Synthetic studies toward the anthrax tetrasaccharide: Alternative synthesis of this antigen

Article abstract of DOI:10.1016/j.carres.2012.01.007

The synthesis of the anthrax tetrasaccharide, amenable for conjugation, has been envisaged by both [2+2] and [1+3] approaches from d-fucose and l-rhamnose. The successful route reported herein relies on a [1+3] strategy in which the 1,2-trans-glycosidic linkages have been secured using a participating group at the 2-position of the donors using conventional thio as well as trichloroacetimidate glycosylation chemistry. The exchange of the ester to benzyl protective groups on the rhamnosyl moiety was key to achieve the final assembly and functionalization of the tetrasaccharide.

Full text of DOI:10.1016/j.carres.2012.01.007

Carbohydrate Research 356 (2012) 115–131
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthetic studies toward the anthrax tetrasaccharide: alternative
synthesis of this antigen
Ophélie Milhomme a, Sandrine G.Y. Dhénin a, Florence Djedaïni-Pilard a, Vincent Moreau a,
Cyrille Grandjean a,b,
⇑
a Laboratoire des Glucides, FRE CNRS 3517, Université de Picardie Jules Verne, 33 rue Saint-Leu, F-80039 Amiens Cedex, France
b Unité de Fonctionnalité et Ingénierie des Protéines, FRE CNRS 4378, LUNAM, 2 rue de la Houssinière, BP92208, F-44322 Nantes Cedex 3, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of the anthrax tetrasaccharide, amenable for conjugation, has been envisaged by both [2+2]
Received 28 November 2011
Received in revised form 15 January 2012
Accepted 17 January 2012
Available online 24 January 2012
and [1+3] approaches from D-fucose and L-rhamnose. The successful route reported herein relies on a
[1+3] strategy in which the 1,2-trans-glycosidic linkages have been secured using a participating group
at the 2-position of the donors using conventional thio as well as trichloroacetimidate glycosylation
chemistry. The exchange of the ester to benzyl protective groups on the rhamnosyl moiety was key to
achieve the final assembly and functionalization of the tetrasaccharide.
Keywords:
Bacillus anthracis
Anthrose
Ó 2012 Elsevier Ltd. All rights reserved.
Exosporium glycoprotein
Oligosaccharide
Kuhn’s methylation
1. Introduction
OH
O
Anthrax is a worldwide disease of sheep, cattle, horses, and
other mammals caused by the spore-forming soil bacterium, Bacil-
lus anthracis. The ease of production and dissemination of the
spores as well as the high lethality that results from their inhala-
tion have designed B. anthracis as a potential bioterrorism weap-
on.1 This threat has stressed the development of alternative
vaccines to the less than optimal currently licensed ones.2–4 Along
this line specific oligosaccharides found at the outermost layer of
the spores could be used as antigens to prepare glycoconjugate
vaccines, known to be efficient and safe in humans,5 to inactivate
the bacterium at an early stage of the infection. Therefore much
attention has been paid to a linear tetrasaccharide 1, abundantly
expressed on the B. anthracis exosporium glycoprotein BclA
(Bacillus collagen-like protein of anthracis), which comprises a rare
sugar called anthrose [4,6-dideoxy-4-(3-hydroxy-3-methylbuta-
HO
HO
O
O
O
HO
OH
O
HO
O
O
1
O
OH
NH
HO
OMe
HO
Anthrose
Chart 1. Structure of the B. anthracis anthrose-terminated tetrasaccharide antigen 1.
ride analogue 2, equipped with a spacer arm for further conjugation
(Scheme 1). All reported syntheses of tetrasaccharide analogues of 1
make use of L-rhamnose and either D-fucose or D-galactose as pre-
namido)-2-methyl-D
-glucopyranose] (Chart 1).6
Several laboratories have succeeded in preparing tetrasaccharide
1
7–13 and further established that it could be used both as a diagnos-
cursors, with the exception of O’Doherty’s fascinating but perhaps
less efficient de novo approach,11 and have been indifferently car-
ried out by adopting either a [2+2]7,12,13 or a [1+3]8–11,13 strategy.
Similarly, and independently of the actual strategy retained, we
have envisioned that unit A of compound 2 could arise from anthro-
syl donor 317 while units B, C, and D could be obtained from a com-
tic probe and as an antigen in vaccine formulations.7a,14–16
We also embarked some years ago on the preparation of frag-
ments and analogues of 117,18 and wish to report herein our own
investigations toward and the synthesis of an anthrax tetrasaccha-
mon precursor such as rhamnopyranosides 418 or 5. Pursuing step-
economy syntheses, the groups of Kovác, Crich, and Wang
⇑
Corresponding author. Tel.: +33 251125732; fax: +33 0251125632.
E-mail address: cyrille.grandjean@univ-nantes.fr (C. Grandjean).
8–10,12,13
ˇ
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2012.01.007

116
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
NH2
O
O
HO
D
HO
O
O
HO
C
O
HO
[2+2]
O
[1+3]
HO
O
O
O
HO
B
NH
HO
OMe
HO
2
A
[2+2]
[1+3]
NHCBZ
O
NHCBZ
O
O
BnO
BnO
O
O
BnO
BnO
O
O
BnO
SX
HO
O
O
R3O
BnO
SX
O
BnO
+
R3O
O
HO
BnO
O
OBz
BnO
O
O
4
5
X = Tol
X = Ar*
R2O
HO
N3
R1O
R2O
OMe
+
CCl3
NH
O
O
N3
R1O
OAc
1
3
R = OBz
1
6
R = OBn
Scheme 1. Retrosynthesis of anthrax tetrasaccharide analogue 2 (⁄Ar = 5-tert-butyl-2-methylphenyl).
elected to secure the
a
-linkage in the rhamnosylation reactions
of a closely related trisaccharide, bearing a benzyl rather than a
benzoyl protecting group at this position (Table 1, entry 3).19
Methylation of 8 was actually achieved with diazomethane but
in low yield (Table 1, entry 2). To circumvent these limitations,
we first decided to design a more efficient synthesis of derivative
3, to prepare the corresponding benzylated compound 6 and to
study the methylation of anthrose-related disaccharides, for which
no data were available, to further determine whether a [2+2] strat-
egy would be more efficient than a [1+3] one for synthesizing 2.
We recently fulfilled our first aim, by synthesizing donors 3 and
without anchimeric assistance. However, in the absence of neigh-
boring group participation, the stereoselectivity seems to strongly
depend on the choice of the glycosylation conditions and has been
fully controlled only by the Kovác’s group.8,9,12 Anomeric mixtures
ˇ
have not been reported by Seeberger, who uses rhamnose building
blocks ester-protected at the 2-position,7 consolidating our initial
choice. Finally, construction of the b-linkage of the terminal anth-
rose unit would analogously rely on the participation of an ester
group since attempts to glycosylate an anthrose precursor already
methylated at the C-2 position has led to a 7:2 or a 4:1 b/
a
anomeric
6 in only nine steps from D-(+)-fucose in 22% and 13% yields,
mixture depending on the conditions.10,19
respectively: The syntheses relied on a key pyranose intermediate
featuring a 3,4-cyclic sulfate function which served successively as
a protecting and a leaving group.21 The preparation of the B and the
CD unit precursors has been undertaken to achieve our second
objective.
2. Results and discussion
2.1. Preliminary data
We have recently reported the preparation of a trisaccharide
analogue from intermediate 3 according to a A + BC (equivalent
to the present 1+3) strategy.18 However, this synthesis suffered
from several drawbacks. Firstly, key intermediate 3 was obtained
2.2. Synthesis of the B precursors and of a CD acceptor
The key 5-tert-butyl-2-methylphenyl 4-O-benzyl-2-O-benzoyl-
1-thio-a-L-rhamnopyranoside 5 was prepared analogously to
known compound 418 from the non-smelling 5-tert-butyl-2-
from
D-galactose following a rather lengthy route, in 13 steps
and 11% overall yield. Secondly, contrasting with our previous
observations made with a simple anthrose-related monosaccha-
ride 7 (Table 1, entry 1),17 methylation of the A residue of interme-
diate 8, after deacetylation, led to an intractable mixture under
Kuhn’s conditions,20 apparently due to instability of at least one
among the three benzoyl groups and, probably, that at the C-3 po-
sition of the A unit: Indeed, no such troubles were noticed by
Boons and coworkers upon methylation under the same conditions
methylthiophenol to compare their respective reactivity (Scheme
2).
Peracetylated aryl 1-thio-L-rhamnopyranoside 10 was obtained
as an
a
/b (9:1) mixture in two steps according to a known proce-
dure in a 91% yield.22 Zemplèn deprotection of compound 10 affor-
ded triol 11 which was next transformed into acceptor 5, following
a one pot three step sequence developed by Field and co-work-
ers,23 comprising the formation of a 2,3-orthoester, the benzylation

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
117
Table 1
Selective deacetylation and methylation of synthetic anthrax tetrasaccharide fragments
Entry
Compound
Deprotection
Methylation
Ref.
17
NHCBZ
O
O
N3
1
AcCl, MeOH, 0 °C?rt, 24 h, 70%
MeI, Ag2O CH2Cl2 8 days, 91%
BzO
AcO
7
NHCBZ
O
O
BnO
O
OBz
2
6 days, 50%
CH2N2, BF3ꢀEt2O CH2Cl2, 0 °C?rt, 3 h, 35–40%
18
O
O
BnO
O
8
OBz
O
N3
BzO
AcO
NHCBZ
O
O
BnO
OBz
3
NH2NH2ꢀHOAc MeOH/CH2Cl2, rt, 93%
MeI, Ag2O, Me2S CH2Cl2, 12 h, 51%
19
O
O
BnO
O
9
OBz
O
N3
BnO
LevO
NHCBZ
O
O
BnO
O
4
6 days, 24 (48%), 20 (14%)
6 days, 25 (20%), 26 (20%), 27 (26%),
This work
OBz
N3
BzO
AcO
20
STol
O
O
BnO
O
5
6
7
8 days, 28 (75%)
8 days, 29 (73%), 28 (12%)
This work
This work
This work
OBz
N3
BzO
AcO
21
SAr
O
O
BnO
O
6 days, 30 (63%), 22 (19%)
Decomposition
OBz
SAr
N3
BzO
AcO
22
O
O
BnO
O
4 days, 23 (17%), 31 (29%), 5 (24%)
Not attempted
OBz
N3
BnO
AcO
23
of the 4-OH and the regioselective opening of the orthoester to
selectively install a benzoyl group at the C-2 position. The orthoes-
ter proved to be rather resistant to acid hydrolysis, perhaps due to
the steric hindrance of the thioaryl substituent, and compound 5
was finally obtained in a markedly lower yield than its analogue
4 (46% vs 75% yield from triol 11).
Acceptor 14 was prepared from intermediate 5 either by direct
condensation of N-CBZ-ethanolamine in the presence of N-iodo-
succinimide (NIS) and silver(I) trifluoromethanesulfonate (50%
yield) or following chloroacetylation to give 12, glycosylation
with N-CBZ-ethanolamine, and selective chloroacetyl deprotec-
tion (59% overall yield from 5). Acceptor 14 was designed to
further prepare a disaccharide analogous to mono- and tri-saccharide
7 and 8 (see Table 1, entries 1 and 2) for the methylation study
(Scheme 2).
sodium methoxide to give diol 15 which was further selectively
monobenzylated at the 3-OH according to Matta’s protocol
(Scheme 3).24
Alcohol 16 was benzoylated, glycosylated with N-CBZ-amino-
ethanol in the presence of N-iodosucinimide and silver(I) trifluoro-
methanesulfonate and deprotected to provide acceptor 17
(precursor of the D unit) in 71% overall yield.
Then NIS/silver(I) trifluoromethanesulfonate-promoted glyco-
sylation of intermediate 12, now considered as a donor, with
acceptor 17 gave disaccharide 18 in 79% yield. The latter was selec-
tively deprotected upon thiourea treatment to provide alcohol 19
in 62% yield.
2.3. Synthesis of the AB disaccharides and deacetylation/
methylation study
Attempts to benzylate 5 to prepare a D acceptor, using benzyl
trichloroacetimidate in the presence of catalytic trifluoromethane-
sulfonic acid, failed. Therefore, compound 5 was treated with
Having prepared some donors and acceptors, we next envisaged
the synthesis of a set of disaccharides to test the [2+2] approach.

118
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
SAr
SAr
a, b
c
O
O
d, e, f
L-Rhamnose
AcO
HO
AcO
HO
11
OAc
OH
10
NHCBZ
SAr
O
g
O
O
OBn
OBn
HO
HO
OBz
OBz
14
5
i
h
NHCBZ
SAr
O
O
g
O
OBn
OBn
ClAcO
ClAcO
OBz
OBz
12
13
Scheme 2. Reagents and conditions: (a) Ac2O, pyridine, 24 h; (b) 2-methyl-5-tert-butylthiophenol, CH2Cl2, BF3ꢀEt2O, ꢁ40?0 °C, 12 h, 91% (2 steps); (c) MeONa 0.2 M, MeOH,
1 h; (d) triethylorthobenzoate, camphorsulfonic acid, DMF, 3 h; (e) NaH, BnBr, DMF, 2 h, 0 °C?rt; (f) aqueous HCl 2 M 46% ( ) (4 steps); (g) N-CBZ-ethanolamine, NIS, AgOTf,
MS 4 Å, CH2Cl2, 30 min, 50% (5?14), 80% (12?14); (h) (ClAc)2O, DMAP, Et3N, CH2Cl2, 0 °C?rt, 2 h, 95%; (i) thiourea, NaHCO3, Bu4NI, CH2Cl2, 55 °C, 24 h, 78%.
a
NHCBZ
SAr
O
SAr
a
c, d, e
b
O
O
O
5
BnO
BnO
BnO
HO
BnO
BnO
OH
OH
OH
15
16
17
NHCBZ
NHCBZ
O
O
O
O
12
+
17
BnO
BnO
f
g
BnO
BnO
O
O
O
O
BnO
BnO
ClAcO
HO
OBz
OBz
19
18
Scheme 3. Reagents and conditions: (a) MeONa 0.2 M, MeOH, 1 h, quantitative; (b) (i) Bu2SnO, MeOH, 6 h, reflux; (ii) BnBr, DMF, 90 °C, 71%; (c) BzCl, pyridine, 0 °C?rt, 12 h,
quantitative; (d) N-CBZ-ethanolamine, NIS, AgOTf, MS 4 Å, CH2Cl2, 30 min, quantitative; (e) MeONa 0.2 M, MeOH, 12 h, quantitative; (f) NIS, AgOTf, MS 4 Å, CH2Cl2, 30 min,
79%; (g) thiourea, NaHCO3, Bu4NI, THF, 55 °C, 12 h, 62%.
Condensation of donor 3 with acceptors 14 and 5 was performed in
the presence of TMSOTf to afford disaccharides 20 and 22 in 48%
and 68% yields, respectively (Scheme 4 and Table 1).
The same conditions were applied to the glycosylation of donor
6 with 5 to give disaccharide 23 in 85% yield.
In particular, the chemical shifts of H-2B and C-2B remain unaf-
fected while H-3A resonances of products 27, 25, and 26 appear
about 1.7 ppm upfield compared to that of parent compound 24,
suggesting the presence and the absence of an ester group at C-
2B and C-3A, respectively. In parallel, the strong deshielding of
the C-2A (respectively C-3A) signal of compound 25 (respectively
compound 26) compared with those of compounds 24 and 27 indi-
cates a possible methylation at this position.
The same reaction sequence was then investigated with disac-
charides 21,26 22 and, notably, 23 for which the labile benzoyl at
the C-3A position was replaced by a benzyl protecting group,
thereby being more likely to survive the methylation reaction in
light of the above mentioned observations and the previously re-
ported data (compare entries 1–3 in Table 1). While methanolysis
of compounds 21 and 22 gave rise to alcohols 28 and 30 in good
yields (75% and 63%, respectively), that of compound 23 proved
impracticable and was accompanied with competitive hydrolysis
as witnessed by the substantial isolation of acceptor 5 (24%). This
observation suggests that the presence of an electron-withdrawing
group like a benzoyl on residue A is crucial to stabilize the
Selective deacetylation of disaccharide 20 was performed by
acid-catalyzed methanolysis25 to give alcohol 24 in a modest 48%
yield together with recovered compound 20 (14%) after 6 days.
Methylation of intermediate 24 according to Kuhn’s procedure
gave rise to a 1:1 mixture of the two monomethylated alcohols
25 and 26 (40%) together with diol 27 (26%), underlining the insta-
bility of the benzoyl group vicinal to the hydroxy group that we
suspected from our earlier results (see Section 2.1).18 The loss of
a benzoyl group has been confirmed by mass spectrometry (m/z
signals at 743 and 729 corresponding to their sodium adducts)
and by NMR spectrometry (in particular, disappearance of five aro-
matic proton signals and of one peak in the carbonyl region). Sites
of debenzoylation and methylation of each product have been de-
duced from the chemical shifts of the H-2, H-3, C-2, and C-3 atoms
(Table 2).

