peritoneal injec-
tion in murin models. Increased ratio of granulocytes in peripheral
blood, potentiation of phagocytosis and production of IL-2 repre-
sent evidences for the expected immunostimulating effects. It
was also interesting to note that impact on the production of inter-
leukin-2 was significantly improved using synthetic glucan-man-
nose conjugates, and more especially the pentasaccharide 1a,
compared to the effects observed with linear laminarihepta- and
pentaose. These results also confirm the biological impact of linear
b-(1,3)-glucans unable to form helical structures. Further efforts to
strengthen the importance of the reducing end of oligo-b-(1,3)-glu-
cans are in due course.
80
*
*
*
60
40
20
0
Macrophages
Control
Laminariheptaose
Laminaripentaose
1a
1b
Figure 5. Potentiation of phagocytosis of synthetic microspheres (HEMA particles)
by different oligosaccharides injected intra-peritoneal 24 h before test. Peritoneal
macrophages with three and more HEMA particles were considered positive. Each
value represents the mean SD. All differences were significant at P <0.05 level
except when marked as *.
4. Experimental
4.1. Material and methods
both peripheral blood monocytes and granulocytes and peritoneal
macrophages. In every case, the highest activity was found with 1a.
It is hypothesized that the immunostimulating actions of oligo-
b-glucans are, at least in part, caused by potentiation of a synthesis
and release of several cytokines.25 To see if our samples influenced
the cytokine production, we measured the immunomodulating
activity through effects on the production of IL-2 by spleen cells.
The production of IL-2 was measured after a 72 h in vitro incuba-
tion of spleen cells isolated from control and oligosaccharide-trea-
ted mice (Fig. 6). Again, 1a was the most active sample, able to
stimulate IL-2 production to the same level as Concanavalin A
(data not shown).
Reactions and chromatographic purifications were monitored
by thin layer chromatography (TLC) analyses conducted on Merck
60 F254 Silica Gel non-activated plates and compounds were re-
vealed using a 5% solution of H2SO4 in EtOH followed by heating.
For column chromatography, Geduran Si 60 (40–63 lm) Silica
Gel was used. 1H, 13C, HMQC and COSY NMR spectra were recorded
on a Bruker ARX 400 spectrometer at 400 MHz for 1H and 100 MHz
for 13C. Chemical shifts are given in d-units (ppm) measured down-
field from Me4Si. Optical rotations were measured on a Perkin–El-
mer 341 polarimeter. The HRMS were measured at the Centre
Régional de Mesures Physiques de l’Ouest (University of Rennes
1, France) with a MS/MS ZabSpec TOF Micromass using m-nitrob-
enzylic alcohol as a matrix and accelerated cesium ions for ioniza-
tion. Elemental analysis was recorded at the CRMPO on a Flash
EA1112 CHN/O microanalyser.
3. Conclusion
The synthesis of small linear neo-oligo-b-(1,3)-glucans was per-
formed in order to evaluate in vivo the impact of structural modi-
fications at the reducing moiety on the activity of macrophages.
Our approach relied on controlled inversion of the C-2a configura-
tion starting from an oligosaccharidic chain. This fine structural
4.2. Benzoylation of oligo-b-(1,3)-glucans
To a solution of unprotected glucan in pyridine cooled at 0 °C
was added benzoyl chloride. After stirring for 2 days at room tem-

352
K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
perature, the reaction media was concentrated, the crude oil di-
luted in CH2Cl2 and the resulting organic layers successively
washed with 10% HCl, a saturated aqueous solution of NaHCO3
and brine. It was further dried (MgSO4) and concentrated. A final
chromatographic purification gave the desired perbenzoylated oli-
go-b-(1,3)-glucan as an inseparable mixture of anomers.
133.0, 132.8, 132.6, 130.0, 129.9, 129.7, 129.6, 129.5, 129.4,
129.3, 129.2, 129.1, 129.0, 128.9, 128.7, 128.6, 128.5, 128.4,
128.2, 128.0, 127.9 (C arom), 101.4, 101.0, 100.9, 100.8, 100.7 (C-
1b, C-1c, C-1d, C-1e, C-1f), 78.3, 78.2, 78.1, 77.9 (C-3b, C-3c, C-3d,
C-3e), 73.3, 73.2, 73.1, 72.9 (C-2), 72.5 (C-3f), 72.1 (C-5f), 71.7,
71.6 (C-5b, C-5c, C-5d, C-5e), 71.3 (C-2f), 70.2 (C-4), 70.0 (C-4),
69.8 (C-4f), 63.7, 63.4, 63.1 (C-6aa, C-6b, C-6c, C-6d, C-6e, C-6f);
4.2.1. Perbenzoylated laminaripentaose (3a)
HRMS found m/z 3093.8418 [M+Na]+, calcd for C176H142NaO51
Compound 3a was obtained according to the general procedure
starting from laminaripentaose (150 mg, 0.181 mmol), pyridine
(15 mL), and benzoyl chloride (4.2 mL, 36 mmol). Chromatography
eluting with petroleum ether/EtOAc (3:2) gave 3a (444 mg,
0.170 mmol) in 94% yield.
3093.8416.
4.3. Bromination of perbenzoylated oligo-b-(1,3)-glucans
To a solution of perbenzoylated glucan in CH2Cl2 cooled at 0 °C
was added a 33 wt % solution of hydrobromic acid in acetic acid.
After stirring for 3.5 h at room temperature, the media was diluted
in CH2Cl2 and washed with a saturated aqueous solution of NaH-
CO3. The resulting organic layer was then dried (MgSO4) and con-
centrated under reduced pressure. The crude oil was subsequently
used without purification.
Rf = 0.4 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data for the
5.47 (t, 1H, J3a,4a = J4a,5a = 9.4 Hz, H-4a), 4.64 (t, 1H, J2a,3a = 9.4 Hz,
H-3a); significant data for the b-anomer: 6.07 (d, 1H,
a-anomer: d 6.65 (d, 1H, J1a,2a = 3.6 Hz, H-1a),
d
J1a,2a = 7.1 Hz, H-1a), 5.57 (t, 1H, J3a,4a = JH4a,H5a = 8.9 Hz, H-4a),
0
4.25 (dt, 1H, J5a,6a = 4.3 Hz, J5a–6 a = 8.9 Hz, H-5a); other signals: d
8.08–7.92 (m, 68H, H arom), 7.61–7.09 (m, 102H, H arom), 5.45–
5.37 (m, 3H, H-2ab, H-3e), 5.26–5.18 (m, 5H, H-2a
5.11–4.83 (m, 14H, H-1b, H-2b, H-4b, H-2c, H-4c, H-2d, H-4d),
4.61–4.35 (m, 10H, H-3ab, H-5a
, H-6ab, H-60ab, H-1c, H-1d, H-
1e), 4.20–3.86 (m, 26H, H-6a
, H-3b, H-6b, H-60b, H-3c,
a
, H-2e, H-4e),
4.3.1. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyrano
syl-(1?3)-2,4,6-tri-O-benzoyl- -glucopyranosyl bromide (4a)
D-glucopyranosyl-(1?3)-2,4,6-
D
a
D
D
a
, H-60a
a
a-D
H-6c, H-60c, H-3d, H-6d, H-60d, H-5e, H-6e, H-60e), 3.80–3.65 (m,
This compound was synthesized starting from 3a (444 mg,
0.170 mmol) and bromhydric acid in acetic acid (0.6 mL,
3.417 mmol), and isolated in a quantitative yield as a colorless oil
(435 mg).