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
119
NHCBZ
O
NHCBZ
O
O
a
b
BnO
c
O
BnO
3
14
+
20*
O
O
OBz
O
O
N3
BzO
OBz
N3
OH
24
HO
OMe
25
+
NHCBZ
NHCBZ
O
O
O
O
BnO
BnO
O
O
O
O
OBz
N3
OBz
STol
N3
MeO
+
HO
OH
27
OH 26
STol
O
O
O
BnO
BnO
c
b
O
O
21$
22
O
OBz
OBz
N3
BzO
N3
BzO
OH
OMe
28
29
SAr
c
O
a
a
b
BnO
3
6
5
+
Decomposition
O
O
OBz
N3
BzO
OH 30
SAr
O
BnO
b
O
O
5
+
+
5
23
OBz
N3
BnO
OH
31
Scheme 4. Reagents and conditions: (a) TMSOTf (0.4 or 0.5 equiv), ꢁ40 °C, CH2Cl2, 30 min; (b) AcCl, MeOH, 0 °C?rt; (c) MeI, Ag2O, CH2Cl2. ⁄See structures, reaction times and
yields in Table 1; $known compound, see Ref. 26.
Table 2
Selected 1H and 13C NMR chemical shifts (ppm) and coupling constants (Hz) of derivatives 24–27
Compound
d H-2A JHH
d H-3A JHH
d H-2B JHH
d C-2A
d C-3A
d C-2B
24
27
25
26
3.63–3.47 m
3.30 dd, 7.8, 9.2
2.95–2.83 m
3.41–3.29 m
5.19 t, 9.5
3.48 t, 9.2
3.41–3.29 m
3.02 t, 9.5
5.42 dd, 1.7, 3.5
5.37 dd, 1.8, 3.5
5.37–5.34 m
73.3
74.6
83.7
75.3
76.4
75.5
74.8
84.7
72.4
72.5
73.1, 72.4
5.37–5.34 m
inter-glycoside bond.27 In view of this latter result, the methylation
was only attempted on intermediates 28 and 30. While compound
28 afforded donor 29 (73%), alcohol 30 decomposed upon methyl
iodide/silver(I) oxide treatment. Immediate purple coloration of
the reaction mixture was observed upon addition of the reagents,
suggesting an activation of sulfur which could reflect a higher reac-
tivity of the 5-tert-butyl-2-methyl-1-thiophenyl group compared
to toluol despite the higher steric hindrance. Final the preparation
of anthrax tetrasaccharide could, in principle, have been achieved
from thio-disaccharide 29. However, this donor did not react with
acceptors like disaccharide 19 in the presence of thiophilic promot-
ers either at 0 °C or rt. In the latter condition, TLC and mass spec-
trometry monitoring of the reaction only revealed the presence of
the intact acceptor and that of hydrolyzed donor 29. We hypothe-
sized that the deactivating benzoyl protecting groups as well as the
poor toluol leaving-group properties of compound 29 were partly
responsible for the absence of reactivity. Exchange of the toluol
to the more reactive trichloroacemididate group was, however,
not attempted so as to not lengthen the synthesis. From this preli-
minary study, we concluded that the synthesis of 2 could not be
efficiently achieved according to a [2+2] strategy and that we
should privilege the use of ether protecting groups to favor both
the final functionalization of the anthrose residue and the glycosyl-
ˇ12
ation steps. Similar reasoning was adopted by Kovác and, more
recently, by Wang.13 The preparation of the tetrasaccharide was
thus revisited from donor 6 following a [1+3] strategy.
2.4. Synthesis of the ABCD tetrasaccharide adopting a [1+3]
strategy
Having donor 6 and acceptor 19, respectively precursors of the
A and CD units, in hands, we focused on the preparation of a novel
donor, precursor of the B unit. To this aim, methyl
a-
L-rhamnopyr-
anoside 32, obtained as a
a
/b (9:1) mixture (70%) according to
Gin’s procedure,28 was further selectively allylated at the 3-OH to
give diol 3329 (Scheme 5). Acetolysis of 33 afforded intermediate
34 which was further activated as the trichloroacetimidate donor
35 (38% for the three steps). Schmidt’s glycosylation30 of donor

120
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
OMe
OMe
a
O
O
b, c
HO
HO
L-Rhamnose
HO
O
OH
OH
33
32
NH
OAc
O
CCl3
d
O
e, f
O
AcO
AcO
O
O
OAc
OAc
34
35
NHCBZ
NHCBZ
BnO
O
NCBZ
Bn
O
O
O
O
O
BnO
BnO
BnO
BnO
BnO
O
O
O
19
g
h
i
O
O
O
BnO
+
BnO
BnO
O
35
O
O
OBz
OH
OBn
O
O
O
AcO
HO
BnO
O
O
RO
38
39
OAc
OH
OBn
R = All
36
37
j, k
R = H
Scheme 5. Reagents and conditions: (a) AcCl, MeOH, ꢁ20 °C?rt, 18 h, 70%; (b) Bu2SnO, MeOH, reflux, 6 h; (c) AllBr, DMF, 48 h, 70 °C, 40% (2 steps); (d) Ac2O/AcOH/H2SO4,
1.5 h, 67%; (e) BnNH2, THF, 12 h, 71%; (f) Cl3CCN, DBU, CH2Cl2, 0 °C?rt, 2 h, 80%; (g) TMSOTf, MS 4 Å, CH2Cl2, 0 °C, 30 min, 99%; (h) MeONa, MeOH, 1 h, rt, 70%; (i) BnBr, NaH,
DMF, 0 °C?rt, 2 h, 87%; (j) (Ph3P)3RhCl, DABCO, EtOH/toluene/H2O, reflux, 16 h; (k) HgCl2, HgO, acetone/H2O, 4 h, rt, 88% (2 steps).
NCBZ
Bn
O
NCBZ
Bn
O
O
O
BnO
BnO
BnO
BnO
O
O
6
a
d, e
f
O
+
O
BnO
2
BnO
39
O
O
OBn
OBn
O
O
BnO
O
OBn
O
O
O
O
OBn
N3
BnO
OBn
HO
NH
BnO
OR
OMe
40
R = Ac
43
b
41 R = H
42
c
R = Me
Scheme 6. Reagents and conditions: (a) TMSOTf, MS 4 Å CH2Cl2, ꢁ40 °C, 30 min, 56%; (b) MeONa, MeOH, 12 h, 85%; (c) MeI, NaH, DMF, 0 °C?rt, 12 h, 96%; (d) NaBH4,
NiCl2ꢀ6H2O, EtOH/CH2Cl2, 1 h; (e) b-hydroxyisovaleric acid, HATU, DIPEA, DMF, 18 h, 40% (2 steps); (f) H2, 10 bars, Pd/C, MeOH/AcOH, 50 °C, 30 min, 88%.
35 with acceptor 19 afforded trisaccharide 36 in 99% yield. The ex-
change of ester to ether protecting groups was then performed
upon treatment of 36 with sodium methoxide leading to interme-
diate 37 followed by perbenzylation to provide compound 38 (48%
for the two steps). Allyl protecting group was finally removed by
double bond isomerisation followed by hydrolysis31 to afford the
trirhamnosyl acceptor 39.
acteristic anthrose amide side-chain (40% for the two steps). Final
catalytic hydrogenolysis of the benzyl and carbamate groups in 43
gave the anthrax tetrasaccharide derivative 2 (88%).
3. Conclusion
In conclusion, we have reported the synthesis of the B. anthracis
Glycosylation of 6 with 39 in the presence of TMSOTf afforded
the tetrasaccharide 40 in 56% yield (Scheme 6). 2-O-Deacetylation
gave alcohol 41 which was further methylated to give 42. Reduc-
tion of azide in 42 was carried out with sodium borohydride in
the presence of nickel chloride32 to provide an intermediate amine
which was not isolated but further acylated to introduce the char-
tetrasaccharide antigen ready for conjugation in 37 steps from
D-
fucose and -rhamnose. The synthesis of the trirhamnan relies on
L
the use of a common thio-rhamnopyranoside precursor while that
of the anthrosyl was achieved from donor 6, efficiently prepared
from a cyclic sulfate fucopyranose intermediate.21 The presence
of an acetate protecting group at the 2-position of donor 6, which

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
121
is mandatory to its preparation, has precluded a [2+2] approach
making the [1+3] approach the strategy of choice.
130.0, 129.8, 126.8, 125.1, 122.8 (SC6H3C(CH3)3CH3), 85.1 (C-1),
71.5 (C-2), 70.9 (C-4), 69.3 (C-3), 67.7 (C-5), 34.2 (SC6H3C(CH3)3
CH3), 31.1 (SC6H3C(CH3)3CH3), 20.5, 20.3, 20.2, 20.0 (3 ꢂ OCOCH3,
SC6H3C(CH3)3CH3), 17.2 (C-6); HR-ESI-MS m/z calcd for C23H32O7S
475.1766 [M+Na]+, found 475.1762.
4. Experimental methods
4.1. General methods
4.2.2. 2-Methyl-5-tert-butylphenyl 1-thio-L-rhamnopyranoside
(11)
All reactions were monitored by TLC on Kieselgel 60 F254 (E.
Merck). Detection was achieved by charring with vanillin. Silica
gel (E. Merck, 240–400 mesh) was used for chromatography.
Optical rotations were measured with a JASCO DIP-370 digital
polarimeter, using a sodium lamp (k = 589 nm) at 20 °C. All NMR
experiments were performed at 300.13 MHz using Bruker
Compound 10 (24.7 g, 54.6 mmol) was treated with NaOMe
(0.2 M solution in MeOH) (546 mL, 109.2 mmol) for 1 h at rt. The
reaction mixture was then neutralized by resin Amberlite 120 H+.
The resin was filtered off and the filtrate was concentrated in vacuo
to give 11 which was used in the next step without any further
purification. Rf 0.20 (cyclohexane/EtOAc 8:2); HR-ESI-MS m/z calcd
for C17H26O4S 349.1450 [M+Na]+, found 349.1460.
DMX300 spectrometer equipped with
a Z-gradient unit for
pulsed-field gradient spectroscopy. Assignments were performed
by stepwise identification using COSY, and HSQC experiments
using standard pulse programs from the Bruker library. Chemical
shifts are given relative to external TMS with calibration involving
the residual solvent signals.
Low-resolution ESI mass spectra were obtained on a hybrid
quadrupole/time-of-flight (Q-TOF) instrument, equipped with a
pneumatically assisted electrospray (Z-spray) ion source (Micro-
mass). High-resolution mass spectra were recorded in positive
mode on a ZabSpec TOF (Micromass, UK) tandem hydrid mass
spectrometer with EBETOF geometry. The compounds were indi-
4.2.3. 2-Methyl-5-tert-butylphenyl 2-O-benzoyl-4-O-benzyl-1-
thio-a-L-rhamnopyranoside (5)
To a suspension of triol 11 (3.0 g, 9.2 mmol) in dry MeCN
(100 mL), camphor-10-sulfonic acid (880 mg, 3.7 mmol) and tri-
ethylorthobenzoate (8.2 mL, 38.6 mmol), were added. The mixture
was stirred at rt for 12 h and Et3N (1.5 mL) was added to neutralize
the solution. The mixture was then evaporated to dryness and the
residue was diluted with DMF (100 mL). NaH (60% dispersion in
mineral oil, 542 mg, 18.4 mmol) was added at 0 °C followed by
dropwise addition of BnBr (1.8 mL, 16.6 mmol). The reaction mix-
ture was stirred at rt for 2 h and the excess of sodium hydride
was neutralized by dropwise addition of MeOH (20 mL) at 0 °C.
The mixture was then evaporated to dryness and the residue was
dissolved with EtOAc. The organic phase was washed with water
and then with 2 M aqueous HCl (ꢂ3). The opening of the orthoester
was checked by TLC. The organic phase was dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was purified by flash
chromatography using cyclohexane/EtOAc (98:2?9:1) as eluent to
give 5 (2.2 g, 46%) and its b-anomer 2-methyl-5-tert-butylphenyl
vidually dissolved in MeOH at a concentration of 10
then infused into the electrospray ion source at a flow rate of
10
L minꢁ1 at 60 °C. The mass spectrometer was operated at
l
g mLꢁ1 and
l
4 kV while scanning the magnet at a typical range of 4000–
100 Da. The mass spectra were collected as continuum profile data.
Accurate mass measurement was achieved using polyethylene gly-
col as internal reference with a resolving power set to a minimum
of 10,000 (10% valley).
4.2. Synthesis of anthrax tetrasaccharide
2-O-benzoyl-4-O-benzyl-1-thio-b-L-rhamnopyranoside (340 mg,
7%) as colorless oils. (
a anomer) Rf 0.42 (cyclohexane/EtOAc 8:2);
ꢁ93 (CHCl3, c 0.7); 1H NMR (300 MHz, CDCl3) d 8.24–7.27
4.2.1. 2-Methyl-5-tert-butylphenyl 2,3,4-tri-O-acetyl-1-thio-L-
rhamnopyranoside (10)
[a]
D
(m, 13H, SC6H3C(CH3)3CH3, OCH2C6H5, OCOC6H5), 5.82 (dd,
1H, J1,2 = 1.3 Hz, J2,3 = 3.3 Hz, H-2), 5.66 (br s, 1H, H-1), 5.11 (d,
1H, JA,B = 11.2 Hz, A part of an AB system, OCHHC6H5), 4.93
(d, 1H, JA,B = 11.2 Hz, B part of an AB system, OCHHC6H5), 4.55–
4.43 (m, 2H, H-3, H-5), 3.78 (t, 1H, J3,4 = J4,5 = 9.2 Hz, H-4), 2.59 (s,
3H, SC6H3C(CH3)3CH3), 1.59 (d, 3H, J5,6 = 5.9 Hz, H-6), 1.46 (s, 9H,
SC6H3C(CH3)3CH3); 13C NMR (75 MHz, CDCl3) d 166.4 (OCOC6H5),
149.8, 138.4, 137.0, 133.5, 133.0, 132.9, 130.2, 130.1, 130.0,
129.7, 128.7, 128.6, 128.5, 128.3, 128.1, 127.6, 125.2, 124.0,
(SC6H3C(CH3)3CH3, OCOC6H5, OCH2C6H5) 85.8 (C-1), 81.8 (C-2),
75.6 (C-4), 75.3 (OCH2C6H5), 71.3 (C-3), 69.0 (C-5), 34.6 (SC6H3C
(CH3)3CH3) 31.5 (SC6H3C(CH3)3CH3), 20.5 (SC6H3C(CH3)3CH3), 18.3
(C-6); HR-ESI-MS m/z calcd for C31H36O5S 543.2175 [M+Na]+, found
543.2160.
To a solution of L-rhamnose (10.0 g, 61 mmol) in pyridine
(70 mL) was added acetic anhydride (70 mL, 635 mmol). The reac-
tion mixture was stirred at rt for 24 h and was then quenched by
MeOH (70 mL). The reaction mixture was concentrated in vacuo.
The residue was dissolved in CH2Cl2 and was washed with satu-
rated aqueous KHSO4, saturated aqueous NaHCO3 and water. The
organic layer was then dried over Na2SO4, filtered, and concen-
trated in vacuo to give 1 as a 8:2 mixture of
material was used in the next step without purification. To a
solution of 1,2,3,4-tetra-O-acetyl-
-rhamnopyranose33 (19.9 g,
a/b anomers. This
L
60.0 mmol) in CH2Cl2 (300 mL) was added 2-methyl-5-tert-butyl-
thiophenol (18.4 mL, 100.0 mmol). The mixture was cooled to
ꢁ40 °C and BF3ꢀEt2O (12.7 mL, 100.0 mmol) was added dropwise.
The reaction mixture was stirred at 0 °C for 12 h and was then
washed with saturated aqueous NaHCO3 and water. The organic
layer was dried over Na2SO4 and filtered. The solvent was evapo-
rated under reduced pressure and the residue was purified by flash
chromatography on silica gel using cyclohexane/EtOAc (95:5?8:2)
4.2.4. 2-Methyl-5-tert-butylphenyl 2-O-benzoyl-4-O-benzyl-3-
O-chloroacetyl-1-thio- a-L-rhamnopyranoside (12)
To a solution of alcohol 5 (1 g, 1.9 mmol) in CH2Cl2 (20 mL) at
0 °C was added chloroacetic anhydride (975 mg, 5.7 mmol), DMAP
as eluent to give 10 as a 9:1 mixture of
oil (24.7 g, 91%). Rf 0.45 (cyclohexane/EtOAc 8:2); 1H NMR
(300 MHz, CDCl3) d ( anomer) 7.61–7.06 (m, 3H, SC6H3C(CH3)3
CH3), 5.59–5.57 (m, 1H, H-2), 5.44–5.40 (m, 2H, H-1, H-3), 5.22
(t, 1H, J4,5 = J5,6 = 9.6 Hz, H-4), 4.46 (dq, 1H, J5,6 = 6.1 Hz, J4,5
a/b anomers as a colorless
(71 mg, 0.6 mmol), and Et3N (800 lL, 8.5 mmol). The reaction mix-
ture was warmed to rt and stirred for 2 h. The mixture was then
diluted with CH2Cl2 and washed with saturated aqueous KHSO4,
saturated aqueous NaHCO3, and water. The organic phase was
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography using cyclohexane/EtOAc
(95:5) as eluent to provide 12 (1.1 g, 95%) as a white solid. Rf
a
=
9.6 Hz, H-5), 2.44 (s, 3H, C6H3C(CH3)3CH3), 2.14 (s, 3H, OCOCH3),
2.09 (s, 3H, OCOCH3), 2.02 (s, 3H, OCH3CO), 1.31 (d, 3H,
J5,6 = 6.1 Hz, H-6), 1.29 (s, 9H, C6H3C(CH3)3CH3); 13C NMR (75
0.65 (cyclohexane/EtOAc 8:2); ½a D20
ꢃ
ꢁ33 (CHCl3, c 0.7); 1H NMR
MHz, CDCl3) d (
a
anomer) 169.5 (3 ꢂ OCOCH3), 149.5, 136.6,
(300 MHz, CDCl3) d 8.18–8.14 (m, 3H, SC6H3C(CH3)3CH3), 7.56–

122
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
7.27 (m, 10H, OCH2C6H5, OCOC6H5), 5.91 (dd, 1H, J1,2 = 1.6 Hz,
J2,3 = 3.3 Hz, H-2), 5.63 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.6 Hz, H-3)
5.54 (d, 1H, J1,2 = 1.6 Hz, H-1), 4.86 (d, 1H, JA,B = 11.4 Hz, A part of
an AB system, OCHHC6H5), 4.81 (d, 1H, JA,B = 11.4 Hz, B part of an
AB system, OCHHC6H5), 4.61–4.52 (m, 1H, H-5), 4.02 (d,
1H, JA,B = 14.9 Hz, A part of an AB system, OCOCHHCl), 3.94
(d, 1H, JA,B = 14.9 Hz, B part of an AB system, OCOCHHCl), 3.86 (t,
1H, J3,4 = J4,5 = 9.6 Hz, H-4), 2.48 (s, 3H, SC6H3C(CH3)3CH3), 1.53
(d, 3H, J5,6 = 6.4 Hz, H-6), 1.40 (s, 9H, SC6H3CH3C(CH3)3CH3); 13C
NMR (75 MHz, CDCl3) d 166.5 and 165.7 (OCOC6H5 and OCOCH2Cl),
149.8, 137.9, 137.3, 133.6, 132.0, 130.2, 130.0, 129.6, 128.7, 128.6,
128.5, 128.1, 128.0, 125.5 (SC6H3C(CH3)3CH3, OCOC6H5, OCH2C6H5)
85.4 (C-1), 78.8 (C-4), 75.4 (OCH2C6H5), 74.4 (C-3), 72.2 (C-2), 69.3
(C-5), 40.7 (OCOCH2Cl), 34.5 (SC6H3C(CH3)3CH3) 31.4 (SC6H3C
(CH3)3CH3), 20.4 (SC6H3C(CH3)3CH3), 18.1 (C-6); HR-ESI-MS m/z
calcd for C33H37ClO6S 619.1897 [M+Na]+, found 619.1902.
filtered, and concentrated in vacuo. The residue was then purified
by flash chromatography using cyclohexane/EtOAc (8:2) as eluent
to give known alcohol 1418 (508 mg, 50%).
4.2.7. 2-Methyl-5-tert-butylphenyl 4-O-benzyl-1-thio-a-L-
rhamnopyranoside (15)
Compound 5 (2.2 g, 4.2 mmol) was treated with NaOMe (0.2 M
solution in MeOH) (42 mL, 8.4 mmol) for 1 h at rt. The reaction mix-
ture was then neutralized by resin Amberlite 120H+. The resin was
filtered off and the filtrate was concentrated in vacuo. The crude
was then purified by flash chromatography using cyclohexane/
EtOAc (7:3) as eluent to give 15 (1.7 g, quantitative). Rf 0.17 (cyclo-
hexane/EtOAc 8:2); [
a]
D
ꢁ63 (CHCl3, c 0.6); 1H NMR (300 MHz,
CDCl3) d 7.65–7.16 (m, 8H, SC6H3C(CH3)3CH3, OCH2C6H5); 5.47 (d,
1H, J1,2 = 1.5 Hz, H-1), 4.84 (d, 1H, JA,B = 11.4 Hz, A part of an AB sys-
tem, OCHHC6H5), 4.80 (d, 1H, JA,B = 11.4 Hz, B part of an AB system,
OCHHC6H5), 4.32 (dq, 1H, J4,5 = 9.4 Hz, J5,6 = 6.2 Hz, H-5), 4.26 (br s,
1H, H-2), 4.06 (m, 1H, H-3), 3.50 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4), 3.09
(br s, 1H, OH-2), 2.85 (br s, 1H, OH-3) 2.43 (s, 3H, SC6H3C(CH3)3CH3),
1.42 (d, 3H, J5,6 = 6.2 Hz, H-6), 1.36 (s, 9H, SC6H3C(CH3)3CH3); 13C
NMR (75 MHz, CDCl3) d 149.8, 138.2, 136.6, 132.9, 130.0, 129.4,
128.7, 128.1, 128.0, 124.7 (SC6H3C(CH3)3CH3, OCH2C6H5) 87.1 (C-
1), 81.2 (C-4), 75.1 (OCH2C6H5), 73.0 (C-2), 72.1 (C-3), 68.7 (C-5),
34.5 (SC6H3C(CH3)3CH3) 31.4 (SC6H3C(CH3)3CH3), 20.2 (SC6H3C
(CH3)3CH3), 18.0 (C-6); ESI-MS m/z calcd for C24H32O4S 439.1919
[M+Na]+, found 439.1926.
4.2.5. 2-[(N-Benzyloxycarbonyl)amino]ethyl 2-O-benzoyl-4-O-
benzyl-3-O-chloroacetyl-a-L-rhamnopyranoside (13)
To a mixture of 12 (1.1 g, 1.8 mmol), 2-(N-benzyloxycarbonyl)-
aminoethanol (448 mg, 2.3 mmol) and 4 Å molecular sieves (1.0 g)
in dry CH2Cl2 (20 mL) was added NIS (562 mg, 2.5 mmol) followed
by AgOTf (180 mg, 0.7 mmol). The reaction mixture was stirred at
rt under inert atmosphere for 30 min and was filtered. The filtrate
was washed by a mixture of saturated aqueous NaHCO3 and satu-
rated aqueous Na2S2O3. The organic phase was dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was then purified
by flash chromatography using cyclohexane/EtOAc (9:1) as eluent
to give 13 (880 mg, 80%) as a white solid. Rf 0.27 (cyclohexane/
EtOAc 8:2); 1H NMR (300 MHz, CDCl3) d 8.12–7.30 (m, 15H,
OCH2C6H5, OCOC6H5, NHCOOCH2C6H5), 5.58 (dd, 1H, J1,2 = 1.7 Hz,
J2,3 = 3.3 Hz, H-2), 5.48 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.7 Hz, H-3),
5.40 (br s, 1H, NH), 5.19 (s, 2H, NHCOOCH2C6H5), 4.90 (d, 1H,
J1,2 = 1.7 Hz, H-1), 4.77 (d, 1H, JA,B = 11.4 Hz, A part of an AB system,
OCHHC6H5), 4.73 (d, 1H, JA,B = 11.4 Hz, B part of an AB system,
OCHHC6H5), 4.00–3.87 (m, 1H, H-5), 3.87–3.77 (m, 1H, A part of
an AB system, OCHHCH2NH), 3.93 (d, 1H, JA,B = 14.9 Hz, A part of
an AB system, OCOCHHCl), 3.86 (d, 1H, JA,B = 14.9 Hz, B part of an
AB system, OCOCHHCl), 3.70 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4), 3.65–
3.52 (m, 1H, B part of an AB system, OCHHCH2NH), 3.52–3.35 (m,
2H, OCH2CH2NH), 1.44 (d, 3H, J5,6 = 6.4 Hz, H-6); 13C NMR
(75 MHz, CDCl3) d 166.5, 165.7 (OCOC6H5, OCOCH2Cl), 156.5
(NHCOOCH2C6H5), 137.8, 136.3, 133.6, 129.9, 129.4, 128.6, 128.5,
128.1, 128.0, 127.9 (COC6H5, OCH2C6H5, NHCOOCH2C6H5), 97.7
(C-1), 78.5 (C-4), 75.3 (OCH2C6H5), 74.0 (C-3), 70.3 (C-2), 68.0 (C-
5), 67.2 (OCH2CH2NH), 66.8 (NHCOOCH2C6H5), 40.7 (OCH2CH2NH
and OCOCH2Cl), 18.1 (C-6); ESI-MS m/z 633.8 [M+Na]+.
4.2.8. 2-Methyl-5-tert-butylphenyl 3,4-di-O-benzyl-1-thio-a-L-
rhamnopyranoside (16)
A mixture of diol 15 (1.0 g, 2.4 mmol) and dibutyltin oxide
(657 mg, 2.6 mmol) in dry MeOH (20 mL) was heated to reflux un-
der inert atmosphere for 6 h and was then evaporated to dryness.
The residue was dried under vacuum for 2 h and was dissolved in
dry DMF (20 mL). BnBr (344 lL, 2.9 mmol) was added dropwise
and the reaction mixture was stirred at 90 °C for 48 h. The mixture
was then concentrated in vacuo and the residue was purified by
flash chromatography using cyclohexane/EtOAc (98:2) to afford
16 (863 mg, 71%) and 2-methyl-5-tert-butylphenyl 2,4-di-O-ben-
zyl-1-thio-
16: Rf 0.64 (cyclohexane/EtOAc 8:2); [
NMR (300 MHz, CDCl3) d 7.73–7.22 (m, 13H, SC6H3C(CH3)3CH3,
a-L-rhamnopyranoside (316 mg, 26%) as colorless oils.
a]
ꢁ149 (CHCl3, c 1.0); 1H
D
2 ꢂ OCH2C6H5), 5.61 (d, 1H, J1,2 = 1.3 Hz, H-1), 5.03 (d, 1H, JA,B
=
11.0 Hz, A part of an AB system, OCHHC6H5), 4.84 (br s, 2H,
OCH2C6H5), 4.78 (d, 1H, JA,B = 11.0 Hz, B part of an AB system,
OCHHC6H5), 4.42–4.35 (m, 2H, H-2, H-5), 4.04 (dd, 1H, J2,3
=
3.1 Hz, J3,4 = 9.4 Hz, H-3), 3.50 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4), 3.03
(br s, 1H, OH-2), 2.50 (s, 3H, SC6H3C(CH3)3CH3), 1.45 (d, 3H,
J5,6 = 6.4 Hz, H-6), 1.42 (s, 9H, SC6H3CH3C(CH3)3CH3); 13C NMR
(75 MHz, CDCl3) d 149.8, 138.4, 137.8, 136.3, 133.0, 130.1, 129.4,
128.8, 128.6, 128.2, 128.1, 127.9, 127.5, 127.3, 127.3, 124.7, 124.0
(SC6H3C(CH3)3CH3, 2 ꢂ OCH2C6H5), 86.6 (C-1), 80.4 (C-3), 80.3 (C-
4), 75.6 (OCH2C6H5), 72.3 (OCH2C6H5), 70.5 (C-2), 68.9 (C-5), 34.6
(SC6H3C(CH3)3CH3), 31.4 (SC6H3C(CH3)3CH3), 20.3 (SC6H3C(CH3)3
CH3), 18.0 (C-6); HR-ESI-MS m/z calcd for C31H38O4S 529.2389
[M+Na]+, found 529.2381.
4.2.6. 2-[(N-Benzyloxycarbonyl)amino]ethyl 2-O-benzoyl-4-O-
benzyl-a-L-rhamnopyranoside (14)
From 13: To a solution of 67 (880 mg, 1.4 mmol) in THF (70 mL)
were added thiourea (212 mg, 2.8 mmol), NaHCO3 (235 mg,
2.8 mmol) and a catalytic amount of Bu4NI (25 mg, 67 lmol). The
reaction mixture was stirred at 55 °C for 24 h and was then cooled
to rt. The mixture was diluted with CH2Cl2 and washed with water.
The organic phase was dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by flash chromatography
using cyclohexane/EtOAc (8:2) as eluent to give alcohol 14 (477
mg, 78%) as a white solid.
From 5: To a mixture of 5 (1.0 g, 1.9 mmol), 2-(N-benzyloxycar-
bonyl)-aminoethanol (370 mg, 1.9 mmol) and 4 Å molecular sieves
(1.0 g) in dry CH2Cl2 (20 mL) was added NIS (600 mg, 2.5 mmol)
followed by AgOTf (196 mg, 0.6 mmol). The reaction mixture was
stirred at rt for 30 min and was then filtered. The filtrate was
washed by a mixture of saturated aqueous NaHCO3 and saturated
aqueous Na2S2O3. The organic phase was dried over Na2SO4,
4.2.9. 2-[(N-Benzyloxycarbonyl)amino]ethyl 3,4-di-O-benzyl-
-rhamnopyranoside (17)
To a solution of 16 (863 mg, 1.7 mmol) in pyridine (20 mL) at
0 °C was added BzCl (391 L, 3.4 mmol) dropwise. The reaction
a-
L
l
mixture was stirred at rt for 12 h and was then quenched by the
addition of MeOH. The mixture was diluted with CH2Cl2 was
washed aq HCl 1 M, with water and brine. The organic phase was
dried over Na2SO4, filtered, and concentrated. The crude was puri-
fied by flash chromatography using cyclohexane/EtOAc (95:5) as
eluent to give 2-methyl-5-tert-butylphenyl 2-O-benzoyl-3,4-di-O-