6H, H-5b, H-5c, H-5d); 13C NMR (CDCl3): significant data for the
a-anomer: d 87.7 (C-1a), 76.3 (C-3a), 72.4 (C-2a), 70.3 (C-5a),
67.6 (C-4a); significant data for the b-anomer: d 89.7 (C-1a), 78.4
(C-3a), 72.4 (C-2a, C-5a), 67.6 (C-4a), 62.0 (C-6a); other chemical
shifts: 166.0, 165.9, 165.8, 165.7, 165.6, 165.4, 164.9, 164.8,
164.7, 164.6, 164.5, 164.3, 164.1, 163.8, 163.6, 163.5 (OCOPh),
133.3, 132.7, 130.0, 129.3, 129.2, 129.1, 129.0, 128.9, 128.7 (Cquat
arom), 133.8, 133.6, 133.4, 133.2, 133.1, 133.0, 132.9, 132.8,
132.6, 129.9, 129.7, 129.6, 129.5, 129.4, 128.6, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.9 (C arom), 101.4, 101.3, 101.0,
100.8, 100.7 (C-1b, C-1c, C-1d, C-1e), 78.1, 77.5, 77.4, 77.2 (C-3b,
C-3c, C-3d), 73.4, 73.3, 73.2 (C-2), 72.7, 72.6, 72.5, 72.4 (C-3e, C-
2), 71.8, 71.7, 71.6, 71.5 (C-5b, C-5c, C-5d, C-5e), 71.3 (C-2e),
70.3, 70.2 (C-4b, C-4c, C-4d), 69.8 (C-4e), 63.5, 63.4, 63.1, 62.7 (C-
6a, C-6b, C-6c, C-6d, C-6e); HRMS found m/z 2619.7110 [M+Na]+,
calcd for C149H120NaO43 2619.7101.
1H NMR (CDCl3): significant data: d 6.55 (d, 1H, J1a,2a = 4.1 Hz, H-
1a), 5.35 (t, 1H, J3a,4a = J4a,5a = 9.9 Hz, H-4a), 4.24 (dd, 1H,
J5a,6 a = 4.6 Hz, J6a,6 a = 12.4 Hz, H-60a); other signals: d 8.92–7.61
0
0
(m, 22H, H arom), 7.48–6.93 (m, 58H, H arom), 5.27 (t, 1H, J2e,3e
=
J3e,4e = 9.7 Hz, H-3e), 5.09 (dd, 1H, J1e,2e = 7.9 Hz, H-2e), 5.08 (t, 1H,
J4e,5e = 9.7 Hz, H-4e), 4.93–4.86 (m, 3H, 2H-2, H-4), 4.80–4.70 (m,
4H, H-2a, H-1, 2H-4), 4.50–4.33 (m, 6H, H-3a, H-5a, H-6a, H-1e,
2H-1), 4.08–3.94 (m, 3H, H-5e, H-3, H-6), 3.87–3.70 (m, 9H, 2H-3,
7H-6), 3.65–3.53 (m, 3H, H-5b, H-5c, H-5d); 13C NMR (CDCl3): se-
lected data: d 87.7 (C-1a), 76.3 (C-3a), 73.4 (C-2a), 72.6 (C-5a),
67.6 (C-4a), 62.0 (C-6a); other signals: d 165.9, 165.8, 165.7,
165.4, 164.9, 164.8, 164.7, 164.6, 164.5, 164.3, 163.8, 163.5
(OCOPh), 133.8, 133.4, 133.2, 133.1, 133.0, 132.9, 132.8, 132.6,
129.9, 129.7, 129.6, 129.5, 129.4, 129.3, 129.2, 129.0, 128.7, 128.6,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9 (C arom), 101.3,
101.0, 100.8, 100.7 (C-1b, C-1c, C-1d, C-1e), 78.4, 78.1, 77.4 (C-3b,
C-3c, C-3d), 73.3, 73.2, 72.7 (C-2b, C-2c, C-2d), 72.5 (C-3e), 71.8,
71.7, 71.6, 71.5 (C-5b, C-5c, C-5d, C-5e), 71.3 (C-2e), 70.2 (C-4b,
C-4c, C-4d), 69.8 (C-4e), 63.4, 63.1 (C-6b, C-6c, C-6d, C-6e); HRMS
found m/z 2577.6011 [M+Na]+, calcd for C142H115BrNaO41
2577.5995.
4.2.2. Perbenzoylated laminarihexaose (3b)
The title compound was obtained according to the general
procedure starting from laminarihexaose (200 mg, 0.202 mmol),
pyridine (20 mL), and benzoyl chloride (5.2 mL, 44.4 mmol). Chro-
matography eluting with petroleum ether/EtOAc (1:1) gave 3b
(545 mg, 0.178 mmol) as a colorless oil in 85% yield.
Rf = 0.4 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data for the
a-anomer: d 6.60 (d, 1H, J1a,2a = 3.8 Hz, H-1a),
5.42 (t, 1H, J3a,4a = J4a,5a = 9.8 Hz, H-4a); selected chemical shifts
for the b-anomer: d 6.02 (d, 1H, J1a,2a = 7.1 Hz, H-1a), 5.52 (t, 1H,
J3a,4a = J4a,5a = 9.0 Hz, H-4a), 4.23–4.18 (m, 1H, H-5a); other signals:
d 8.01–7.65 (m, 60H, H arom), 7.55–7.01 (m, 140H, H arom), 5.36–
4.3.2. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyran
osyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-
2,4,6-tri-O-benzoyl- -glucopyranosyl bromide (4b)
D-glucopyranosyl-(1?3)-2,4,6-
D
D
D
D
5.30 (m, 3H, H-2ab, H-3f), 5.20–5.12 (m, 5H, H-2a
5.07–4.89 (m, 8H, 2H-2, 2H-4), 4.88–4.72 (m, 10H, H-1b, 2H-2,
2H-4), 4.61–4.30 (m, 13H, H-3a , H-3ab, H-5a
, H-6ab, H-60ab,
, H-60a
, H-3b,
a, H-2f, H-4f),
a-D
The title compound was prepared from 3b (643 mg,
0.209 mmol) and HBr in AcOH (1.1 mL, 6.364 mmol) and obtained
as a colorless oil in a quantitative yield (633 mg).
a
a
H-1c, H-1d, H-1e, H-1f), 4.12–3.78 (m, 32H, H-6a
a
a
H-6b, H-60b, H-3c, H-6c, H-60c, H-3d, H-6d, H-60d, H-3e, H-6e, H-
1H NMR (CDCl3): significant data: d 6.68 (d, 1H, J1a,2a = 3.8 Hz,
H-1a), 5.48 (t, 1H, J3a,4a = J4a,5a = 9.7 Hz, H-4a), 4.82 (dd, 1H,
J2a,3a = 9.7 Hz, H-2a), 4.61 (t, 1H, H-3a), 4.60 (dd, 1H, J5a,6a = 3.8 Hz,
60e, H-5f, H-6f, H-60f), 3.73–3.56 (m, 8H, H-5b, H-5c, H-5d, H-5e);
13C NMR (CDCl3): significant data for the
a-anomer: d 89.7 (C-
0
1a), 76.3 (C-3a), 72.7 (C-2a), 70.2 (C-5a), 68.8 (C-4a); selected data
connected with the b-anomer: d 92.0 (C-1a), 78.3 (C-3a), 72.9 (C-
5a), 72.5 (C-2a), 68.8 (C-4a), 62.7 (C6a); further chemical shifts: d
166.0, 165.9, 165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.4,
164.3, 164.1, 163.8, 163.6, 163.5 (OCOPh), 133.5, 133.2, 133.1,
J6a,6 a = 12.2 Hz, H-6a), 4.55–4.51 (m, 1H, H-5a), 4.37 (dd, 1H,
J5a,6 a = 4.2 Hz, H60a); other signals: d 8.00–7.71 (m, 26H, H arom),
0
7.61–7.03 (m, 69H, H arom), 5.39 (t, 1H, J2f,3f = J3f,4f = 9.7 Hz, H-
3f), 5.22 (dd, 1H, J1f,2f = 7.8 Hz, H-2f), 5.21 (t, 1H, J4f,5f = 9.7 Hz, H-
4f), 5.05–4.97 (m, 4H, 3H-2, H-4), 4.94 (t, 1H, J4–5 = 9.3 Hz, H-4),

K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
353
4.88 (d, 1H, J1,2 = 8.2 Hz, H-1), 4.87–4.77 (m, 3H, H-2, 2H-4), 4.55
4.4.2. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyran
osyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,
6-tri-O-benzoyl-2- -glu-hex-1-enopyranose (5b)
D-glucopyranosyl-(1?3)-2,4,6-
(d, 1H, H-1f), 4.49 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.42 (d, 1H,
J1,2 = 7.5 Hz, H-1), 4.40 (d, 1H, J1,2 = 7.5 Hz, H-1), 4.19–4.12 (m,
2H, H-3, H-6), 4.08–3.81 (m, 12H, 3H-3, 9H-6), 3.77–3.71 (m, 1H,
H-5f), 3.71–3.60 (m, 4H, H-5b, H-5c, H-5d, H-5e); 13C NMR (CDCl3):
selected data: d 87.7 (C-1a), 76.3 (C-3a), 73.4 (C-2a), 72.7 (C-5a),
67.6 (C-4a), 62.0 (C-6a); other signals: d 165.9, 165.8, 165.7,
165.4, 164.9, 164.8, 164.7, 164.6, 164.5, 164.3, 163.8, 163.5
(OCOPh), 133.8, 133.4, 133.2, 133.1, 133.0, 132.8, 132.6, 129.9,
129.7, 129.6, 129.5, 129.4, 129.3, 129.2, 129.1, 129.0, 128.9,
128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9 (C
arom), 101.3, 101.0, 100.8, 100.7 (C-1b, C-1c, C-1d, C-1e, C-1f),
78.4, 78.1, 77.8, 77.4 (C-3b, C-3c, C-3d, C-3e), 73.3, 73.2, 72.9 (C-
2b, C-2c, C-2d, C-2e), 72.5 (C-3f), 71.8, 71.6 (C-5b, C-5c, C-5d, C-
5e, C-5f), 71.3 (C-2f), 70.2 (C-4b, C-4c, C-4d, C-4e), 69.8 (C-4f),
63.5, 63.4, 63.1 (C-6b, C-6c, C-6d, C-6e, C-6f); HRMS found m/z
3051.7334 [M+Na]+, calcd for C169H137BrNaO49 3051.7310.
D
D
D
D
D
The hexasaccharidic derivative 5b was prepared from 4b
(550 mg, 0.181 mmol) and DBU (35 mL, 0.235 mmol) in CH2Cl2
(10 mL). After flash chromatography eluting with 3:2 petroleum
ether/EtOAc, a white solid was isolated (458 mg, 85%).
Rf = 0.5 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data: d 6.54 (s, 1H, H-1a), 5.69 (d, 1H, J3a,4a = 1.6 Hz, H-3a),
0
4.64 (dd, 1H, JH5a–H6a = 8.8 Hz, J6a,6 a = 12.4 Hz, H-6a), 4.32 (dd,
1H, J5a,6 a = 2.4 Hz, H-60a); other signals: d 7.95–7.62 (m, 25H, H
0
arom), 7.51–6.93 (m, 70H, H arom), 5.29 (t, 1H, J2f,3f = J3f,4f = 9.7 Hz,
H-3f), 5.12 (dd, 1H, J1f,2f = 8.0 Hz, H-2f), 5.11 (t, 1H, J4f,5f = 9.7 Hz, H-
4f), 5.10–5.12 (m, 1H, H-2b, H-4b), 4.96–4.80 (m, 4H, H-1, 2H-2, H-
4), 4.78–4.71 (m, 3H, H-2, 2H-4), 4.60 (d, 1H, J1,2 = 8.0 Hz, H-1),
4.52–4.44 (m, 3H, H-4a, H-5a, H-1), 4.42–4.35 (m, 3H, 2H-1, H-
0
4.4. Synthesis of hexenopyranoses
6), 4.07–4.01 (m, 1H, H-5b), 3.98 (dd, 1H, J5,6 = 3.5 Hz, J6,6 = 11.9
H, H-6z), 3.93–3.75 (m, 1H, 3H-3c, 8H-6), 3.74–3.55 (m, 4H, H-
5c, H-5d, H-5e, H-5f); 13C NMR (CDCl3): selected data: d 138.2
(C-1a), 74.1 (C-5a), 72.3 (C-4a), 68.6 (C-3a), 61.6 (C-6a); other sig-
nals: d 165.8, 165.7, 165.4, 165.2, 165.1, 164.9, 164.7, 164.6, 163.8,
163.7, 163.6 (19C, OCOPh), 133.3, 133.2, 133.1, 133.0, 132.9, 132.8,
132.7, 132.6, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.2,
129.1, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.9, 127.7 (C-2a, C arom), 101.2 (C-1b), 100.9, 100.8, 100.7 (C-1c,
C-1d, C-1e, C-1f), 78.2, 78.0 (C-3b, C-3c, C-3d, C-3e), 73.3 (C-2), 73.1
(C-2b), 73.0 (C-2), 72.7 (C-5b), 72.6 (C-3f, C-2), 72.0, 71.8, 71.6 (C-
5c, C-5d, C-5e, C-5f), 71.3 (C-2f), 70.2, 70.1, 69.8, 69.7 (C-4b, C-4c,
C-4d, C-4e, C-4f), 63.5, 63.3, 63.1 (C-6b, C-6c, C-6d, C-6e, C-6f);
HRMS found m/z 2971.8034 [M+Na]+, calcd for C169H136NaO49
2971.8048; HRMS found m/z 2987.7811 [M+K]+, calcd for
C169H136KO49 2987.7787.
To a solution of the crude oil previously obtained in CH2Cl2 was
added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was further
stirred for 5.5 h, diluted with CH2Cl2, and successively washed
with a 10% aqueous solution of HCl, a saturated solution of NaHCO3
and brine. The organic layer was dried over MgSO4 and the
solvent removed under reduced pressure before chromatographic
purification.
4.4.1. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyran
osyl-(1?3)-2,4,6-tri-O-benzoyl-2- -glu-hex-1-enopyranose (5a)
D-glucopyranosyl-(1?3)-2,4,6-
D
D
D
D
The pentasaccharidic derivative 5a was obtained according to
the general procedure starting from 4a (435 mg, 0.170 mmol) in
CH2Cl2 (10 mL) and using DBU (40 lL, 0.270 mmol). Chromato-
graphic purification (3:2 petroleum ether/EtOAc) gave 5a
4.5. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyran
-mannopyran
D-glucopyranosyl-(1?3)-2,4,6-
(343 mg, 81%) as a white solid.
D
Rf = 0.4 (3:2 petroleum ether/EtOAc); 1H NMR (CDCl3): signif-
icant data: d 6.63 (s, 1H, H-1a), 5.77 (d, 1H, J3a,4a = 3.3 Hz, H-3a),
D
D
osyl-(1?3)-2,4,6-tri-O-benzoyl-2-C-bromo-a-D
0
4.73 (dd, 1H, J5a,6a = 9.0 Hz, J6a,6 a = 12.6 Hz, H-6a), 4.40 (dd, 1H,
osyl bromide (6)
J5a,6 a = 3.1 Hz, H-60a); other signals: d 8.14–7.70 (m, 26H, H
0
arom), 7.63–7.03 (m, 54H,
H
arom), 5.38 (t, 1H, J2e,3e
=
To a solution of glucal 5a (90 mg, 0.036 mmol) in CH2Cl2
(0.5 mL) cooled at 0 °C were successively added molecular sieves
(100 mg), ethanethiol (10 lL, 0.171 mmol), and N-bromosuccini-
J3e,4e = 9.5 Hz, H-3e), 5.21 (dd, 1H, J1e,2e = 8.0 Hz, H-2e), 5.20 (t,
1H, H-4e, J4e,5e = 9.5 Hz), 5.17 (t, 1H, J1b,2b = J2b,3b = 8.8 Hz, H-2b),
5.12 (t, 1H, J3b,4b = J4b,5b = 10.5 Hz, H-4b), 5.05–4.97 (m, 2H, H-
4c, H-2d), 4.94 (d, 1H, H-1b), 4.94–4.88 (m, 2H, H-2c, H-4d),
4.71 (d, 1H, J1c,2c = 6.7 Hz, H-1c), 4.59 (d, 1H, J1e,2e = 8.0 Hz, H-
1e), 4.58–4.53 (m, 3H, H-4a, H-5a, H-1d), 4.46 (dd, 1H,
mide (NBS, 8.5 mg, 0.048 mmol). After stirring for 30 min at 0 °C
and 30 min at room temperature, the media was diluted (CH2Cl2,
20 mL), washed with a 10% aqueous solution of Na2S2O4, and final-
ly with cooled brine. The solvent was then dried (MgSO4), removed
under reduced pressure to afford the target dibromide derivative 6
(96 mg, 100%).