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
123
benzyl-1-thio-
white solid. Rf 0.68 (cyclohexane/EtOAc 8:2); [
0.6); 1H NMR (300 MHz, CDCl3) d 8.25–7.28 (m, 18H, SC6H3C(CH3)3
a
-
L
-rhamnopyranoside (1.0 g, quantitative) as
a
J3,4 = 9.1 Hz, H-3), 3.79–3.70 (m, 2H, H-5, A part of an AB system,
OCHHCH2NH), 3.59–3.32 (m, 3H, part of an AB system,
a
]
D
ꢁ59 (CHCl3, c
B
OCHHCH2NH, OCH2CH2NH), 3.51 (t, 1H, J3,4 = J4,5 = 9.1 Hz, H-4),
2.87 (s, 1H, OH-2), 1.35 (d, 3H, J5,6 = 6.4 Hz, H-6); 13C NMR
(75 MHz, CDCl3) d 156.4 (NHCOOCH2C6H5), 138.3, 137.9, 136.6,
128.6, 128.5, 128.2, 128.1, 127.9 (2 ꢂ OCH2C6H5, NHCOOCH2C6H5),
99.4 (C-1), 80.0 (C-4), 79.8 (C-3), 75.5 and 72.1 (2 ꢂ OCH2C6H5),
68.5 (C-2), 67.7 (C-5), 66.9 and 66.8 (NHCOOCH2C6H5 and
OCH2CH2NH), 40.9 (OCH2CH2NH), 18.0 (C-6); HR-ESI-MS m/z calcd
for C30H35NO7 544.2306 [M+Na]+, found 544.2292.
CH3, 2 ꢂ OCH2C6H5, OCOC6H5), 6.00 (dd, 1H, J1,2 = 1.6 Hz, J2,3
=
=
3.3 Hz, H-2), 5.58 (d, 1H, J1,2 = 1.6 Hz, H-1), 5.04 (d, 1H, JA,B
11.0 Hz,
A part of an AB system, OCHHC6H5), 4.91 (d,
1H, JA,B = 11.4 Hz, A part of an AB system, OCHHC6H5), 4.77
(d, 1H, JA,B = 11.0 Hz, B part of an AB system, OCHHC6H5), 4.71 (d,
1H, JA,B = 11.4 Hz, B part of an AB system, OCHHC6H5), 4.55 (dq,
1H, J4,5 = 9.2 Hz, J5,6 = 6.2 Hz, H-5), 4.20 (dd, 1H, J2,3 = 3.3 Hz,
J3,4 = 9.2 Hz, H-3), 3.76 (t, 1H, J3,4 = J4,5 = 9.2 Hz, H-4), 2.51 (s, 3H,
SC6H3C(CH3)3CH3), 1.49 (d, 3H, J5.6 = 6.2 Hz, H-6), 1.35 (s, 9H,
SC6H3C(CH3)3CH3); 13C NMR (75 MHz, CDCl3) d 165.8 (OCOC6H5),
149.9, 138.5, 138.0, 137.1, 133.4, 132.9, 132.9,132.7, 130.5, 130.2,
130.1, 128.6, 128.5, 128.3, 128.2, 127.7, 125.3 (SC6H3C(CH3)3CH3,
OCH2C6H5, OCOC6H5), 86.0 (C-1), 80.3 (C-4), 78.8 (C-3), 75.5
(OCH2C6H5), 71.8 (OCH2C6H5) 71.5 (C-2), 69.3 (C-5), 34.6 (SC6H3C
(CH3)3CH3), 31.5 (SC6H3C(CH3)3CH3), 20.5 (SC6H3C(CH3)3CH3), 18.3
(C-6); HR-ESI-MS m/z calcd for C38H42O5S 633.2632 [M+Na]+, found
633.2645.
4.2.10. 2-[(N-Benzyloxycarbonyl)amino]ethyl 2-O-benzoyl-4-O-
benzyl-3-O-chloroacetyl-a-L-rhamnopyranosyl-(1?2)-3,4-di-O-
benzyl- -rhamnopyranoside (18)
a-L
To a mixture of 17 (100 mg, 0.17 mmol), 12 (113 mg, 0.19 mmol)
and 4 Å molecular sieves (100 mg) in dry CH2Cl2 (5 mL) was added
NIS (51 mg, 0.22 mmol) followed by AgOTf (15 mg, 0.06 mmol). The
reaction mixture was stirred at rt for 30 min and was then filtered.
The filtrate was washed by a mixture of saturated aqueous NaHCO3
and saturated aqueous Na2S2O3. The organic phase was dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was then
purified by flash chromatography using cyclohexane/EtOAc (8:2)
as eluent to give 18 (126 mg, 79%). Rf 0.33 (cyclohexane/EtOAc
To a mixture of 2-methyl-5-tert-butylphenyl 2-O-benzoyl-3,4-
di-O-benzyl-1-thio-a-L-rhamnopyranoside (1.0 g, 1.7 mmol), 2-
(N-benzyloxycarbonyl)-aminoethanol (332 mg, 1.7 mmol) and 4 Å
molecular sieves (1.0 g) in dry CH2Cl2 (20 mL) was added NIS
(495 mg, 2.2 mmol) followed by AgOTf (128 mg, 0.5 mmol). The
reaction mixture was stirred at rt for 30 min and was then filtered.
The filtrate was washed by a mixture of saturated aqueous NaHCO3
and saturated aqueous Na2S2O3. The organic phase was dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was then
purified by flash chromatography using cyclohexane/EtOAc (8:2)
as eluent to give 2-[(N-benzyloxycarbonyl)amino]ethyl 2-O-ben-
8:2); [a]
+6 (CHCl3, c 1.1); 1H NMR (300 MHz, CDCl3) d 8.13–7.11
D
(m, 25H, NHCOOCH2C6H5, 3 ꢂ OCH2C6H5, OCOC6H5), 5.79 (dd, 1H,
J1C,2C = 1.9 Hz, J2C,3C = 3.3 Hz, H-2C), 5.59 (dd, 1H, J2C,3C = 3.3 Hz,
J3C,4C = 9.7 Hz, H-3C), 5.18 (s, 2H, NHCOOCH2C6H5), 5.15 (d, 1H,
J1C,2C = 1.9 Hz, H-1C), 4.97 (d, 1H, JA,B = 11.0 Hz, A part of an AB sys-
tem, OCHHC6H5), 4.82 (d, 1H, J1D,2D = 1.5 Hz, H-1D), 4.81–4.66 (m,
5H, B part of an AB system, OCHHC6H5, 2 ꢂ OCH2C6H5), 4.15–4.08
(m, 1H, H-5C), 4.02 (d, 1H, JA,B = 14.9 Hz, A part of an AB system,
OCOCHHCl), 4.04 (br s, 1H, H-2D), 3.92 (d, 1H, JA,B = 14.9 Hz, B part
of an AB system, OCOCHHCl), 3.91–3.85 (m, 1H, H-3D), 3.82–3.61
(m, 4H, H-4D, H-5D, H-4C, A part of an AB system, OCHHCH2NH),
zoyl-3,4-di-O-benzyl-
Rf 0.31 (cyclohexane/EtOAc 8:2); [
a
-
L
-rhamnopyranoside (1.1 g, quantitative).
a]
+14 (CHCl3, c 1.1); 1H NMR
D
(300 MHz, CDCl3) d 8.19–7.30 (m, 20H, NHCOOCH2C6H5, 2 ꢂ
OCH2C6H5, OCOC6H5), 5.67 (dd, 1H, J1,2 = 1.6 Hz, J2,3 = 3.2 Hz, H-2),
5.26 (br s, 1H, NH), 5.20 (s, 2H, NHCOOCH2C6H5), 5.00 (d, 1H,
JA,B = 11.0 Hz, A part of an AB system, OCHHC6H5), 4.95 (d, 1H,
J1,2 = 1.6 Hz, H-1), 4.86 (d, 1H, JA,B = 11.2 Hz, A part of an AB system,
OCHHC6H5), 4.73 (d, 1H, JA,B = 11.0 Hz, B part of an AB system,
OCHHC6H5), 4.65 (d, 1H, JA,B = 11.2 Hz, B part of an AB system,
OCHHC6H5), 4.11 (dd, 1H, J2,3 = 3.2 Hz, J3,4 = 9.2 Hz, H-3), 3.91–
3.83 (m, 2H, H-5, A part of an AB system, OCHHCH2NH), 3.65 (t,
1H, J3,4 = J4,5 = 9.2 Hz, H-4), 3.62–3.57 (m, 1H, B part of an AB sys-
tem, OCHHCH2NH), 3.55–3.37 (m, 2H, OCH2CH2NH), 1.45 (d, 3H,
J5,6 = 6.4 Hz, H-6); 13C NMR (75 MHz, CDCl3) d 165.9 (OCOC6H5),
156.4 (NHCOOCH2C6H5), 138.4, 138.1, 136.6, 133.3, 130.0, 128.6,
128.5, 128.4, 128.2, 128.1, 127.8, 127.7 (2 ꢂ OCH2C6H5, OCOC6H5,
NHCOOCH2C6H5), 98.0 (C-1), 80.0 (C-4), 78.1 (C-3), 75.5
(OCH2C6H5), 71.7 (OCH2C6H5), 69.4 (C-2), 68.1 (C-5), 67.1 and
66.9 (NHCOOCH2C6H5, OCH2CH2NH), 40.9 (OCH2CH2NH), 18.2 (C-
6); HR-ESI-MS m/z calcd for C37H39NO8 648.2573 [M+Na]+, found
648.2582.
3.60–3.34 (m, 3H,
B part of an AB system, OCH2CHHNH,
OCH2CH2NH), 1.47 (d, 3H, J5C,6C = 6.1 Hz, H-6C), 1.39 (d, 3H,
J5D,6D = 6.1 Hz, H-6D); 13C NMR (75 MHz, CDCl3) d 166.4 and 165.5
(OCOCH2Cl and OCOC6H5), 156.4 (NHCOOCH2C6H5), 138.5, 138.2,
137.9, 136.5, 133.5, 128.6, 128.4, 127.9, 127.7, 127.5
(3 ꢂ OCH2C6H5, OCOC6H5, NHCOOCH2C6H5), 99.2 (C-1C), 99.0 (C-
1D), 80.2 (C-4C), 79.7 (C-3D), 78.7 (C-4D), 75.7 (C-2D), 75.6
(OCH2C6H5), 75.3 (OCH2C6H5), 74.0 (C-3C), 72.6 (OCH2C6H5), 70.4
(C-2C), 68.6 (C-5D), 68.3 (C-5C), 66.9 (NHCOOCH2C6H5,
OCH2CH2NH), 40.8 (OCH2CH2NH, COCH2Cl), 18.2 (C-6C), 18.0 (C-
6D); HR-ESI-MS m/z calcd for C52H56ClNO13 960.3338 [M+Na]+,
found 960.3347.
4.2.11. 2-[(N-Benzyloxycarbonyl)amino]ethyl 2-O-benzoyl-
4-O-benzyl-a-L-rhamnopyranosyl-(1?2)-3,4-di-O-benzyl-
a-
L-rhamnopyranoside (19)
To a solution of 18 (126 mg, 0.13 mmol) in THF (5 mL) were
added thiourea (20 mg, 0.26 mmol), NaHCO3 (22 mg, 0.26 mmol)
and a catalytic amount of tetrabutylammonium iodide (2.4 mg,
2-[(N-Benzyloxycarbonyl)amino]ethyl 2-O-benzoyl-3,4-di-O-
benzyl-a-
L-rhamnopyranoside (1.1 g, 1.7 mmol) was treated with
6.5 lmol). The solution was stirred for 12 h at 55 °C. The reaction
NaOMe (0.2 M solution in MeOH) (17 mL, 3.4 mmol) overnight at
rt. The reaction mixture was then neutralized by resin Amberlite
120 H+. The resin was filtered off and the filtrate was concentrated
in vacuo. The residue was then purified by flash chromatography
using cyclohexane/EtOAc (8:2) as eluent to give 17 (887 mg, quan-
mixture was then cooled to room temperature, diluted with
CH2Cl2, and washed with water. The organic layer was dried over
Na2SO4, filtered, and concentrated. The crude residue was purified
by flash chromatography using cyclohexane/EtOAc (8:2) as eluent
to give 19 (70 mg, 62%) as a pale yellow foam. Rf 0.20 (cyclohexane/
titative). Rf 0.19 (cyclohexane/EtOAc 8:2); [
a]
ꢁ23 (CHCl3, c 1.0);
EtOAc 8:2); [
a
]
D
ꢁ8 (CHCl3, c 1.0); 1H NMR (300 MHz, CDCl3) d
D
1H NMR (300 MHz, CDCl3) d 7.42–7.30 (m, 15H, NHCOOCH2C6H5,
2 ꢂ OCH2C6H5), 5.23 (m, 1H, NH), 5.16 (s, 2H, NHCOOCH2C6H5),
4.93 (d, 1H, JA,B = 10.8 Hz, A part of an AB system, OCHHC6H5),
4.84 (d, 1H, J1,2 = 1.5 Hz, H-1), 4.74 (br s, 2H, OCH2C6H5), 4.69 (d,
1H, JA,B = 10.8 Hz, B part of an AB system, OCHHC6H5), 4.05 (dd,
1H, J1,2 = 1.5 Hz, J2,3 = 3.2 Hz, H-2), 3.86 (dd, 1H, J2,3 = 3.2 Hz,
8.13–7.19 (m, 25H, NHCOOCH2C6H5, 3 ꢂ OCH2C6H5, OCOC6H5),
5.58 (dd, 1H, J1C,2C = 1.7 Hz, J2C,3C = 3.5 Hz, H-2C), 5.16 (br s, 3H,
H-1C, NHCOOCH2C6H5), 5.11 (br s, 1H, NH), 4.94 (d, 1H, JA,B
10.9 Hz, A part of an AB system, OCHHC6H5), 4.90 (d, 1H, JA,B
11.0 Hz, A part of an AB system, OCHHC6H5), 4.80 (d, 1H, J1D,2D
=
=
=
1.7 Hz, H-1D), 4.78 (d, 1H, JA,B = 11.0 Hz, B part of an AB system,

124
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
OCHHC6H5), 4.72 (s, 2H, OCH2C6H5), 4.67 (d, 1H, JA,B = 10.9 Hz, B
part of an AB system, OCHHC6H5), 4.36 (m, 1H, H-3C), 4.00–3.91
(m, 1H, H-2D, H-5C), 3.88 (dd, 1H, J2D,3D = 3.1 Hz, J3D,4D = 9.2 Hz,
OCH2C6H5, NHCOOCH2C6H5, SC6H3C(CH3)3CH3), 5.72 (dd, 1H,
J1B,2B = 1.7 Hz, J2B,3B = 3.3 Hz, H-2B), 5.60 (d, 1H, J1B,2B = 1.7 Hz, H-
1B), 5.44 (t, 1H, J2A,3A = J3A,4A = 9.7 Hz, H-3A), 5.21 (dd, 1H,
J1A,2A = 7.8 Hz, J2A,3A = 9.6 Hz, H-2A), 4.94 (d, 1H, JA,B = 11.6 Hz, A
part of an AB system, OCHHC6H5), 4.91 (d, 1H, J1A,2A = 7.9 Hz, H-
1A), 4.71 (d, 1H, JA,B = 11.6 Hz, B part of an AB system, OCHHC6H5),
4.41 (dq, 1H, J4B,5B = 9.4 Hz, J5B,6B = 6.3 Hz, H-5B), 4.34 (dd, 1H,
H-3D), 3.79–3.65 (m, 2H, H-5D,
OCHHCH2NH), 3.56–3.31 (m, 3H,
A
B
part of an AB system,
part of an AB system,
OCHHCH2NH, OCH2CH2NH), 3.53 (t, 1H, J3D,4D = J4D,5D = 9.2 Hz, H-
4D), 3.52 (t, 1H, J3C,4C = J4C,5C = 9.4 Hz, H-4C), 1.43 (d, 3H, J5C,6C
6.1 Hz, H-6C), 1.34 (d, 3H, J5D,6D = 6.1 Hz, H-6D); 13C NMR
(75 MHz, CDCl3) 166.3 (COC6H5), 156.4 (NHCOOCH2C6H5),
=
J2B,3B = 3.3 Hz, J3B,4B = 9.4 Hz, H-3B), 3.74 (t, 1H, J3B,4B = J4B,5B
=
d
9.4 Hz, H-4B), 3.52 (dq, 1H, J4A,5A = 9.7 Hz, J5A,6A = 5.9 Hz, H-5A),
3.35 (t, 1H, J3A,4A = J4A,5A = 9.7 Hz, H-4A), 2.50 (s, 3H, SC6H3C(CH3)3
CH3), 1.71 (s, 3H, OCOCH3), 1.38 (d, 3H, J5B,6B = 6.3 Hz, H-6B), 1.35
(d, 3H, J5A,6A = 5.9 Hz, H-6A), 1.34 (s, 9H, SC6H3CH3C(CH3)3CH3);
13C NMR (75 MHz, CDCl3) d 169.5 (OCOCH3), 165.9 (OCOC6H5),
165.7 (OCOC6H5), 149.8, 138.1, 137.0, 133.6, 133.2, 132.5, 130.3,
130.2, 129.9, 129.0, 128.6, 128.5, 128.0, 127.9, 125.2 (SC6H3C(CH3)3
CH3, 2 ꢂ OCOC6H5, OCH2C6H5), 100.7 (C-1A), 85.3 (C-1B), 79.8 (C-
4B), 79.6 (C-3B), 75.2 (OCH2C6H5), 74.5 (C-2B), 74.1 (C-3A), 72.0
(C-2A), 70.9 (C-5A), 69.0 (C-5B), 65.8 (C-4A), 34.5 (SC6H3C(CH3)3
CH3), 31.4 (SC6H3C(CH3)3CH3), 20.4 (SC6H3C(CH3)3CH3, OCOCH3),
138.4, 138.3, 133.4, 130.0, 128.6, 128.5, 128.4, 128.0, 127.8
(3 ꢂ OCH2C6H5, OCOC6H5, NHCOOCH2C6H5), 99.2 (C-1C), 99.1 (C-
1D), 81.7 (C-4C), 80.2 (C-4D), 79.5 (C-3D), 75.5 (OCH2C6H5), 75.3
(OCH2C6H5), 75.2 (C-2D), 73.3 (C-2C), 72.4 (OCH2C6H5), 70.5 (C-
3C), 68.3 and 68.2 (C-5C and C-5D), 66.9 (NHCOOCH2C6H5,
OCH2CH2NH), 40.9 (OCH2CH2NH), 18.3 (C-6C), 18.1 (C-6D); HR-
ESI-MS m/z calcd for
C
50H55NO12 884.3622 [M+Na]+, found
884.3607.
4.2.12. 2-[(N-Benzyloxycarbonyl)amino]ethyl 2-O-acetyl-4-
azido-3-O-benzoyl-4,6-dideoxy-b- -glucopyranosyl-(1?3)-
2-O-benzoyl-4-O-benzyl- -rhamnopyranoside (20)
D
18.1, 18.0 (C-6A, C-6B); HR-ESI-MS m/z calcd for C46H51N3O10S
a-L
860.3193 [M+Na]+, found 860.3184.
To a solution of 3 (184 mg, 0.38 mmol), 14 (184 mg, 0.34 mmol)
and 4 Å molecular sieves (200 mg) in dry CH2Cl2 (10 mL) under in-
4.2.14. 2-Methyl-5-tert-butylphenyl 2-O-acetyl-4-azido-3-O-
ert atmosphere at ꢁ40 °C was added TMSOTf (33
l
L, 0.19 mmol).
benzyl-4,6-dideoxy-b-
O-benzyl-1-thio- -rhamnopyranoside (23)
TMSOTf (18 L, 0.1 mmol) was added to a solution of donor 6
D-glucopyranosyl-(1?3)-2-O-benzoyl-4-
After 30 min stirring at this temperature, the reaction mixture
was neutralized by Et3N and filtered. The filtrate was washed with
water, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography using cyclohexane/
EtOAc (8:2) as eluent to give disaccharide 20 (157 mg, 48%) as a
a-D
l
(116 mg, 0.25 mmol) and acceptor 5 (104 mg, 0.2 mmol) in CH2Cl2
(4 mL) containing 4 Å molecular sieves at ꢁ30 °C under an inert
atmosphere. The reaction mixture was stirred at ꢁ30 °C over 1 h
and then quenched upon addition of excess Et3N. The crude mix-
ture was filtered over a CeliteÒ pad which was further washed with
CH2Cl2. The filtrate was next evaporated under reduced pressure
and the residue purified by flash chromatography on silica gel
using cyclohexane/EtOAc (95:5) as eluent to give disaccharide 23
colorless oil. Rf 0.41 (cyclohexane/EtOAc 7:3); [
a
]
D
ꢁ3 (CHCl3, c
0.9); 1H NMR (300 MHz, CDCl3) d 8.10–7.29 (m, 20H, 2 ꢂ OCOC6H5,
OCH2C6H5, NHCOOCH2C6H5), 5.42 (dd, 1H, J1B,2B = 1.8 Hz, J2B,3B
=
3.5 Hz, H-2B), 5.35 (t, 1H, J2A,3A = J3A,4A = 9.6 Hz, H-3A), 5.33 (br s,
1H, NH), 5.16 (dd, 1H, J1A,2A = 7.8 Hz, J2A,3A = 9.6 Hz, H-2A), 5.15
(s, 2H, NHCOOCH2C6H5), 4.90–4.85 (m, 2H, H-1B, A part of an AB
system, OCHHC6H5), 4.83 (d, 1H, J1A,2A = 7.8 Hz, H-1A), 4.66 (d,
1H, JA,B = 11.2 Hz, B part of an AB system, OCHHC6H5), 4.26 (dd,
1H, J2B,3B = 3.5 Hz, J3B,4B = 9.4 Hz, H-3B), 3.85–3.72 (m, 2H, H-5B, A
(170 mg, 85%). Rf 0.54 (cyclohexane/EtOAc 9:1); [
a
]
D
ꢁ62 (CHCl3,
c 1.0); 1H NMR (300 MHz, CDCl3) d 8.16–7.20 (m, 18H, OCOC6H5,
2 ꢂ OCH2C6H5, SC6H3C(CH3)3CH3), 5.70 (dd, 1H, J1B,2B = 1.5 Hz,
J2B,3B = 3.3 Hz, H-2B), 5.61 (d, 1H, J1B,2B = 1.5 Hz, H-1B), 5.14 (dd,
1H, J1A,2A = 8.1 Hz, J2A,3A = 9.4 Hz, H-2A), 5.00 (d, 1H, JA,B = 11.2 Hz,
A part of an AB system, OCHHC6H5), 4.87 (d, 1H, JA,B = 11.2 Hz, A
part of an AB system, OCHHC6H5), 4.75 (d, 1H, JA,B = 11.2 Hz, B part
of an AB system, OCHHC6H5), 4.73 (d, 1H, J1A,2A = 8.1 Hz, H-1A),
4.69 (d, 1H, JA,B = 11.2 Hz, B part of an AB system, OCHHC6H5),
4.49–4.38 (m, 1H, H-5B), 4.30 (dd, 1H, J2B,3B = 3.3 Hz, J3B,4B = 9.5 Hz,
H-3B), 3.76 (t, 1H, J3B,4B = J4B,5B = 9.4 Hz, H-4B), 3.56 (t, 1H, J2A,3A = -
J3A,4A = 9.4 Hz, H-3A), 3.42–3.28 (m, 1H, H-5A), 3.21 (t, 1H, J3A,4A = -
J4A,5A = 9.4 Hz, H-4A), 2.54 (s, 3H, CH3), 1.79 (s, 3H, COCH3), 1.38 (s,
9H, SC6H3C(CH3)3CH3), 1.37 (d, 3H, J5B,6B = 6.3 Hz, H-6B), 1.32 (d,
3H, J5A,6A = 6.1 Hz, H-6A); 13C NMR (75 MHz, CDCl3) d 169.3
(OCOCH3), 166.0 (OCOC6H5), 149.8, 138.3, 137.2, 136.9, 133.1,
132.5, 130.4, 130.1, 129.9, 128.6, 128.5, 128.4, 128.1, 128.0,
127.8, 127.7 (SC6H3C(CH3)3CH3, 2 ꢂ OCH2C6H5, OCOC6H5), 101.0
(C-1A), 85.3 (C-1B), 81.5 (C-3A), 79.8 (C-4B), 79.6 (C-3B), 75.1
(OCH2C6H5), 74.8 (OCH2C6H5), 74.4 (C-2B), 73.1 (C-2A), 70.9 (C-
5A), 69.0 (C-5B), 67.5 (C-4A), 34.5 (SC6H3C(CH3)3CH3), 31.4
(SC6H3C(CH3)3CH3), 20.7 (OCOCH3), 20.4 (SC6H3C(CH3)3CH3), 18.2
part of an AB system, OCHHCH2NH), 3.63 (t, 1H, J3B,4B = J4B,5B
=
9.4 Hz, H-4B), 3.60–3.50 (m, 1H, part of an AB system,
B
OCHHCH2NH), 3.49–3.39 (m, 3H, H-5A, OCH2CH2NH), 3.27 (t, 1H,
J3A,4A = J4A,5A = 9.6 Hz, H-4A), 1.65 (s, 3H, OCOCH3), 1.33 (d, 3H,
J5B,6B = 6.1 Hz, H-6B), 1.27 (d, 3H, J5A,6A = 5.9 Hz, H-6A); 13C NMR
(75 MHz, CDCl3)
d 169.5 (OCOCH3), 166.0 (OCOC6H5), 165.6
(OCOC6H5), 156.4 (NHCOOCH2C6H5), 138.0, 136.4, 133.2, 133.1,
129.8, 128.5, 128.4, 128.1, 127.8, 127.7 (NHCOOCH2C6H5,
2 ꢂ OCOC6H5, OCH2C6H5), 100.4 (C-1A), 97.2 (C-1B), 79.4 (C-4B),
78.8 (C-3B), 75.0 (OCH2C6H5), 74.0 (C-3A), 72.1 (C-2B), 71.9 (C-
2A), 70.7 (C-5A), 67.7 (C-5B) 67.0 and 66.8 (NHCOOCH2C6H5,
OCH2CH2NH), 65.7 (C-4A), 40.8 (OCH2CH2NH), 20.2 (OCOCH3),
17.9 (C-6A and C-6B); HR-ESI-MS m/z calcd for C45H48N4O13
875.3116 [M+Na]+, found 875.3126.
4.2.13. 2-Methyl-5-tert-butylphenyl 2-O-acetyl-4-azido-3-O-
benzoyl-4,6-dideoxy-b-
D-glucopyranosyl-(1?3)-2-O-benzoyl-
4-O-benzyl-1-thio- -rhamnopyranoside (22)
a-L
To a solution of 3 (184 mg, 0.38 mmol), 5 (177 mg, 0.34 mmol)
and 4 Å molecular sieves (200 mg) in dry CH2Cl2 (10 mL) under in-
(C-6A), 18.0 (C-6B); HR-ESI-MS m/z calcd for C46H53N3O9S
[M+Na]+ 846.3400, found 846.3411.
ert atmosphere at ꢁ40 °C was added TMSOTf (33
lL, 0.19 mmol).
After 30 min stirring at this temperature, the reaction mixture
was neutralized by Et3N, and filtered. The filtrate was washed with
water, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography using cyclohexane/
EtOAc (8:2) as eluent to give disaccharide 22 (216 mg, 68%) as a
4.2.15. 2-[(N-Benzyloxycarbonyl)amino]ethyl 4-azido-3-O-
benzoyl-4,6-dideoxy-b- -glucopyranosyl-(1?3)-2-O-benzoyl-
4-O-benzyl- -rhamnopyranoside (24)
To a solution of ester 20 (157 mg, 0.19 mmol) in CH2Cl2 (5 mL)
was added a solution of acetyl chloride (396 L, 5.5 mmol) in
D
a-L
l
white solid. Rf 0.62 (cyclohexane/EtOAc 7:3); [
a
]
D
ꢁ57 (CHCl3, c
MeOH (8 mL) dropwise. The reaction mixture was stirred at rt
for 6 days and was then poured in saturated aqueous NaHCO3.
1.0); 1H NMR (300 MHz, CDCl3) d 8.12–7.13 (m, 18H, 2 ꢂ OCOC6H5,