0
J5b,6b = 3.1 Hz, J6b,6 b = 12.2 Hz, H-6b), 4.31 (dd, 1H, H-3b), 4.25
(dd, 1H, J5b,6 b = 6.6 Hz, H-60b), 4.15–3.87 (m, 9H, H-5b, H-3c, H-
0
6c, H-60c, H-3d, H-6d, H-60d, H-6e, H-60e), 3.80 (dt, 1H,
1H NMR (CDCl3): significant data: d 5.81 (dd, 1H, J3a,4a = 9.3 Hz,
J4a,5a = 10.0 Hz,, H-4a), 4.62–4.54 (m, 1H, H-5a), 4.43 (dd, 1H,
0
J5d,6d = 4.9 Hz, J5d,6 d = 9.3 Hz, H-5d), 3.76–3.68 (m, 2H, H-5c,
H-5e); 13C NMR (CDCl3): selected data: d 138.2 (C-1a), 74.1 (C-
5a), 72.3 (C-4a), 68.7 (C-3a), 61.6 (C-6a); other signals: d 165.9,
165.8, 165.7, 165.4, 165.3, 165.1, 165.0, 164.8, 164.7, 164.6,
163.9, 163.7 (OCOPh), 133.3, 133.2, 133.0, 132.9, 132.7, 130.1,
130.0, 129.9, 129.7, 129.6, 129.5, 129.3, 129.2, 129.1,
128.9, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.8 (C-2a, C arom), 101.2 (C-1b), 100.9 (C-1c), 100.8 (C-1e),
100.7 (C-1d), 78.2, 78.0, 77.7 (C-3b, C-3c, C-3d), 73.5 (C-2c),
73.2 (C-2b), 72.8 (C-2d), 72.7 (C-5b), 72.6 (C-3e), 72.1 (C-5d),
71.6 (C-5c, C-5e), 71.3 (C-2e), 70.3 (C-4c), 70.0 (C-4d), 69.9,
69.8 (C-4b, C-4e), 63.5, 63.4, 63.2 (C-6c, C-6d, C-6e), 63.1 (C-
6b); HRMS found m/z 2497.6712 [M+Na]+, calcd for
C142H114NaO49 2497.6733.
J5a,6a = 2.5 Hz, J6a,6 a = 12.8 Hz,, H-6a), 4.24 (dd, 1H, J5a,6 a = 3.3 Hz,
H-60a); other signals: d 8.06–7.69 (m, 32H, H arom), 7.60–7.10
(m, 49H, H-1a, H arom), 5.40 (t, 1H, J2e,3e = J3e,4e = 9.7 Hz, H-3e),
5.24–5.16 (m, 5H, H-1b, H-2b, H-4b, H-2e, H-4e), 5.06–4.98 (m,
2H, H-4c, H-2d), 4.93–4.84 (m, 3H, H-3a, H-2c, H-4d), 4.66 (d,
1H, JH1c–H2c = 8.0 Hz, H-1c), 4.59 (d, 1H, J1e,2e = 8.0 Hz, H-1e), 4.51
(d, 1H, J1d,2d = 8.0 Hz, H-1d), 4.35–4.26 (m, 1H, H-3b), 4.07–3.64
(m, 14H, H-5b, H-6b, H-60b, H-3c, H-5c, H-6c, H-60c, H-3d, H-5d,
H-6d, H-60d, H-5e, H-6e, H-60e); 13C NMR (CDCl3): selected data:
d 89.9 (C-2a), 87.8 (C-1a), 79.5 (C-3a), 72.9 (C-5a), 67.3 (C-4a),
61.6 (C-6a); other signals: d 166.6, 165.8, 165.7, 165.4, 164.9,
164.8, 164.7, 164.6, 164.5, 163.9, 163.6, 163.4, 162.5 (16C–,
OCOPh), 133.3, 133.1, 133.0, 132.9, 132.8, 130.4, 130.3, 130.0,
0
0

354
K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
129.7, 129.6, 129.5, 129.4, 129.3, 129.2, 129.0, 128.9, 128.8, 128.6,
128.5, 128.3, 128.2, 128.1, 128.0, 127.8, 127.6 (C arom), 101.6 (C-
1b), 101.0 (C-1c), 100.9 (C-1e), 100.7 (C-1d), 78.1 (C-3b, C-3c, C-
3d), 73.5 (C-2c), 73.1 (C-2b), 72.9 (C-2d), 72.6 (C-3e), 71.6, 71.3
(C-5b, C-5c, C-5d, C-2e, C-5e), 70.1 (C-4c, C-4d), 69.8 (C-4b, C-4e),
63.6, 63.4, 63.3, 63.1 (4C, C-6b, C-6c, C-6d, C-6e); HRMS found m/
z 2655.5017 [M+Na]+, calcd for C142H114Br2NaO41 2655.5101;
HRMS found m/z 2671.4854 [M+K]+, calcd for C169H136 Br2KO49
2671.4839.
d 8.09–7.70 (m, 25H, H arom), 7.60–6.84 (m, 65H, H arom), 5.37
(t, 1H, J2f,3f = J3f,4f = 9.7 Hz, H-3f), 5.20 (dd, 1H, J1f,2f = 8.0 Hz, H-2f),
5.19 (t, 1H, J4f,5f = 9.7 Hz, H-4f), 5.10 (dd, 1H, J1b,2b = 8.1 Hz,
J2b,3b = 9.1 Hz, H-2b), 5.07–4.96 (m, 4H, H-1b, H-2, 2H-4), 4.92–
4.78 (m, 4H, 2H-2, 2H-4), 4.73 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.66 (d,
1H, J1,2 = 8.0 Hz, H-1), 4.65–4.57 (m, 2H, H-5a, H-6), 4.54 (d, 1H,
J1,2 = 8.0 Hz, H-1), 4.49 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.47–4.41 (m,
0
1H, H-6), 4.35 (dd, 1H, J5,6 = 4.9 Hz, J6,6 = 12.4 Hz, H-6), 4.28 (t,
0
1H, J3b,4b = 9.1 Hz, H-3b), 4.19 (dd, 1H, J5,6 = 3.5 Hz, J6,6 = 12.2 Hz,
0
H-6), 4.12 (dd, 1H, J5,6 = 6.2 Hz, J66 = 12.2 Hz, H-6), 4.05–3.81 (m,
4.6. Synthesis of ulosyl bromides
11H, H5-b, H-3c, H-3d, H-3e, 7H-6), 3.80–3.62 (m, 4H, H-5c, H-
5d, H-5e, H-5f); 13C NMR (CDCl3): significant data: d 191.5 (C-
2a), 83.9 (C-1a), 76.3 (C-3a), 72.6 (C-5a), 69.0 (C-4a), 61.7 (C-6a);
other signals: d 165.9, 165.8, 165.7, 165.4, 164.9, 164.7, 164.6,
164.3, 163.8, 163.6 (OCOPh), 133.6, 133.3, 133.2, 133.1, 133.0,
132.9, 132.6, 130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3,
129.2, 129.1, 129.0, 128.8, 128.6, 128.4, 128.3, 128.2, 128.1, 127.9
(C arom), 101.0, 100.9, 100.7, 100.6 (C-1b, C-1c, C-1d, C-1e, C-1f),
78.3, 78.2, 78.1, 78.0 (C-3b, C-3c, C-3d, C-3e), 73.5, 73.1, 73.0,
72.8 (C-2b, C-2c, C-2d, C-2e), 72.6 (C-3f), 71.9, 71.8, 71.6 (C-5b,
C-5c, C-5d, C-5e, C-5f), 71.3 (C-2f), 70.2, 70.1, 70.0, 69.8 (C-4b, C-
4c, C-4d, C-4e, C-4f), 63.6, 63.5, 63.4, 63.3, 63.2 (C-6b, C-6c, C-6d,
C-6e, C-6f); HRMS found m/z 2945.6917 [M+Na]+, calcd for
C162H131BrNaO48 2945.6891.
A solution of hexenopyranose in MeOH was stirred at room
temperature for 10 min and cooled at 0 °C. N-Bromosuccinimide
(NBS) was then added and the reaction monitored by TLC. After
completion of the reaction (2 h), the media was diluted with
CH2Cl2, successively washed with an aqueous saturated solution
of Na2S2O3 and water, dried (MgSO4), and concentrated. The result-
ing crude oil was used without further purification for the follow-
ing step.
4.6.1. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopy
-glucopyranos-2-ulosyl
D-glucopyranosyl-(1?3)-2,4,6-
D
D
D
ranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
D
bromide (7a)
4.7. Benzyl 2,3,4,6-tetra-O-benzoyl-b-
2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1,3)-2,4,6-tri-O-ben
zoyl-b- -glucopyranosyl-(1,3)-2,4,6-tri-O-benzoyl-b- -glucopy
-gluco-hex-3-enopy
D-glucopyranosyl-(1,3)-
This compound was obtained starting from 5a (48 mg,
0.019 mmol) diluted in CH2Cl2 (5 mL) and anhydrous MeOH
D
D
D
(1.6
lL, 0.040 mmol), and NBS (10.8 mg, 0.060 mmol). Removal of
ranosyl-(1,3)-6-O-benzoyl-4-deoxy-b-D
the solvent afforded 7a (47 mg) in a quantitative yield as a color-
ranos-2-uloside (8)
less oil.
1H NMR (CDCl3): significant data: d 6.25 (s, 1H, H-1a), 5.44 (t,
1H, J3a,4a = J4a,5a = 10.1 Hz, H-4a), 5.17 (d, 1H, H-3a); other signals:
d 8.06–7.71 (m, 20H, H arom), 7.61–7.12 (m, 55H, H arom), 5.39
(t, 1H, J2e,3e = J3e,4e = 9.5 Hz, H-3e), 5.22 (dd, 1H, J1e,2e = 8.0 Hz, H-
2e), 5.21 (t, 1H, J4e,5e = 9.5 Hz, H-4e), 5.10 (dd, 1H, J1b,2b = 8.2 Hz,
J2b,3b = 9.3 Hz, H-2b), 5.06–4.98 (m, 4H, H-1b, H-4b, H-4c, H-2d),
4.89 (t, 1H, J3d,4d = J4d,5d = 9.5 Hz, H-4d), 4.88 (dd, 1H, J1c,2c = 7.7 Hz,
J2c,3c = 8.6 Hz, H-2c), 4.74 (d, 1H, J1c,2c = 8.2 Hz, H-1c), 4.69 (d, 1H,
H-1e), 4.66–4.55 (m, 3H, H-5a, H-6a, H-1d), 4.39–4.26 (m, 2H, H-
3b, H-6), 4.24–4.10 (m, 3H, 3H-6), 4.05–3.86 (m, 8H, H-5b, H-3c,
H-3d, 5H-6), 3.82–3.70 (m, 3H, H-5c, H-5d, H-5e); 13C NMR
(CDCl3): significant data: d 191.5 (C-2a), 83.8 (C-1a), 76.3 (C-3a),
72.6 (C-5a), 69.0 (C-4a), 61.7 (C-6a); other signals: d 167.1, 165.9,
165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.3, 164.1, 163.9, 163.6
(OCOPh), 133.6, 133.3, 133.2, 133.1, 133.0, 132.9, 132.8, 132.7,
130.3, 130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.2,
129.1, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.8, 127.6 (C arom), 101.0, 100.9, 100.7, 100.6 (C-
1b, C-1c, C-1d, C-1e), 78.2, 78.0, 77.7 (C-3b, C-3c, C-3d), 73.6,
73.1, 72.8 (C-2b, C-2c, C-2d), 72.6 (C-3e), 71.9, 71.6 (C-5b, C-5c,
C-5d, C-5e), 71.3 (C-2e), 70.1, 69.9, 69.8 (C-4b, C-4c, C-4d, C-4e),
63.5, 63.4, 63.1 (C-6b, C-6c, C-6d, C-6e); HRMS found m/z
2471.5595 [M+Na]+, calcd for C135H109BrNaO40 2471.5576.
To a solution of ulosyl bromide 7a (50 mg, 0.020 mmol) in
CH2Cl2 (0.75 mL) were successively added at 0 °C benzyl alcohol
(4.4 lL, 0.040 mmol) and silver triflate (5.2 mg, 0.020 mmol). After
stirring for 3 h at room temperature, the reaction media was neu-
tralized by adding few drops of triethylamine, and then filtered
over a bed of Celite. The concentrated substrate was finally purified
by flash chromatography eluting with 3:2 petroleum ether/EtOAc
to give the enone 8 (38 mg, 75%).
An alternative procedure consisted in substituting silver triflate
by silver carbonate. To a suspension of benzyl alcohol (4.4 lL,
0.040 mmol), silver carbonate (11.6 mg, 0.042 mmol) and molecu-
lar sieves (50 mg) in dichloromethane (1 mL) stirred for 15 min
was further added ulosyl bromide 7a (50 mg, 0.020 mmol). The
glycosylation was performed for 2 h at room temperature and fil-
tered over a bed of Celite. Subsequent purification as indicated pre-
viously afforded the enone 8 in a similar yield (38 mg, 75%).
Rf = 0.5 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data: d 6.26 (d, 1H, H-4a, J4a,5a = 3.6 Hz, H-4a), 4.87 (s, 1H,
0
H-1a, 4.83 (d, 1H, J7a,7 a = 11.7 Hz, H-7a), 4.68–4.61 (m, 1H, H-5a),
4.60 (d, 1H, H-70a); Complementary signals: d 8.10–7.70 (m, 20H,
H
arom), 7.60–7.12 (m, 55H, H arom), 5.39 (t, 1H, J2e,3e =
J3e,4e = 9.7 Hz, H-3e), 5.28–5.17 (m, 5H, H-1b, H-2b, H-4b, H-2e,
H-4e), 5.09–5.02 (m, 4H, H-2c, H-4c, H-2d, H-4d), 4.95 (d, 1H,
J1c,2c = 8.0 Hz, H-1c), 4.63 (d, 1H, J1d,2d = 7.9 Hz, H-1d), 4.61 (d, 1H,
J1e,2e = 8.0 Hz, H-1e), 4.53–4.40 (m, 3H, H-6a, 2H-6), 4.38–4.32
(m, 2H, H-3b, H-6), 4.25–4.18 (m, 2H, H-60a, H-3), 4.15–3.90 (m,
8H, H-5b, H-3, H-5, 5H-6), 3.80–3.70 (m, 2H, H-5e, H-5); 13C
NMR (CDCl3): significant data: d 183.5 (C-2a), 144.9 (C-3a), 126.2
(C-4a), 97.7 (C-1a), 71.0 (C-5a), 70.6 (C-7a), 66.1 (C-6a); other sig-
nals: d 165.8, 165.7, 165.4, 164.9, 164.6, 164.5, 163.9, 163.8
(OCOPh), 136.1 (C quat. arom OCH2Ph), 133.3, 133.2, 133.1,
133.0, 132.9, 132.7, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4,
129.3, 129.2, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.8 (C arom), 100.9 (C-1d, C-1e), 100.1 (C-1c),
98.3 (C-1b), 78.3, 78.0 (C-3c, C-3d), 76.8 (C-3b), 73.7 (C-2c, C-2d),
4.6.2. 2,3,4,6-Tetra-O-benzoyl-b-
tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
-glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyran
osyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-
2,4,6-tri-O-benzoyl-b- -glucopyranos-2-ulosyl bromide (7b)
D-glucopyranosyl-(1?3)-2,4,6-
D
D
D
D
D
As described in the general procedure, compound 7b was syn-
thesized from 5b (124 mg, 0.042 mmol), anhydrous MeOH
(3.4 lL, 0.084 mmol), CH2Cl2 (1.5 mL), NBS (22.4 mg, 0.126 mmol),
and obtained as a colorless oil in a quantitative yield (123 mg).
1H NMR (CDCl3): significant data: d 6.25 (s, 1H, H-1a), 5.44 (t,
1H, J3a,4a = J4a,5a = 10.2 Hz, H-4a), 5.16 (d, 1H, H-3a); other signals:

K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
355
1
73.0 (C-2b), 72.8 (C-5b), 72.6 (C-3e), 72.1 (C-5), 71.6 (C-2e), 71.3
(C-5e, C-5), 69.9, 69.8 (C-4c, C-4d, C-4e), 69.3 (C-4b), 63.4 (C-6e,
C-6), 63.1 (C-6b, C-6); HRMS found m/z 2377.6516 [M+Na]+, calcd
for C1359H110NaO39 2377.6522.