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
125
The layers were separated and the organic phase was washed
with water, dried over Na2SO4, filtered, and concentrated in va-
cuo. The residue was purified by flash chromatography using
cyclohexane/EtOAc (8:2) as eluent to provide recovered 20
(22 mg, 14%) and 24 (72 mg, 48%) and as a colorless oil. Rf 0.26
138.0, 133.3, 130.0, 129.9, 128.7, 128.5, 128.3, 128.2 (NHCO-
OCH2C6H5 I and II, OCOC6H5 I and II, OCH2C6H5 I and II), 103.0
(C-1AII), 102.9 (C-1AI), 97.6 (C-1BI and C-1BII), 84.7 (C-3AII), 83.7
(C-2AI), 81.0, 80.3 (C-4BI, C-4BII), 78.0, 76.1 (C-3BI, C-3BII), 75.7,
75.3 (NHCOOCH2C6H5 I and II), 75.3 (C-2AII), 74.8 (C3AI), 73.1,
72.4 (C-2BI, C-2BII), 70.9, 70.7 (C-5AI, C-5AII), 68.1 (C-5BI and C-
5BII), 67.4, 67.3 (OCH2CH2NH I and II, OCH2C6H5 I and II), 67.0,
66.9 (C-4AI, C-4AII), 41.1 (OCH2CH2NH I and II), 18.3, 18.2 (C-6AI,
C-6AII, C-6BI, C-6BII); ESI-MS m/z 743.1 [M+Na]+.
(cyclohexane/EtOAc 7:3);
½
a 2D0 0.3); 1H NMR
+8 (CHCl3, c
ꢃ
(300 MHz, CDCl3) d 8.08–7.29 (m, 20H, 2 ꢂ OCOC6H5, OCH2C6H5,
NHCOOCH2C6H5), 5.42 (dd, 1H, J1B,2B = 1.7 Hz, J2B,3B = 3.5 Hz, H-
2B), 5.19 (t, 1H, J2A,3A = J3A,4A = 9.5 Hz, H-3A), 5.12 (br s, 2H,
NHCOOCH2C6H5), 4.96 (d, 1H, JA,B = 10.6 Hz, A part of an AB sys-
tem, OCHHC6H5), 4.84 (d, 1H, J1B,2B = 1.7 Hz, H-1B), 4.69 (d, 1H,
JA,B = 11.2 Hz, B part of an AB system, OCHHC6H5), 4.64 (d, 1H,
J1A,2A = 7.7 Hz, H-1A), 4.26 (dd, 1H, J2B,3B = 3.5 Hz, J3B,4B = 9.5 Hz,
Compound 27: Rf 0.20 (cyclohexane/EtOAc 7:3); [
a]
D
+21
(CHCl3, c 0.2); 1H NMR (300 MHz, CDCl3) d 7.98–7.19 (m, 15H,
OCOC6H5, OCH2C6H5, NHCOOCH2C6H5), 5.37 (dd, 1H, J1B,2B = 1.8 Hz,
J2B,3B = 3.5 Hz, H-2B), 5.18 (br s, 1H, NH), 5.11 (s, 2H,
NHCOOCH2C6H5), 4.89 (d, 1H, JA,B = 10.8 Hz, A part of an AB system,
OCHHC6H5), 4.83 (d, 1H, J1B,2B = 1.8 Hz, H-1B), 4.72 (d, 1H,
JA,B = 10.8 Hz, B part of an AB system, OCHHC6H5), 4.41 (d, 1H,
J1A,2A = 7.8 Hz, H-1A), 4.20 (dd, 1H, J2B,3B = 3.5 Hz, J3B,4B = 9.4 Hz,
H-3B), 3.82–3.70 (m, 2H, H-5B,
A part of an AB system,
OCHHCH2NH), 3.64 (t, 1H, J3B,4B = J4B,5B = 9.5 Hz, H-4B), 3.63–3.47
(m, 2H, H-2A, B part of an AB system, OCHHCH2NH), 3.47–3.35
(m, 3H, H-5A, OCH2CH2NH), 3.24 (t, 1H, J3A,4A = J4A,5A = 9.5 Hz, H-
4A), 1.35 (d, 3H, J5B,6B = 6.1 Hz, H-6B), 1.29 (d, 3H, J5A,6A = 6.2 Hz,
H-3B), 3.83–3.74 (m, 2H, H-5B,
A part of an AB system,
H-6A); 13C NMR (75 MHz, CDCl3)
d
166.6 (OCOC6H5), 165.9
OCHHCH2NH), 3.63 (t, 1H, J3B,4B = J4B,5B = 9.4 Hz, H-4B), 3.57–3.49
(m, 1H, B part of an AB system, OCHHCH2NH), 3.48 (t, 1H, J2A,3A = -
J3A,4A = 9.2 Hz, H-3A), 3.43–3.38 (m, 2H, OCH2CH2NH), 3.30 (dd, 1H,
J1A,2A = 7.8 Hz, J2A,3A = 9.2 Hz, H-2A) 3.22 (dq, 1H, J4A,5A = 9.2 Hz,
J5A,6A = 6.2 Hz, H-5A), 2.95 (t, 1H, J3A,4A = J4A,5A = 9.2 Hz, H-4A),
2.87 (br s, 1H, OH), 2.74 (br s, 1H, OH), 1.37 (d, 3H, J5B,6B = 6.1 Hz,
H-6B), 1.19 (d, 3H, J5A,6A = 6.2 Hz, H-6A); 13C NMR (75 MHz, CDCl3)
d 165.9 (OCOC6H5), 156.4 (NHCOOCH2C6H5), 137.8, 133.3, 129.9,
128.6, 128.4, 128.2, 128.1 (NHCOOCH2C6H5, OCOC6H5, OCH2C6H5),
102.6 (C-1A), 97.4 (C-1B), 80.5 (C-4B), 78.0 (C-3B), 75.8
(OCH2C6H5), 75.5 (C-3A), 74.6 (C-2A), 72.5 (C-2B), 71.2 (C-5A),
(OCOC6H5), 156.4 (NHCOOCH2C6H5), 137.9, 136.5, 133.6, 130.1,
130.0, 129.9, 129.2, 128.6, 128.5, 128.3, 128.2, 128.1 (NHCO-
OCH2C6H5, 2 ꢂ OCOC6H5, OCH2C6H5), 103.2 (C-1A), 97.4 (C-1B),
80.0 (C-4B), 78.2 (C-3B), 76.4 (C-3A), 75.5 (OCH2C6H5), 73.3 (C-
2A), 72.4 (C-2B), 70.7 (C-5A), 68.0 (C-5B) 67.1 and 66.9
(NHCOOCH2C6H5, OCH2CH2NH), 65.8 (C-4A), 40.9 (OCH2CH2NH),
18.2 (C-6A), 18.1 (C-6B); HR-ESI-MS m/z calcd for
C
43H46N4O12 833.3010 [M+Na]+, found 833.3018.
4.2.16. 2-[(N-Benzyloxycarbonyl)aminoethyl 4-azido-4,6-dideo
xy-2-O-methyl- -glucopyranosyl-(1?3)-2-O-benzoyl-4-O-be
nzyl- -rhamnopyranoside (25) and 2-[(N-benzyloxycarbonyl)
amino]ethyl 4-azido-4,6-dideoxy-3-O-methyl-b- -glucopyrano
syl-(1?3)-2-O-benzoyl-4-O-benzyl- -rhamnopyranoside (26),
2-[(N-Benzyloxycarbonyl)amino]ethyl 4-azido-4,6-dideoxy-b-
68.2
(C-5B),
67.4
(C-4A),
67.3
(OCH2CH2NH),
67.1
a-D
(NHCOOCH2C6H5), 41.1 (OCH2CH2NH), 18.4 (C-6A and C-6B); HR-
a-L
ESI-MS m/z calcd for
C
36H42N4O11 729.2748 [M+Na]+, found
D
729.2759.
a-L
D
-
glucopyranosyl-(1?3)-2-O-benzoyl-4-O-benzyl-b-
L
-rhamnopyr
4.2.17. p-Methylphenyl 4-azido-3-O-benzoyl-4,6-dideoxy-b-D-
anoside (27)
glucopyranosyl-(1?3)-2-O-benzoyl-4-O-benzyl-1-thio-a-L-
To a solution of 24 (60 mg, 0.07 mmol) in dry CH2Cl2 (1 mL) was
added silver (I) oxide (100 mg, 0.42 mmol). The reaction mixture
rhamnopyranoside (28)
To a solution of 2126 (220 mg, 0.28 mmol) in CH2Cl2 (8 mL) was
was stirred at rt for 1 h. Iodomethane (43
l
L, 0.70 mmol) was then
added a solution of acetyl chloride (600 lL, 8.4 mmol) in MeOH
added dropwise and the reaction mixture was stirred at rt for
3 days. Silver (I) oxide was filtered through a CeliteÒ pad and the
filtrate was concentrated. The residue was purified by flash chro-
matography using cyclohexane/EtOAc (7:3) as eluent to give a
1:1 mixture of 25 and 26 (20 mg, 40%) as a colorless oil and 27
(13 mg, 26%) as a colorless oil. 25 (I) and 26 (II): Rf 0.39 (cyclohex-
ane/EtOAc 7:3); 1H NMR (300 MHz, CDCl3) d 8.15–7.33 (m, 30H,
OCOC6H5 I and II, OCH2C6H5 I and II, NHCOOCH2C6H5 I and II),
5.37–5.34 (m, 2H, H-2BI, H-2BII), 5.12 (br s, 4H, NHCOOCH2C6H5
I and II), 4.91 and 4.93 (2 d, 2H, JA,B = 10.0 Hz and JA,B = 10.3 Hz,
2 ꢂ A part of an AB systems, OCHHC6H5 I and II), 4.86 and 4.84
(2 d, 2H, J1BI,2BI = J1BII,2BII = 1.8 Hz, H-1BI, H-1BII), 4.72 and 4.68 (2
d, 2H, JA,B = 10.0 Hz and JA,B = 10.3 Hz, B part of an AB system,
OCHHC6H5 I and II), 4.51 (d, 1H, J1AI,2AI = 7.8 Hz, H-1AI), 4.35 (d,
1H, J1AII,2AII = 7.8 Hz, H-1AII), 4.18–4.09 (m, 2H, H-3BI, H-3BII),
3.75–3.62 (m, 4H, H-5BI, H-5BII, 2 ꢂ A parts of an AB systems,
OCHHCH2NH I and II), 3.59–3.47 (m, 4H, H-4BI, H-4BII, 2 ꢂ B parts
of an AB systems, OCHHCH2NH I and II), 3.55 and 3.42 (2s, 2 ꢂ 3H,
OCH3), 3.41–3.29 (m, 6H, H-3AI, H-2AII, OCH2CH2NH I and II), 3.20–
3.07 (m, 2H, H-5AI, H-5AII), 3.02 (t, 1H, J3AII,4AII = J4AII,5AII = 9.5 Hz,
H-3AII), 2.95–2.83 (m, 3H, H-4AI, H-4AII, H-2AI), 2.71 (br s, 1H,
OH), 2.51 (br s, 1H, OH), 1.37 and 1.36 (2 d, 2 ꢂ 3H, J5BI,6BI = J5BII,6-
(13 mL) dropwise. The reaction mixture was stirred at rt for 8 days
and was then poured in saturated aqueous NaHCO3. The layers
were separated and the organic phase was washed with water,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography using cyclohexane/EtOAc
(9:1) as eluent to provide 28 (156 mg, 75%) as a white powder. Rf
0.50 (cyclohexane/EtOAc 8:2); [
a]
ꢁ63 (CHCl3, c 0.05); 1H NMR
D
(300 MHz, CDCl3) d 8.11–7.32 (m, 17H, 2 ꢂ OCOC6H5, OCH2C6H5,
SC6H2H2CH3), 7.16 (d, 2H, J = 8.1 Hz, SC6H2H2CH3), 5.73 (dd, 1H,
J1B,2B = 1.6 Hz, J2B,3B = 3.0 Hz, H-2B), 5.54 (d, 1H, J1B,2B = 1.6 Hz, H-
1B), 5.26 (t, 1H, J2A,3A = J3A,4A = 9.5 Hz, H-3A), 5.05 (d,
1H, JA,B = 10.6 Hz, A part of an AB system, OCHHC6H5), 4.76
(d, 1H, JA,B = 10.6 Hz, B part of an AB system, OCHHC6H5), 4.71 (d,
1H, J1A,2A = 7.7 Hz, H-1A), 4.41–4.31 (m, 2H, H-3B, H-5B), 3.76 (t,
1H, J3B,4B = J4B,5B = 9.4 Hz, H-4B), 3.68–3.64 (m, 1H, H-2A), 3.45
(m, 1H, H-5A), 3.32 (t, 1H, J3A,4A = J4A,5A = 9.5 Hz, H-4A), 2.90 (d,
1H, J2A,OH = 3.4 Hz, OH), 2.36 (s, 3H, SC6H4CH3), 1.43 (d, 3H,
J5B,6B = 6.2 Hz, H-6B), 1.39 (d, 3H, J5A,6A = 6.0 Hz, H-6A), 1.34 (s,
9H, SC6H3CH3C(CH3)3CH3); 13C NMR (75 MHz, CDCl3) d 166.5
(OCOC6H5), 165.6 (OCOC6H5), 137.9–128.0 (SC6H4CH3, 2 ꢂ
OCOC6H5, OCH2C6H5), 103.2 (C-1A), 85.9 (C-1B), 80.1 (C-4B), 78.4
(C-3B), 76.4 (C-3A), 75.4 (OCH2C6H5), 74.4 (C-2B), 73.2 (C-2A),
70.7 (C-5A), 69.0 (C-5B), 65.8 (C-4A), 21.0 (SC6H4CH3), 18.2, 17.9
(C-6A, C-6B); HR-ESI-MS m/z calcd for C40H41N3O9S 762.2461
[M+Na]+, found 762.2444.
BII = 6.1 Hz, H-6BI, H-6BII), 1.22 and 1.18 (d, 2 ꢂ 3H, J5AI,6AI
=
J5AII,6AII = 6.1 Hz, H-6AI, H-6AII); 13C NMR (75 MHz, CDCl3)
d
166.0, 165.9 (OCOC6H5 I and II), 156.4 (NHCOOCH2C6H5 I and II),