98.7 (C-1a, JH1a,C1a = 171 Hz), 93.0 (C-2a), 81.7 (C-3a), 70.7 (C-
7a), 69.8 (C-4a); complementary data: d 166.1, 165.8, 165.7,
165.4, 164.9, 164.8, 164.7, 164.6, 163.8, 163.6 (OCOPh), 133.4,
133.3, 133.2, 133.1, 133.0, 132.6, 132.1, 132.0, 129.9, 129.8,
129.6, 129.4, 129.3, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7,
128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 127.9, 127.6, 126.9, 126.5
(C arom), 102.4 (C-1b), 100.9, 100.8 (C-1c, C-1d, C-1e, C-1f), 78.3,
78.2, 78.1 (C-3b, C-3c, C-3d, C-3e), 73.1, 73.0 (C-2), 72.6 (C-3f, C-
5a, 2 C-2), 71.8, 71.6 (C-5b, C-5c, C-5d, C-5e, C-5f), 71.3 (C-2f),
70.3, 70.2, 70.1, 69.9 (C-4b, C-4c, C-4d, C-4e, C-4f), 63.6, 63.5,
63.4, 63.2, 63.1 (C-6a, C-6b, C-6c, C-6d, C-6e, C-6f); HRMS found
m/z 2973.8163 [M+Na]+, calcd for C169H138NaO49 2973.8204; HRMS
found m/z 2991.8106 [M+Na]+, calcd for C169H140KO50 2991.8310.
4.8. Glycosylation of benzyl alcohol with ulosyl bromide as
donor
A suspension of ulosyl bromide, benzyl alcohol and molecular
sieves in CH2Cl2 was vigorously stirred for 5 min at room temper-
ature. Triphenylphosphine oxide was then added. After 4 days, the
molecular sieves were removed by filtration over Celite, and the
organic layer concentrated under reduced pressure. A flash chro-
matography afforded the desired glycoside in its hydrated form.
4.9. Reduction with L-Selectride
4.8.1. Benzyl 2,3,4,6-tetra-O-benzoyl-b-
(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-
O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
glucopyranosyl-(1?3)-4,6-di-O-benzoyl-2-C-hydroxyl-b-D-
glucopyranoside (9a)
D-glucopyranosyl-
D
To a solution of benzylic glycoside in THF cooled at ꢀ78 °C was
added L-Selectride. After stirring for 1.5 h at the same temperature,
the reaction was quenched by adding few drops of HOAc. The med-
ia was further diluted with CH2Cl2, successively washed with a 10%
aqueous solution of HCl, a saturated aqueous solution of NaHCO3,
and finally with brine. The desired glucanyl-mannose derivative
was then obtained by drying the organic layer (MgSO4) and re-
moval of the solvent.
D
D-
Compound 9a was prepared from 7a (94 mg, 0.038 mmol), ben-
zyl alcohol (8 L, 0.077 mmol), triphenylphosphine oxide (21 mg,
l
0.077 mmol) in CH2Cl2 (1.5 mL). A chromatographic purification
(3:2 petroleum ether/EtOAc) gave the target pentasaccharide 9a
(83 mg, 87%) as a colorless oil.
Rf = 0.5 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data: d 4.45–4.34 (m, 3H, H-5a, H-6a, H-60a), 4.25 (s, 1H, H-
1a); other signals: d 8.00–7.10 (m, 80H, H arom), 5.42–5.16 (m,
5H, H-4a, H-2e, H-3e, H-4e, H-2), 5.12–4.79 (m, 6H, H-7a, H-4b,
H-4c, H-4d, 2H-2), 4.78–4.68 (m, 3H, H-70a, 2H-1), 4.64–4.46 (m,
2H, 2H-1), 4.26 (t, 1H, J2b,3b = J3b,4b = 9.0 Hz, H-3b), 4.15–3.69 (m,
15H, H-3a, H-5b, H-6b, H-60b, H-3c, H-5c, H-6c, H-60c, H-3d, H-
5d, H-6d, H-60d, H-5e, H-6e, H-60e); 13C NMR (CDCl3): significant
4.9.1. Benzyl 2,3,4,6-tetra-O-benzoyl-b-
(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-
O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
glucopyranosyl-(1?3)-4,6-di-O-benzoyl-b-D-mannopyranoside
(10a)
D-glucopyranosyl-
D
D
D-
Reduction of 9a (83 mg, 0.034 mmol) required L-Selectride (1 M
in THF, 34 L, 0.034 mmol) in THF (0.7 mL). Pentasaccharide 10a
l
was isolated in a quantitative yield (83 mg) as a colorless oil.
Rf = 0.5 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data: d 5.53 (t, 1H, J3a,4a = J4a,5a = 7.3 Hz, H-4a), 4.82 (d, 1H,
1
data: d 98.7 (C-1a, JH1a,C1a = 168 Hz), 93.0 (C-2a), 81.7 (C-3a),
70.7 (C-7a), 69.3 (C-4a), 63.1 (C-6a); other signals: d 166.1, 165.8,
165.7, 165.4, 165.0, 164.9, 164.7, 164.2, 163.8, 163.7, 163.6
(OCOPh), 133.4, 133.2, 133.1, 133.0, 132.7, 130.0, 129.9, 129.7,
129.6, 129.5, 129.3, 129.2, 129.1, 129.0, 128.8, 128.7, 128.6,
128.5, 128.3, 128.2, 128.1, 128.0 (C arom), 102.4 (C-1b), 101.0,
100.9 (C-1c, C-1d, C-1e), 78.2, 78.0, 77.7 (C-3b, C-3c, C-3d), 73.4,
72.8, 72.6 (C-5a, C-2b, C-2c, C-2d, C-3e), 71.8, 71.6 (C-5b, C-5c, C-
5d, C-5e), 71.3 (C-2e), 70.2, 70.0, 69.9, 69.7 (C-4b, C-4c, C-4d, C-
4e), 63.6, 63.5, 63.4 (C-6b, C-6c, C-6d, C-6e); HRMS found m/z
2499.6898 [M+Na]+, calcd for C142H116NaO41 2499.6890; HRMS
found m/z 2517.6991 [M+K]+, calcd for C142H118KO42 2517.6991.
0
J7a,7 a = 12.4 Hz, H-7a), 4.49 (d, 1H, J1a,2a = 2.0 Hz, H-1a); Comple-
mentary signals: d 8.04–7.10 (m, 80H, H arom), 5.41 (t, 1H, J2e,3e
=
J3e,4e = 9.5 Hz, H-3e), 5.24 (dd, 1H, J1e,2e = 8.0 Hz, H-2e), 5.22 (t, 1H,
J4e,5e = 9.5 Hz, H-4e), 5.15–4.85 (m, 6H, H-2b, H-4b, H-2c, H-4c,
H-2d, H-4d), 4.76 (d, 1H, J1b,2b = 7.7 Hz, H-1b), 4.65–4.50 (m, 6H,
H-70a, H-6a, H-60a, H-1c, H-1d, H-1e), 4.34–3.83 (m, 14H, H-2a, H-
3a, H-3b, H-6b, H-60b, H-3c, H-6c, H-60c, H-3d, H-6d, H-60d, H-6e,
H-60e, 2H-5), 3.80–3.71 (m, 3H, 3H-5); 13C NMR (CDCl3): significant
data: d 96.5 (C-1a), 70.1 (C-7a), 68.4 (C-4a), 67.1 (C-2a), 64.1 (C-6a);
complementary data: d 166.0, 165.8, 165.7, 165.4, 165.0, 164.9,
164.7, 164.6, 164.1, 163.8, 163.6 (OCOPh), 136.3 (C quat arom
OCH2Ph), 133.2, 133.1, 133.0, 132.9, 132.6, 129.9, 129.7, 129.6,
129.5, 129.4, 129.3, 129.2, 129.1, 129.0, 128.7, 128.6, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.9, 127.7 (C arom), 100.8, 100.7,
100.6 (C-1b, C-1c, C-1d, C-1e), 78.1, 78.0, 77.7 (C-3a, C-3b, C-3c, C-
3d), 73.6, 73.5, 72.8 (C-2b, C-2c, C-2d) 72.5 (C-3e), 72.2, 72.0, 71.9,
71.6 (C-5a, C-5b, C-5c, C-5d, C-5e), 71.3 (C-2e), 70.0, 69.8, 69.7 (C-
4b, C-4c, C-4d, C-4e), 63.4, 63.2, 63.1, 62.7 (C-6b, C-6c, C-6d, C-
6e); HRMS found m/z 2501.7064 [M+Na]+, calcd for C142H118NaO49
2501.7046; HRMS found m/z 2517.6991 [M+K]+, calcd for
C142H118KO41 2517.6786.