126
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
4.2.18. p-Methylphenyl 4-azido-3-O-benzoyl-4,6-dideoxy-2-O-
methyl-b- -glucopyranosyl-(1?3)-2-O-benzoyl-4-O-benzyl-1-
thio- -rhamnopyranoside (29)
To a solution of 28 (136 mg, 0.18 mmol) in dry CH2Cl2 (1.5 mL)
was added silver (I) oxide (256 mg, 1.10 mmol). The reaction mix-
4.2.20. 2-Methyl-5-tert-butylphenyl 4-azido-3-O-benzyl-4,6-
dideoxy-b- -glucopyranosyl-(1?3)-2-O-benzoyl-4-O-benzyl-1-
thio- -rhamnopyranoside (31)
To a solution of b23 (160 mg, 0.19 mmol) in CH2Cl2 (5 mL) was
added a solution of acetyl chloride (396 L, 5.5 mmol) in MeOH
D
D
a-
L
a-D
l
ture was stirred at rt for 1 h. Iodomethane (137
l
L, 2.21 mmol) was
(8 mL) dropwise. The reaction mixture was stirred at rt for 4 days
and was then poured in saturated aqueous NaHCO3. The layers were
separated and the organic phase was washed with water, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by flash chromatography using cyclohexane/EtOAc
(95:5?9:1) as eluent to provide 23 (27 mg, 17%), 31b (43 mg,
then added dropwise and the reaction mixture was stirred at rt for
8 days. Silver (I) oxide was filtered through a CeliteÒ pad and the
filtrate was concentrated. The residue was purified by flash chro-
matography using cyclohexane/EtOAc (9:1) as eluent to give 29
(104 mg, 73%) as a colorless oil and 28 (16 mg, 12%). Rf 0.65 (cyclo-
hexane/EtOAc 8:2); [
a]
ꢁ72 (CHCl3, c 0.1); 1H NMR (600 MHz,
29%), and 5 (24 mg, 24%). Rf 0.53 (cyclohexane/EtOAc 8:2); [a]
D
D
CDCl3) d 8.02–6.96 (m, 19H, 2 ꢂ OCOC6H5, OCH2C6H5, SC6H4CH3),
5.52 (dd, 1H, J1B,2B = 1.8 Hz, J2B,3B = 3.6 Hz, H-2B), 5.43 (br s, 1H,
H-1B), 4.74 (t, 1H, J2A,3A = J3A,4A = 9.6 Hz, H-3A), 4.74 (d, 1H,
J1A,2A = 7.8 Hz, H-1A), 4.71 (d, 1H, JA,B = 10.8 Hz, A part of an AB sys-
tem, OCHHC6H5), 4.68 (d, 1H, JA,B = 10.8 Hz, B part of an AB system,
OCHHC6H5), 3.12 (dd, 1H, J2B,3B = 3.6 Hz, J3B,4B = 9.2 Hz, H-3B),
4.14–4.09 (m, 1H, H-5B), 3.48 (t, 1H, J3B,4B = J4B,5B = 9.2 Hz), 3.38–
3.33 (m, 1H, H-5A), 3.31 (dd, 1H, J1A,2A = 7.8 Hz, J2A,3A = 9.6 Hz, H-
2A) 2.30 (s, 3H, SC6H4CH3), 1.36 (d, 3H, J5B,6B = 6.2 Hz, H-6B), 1.29
(d, 3H, J5A,6A = 6.0 Hz, H-6A); 13C NMR (150 MHz, CDCl3) d 166.5
(OCOC6H5), 166.0 (OCOC6H5), 138.4, 129.9, 129.4, 128.7, 128.1,
128.0, 127.5 (SC6H4CH3, 2 ꢂ OCOC6H5, OCH2C6H5), 101.6 (C-1A),
86.2 (C-1B), 83.5 (C-2A), 79.9 (C-4B), 78.9 (C-3B), 75.6 (OCH2C6H5),
75.4 (C-3A), 74.6 (C-2B), 70.6 (C-5A), 69.0 (C-5B), 66.8 (C-4A), 20.4
(SC6H4CH3), 18.1, 17.5 (C-6A, C-6B).
ꢁ90 (CHCl3, c 1.0); 1H NMR (300 MHz, CDCl3) d 8.16–7.18 (m,
18H, OCOC6H5, 2 ꢂ OCH2C6H5, SC6H3C(CH3)3CH3), 5.71 (dd, 1H,
J1B,2B = 1.5 Hz, J2B,3B = 3.3 Hz, H-2B), 5.50 (d, 1H, J1B,2B = 1.5 Hz, H-
1B), 5.00 (d, 1H, JA,B = 10.6 Hz, A part of an AB system, OCHHC6H5),
4.94 (d, 1H, JA,B = 11.2 Hz, A part of an AB system, OCHHC6H5), 4.87
(d, 1H, JA,B = 11.2 Hz, B part of an AB system, OCHHC6H5), 4.80 (d,
1H, JA,B = 10.6 Hz, B part of an AB system, OCHHC6H5), 4.53 (d, 1H,
J1A,2A = 7.7 Hz, H-1A), 4.46–4.33 (m, 1H, H-5B), 4.32 (dd, 1H,
J2B,3B = 3.3 Hz, J3B,4B = 9.5 Hz, H-3B), 3.95 (s, 1H, OH), 3.77 (t, 1H,
J3B,4B = J4B,5B = 9.3 Hz, H-4B), 3.54 (dd, 1H, J1A,2A = 7.7 Hz, J2A,3A
=
9.3 Hz, H-2A), 3.43 (t, 1H, J2A,3A = J3A,4A = 9.3 Hz, H-3A), 3.36–3.26
(m, 1H, H-5A), 3.11 (t, 1H, J3A,4A = J4A,5A = 9.3 Hz, H-4A), 2.48 (s,
3H, SC6H3C(CH3)3CH3), 1.34 (s, 9H, SC6H3CH3C(CH3)3CH3), 1.33 (d,
3H, J5B,6B = 6.3 Hz, H-6B), 1.30 (d, 3H, J5A,6A = 6.1 Hz, H-6A); 13C
NMR (75 MHz, CDCl3) d 165.8 (OCOC6H5), 149.8, 138.1, 138.0,
137.0, 133.2, 132.4, 130.4, 130.1, 129.9, 128.7, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 125.2 (SC6H3C(CH3)3CH3, 2 ꢂ OCH2C6H5,
OCOC6H5), 103.1 (C-1A), 85.4 (C-1B), 82.5 (C-3A), 80.6 (C-4B), 78.5
(C-3B), 75.6 (OCH2C6H5), 75.1 (C-2A), 75.0 (OCH2C6H5), 74.6 (C-
2B), 71.0 (C-5A), 69.1 (C-5B), 67.2 (C-4A), 34.5 (SC6H3C(CH3)3CH3),
31.4 (SC6H3C(CH3)3CH3), 20.4 (SC6H3C(CH3)3CH3), 18.3 (C-6A),
18.1 (C-6B); ESI-MS calcd for C44H51N3O8S [M+Na]+ 804.3295,
found 804.3288.
HR-ESI-MS m/z calcd for C40H41N3O9S 776.2618 [M+Na]+, found
776.2627.
4.2.19. 2-Methyl-5-tert-butylphenyl 4-azido-3-O-benzoyl-4,6-
dideoxy-b-
thio- -rhamnopyranoside (30)
To a solution of ester 22 (216 mg, 0.26 mmol) in CH2Cl2 (6 mL)
was added a solution of acetyl chloride (523 L, 7.8 mmol) in
D-glucopyranosyl-(1?3)-2-O-benzoyl-4-O-benzyl-1-
a-L
l
MeOH (10 mL) dropwise. The reaction mixture was stirred at rt
for 6 days and was then poured in saturated aqueous NaHCO3.
The layers were separated and the organic phase was washed with
water, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography using cyclohexane/
EtOAc (8:2) as eluent to provide recovered 22 (41 mg, 19%) and
30 (130 mg, 63%) as a colorless oil. Rf 0.58 (cyclohexane/EtOAc
4.2.21. Methyl
To a solution of MeOH (7.0 mL) and acetyl chloride (4.3 mL,
-rhamnose (5.0 g, 30.4 mmol).
The reaction mixture was then stirred for 12 h at rt before NaHCO3
in excess was added. The solvent was then evaporated in vacuo and
the residue was purified by flash chromatography on silica gel
L-rhamnopyranoside (32)
60.8 mmol) at ꢁ10 °C was added
L
using CH2Cl2/MeOH (9:1) as eluent to afford methyl-
anoside 32 as a 9:1 mixture of /b anomers as a white solid (3.8 g,
70%). Rf 0.47 (
) and 0.56 (b) (CH2Cl2/MeOH 8:2); 1H NMR
(300 MHz, CD3OD) ( anomer) d 4.61 (s, 1H, H-1), 3.86–3.83 (m,
D-rhamnopyr-
7:3); [
a]
D
ꢁ25 (CHCl3, c 0.3); 1H NMR (300 MHz, CDCl3) d 8.10–
a
7.15 (m, 18H, 2 ꢂ OCOC6H5, OCH2C6H5 and SC6H3C(CH3)3CH3),
5.72 (dd, 1H, J1B,2B = 1.7 Hz, J2B,3B = 3.2 Hz, H-2B), 5.49 (d, 1H,
J1B,2B = 1.7 Hz, H-1B), 5.24 (t, 1H, J2A,3A = J3A,4A = 9.6 Hz, H-3A),
5.02 (d, 1H, JA,B = 10.6 Hz, A part of an AB system, OCHHC6H5),
4.76 (d, 1H, JA,B = 10.6 Hz, B part of an AB system, OCHHC6H5),
4.71 (d, 1H, J1A,2A = 7.7 Hz, H-1A), 4.41–4.31 (m, 2H, H-3B, H-5B),
3.75 (t, 1H, J3B,4B = J4B,5B = 9.4 Hz, H-4B), 3.64 (dd, 1H, J1A,2A = 7.7 Hz,
J2A,3A = 9.6 Hz, H-2A), 3.45 (dq, 1H, J4A,5A = 9.6 Hz, J5A,6A = 6.0 Hz, H-
5A), 3.32 (t, 1H, J3A,4A = J4A,5A = 9.6 Hz, H-4A), 2.50 (s, 3H, SC6H3C
a
a
1H, H-2), 3.65 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.4 Hz, H-3), 3.56 (dq,
1H, J4,5 = 9.4 Hz, J5,6 = 6.2 Hz, H-5), 3.41 (t, 1H, J3,4 = J4,5 = 9.4 Hz,
H-4), 3.37 (s, 1H, OCH3), 1.30 (d, 1H, J5,6 = 9.4 Hz, H-6); 13C NMR
(75 MHz, CD3OD) (a anomer) d 101.3 (C-1), 72.5 (C-4), 71.1 (C-3),
70.7 (C-2), 68.2 (C-5), 53.9 (OCH3), 16.8 (C-6); ESI-MS m/z 201.9
[M+Na]+.
(CH3)3CH3), 1.41 (d, 3H, J5B,6B = 6.1 Hz, H-6B), 1.35 (d, 3H, J5A,6A
=
4.2.22. Methyl 3-O-allyl-L-rhamnopyranoside (33)
6.1 Hz, H-6A), 1.34 (s, 9H, SC6H3CH3C(CH3)3CH3); 13C NMR
(75 MHz, CDCl3) d 166.8 (OCOC6H5), 165.8 (OCOC6H5), 149.8,
138.0, 137.0, 133.7, 133.3, 132.5, 131.0, 130.4, 130.3, 130.2,
130.1, 130.0, 129.3, 129.0, 128.6, 128.5, 128.2, 125.2 (SC6H3C
(CH3)3CH3, 2 ꢂ OCOC6H5, OCH2C6H5), 103.4 (C-1A), 85.5 (C-1B),
80.4 (C-4B), 78.8 (C-3B), 76.6 (C-3A), 75.6 (OCH2C6H5), 74.9 (C-
2B), 73.4 (C-2A), 70.9 (C-5A), 69.2 (C-5B), 66.0 (C-4A), 34.5
(SC6H3C(CH3)3CH3), 31.4 (SC6H3C(CH3)3CH3), 20.4 (SC6H3C(CH3)3
CH3), 18.4, 18.2 (C-6A, C-6B); HR-ESI-MS m/z calcd for
A mixture of triol 32 (3.8 g, 21.3 mmol) and dibutyltin oxide
(5.8 g, 23.4 mmol) in dry MeOH (60 mL) was heated to reflux under
inert atmosphere for 6 h and was then evaporated to dryness. The
residue was dried under vacuum for 2 h and was dissolved in dry
DMF (40 mL). Allyl bromide (2.2 mL, 25.6 mmol) was added drop-
wise and the reaction mixture was stirred at 90 °C for 48 h. The
mixture was then concentrated in vacuo and the residue was puri-
fied by flash chromatography using cyclohexane/EtOAc (5:5) to
afford 3329 (1.9 g, 40%) as a 9:1
0.33 (cyclohexane/EtOAc 5:5); 1H NMR (300 MHz, CDCl3) (
a/b mixture as a colorless oil. Rf
C
44H49N3O9S 818.3087 [M+Na]+, found 818.3082.
a
ano-