4.8.2. Benzyl 2,3,4,6-tetra-O-benzoyl-b-
(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-
O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-
(1?3)-4,6-di-O-benzoyl-2-C-hydroxyl-b- -glucopyranoside (9b)
Synthesis of compound 9b required 7b (51 mg, 0.017 mmol),
CH2Cl2 (0.7 mL), benzyl alcohol (3.6 L, 0.035 mmol), triphenyl-
D-glucopyranosyl-
D
D
D-
D
D
l
phosphine oxide (9.7 mg, 0.035 mmol). A flash chromatography
eluting with 3:2 petroleum ether/EtOAc gave 9b as a colorless oil
(43.8 mg, 85%).
Rf = 0.3 (3:2 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data: d 4.61 (d, 1H, J7a,7 a = 12.2 Hz, H-70a), 4.57–4.47 (m, 1H,
0
4.9.2. Benzyl 2,3,4,6-tetra-O-benzoyl-b-
(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-
O-benzoyl-b- -glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b-
glucopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-b- -glucopyranosyl-
(1?3)-4,6-di-O-benzoyl-b- -mannopyranoside (10b)
Reduction of 9b (83 mg, 0.034 mmol) required L-Selectride (1 M
in THF, 18 L, 0.018 mmol) in THF (0.5 mL). Hexasaccharide 10b
was isolated in 98% yield (50 mg) as a colorless oil.
D-glucopyranosyl-
H-6a), 4.25 (s, 1H, H-1a); complementary data: d 8.03–7.69 (m,
D
25H,
H arom), 7.58–7.08 (m, 70H, H arom), 5.36 (t, 1H,
D
D-
J2f,3f = 9.7 Hz, H-3f), 5.30–5.10 (m, 3H, H-2f, H-4a, H-4f), 5.08–
4.94 (m, 4H, 2H-2, 2H-4), 4.92–4.73 (m, 5H, H-7a, 2H-2, 2H-4),
4.45–4.32 (m, 5H, H-5a, H-60a, 2H-1), 4.24–4.19 (m, 1H, H-3b),
4.15–3.77 (m, 14H, H-3a, H-3c, H-3d, H-3e, H-6b, H-60b, H-6c, H-
60c, H-6d, H-60d, H-6e, H-60e, H-6f, H-60f), 3.74–3.65 (m, 5H, H-
5b, H-5c, H-5d, H-5e, H-5f); 13C NMR (CDCl3): significant data: d
D
D
l

356
K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
Rf = 0.5 (1:1 petroleum ether/EtOAc); 1H NMR (CDCl3): signifi-
cant data: d 5.48 (t, 1H, J3a,4a = J4a,5a = 7.3 Hz, H-4a), 4.82 (d, 1H,
signals: d: 7.22–7.10 (m, 5H, H arom), 4.48 (d, 3H, J1,2 = 8.0 Hz, 3H-
1), 4.39 (d, 2H, J1,2 = 7.5 Hz, 2H-1), 3.76–3.53 (m, 8H, H-3a, H-4a, H-
6a, H-6b, H-6c, H-6d, H-6e, H-6f), 3.55–3.36 (m, 12H, H-2a, H-3b,
H-3c, H-3d, H-3e, H-5f, H-60a, H-60b, H-60c, H-60d, H-60e, H-60f),
3.35–3.25 (m, 4H, H-2b, H-2c, H-2d, H-2e), 3.24–3.05 (m, 12H, H-
2f, H-3f, H-4b, H-4c, H-4d, H-4e, H-4f, H-5a, H-5b, H-5c, H-5d, H-
5e); 13C NMR (CDCl3): significant data: d 99.9 (C-1a), 85.3 (C-3a),
73.1 (C-2a), 72.2 (C-4a), 71.7 (C-7a); complementary data: d
131.2 (C quat arom), 129.4, 128.7 (C arom), 105.2, 104.7 (C-1b, C-
1c, C-1d, C-1e, C-1f), 87.4, 87.1, 87.0 (C-3b, C-3c, C-3d, C-3e),
82.1 (C-5f), 78.2, 78.1, 77.8 (C-3f, C-5a, C-5b, C-5c, C-5d, C-5e),
75.5 (C-2f), 75.0, 74.9, 74.4 (C-2b, C-2c, C-2d, C-2e), 71.5 (C-4f),
70.0, 69.9 (C-4b, C-4c, C-4d, C-4e), 62.8, 62.6, 62.5 (C-6a, C-6b, C-
6c, C-6d, C-6e, C-6f); HRMS found m/z 1103.3639 [M+Na]+, calcd
for C43H68NaO31 1119.3338; HRMS found m/z 957.2892 [M+K]+,
calcd for C43H68KO31 1119.3382.
0
J7a,7 a = 12.4 Hz, H-7a), 4.45 (d, 1H, J1a,2a = 2.2 Hz, H-1a); other sig-
nals: d 8.00–7.70 (m, 25H, H arom), 7.55–7.10 (m, 90H, H arom),
5.35 (t, 1H, J2f,3f = J3f,4f = 9.5 Hz, H-3f), 5.17 (dd, 1H, J1f,2f = 8.0 Hz,
H-2f), 5.16 (t, 1H, J4f,5f = 9.5 Hz, H-4f), 5.15–5.10 (m, 1H, H-4),
5.08 (dd, 1H, J1,2 = 8.2 Hz, J2,3 = 9.1 Hz, H-2), 5.02–4.90 (m, 3H, H-
2, 2H-4d), 4.89–4.75 (m, 3H, 2H-2, H-4), 4.71 (d, 1H, J1,2 = 8.0 Hz,
H-1), 4.68 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.57–4.36 (m, 6H, H-70a, 3H-
1, 2H-6), 4.32–4.19 (m, 2H, H-3, H-6), 3.99–3.78 (m, 16H, H-2a,
4H-3, 2H-5, 9H-6), 3.76–3.63 (m, 4H, 4H-5); 13C NMR (CDCl3): sig-
nificant data: d 96.6 (C-1a), 69.8 (C-7a), 68.5 (C-4a), 67.1 (C-2a),
64.2 (C-6a); complementary data: d 165.8, 165.7, 165.4, 165.1,
164.9, 164.7, 164.1, 163.8, 163.6 (OCOPh), 136.4 (C quat arom
OCH2Ph), 133.2, 133.1, 133.0, 132.6, 130.0, 129.7, 129.6, 129.5,
129.4, 129.3, 129.2, 129.1, 129.0, 128.8, 128.6, 128.5, 128.3,
128.2, 128.1, 128.0, 127.9 (C arom), 100.9, 100.8 (C-1b, C-1c, C-
1d, C-1e, C-1f), 78.4, 78.3, 78.2, 78.0 (C-3a, C-3b, C-3c, C-3d, C-
3e), 73.6, 73.5, 73.3 (C-2), 73.0 (C-3f, C-2), 72.2, 72.0, 71.7, 71.6
(C-5a, C-5b, C-5c, C-5d, C-5e, C-5f), 71.3 (C-2f), 70.2, 70.1 (C-4b,
C-4c, C-4d, C-4e), 69.3 (C-4f), 63.6, 63.5, 63.3, 63.2 (C-6b, C-6c, C-
6d, C-6e, C-6f); HRMS found m/z 2975.8366 [M+Na]+, calcd for
C169H140NaO49 2975.8361.
4.11. Hydrolytic debenzylation
To a solution of benzyl oligoglucan in methanol was added
Pd(OAc)2. After stirring for 7 days at room temperature under
hydrogen atmosphere, the catalyst was removed by filtration and
the solvent under reduced pressure. Elution on a Sephadex G-10
column eluting with water, followed by freeze drying, yield the de-
sired glucan-mannose conjugate.
4.10. Zemplén debenzoylation
The oil previously obtained was then diluted in a 2:1 mixture of
HOMe/CH2Cl2 containing 2 equiv of NaOMe (0.1 M MeONa in
HOMe). After stirring for 6 h at room temperature, neutralization
was performed with the help of Amberlite IR 120 (H+-form). The
resin was further filtered off, and the solvent removed under re-
duced pressure. The oligosaccharide was finally purified by flash
chromatography (99:1 CH2Cl2/HOMe).
4.11.1. b-
-glucopyranosyl-(1?3)-b-
mannopyranose (1a)
Debenzylation of 11a (25.2 mg, 0.027 mmol) in the presence of
Pd(OAc)2 quantitatively gave 1a (22.5 mg) as a white foam.