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
127
mer) d 5.99–5.82 (m, 1H, OCH2CHCH2), 5.33–5.26 (m, 1H, A part of
an AB system, OCH2CHCHH), 5.22–5.18 (m, 1H, B part of an AB sys-
tem, OCH2CHCHH), 4.68 (br s, 1H, H-1), 4.19–4.13 (m, 1H, A part of
an AB system, OCHHCHCH2), 4.07–4.01 (m, 1H, B part of an AB sys-
tem, OCHHCHCH2), 3.97 (br s, 1H, H-2), 3.62 (dq, 1H, J4,5 = 9.6 Hz,
J5,6 = 5.9 Hz, H-5), 3.51–3.49 (m, 2H, H-3, H-4), 3.33 (s, 3H, OCH3),
5:5); 1H NMR (300 MHz, CDCl3) d (
a
anomer) 8.69 (br s, 1H, NH),
6.14 (d, 1H, J1,2 = 2.0 Hz, H-1), 5.76–5.71 (m, 1H, OCH2CHCH2),
5.39 (dd, 1H, J1,2 = 2.0 Hz, J2,3 = 3.5 Hz, H-2), 5.21–5.15 (m, 1H, A
part of an AB system, OCH2CHCHH), 5.14–5.09 (m, 1H, B part of
an AB system, OCH2CHCHH), 5.03 (t, 1H, J3,4 = J4,5 = 9.9 Hz, H-4),
4.08–4.01 (m, 1H, A part of an AB system, OCHHCHCH2), 3.96–
3.86 (m, 2H, B part of an AB system, OCHHCHCH2, H-5), 3.80 (dd,
1H, J2,3 = 3.3 Hz, J3,4 = 9.9 Hz, H-3), 2.11 (s, 3H, OCOCH3), 2.04 (s,
3H, OCOCH3), 1.18 (d, 3H, J5,6 = 6.4 Hz, H-6); 13C NMR (75 MHz,
1.28 (d, 3H, J5,6 = 5.9 Hz, H-6); 13C NMR (75 MHz, CDCl3) (
a ano-
mer) d 134.4 (OCH2CHCH2), 117.6 (OCH2CHCH2), 100.3 (C-1), 79.1
(C-3), 71.0 (C-4), 70.5 (OCH2CHCH2), 67.7 (C-2 and C-5), 54.5
(OCH3), 17.5 (C-6); ESI-MS m/z 240.9 [M+Na]+.
CDCl3) d (a anomer) 169.9 (OCOCH3), 169.8 (OCOCH3), 159.7
(OCNHCCl3), 134.1 (OCH2CHCH2), 117.6 (OCH2CHCH2), 95.0 (C-1),
77.4 (OCNHCCl3), 73.9 (C-3), 71.7 (C-4), 70.7 (OCH2CHCH2), 69.4
(C-5), 67.2 (C-2), 20.9 (2 ꢂ OCOCH3), 17.5 (C-6); ESI-MS m/z
453.7 [M+Na]+.
4.2.23. 1,2,4-Tri-O-acetyl-3-O-allyl-
A solution of 33 (1.9 g, 8.7 mmol) in Ac2O/AcOH/H2SO4 (12 mL/
2 mL/46 L) was stirred at rt for 1.5 h. The reaction mixture was then
quenched by NaHCO3 in excess. The residue was dissolved in EtOAc
andwaswashedby saturatedaqueousNaHCO3 andbrine. Theorganic
phase was then dried over Na2SO4, filtered, and concentrated in va-
cuo. The crude residue was purified by flash chromatography using
cyclohexane/EtOAc (8:2) as eluent to give 34 (1.9 g, 67%) as a 10:1
L-rhamnopyranoside (34)
l
4.2.25. 2-[(N-Benzyloxycarbonyl)amino]ethyl 2,4-di-O-acetyl-3-
O
-allyl-
-rhamnopyranosyl-(1?2)-3,4-di-O-benzyl-
anoside (36)
To a solution of donor 35 (45 mg, 0.10 mmol) and acceptor 19
(69 mg, 80 mol) in dry CH2Cl2 (5 mL) containing molecular sieves
4 Å (20 mg) was added TMSOTf (9 L, 50 mol) under argon at
a
-
L
-rhamnopyranosyl-(1?3)-2-O-benzoyl-4-O-benzyl-
a-
L
a-L-rhamnopyr
mixture of
a
/b anomers as a colorless oil. Rf 0.74 (
a
) and 0.67 (b)
(cyclohexane/EtOAc 5:5); 1H NMR (300 MHz, CDCl3) (
a
anomer) d
l
5.81 (d, 1H, J1,2 = 1.9 Hz, H-1), 5.68–5.56 (m, 1H, OCH2CHCH2), 5.08
(dd, 1H, J1,2 = 1.9 Hz, J2,3 = 3.5 Hz, H-2), 5.09–5.01 (m, 1H, A part of
anABsystem, OCH2CHCHH), 4.98–4.94(m, 1H, BpartofanABsystem,
OCH2CHCHH), 4.82 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 3.94–3.87 (m, 1H, A
part of an AB system, OCHHCHCH2), 3.78–3.72 (m, 1H, B part of an AB
system, OCHHCHCH2), 3.67 (dq, 1H, J4,5 = 9.8 Hz, J5,6 = 6.2 Hz, H-5),
3.60 (dd, 1H, J2,3 = 3.5 Hz, J3.4 = 9.8 Hz, H-3), 1.94 (s, 3H, OCOCH3),
1.93 (s, 3H, OCOCH3), 1.89 (s, 3H, OCOCH3), 1.00 (d, 3H, J5,6 = 6.2 Hz,
l
l
0 °C. After 30 min of stirring, the solution was neutralized with
Et3N and the molecular sieves were removed by filtration. The
reaction mixture was diluted with CH2Cl2, washed with water,
dried over Na2SO4, and then concentrated in vacuo. The residue
was purified by flash chromatography on silica gel using cyclohex-
ane/EtOAc (7:3) as eluent to provide 36 (116 mg, 99%) as a color-
less oil. Rf 0.10 (cyclohexane/EtOAc 8:2); [
a
]
D
ꢁ8 (CHCl3, c 1.0);
H-6); 13C NMR (75 MHz, CDCl3) (
a
anomer) d 169.3 (OCOCH3),
1H NMR (300 MHz, CDCl3) d 8.12–7.16 (m, 25H, NHCOOCH2C6H5,
3 ꢂ OCH2C6H5, OCOC6H5), 5.77–5.64 (m, 1H, OCH2CHCH2), 5.54
(dd, 1H, J1C,2C = 2.0 Hz, J2C,3C = 3.3 Hz, H-2C), 5.33 (dd, 1H,
J1B,2B = 1.8 Hz, J2B,3B = 3.3 Hz, H-2B), 5.12–5.11 (m, 4H, H-1B, H-
1C, NHCOOCH2C6H5), 5.08–4.98 (m, 3H, H-4B, OCH2CHCH2), 4.87
(d, 1H, JA,B = 10.7 Hz, A part of an AB system, OCHHC6H5), 4.82 (d,
1H, JA,B = 10.7 Hz, A part of an AB system, OCHHC6H5), 4.73 (d,
1H, J1D,2D = 1.6 Hz, H-1D), 4.69 (d, 1H, JA,B = 10.7 Hz, B part of an
AB system, OCHHC6H5), 4.60 (d, 1H, JA,B = 10.7 Hz, B part of an AB
system, OCHHC6H5), 4.67 (s, 2H, OCH2C6H5), 4.31 (dd, 1H,
J2C,3C = 3.2 Hz, J3C,4C = 9.5 Hz, H-3C), 3.99–3.88 (m, 4H, H-2D, H-
5B, H-5C, A part of an AB system, OCHHCHCH2), 3.82 (dd, 1H,
J2D,3D = 2.9 Hz, J3D,4D = 9.3 Hz, H-3D), 3.79–3.74 (m, 1H, B part of
an AB system, OCHHCHCH2), 3.71–3.63 (m, 3H, H-3B, H-5D, A part
of an AB system, OCHHCH2NH), 3.61 (t, 1H, J3C,4C = J4C,5C = 9.5 Hz,
H-4C), 3.46 (t, 1H, J3D,4D = J4D,5D = 9.3 Hz, H-4D), 3.51–3.28 (m, 3H,
B part of an AB system, OCHHCH2NH, OCH2CH2NH), 2.08 (s, 3H,
OCOCH3), 2.03 (s, 3H, OCOCH3), 1.35 (d, 3H, J5C,6C = 6.1 Hz, H-6C),
1.29 (d, 3H, J5D,6D = 6.2 Hz, H-6D), 1.09 (d, 3H, J5B,6B = 6.3 Hz, H-
6B); 13C NMR (75 MHz, CDCl3) d 170.2 (2 ꢂ OCOCH3), 165.6
(OCOC6H5), 156.4 (NHCOOCH2C6H5), 138.5, 137.8, 133.4, 129.9,
128.6, 128.5, 128.5, 128.2, 128.2, 128.1, 127.8, 127.6, 123.1
(3 ꢂ OCH2C6H5, OCOC6H5, NHCOOCH2C6H5), 134.4 (OCH2CHCH2),
117.0 (OCH2CHCH2), 99.1, 99.0 (C-1B, C-1C, C-1D), 80.9 (C-4C),
80.3 (C-4D), 79.4 (C-3D), 75.6 (C-3C, C-2D, 2 ꢂ OCH2C6H5), 74.2
(C-3B), 72.7 (C-2C), 72.4 (C-4B), 72.3 (OCH2C6H5), 70.6
(OCH2CHCH2), 69.0 (C-2B), 68.6 (C-5D), 68.4 (C-5C), 67.6 (C-5B),
66.9 (NHCOOCH2C6H5, OCH2CH2NH), 41.0 (OCH2CH2NH), 21.0
(OCOCH3), 18.3 (C-6C), 18.1 (C-6D), 17.3 (C-6B); HR-ESI-MS m/z
calcd for C63H73NO18 1154.4725 [M+Na]+, found 1154.4755.
169.3 (OCOCH3), 167.8 (OCOCH3), 134.0 (OCH2CHCH2), 116.5
(OCH2CHCH2), 100.3 (C-1), 73.8 (C-3), 71.5 (C-4), 70.5 (OCH2CHCH2),
68.4 (C-5), 67.3 (C-2), 20.4 (3 ꢂ OCOCH3), 17.1 (C-6); HR-ESI-MS m/z
calcd for C15H22O8 353.1223 [M+Na]+, found 353.1201.
4.2.24. 2,4-Di-O-acetyl-3-O-allyl-L-rhamnopyranosyl
trichloroacetimidate (35)
To a solution of acetate 34 (1.9 g, 5.7 mmol) in dry THF (30 mL)
was added benzylamine (688 L, 6.3 mmol). The reaction mixture
l
was stirred at rt under inert atmosphere overnight and then
quenched with 1 M aqueous HCl. The solvent was evaporated un-
der reduced pressure and the crude residue was purified by flash
chromatography using cyclohexane/EtOAc (7:3) as eluent to pro-
vide 2,4-di-O-acetyl-3-O-allyl-
9:1 /b mixture as a white solid. Rf 0.50 (cyclohexane/EtOAc
5:5); 1H NMR (300 MHz, CDCl3) (
anomer) d 5.74–5.63 (m, 1H,
L-rhamnopyranoside (1.2 g, 71%) as
a
a
OCH2CHCH2), 5.16 (dd, 1H, J1,2 = 1.5 Hz, J2,3 = 3.3 Hz, H-2), 5.11–
5.10 (m, 1H, A part of an AB system, OCH2CHCHH), 5.07–5.06 (m,
1H, B part of an AB system, OCH2CHCHH), 5.03 (br s, 1H, H-1),
4.86 (t, 1H, J3,4 = J4,5 = 9.9 Hz, H-4), 4.55 (br s, 1H, OH), 4.04–3.96
(m, 1H, A part of an AB system, OCHHCHCH2), 3.92 (dq, 1H,
J4,5 = 9.9 Hz, J5,6 = 6.4 Hz, H-5), 3.86–3.79 (m, 1H, B part of an AB
system, OCHHCHCH2), 3.74 (dd, 1H, J2,3 = 3.3 Hz, J3,4 = 9.9 Hz,
H-3), 2.03 (s, 3H, OCOCH3), 1.98 (s, 3H, OCOCH3), 1.07 (d,
3H, J5,6 = 6.4 Hz, H-6); 13C NMR (75 MHz, CDCl3) (
a anomer) d
170.6 (OCOCH3), 170.3 (OCOCH3), 134.2 (OCH2CHCH2), 117.0
(OCH2CHCH2), 92.0 (C-1), 73.9 (C-3), 72.6 (C-4), 70.4 (OCH2CHCH2),
69.5 (C-2), 66.1 (C-5), 20.9 and 20.8 (3 ꢂ OCOCH3), 17.4 (C-6); ESI-
MS m/z 310.8 [M+Na]+. To a solution of 2,4-di-O-acetyl-3-O-allyl-
L-
rhamnopyranoside (1.2 g, 4.2 mmol) in CH2Cl2 (20 mL) were added
trichloroacetonitrile (8.4 mL, 84 mmol) and DBU (250, 1.7 mmol)
dropwise at 0 °C. The mixture was then strirred at rt for 2 h and
then concentrated in vacuo. The residue was purified by flash chro-
matography on silica gel using 2% Et3N in cyclohexane/EtOAc (8:2)
as eluent to give trichloroacetimide 103 (1.4 mg, 80%) as a white
4.2.26. 2-[(N-Benzyloxycarbonyl)amino]ethyl 3-O-allyl-
rhamnopyranosyl-(1?3)-4-O-benzyl- -rhamnopyranosyl-
(1?2)-3,4-di-O-benzyl- -rhamnopyranoside (37)
a-L-
a-L
a-L
Compound 36 (116 mg, 0.10 mmol) was treated with NaOMe
(0.2 M solution in MeOH) (3 mL, 0.60 mmol) for 1 h at rt. The reac-
tion mixture was then neutralized by resin Amberlite 120 H+. The
solid as a 20:1 mixture of a/b anomers. Rf 0.76 (cyclohexane/EtOAc