Rf = 0.2 (3:2:2 EtOAc/HOiPr/H2O); 1H NMR (CDCl3): significant
D
-Glucopyranosyl-(1?3)-b-
D
-glucopyranosyl-(1?3)-b-
,b-
D
D-glucopyranosyl-(1?3)-
a
D-
data: d 5.20 (d, 1H, J1a,2a = 1.8 Hz, H-1aa); complementary signals:
d 4.75 (d, 2H, J1,2 = 8.2 Hz, 2H-1), 4.71 (d, 2H, J1,2 = 8.0 Hz, 2H-1,;
3.91–3.80 (m, 7H, H-3a, H-4a, H-6a, H-6b, H-6c, H-6d, H-6e),
3.78–3.59 (m, 9H, H-2a, H-60a, H-3b, H-60b, H-3c, H-60c, H-3d, H-
60d, H-60e), 3.58–3.40 (m, 12H, H-5a, H-2b, H-4b, H-5b, H-2c, H-
4.10.1. Benzyl b-
(1?3)-b- -glucopyranosyl-(1?3)-b-
-mannopyranoside (11a)
Deacylation of 10a (83 mg, 0.034 mmol) in NaOMe, HOMe
D
-glucopyranosyl-(1?3)-b-
D-glucopyranosyl-
D
D
-glucopyranosyl-(1?3)-b-
D
4c, H-5c, H-2d, H-4d, H-5d, H-3e, H-5e), 3.36 (t, 1H, J3e,4e
=
(0.1 M solution, 0.7 mL, 0.070 mmol) afforded 11a (28 mg) in 91%
yield as a colorless oil.
J4e,5e = 9.7 Hz, H-4e), 3.31 (dd, 1H, J1e,2e = 8.0 Hz, J2e,3e = 9.3 Hz,
H-2e; HRMS found m/z 851.2634 [M+Na]+, calcd for C303H52NaO26
851.2644.
1H NMR (CDCl3): significant data: d 5.03 (d, 1H, J1a,2a = 3.6 Hz, H-
1a), 4.81 (d, 1H, J7a,7 a = 12.2 Hz, H-7a), 4.58 (d, 1H, H-70a); comple-
0
mentary signals: d 7.88–7.83 (m, 2H, H arom), 7.33–7.13 (m, 3H, H
arom), 4.56 (d, 2H, J1,2 = 8.0 Hz, 2H-1), 4.46 (d, 2H, J1,2 = 7.7 Hz, 2H-
1), 3.3–3.4 (m, 7H, H-3a, H-4a, H-6a, H-6b, H-6c, H-6d, H-6e), 3.64–
3.46 (m, 10H, H-2a, H-60a, H-3b, H-60b, H-3c, H-60c, H-3d, H-60d, H-
5e, H-60e), 3.44–3.34 (m, 3H, H-2b, H-2c, H-2d), 3.33–3.13 (m, 10H,
H-5a, H-4b, H-5b, H-4c, H-5c, H-4d, H-5d, H-2e, H-3e, H-4e); 13C
NMR (CDCl3): significant data: d 99.9 (C-1a), 85.3 (C-3a), 73.1 (C-
2a), 72.9 (C-4a), 71.5 (C-7a); complementary data: d 131.0 (C quat
arom), 129.4, 128.9 (C arom), 105.1, 104.7 (C-1b, C-1c, C-1d, C-1e),
87.4, 87.1 (C-3b, C-3c, C-3d), 82.1 (C-5e), 78.1, 77.7 (C-5a, C-5b, C-
5c, C-5d, C-3e), 75.5 (C-2e), 75.0, 74.9 (C-2b, C-2c, C-2d), 71.6 (C-
4e), 70.0, 69.9 (C-4b, C-4c, C-4d), 62.6, 62.5 (C-6a, C-6b, C-6c, C-
6d, C-6e); HRMS found m/z 941.3119 [M+Na]+, calcd for
C37H58NaO26 941.3114; HRMS found m/z 957.2892 [M+K]+, calcd
for C37H58KO26 957.2853.
4.11.2. b-
-glucopyranosyl-(1?3)-b-
glucopyranosyl-(1?3)- ,b-
D-Glucopyranosyl-(1?3)-b-D-glucopyranosyl-(1?3)-b-
D
D-glucopyranosyl-(1?3)-b-D-
a
D
-mannopyranose (1b)
Debenzylation of 11b (15.7 mg, 0.015 mmol) in the presence of
Pd(OAc)2 (16 mg, 0.071 mmol) afforded the desired hexasaccharide
1b (14.3 mg, 100%) as a white foam.
Rf = 0.2 (3:2:2 EtOAc/HOiPr/H2O); 1H NMR (CDCl3): significant
data: 5.19 (d, 1H, J1a,2a = 2.2 Hz, H-1aa); complementary signals:
d 4.75 (d, 3H, J1,2 = 8.0 Hz, 3H-1), 4.71 (d, 2H, J1,2 = 7.8 Hz, 2H-1),
3.93–3.81 (m, 8H, H-3a, H-4a, H-6a, H-6b, H-6c, H-6d, H-6e, H-
6f), 3.80–3.63 (m, 11H, H-2a, H-3b, H-3c, H-3d, H-3e, H-60a, H-
60b, H-60c, H-60d, H-60e, H-60f), 3.55–3.40 (m, 11H, H-2b, H-2c, H-
2d, H-2e, H-3f, H-4b, H-4c, H-4d, H-4e, H-5a, H-5b, H-5c, H-5d,
H-5e, H-5f), 3.36 (t, 1H, J3f,4f = J4f,5f = 9.3 Hz, H-4f), 3.31 (dd, 1H,
J1f,2f = 8.0 Hz, J2f,3f = 9.3 Hz, H-2f); HRMS found m/z 1013.3172
[M+Na]+, calcd for C36H62NaO31 1013.3173; HRMS found m/z
1029.2920 [M+K]+, calcd for C36H62KO31 1029.2912.
4.10.2. Benzyl b-
(1?3)-b- -glucopyranosyl-(1?3)-b-
-glucopyranosyl-(1?3)-b- -mannopyranoside (11b)
Deacylation was performed from 10b (44 mg, 0.015 mmol) and
D
-glucopyranosyl-(1?3)-b-D-glucopyranosyl-
D
D
-glucopyranosyl-(1?3)-b-
D
D
4.12. Biological tests
NaOMe, HOMe (0.1 M, 0.3 mL, 0.030 mmol) and gave the hexasac-
charide 11b (15.7 mg, 96%) as a colorless oil.
4.12.1. Evaluation of cytotoxicity
The cytotoxicity was studied on five distinct cell lines obtained
from the european ECAC collection and on skin diploid fibroblasts
which were provided by BIOPREDIC International Company. They
1H NMR (CDCl3): significant data: d 4.95 (d, 1H, J1a,2a = 3.8 Hz,
H-1a), 4.75 (d, 1H, J7a,7 a = 12.2 Hz, H-7a), 4.50 (d, 1H, H-70a); other
0

K. Descroix et al. / Bioorg. Med. Chem. 18 (2010) 348–357
357
included two human colon carcinoma cells Caco2 and HCT 116
filtered through 0.45
l
m filters and tested for the presence of IL-
representative of two distinct differentiated and highly colon
tumorigenic tumors, respectively, a differentiated highly growing
HUH7 hepatocarcinoma cells, the NCI lung and PC3 prostate
tumor cells. They were grown according to the providers
recommendations.
2.26 Levels of the IL-2 were measured using a Quantikine mouse
IL-2 kit (R&D Systems, Minneapolis, MN).
Acknowledgements
The cytotoxicity assay was based on an automated imaging
analysis as followed: 4 ꢁ 103 cells were seeded in 96-multiwell
plates and led for 24 h for attachment, spreading and growth. Then,
they were exposed for 24 and 48 h to increasing concentrations of
We thank the Cancéropole Grand Ouest (France) for financial
support, Rémy Le Guével (University of Rennes 1, France) for assis-
tance onto the ImPACcell platform from Biogenouest, and Ms.
Rosemary Williams for editing assistance.
the compounds, ranging from 0.1 to 25
lM in a final volume of
80 L of culture medium. They were fixed with 4% paraformalde-
l
Supplementary data
hyde solution and nuclei were stained with Hoechst 3342 and
counted according to automated imaging quantification. Four pic-
tures per well were obtained with a high speed camera and statis-
tical analyses were established using the Simple PCI software. In
addition, imaging analysis allowed detection of possible cell mor-
phology changes.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.10.053.
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