128
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
resin was filtered off and the filtrate was concentrated in vacuo. The
residue was purified by flash chromatography on silica gel using
cyclohexane/EtOAc (7:3) as eluent to provide 37 (66 mg, 70%) as a
99.7 (C-1B), 99.2, 99.1 (C-1C, C-1D), 81.0 (C-4C), 80.5 (C-3D, C-4D),
79.9 (C-4B), 79.8 (C-3B), 78.1 (C-2C), 76.9 (C-3C), 75.7 (C-2B)4,
75.6 (OCH2C6H5), 75.3 (OCH2C6H5), 74.8 (OCH2C6H5), 74.7 (C-2D),
72.7 (OCH2C6H5), 72.5 (OCH2C6H5), 72.4 (OCH2C6H5), 71.1
(OCH2CHCH2), 68.8, 68.7 (C-5C, C-5D), 68.3 (C-5B), 67.5
(NCOOCH2C6H5), 65.7 (OCH2CH2N), 51.8 (NCH2C6H5), 47.0 and
45.9 (OCH2CH2N), 18.1 (C-6B, C-6C, C-6D); HR-ESI-MS m/z calcd
for C80H89NO15 1326.6130 [M+Na]+, found 1326.6152.
white solid. Rf 0.36 (cyclohexane/EtOAc 5:5); [
a
]
D
ꢁ26 (CHCl3, c
0.9); 1H NMR (300 MHz, CDCl3) d 7.37–7.27 (m, 20H, NHCO-
OCH2C6H5, 3 ꢂ OCH2C6H5), 5.99–5.86 (m, 1H, OCH2CHCH2), 5.31–
5.24 (m, 1H, A part of an AB system, OCH2CHCHH), 5.22–5.17 (m,
1H, B part of an AB system, OCH2CHCHH), 5.18 (br s, 1H, H-1B),
5.11 (br s, 2H, NHCOOCH2C6H5), 5.03 (br s, 1H, NHCOOCH2C6H5),
5.00 (d, 1H, J1C,2C = 1.5 Hz, H-1C), 4.82 (d, 1H, JA,B = 11.0 Hz, A part
of an AB system, OCHHC6H5), 4.74 (d, 1H, JA,B = 11.0 Hz, A part of
an AB system, OCHHC6H5), 4.70 (d, 1H, J1D,2D = 1.6 Hz, H-1D), 4.63
(d, 1H, JA,B = 11.0 Hz, B part of an AB system, OCHHC6H5), 4.59 (d,
1H, JA,B = 11.0 Hz, B part of an AB system, OCHHC6H5), 4.16 (dd,
1H, J1C,2C = 1.5 Hz, J2C,3C = 3.2 Hz, H-2C), 4.12–4.10 (m, 1H, A part
of an AB system, OCHHCHCH2), 4.06 (dd, 1H, J2C,3C = 3.2 Hz,
J3C,4C = 9.3 Hz, H-3C), 4.05–4.01 (m, 1H, B part of an AB system,
OCHHCHCH2), 4.00 (dd, 1H, J1B,2B = 1.6 Hz, J2B,3B = 2.4 Hz, H-2B),
3.96–3.94 (m, 1H, H-2D), 3.88–3.79 (m, 2H, H-5B, H-5C), 3.81 (dd,
1H, J2D,3D = 2.8 Hz, J3D,4D = 9.2 Hz, H-3D), 3.72–3.62 (m, 2H, H-5D,
A part of an AB system, OCHHCH2NH), 3.59–3.57 (m, 2H, H-3B, H-
4.2.28. 2-[(N,N0-Benzyl-benzyloxycarbonyl)amino]ethyl 2,4-di-
O-benzyl-
a
-L
-rhamnopyranosyl-(1?3)-2,4-di-O-benzyl-
a-L
-rha
mnopyranosyl-(1?2)-3,4-di-O-benzyl-a-L-rhamnopyranoside
(39)
A
solution of 38 (79 mg, 60
lmol), (Ph3P)3RhCl (4.4 mg,
4.8
l
mol), and DABCO (1.3 mg, 12 lmol), in a 7:3:1 mixture of
EtOH/toluene/H2O (4 mL) was stirred at reflux for 16 h. After filtra-
tion through a CeliteÒ pad, the reaction mixture was concentrated
to dryness and dissolved in a 9:1 mixture of acetone/H2O (1 mL).
HgCl2 (49 mg, 180 lmol) and HgO (1.3 mg, 6 lmol) were then
added. After stirring for 4 h at rt, the reaction mixture was concen-
trated and the residue was dissolved in EtOAc. The organic phase
was washed with brine, dried over Na2SO4, filtered, and concen-
trated. The residue was purified by flash chromatography using
cyclohexane/EtOAc (8:2) as eluent to give 39 (67 mg, 88%) as a white
4B), 3.47 (t, 1H, J3C,4C = J4C,5C = 9.3 Hz, H-4C), 3.42 (t, 1H, J3D,4D
=
J4D,5D = 9.3 Hz, H-4D), 3.50–3.29 (m, 3H, B part of an AB system,
OCHHCH2NH, OCH2CH2NH), 2.27 (m, 3H, 3 ꢂ OH), 1.32–1.26 (m,
9H, H-6B, H-6C, H-6D); 13C NMR (75 MHz, CDCl3)
d
156.4
solid (mixture of conformers). Rf 0.24 (cyclohexane/EtOAc 8:2); [a]
D
(NHCOOCH2C6H5), 138.4, 138.2, 136.5, 128.7, 128.6, 128.5, 128.3,
128.2, 128.0, 127.9, 127.8 (3 ꢂ OCH2C6H5, NHCOOCH2C6H5), 134.3
(OCH2CHCH2), 118.1 (OCH2CHCH2), 101.2, 101.1 (C-1B, C-1C), 99.2
(C-1D), 80.4, 80.3 (C-4C, C-4D), 79.7 (C-3D), 79.5 (C-3B), 79.3 (C-
3C), 75.6, 75.5 (2 ꢂ OCH2C6H5), 75.2 (C-2D), 72.5 (OCH2C6H5), 71.5
(C-4B), 70.9 (C-2C), 70.7 (OCH2CHCH2), 68.7, 68.4, 68.3 (C-5B, C-
5C, C-5D), 68.2 (C-2B), 67.0, 66.8 (NHCOOCH2C6H5, OCH2CH2NH),
41.0 (OCH2CH2NH), 18.1, 17.8 (C-6B, C-6C, C-6D); HR-ESI-MS m/z
calcd for C52H65NO15 966.4252 [M+Na]+, found 966.4240.
+2 (CHCl3, c 0.4); 1H NMR (600 MHz, CDCl3) d 7.27–7.15 (m, 40H,
NCOOCH2C6H5, 6 ꢂ OCH2C6H5, NCH2C6H5), 5.15 (br s, 1H, H-1B),
5.11 (br s, 2H, NCOOCH2C6H5), 5.02 (br s, 1H, H-1C), 4.80 (d,
1H, JA,B = 11.1 Hz, A part of an AB system, OCHHC6H5), 4.78 (d, 1H,
JA,B = 10.5 Hz, A part of an AB system, OCHHC6H5), 4.71 (d,
1H, JA,B = 11.7 Hz, A part of an AB system, OCHHC6H5), 4.64 (d, 1H,
JA,B = 11.7 Hz, B part of an AB system, OCHHC6H5), 4.61 (br s, 1H,
H-1D), 4.55 (d, 1H, JA,B = 11.1 Hz, B part of an AB system, OCHHC6H5),
4.50 (d, 1H, JA,B = 10.5 Hz, B part of an AB system, OCHHC6H5), 4.56–
4.39 (m, 6H, 2 ꢂ OCH2C6H5, NCH2C6H5), 4.26 (d, 1H, JA,B = 11.6 Hz,
A part of an AB system, OCHHC6H5), 4.09 (dd, 1H, J2C,3C = 2.4 Hz,
J3C,4C = 9.6 Hz, H-3C), 4.05–4.02 (m, 1H, B part of an AB system,
OCHHC6H5), 3.89–3.86 (m, 2H, H-3B, H-2D), 3.72 (br s, 1H, H-2C),
3.64 (br s, 1H, H-2B), 3.75–3.62 (m, 3H, H-5C, H-3D, H-5B), 3.62–
3.50 (m, 2H, H-4B, A part of an AB system, OCHHCH2N), 3.50–3.41
(m, 2H, H-5D, H-4C, B part of an AB system, OCHHCH2N), 3.36–
3.25 (m, 3H, H-4D, OCH2CH2NH), 3.24 (t, 1H, J3B,4B = J4B,5B = 9.4 Hz,
H-4B), 1.22 (d, 3H, J5C,6C = 5.9 Hz, H-6C), 1.18–1.16 (m, 9H, H-6B,
H-6D); 13C NMR (75 MHz, CDCl3) d 156.6 and 156.3 (NCOOCH2C6H5),
138.7, 138.3, 137.9, 128.7, 128.6, 128.5, 128.1, 128.0, 127.8, 127.0
(NCOOCH2C6H5, 6 ꢂ OCH2C6H5, NCH2C6H5), 99.3, 99.2 (C-1C, C-
1D), 98.6 (C-1B), 82.3 (C-4B), 81.1 (C-4C), 80.6 (C-4D), 79.8 (C-3D),
79.3 (C-2B), 78.0 (C-2C), 76.7 (C-3C), 75.6 (OCH2C6H5), 75.0
(OCH2C6H5), 74.8 (OCH2C6H5), 74.9 (C-2D), 72.7 (OCH2C6H5), 72.6
(OCH2C6H5), 72.4 (OCH2C6H5), 71.8 (C-3B), 69.0 (C-5D), 68.3 (C-
5B), 67.9 (C-5C), 67.5 (NCOOCH2C6H5), 66.3 (OCH2CH2N), 51.8
(NCH2C6H5), 47.0 and 46.0 (OCH2CH2N), 18.2, 18.1 (C-6B, C-6C, C-
6D); HR-ESI-MS m/z calcd for C77H85NO15 1286.5817 [M+Na]+, found
1286.5774.
4.2.27. 2-[(N,N0-Benzyl-benzyloxycarbonyl)amino]ethyl 3-O-
allyl-2,4-di-O-benzyl-a-L-rhamnopyranosyl-(1?3)-2,4-di-O-
benzyl- -rhamnopyranosyl-(1?2)-3,4-di-O-benzyl-a-L-
a-
L
rhamnopyranoside (38)
To a solution of 37 (66 mg, 70 lmol) in DMF (1 mL) at 0 °C was
added NaH (60% dispersion in mineral oil, 22 mg, 0.56 mmol) fol-
lowed by dropwise addition of BnBr (40 L, 0.34 mmol). The reac-
tion mixture was stirred at rt for 2 h and the excess of NaH was
neutralized by dropwise addition of MeOH (500 L) at 0 °C. The mix-
l
l
ture was concentrated in vacuo and the residue was purified by flash
chromatography using cyclohexane/EtOAc (9:1?7:3) as eluent to
give 39 (79 mg, 87%) as a colorless oil (mixture of conformers). Rf
0.60 (cyclohexane/EtOAc 8:2); [
(600 MHz, CDCl3)
a
]
ꢁ13 (CHCl3, c 1.7); 1H NMR
D
d
7.38–7.17 (m, 40H, NCOOCH2C6H5, 6 ꢂ
OCH2C6H5, NCH2C6H5), 5.93–5.86 (m, 1H, OCH2CHCH2), 5.29–5.26
(m, 1H, A part of an AB system, OCH2CHCHH), 5.19–5.15 (m, 3H,
H-1B, NCOOCH2C6H5), 5.13–5.11 (m, 1H, B part of an AB system,
OCH2CHCHH), 5.06 (d, 1H, J1C,2C = 1.1 Hz, H-1C), 4.95 (d,
1H, JA,B = 11.0 Hz, A part of an AB system, OCHHC6H5), 4.86
(d, 1H, JA,B = 10.9 Hz, A part of an AB system, OCHHC6H5), 4.73 (d,
1H, JA,B = 11.5 Hz, A part of an AB system, OCHHC6H5), 4.67–4.46
(m, 12H, H-1D, 3 ꢂ B part of 3 ꢂ AB system, 3 ꢂ OCHHC6H5,
NCH2C6H5), 4.15 (dd, 1H, J2C,3C = 2.7 Hz, J3C,4C = 9.3 Hz, H-3C), 4.03
(m, 2H, OCH2CHCH2), 3.92 (br s, 1H, H-2D), 3.81–3.76 (m, 7H, H-
2B, H-3B, H-2C, H-5C, H-3D, H-5B, A part of an AB system,
OCHHCH2N), 3.59–3.47 (m, 4H, H-4B, H-5D, H-4C, B part of an AB
system, OCHHCH2N), 3.46–3.30 (m, 3H, H-4D, OCH2CH2NH), 1.28–
1.21 (m, 9H, H-6B, H-6C, H-6D); 13C NMR (75 MHz, CDCl3) d 156.6
and 156.3 (NCOOCH2C6H5), 139.1, 138.6, 138.3, 128.7, 128.6,
128.4, 128.3, 128.0, 127.8, 127.7, 127.6, 127.1 (NCOOCH2C6H5, 6 ꢂ
OCH2C6H5, NCH2C6H5), 135.2 (OCH2CHCH2), 116.5 (OCH2CHCH2),
0
4.2.29. 2-[(N,N -Benzyl-benzyloxycarbonyl)amino]ethyl 2-O-acet
yl-4-azido-3-O-benzyl-4,6-dideoxy-b-
2,4-di-O-benzyl- -rhamnopyranosyl-(1?3)-2,4-di-O-benzyl-
-rhamnopyranosyl-(1?2)-3,4-di-O-benzyl- -rhamnopyra
noside (40)
To a solution of 6 (28 mg, 51.3
and 4 Å molecular sieves (50 mg) in dry CH2Cl2 (2 mL) under inert
L, 25.6 mol). After
D-glucopyranosyl-(1?3)-
a-L
a-L
a-L
l
mol), 39 (50 mg, 39.5 mmol)
atmosphere at ꢁ40 °C was added TMSOTf (1.2
l
l
30 min stirring at this temperature, the reaction mixture was neu-
tralized by Et3N and filtered. The filtrate was washed with water,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
129
was purified by flash chromatography using cyclohexane/EtOAc
(9:1?7:3) as eluent to give 40 (35 mg, 56%) as a colorless oil (mix-
99.2 (C-1C), 99.1 (C-1D), 82.4 (C-3A), 80.9, 80.8, 80.7 (C-4B, C-4C
and C-4D), 80.3 (C-3B), 80.0 (C-3D), 78.6 (C-2B), 78.1 (C-2C), 77.5
(C-3C), 76.0 (C-2A), 75.7 (OCH2C6H5), 75.5 (OCH2C6H5), 75.1
(2 ꢂ OCH2C6H5), 74.8 (C-2D), 73.5 (OCH2C6H5), 72.6 (OCH2C6H5),
72.4 (OCH2C6H5), 71.0 (C-5A), 68.9, 68.8 (C-5B and C-5C), 68.4
(C-5D), 67.6 (NCOOCH2C6H5), 67.4 (C-4A), 65.9 (OCH2CH2N), 51.9
(NCH2C6H5), 47.1 and 46.1 (OCH2CH2N), 18.8 (C-6A), 18.8, 18.3,
18.2 (C-6B, C-6C and C-6D); HR-ESI-MS m/z calcd for
ture of conformers). Rf 0.45 (cyclohexane/EtOAc 7:3); [
a
]
ꢁ4
D
(CHCl3, c 0.5); 1H NMR (600 MHz, CDCl3) d 7.29–7.12 (m, 45H,
NCOOCH2C6H5, 7 ꢂ OCH2C6H5, NCH2C6H5), 5.11 (br s, 2H,
NCOOCH2C6H5), 5.02–5.01 (m, 2H, H-1B, H-2A), 4.99 (br s, 1H, H-
1C), 4.77 (d, 1H, JA,B = 10.8 Hz, A part of an AB system, OCHHC6H5),
4.70 (d, 2H, JA,B = 11.2 Hz, 2 ꢂ A part of an 2 ꢂ AB system,
2 ꢂ OCHHC6H5), 4.62–4.38 (m, 13H, H-1A, H-1D, 3 ꢂ B part of an
3 ꢂ AB system, 3 ꢂ OCHHC6H5), 4.38–4.37 (m, 2H, NCH2C6H5),
4.02 (dd, 1H, J2C,3C = 2.4 Hz, J3C,4C = 9.9 Hz, H-3C), 4.01 (dd, 1H,
J2B,3B = 2.9 Hz, J3B,4B = 9.9 Hz, H-3B), 3.84 (br s, 1H, H-2D), 3.81 (br
s, 1H, H-2B), 3.74 (br s, 1H, H-2C), 3.71–3.67 (m, 4H, H-5B, H-5C,
H-3D, A part of an AB system, OCHHCH2NH), 3.51–3.42 (m, 4H,
H-4B, H-4C, H-5D, B part of an AB system, OCHHCH2N), 3.35–
C
90H100N4O18 1547.6930 [M+Na]+, found 1547.6984.
4.2.31. 2-[(N,N0-Benzyl-benzyloxycarbonyl)amino]ethyl 4-azido-
3-O-benzyl-4,6-dideoxy-2-O-methyl-b- -glucopyranosyl-(1?3)-
2,4-di-O-benzyl- -rhamnopyranosyl-(1?3)-2,4-di-O-benzyl-
-rhamnopyranosyl-(1?2)-3,4-di-O-benzyl- -rhamnopyr
anoside (42)
To a solution of 41 (29 mg, 19.0
NaH (60% dispersion in mineral oil, 1.5 mg, 38.0
lowed by dropwise addition of MeI (12 L, 190
mixture was stirred at rt overnight and the excess of NaH was neu-
tralized by dropwise addition of MeOH (500 L) at 0 °C. The mix-
D
a
-L
a-
L
a-L
3.28 (m, 4H, H-3A, H-4D, OCH2CH2NH), 3.08 (t, 1H, J3A,4A
=
l
mol) in DMF (1 mL) was added
mol) at 0 °C, fol-
mol). The reaction
J4A,5A = 9.4 Hz, H-4A), 3.04–3.02 (m, 1H, H-5A), 1.71 (s, 3H,
OCOCH3), 1.20–1.17 (m, 6H, H-6C, H-6D) 1.14 (d, 3H, J5B,6B = 6.3 Hz,
H-6B), 1.06 (d, 3H, J5A,6A = 5.9 Hz, H-6A); 13C NMR (75 MHz, CDCl3)
d 169.3 (OCOCH3), 156.6 and 156.3 (NCOOCH2C6H5), 138.7, 138.5,
138.4, 128.7, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9,
127.8, 127.7, 127.6, 127.5 (NCOOCH2C6H5, 7 ꢂ OCH2C6H5,
NCH2C6H5), 101.4 (C-1A), 100.5 (C-1B), 99.1 (C-1C and C-1D),
81.6 (C-3A), 80.6 (C-4B, C-4C and C-4D), 79.9 (C-3D), 79.5 (C-3B),
78.9 (C-2B), 78.2 (C-2C), 78.0 (C-3C), 75.6 (OCH2C6H5), 74.8
(2 ꢂ OCH2C6H5), 74.7 (OCH2C6H5), 74.1 (C-2D), 73.6 (C-2A), 72.5
(OCH2C6H5), 72.5 (OCH2C6H5), 72.3 (OCH2C6H5), 70.7 (C-5A), 68.8,
68.6 (C-5B and C-5C), 68.3 (C-5D), 67.8 (C-4A), 67.5
(NCOOCH2C6H5), 65.7 (OCH2CH2N), 51.8 (NCH2C6H5), 47.0 and
46.0 (OCH2CH2N), 18.6, 18.5, 18.2, 18.1 (C-6A, C-6B, C-6C and C-
6D); HR-ESI-MS m/z calcd for C92H102N4O19 1589.7036 [M+Na]+,
found 1589.7084.
l
l
l
l
ture was then evaporated to dryness and the residue was
purified by flash chromatography on silica gel using cyclohexane/
EtOAc (8:2) as eluent to give 42 (29 mg, 96%) as a colorless oil
(mixture of conformers). Rf 0.59 (cyclohexane/EtOAc 7:3); [a] +2
D
(CHCl3, c 1.1); 1H NMR (600 MHz, CDCl3) d 7.37–7.19 (m, 45H,
NCOOCH2C6H5, 7 ꢂ OCH2C6H5, NCH2C6H5), 5.16 (br s, 2H,
NCOOCH2C6H5), 5.11 (br s, 1H, H-1B), 5.06 (br d, 1H, J1C,2C = 1.6 Hz,
H-1C), 4.96 (d, 1H, JA,B = 10.5 Hz,
A part of an AB system,
OCHHC6H5), 4.90 (d, 1H, JA,B = 10.7 Hz, A part of an AB system,
OCHHC6H5), 4.85 (d, 1H, JA,B = 11.0 Hz, A part of an AB system,
OCHHC6H5), 4.79 (d, 1H, JA,B = 10.7 Hz, B part of an AB system,
OCHHC6H5), 4.69 (d, 1H, JA,B = 11.4 Hz, A part of an AB system,
OCHHC6H5), 4.65–4.44 (m, 13H, H-1A, H-1D, 3 ꢂ B part of an
3 ꢂ AB system, 3 ꢂ OCHHC6H5, 3 ꢂ OCH2C6H5, NCH2C6H5), 4.11
(br dd, 2H, J2B,3B = J2C,3C = 3.0 Hz, J3B,4B = J3C,4C = 9.6 Hz, H-3B, H-
3C), 3.84–3.82 (m, 1H, H-2D), 3.82–3.80 (m, 1H, H-2C), 3.81–3.79
(m, 1H, H-2B), 3.79–3.69 (m, 4H, H-5B, H-5C, H-3D, A part of an
AB system, OCHHCH2NH), 3.66–3.49 (m, 4H, H-4B, H-4C, H-5D, B
part of an AB system, OCHHCH2N), 3.63 (s, 3H, OCH3), 3.49–3.33
(m, 4H, H-3A, H-4D, OCH2CH2NH), 3.11 (dd, 1H, J1A,2A = 7.9 Hz,
J2A,3A = 9.0 Hz, H-2A), 3.04–3.02 (m, 2H, H-4A, H-5A), 1.28–1.23
(m, 9H, H-6B, H-6C, H-6D), 1.09 (d, 3H, J5A,6A = 5.7 Hz, H-6A); 13C
NMR (75 MHz, CDCl3) d 156.9 and 156.5 (NCOOCH2C6H5), 138.9,
138.6, 138.3, 128.7, 128.6, 128.4, 128.2, 128.1, 128.0, 127.8,
127.7, 127.5 (NCOOCH2C6H5, 7 ꢂ OCH2C6H5, NCH2C6H5), 103.7 (C-
1A), 100.4 (C-1B), 99.2 (C-1C and C-1D), 85.0 (C-2A), 82.4 (C-3A),
81.1, 80.7, 80.6 (C-4B, C-4C and C-4D), 79.9 (C-3D), 79.5 (C-2B),
78.7 (C-3B), 78.2 (C-2C), 77.4 (C-3C), 75.6 (2 ꢂ OCH2C6H5), 75.0
(C-2D), 74.8 (2 ꢂ OCH2C6H5), 73.5 (OCH2C6H5), 72.5 (OCH2C6H5),
72.3 (OCH2C6H5), 70.4 (C-5A), 68.9, 68.6 (C-5B and C-5C), 68.3
(C-5D), 67.8 (C-4A), 67.5 (NCOOCH2C6H5), 65.7 (OCH2CH2N), 60.9
(OCH3), 51.8 (NCH2C6H5), 47.0 and 45.9 (OCH2CH2N), 18.6 (C-6A),
18.1 (C-6B, C-6C and C-6D); HR-ESI-MS m/z calcd for
0
4.2.30. 2-[(N,N -Benzyl-benzyloxycarbonyl)amino]ethyl 4-azido-
3-O-benzyl-4,6-dideoxy-b-
nzyl- -rhamnopyranosyl-(1?3)-2,4-di-O-benzyl-
pyranosyl-(1?2)-3,4-di-O-benzyl- -rhamnopyranoside (41)
Compound 40 (35 mg, 22.3 mol) was treated by NaOMe (0.2 M
solution in MeOH) (223 L, 44.6 mol) for 12 h at rt. The reaction
D-glucopyranosyl-(1?3)-2,4-di-O-be
a-
L
a-L-rhamno
a-L
l
l
l
mixture was then neutralized by resin Amberlite IR120 H+. The re-
sin was filtered off and the filtrate was concentrated in vacuo. The
residue was purified by flash chromatography on silica gel using
cyclohexane/EtOAc (8:2) as eluent to give 41 (29 mg, 85%) as a col-
orless oil (mixture of conformers). Rf 0.25 (cyclohexane/EtOAc
8:2); [
a]
+8 (CHCl3, c 1.0); 1H NMR (600 MHz, CDCl3) d 7.42–
D
7.19 (m, 45H, NCOOCH2C6H5, 7 ꢂ OCH2C6H5, NCH2C6H5), 5.20 (br
s, 2H, NCOOCH2C6H5), 5.10 (br s, 2H, H-1B, H-1C), 4.91 (d,
1H, JA,B = 11.0 Hz, A part of an AB system, OCHHC6H5), 4.88
(d, 1H, JA,B = 10.9 Hz, A part of an AB system, OCHHC6H5), 4.83
(d, 1H, JA,B = 10.7 Hz, A part of an AB system, OCHHC6H5), 4.80 (d,
1H, JA,B = 11.0 Hz, B part of an AB system, OCHHC6H5), 4.68–4.41
(m, 13H, H-1D, 2 ꢂ B part of an 2 ꢂ AB system, 2 ꢂ OCHHC6H5,
4 ꢂ OCH2C6H5, NCH2C6H5), 4.40 (d, 1H, J1A,2A = 7.7 Hz, H-1A), 4.09
(dd, 1H, J2C,3C = 2.8 Hz, J3C,4C = 9.5 Hz, H-3C), 4.08 (dd, 1H,
J2B,3B = 1.5 Hz, J3B,4B = 9.4 Hz, H-3B), 3.94 (br s, 1H, H-2D), 3.89 (br
s, 1H, H-2B), 3.81 (br s, 1H, H-2C), 3.81–3.71 (m, 4H, H-5B, H-5C,
C
91H102N4O18 1561.7087 [M+Na]+, found 1561.7028.
4.2.32. 2-[(N,N0-Benzyl-benzyloxycarbonyl)amino]ethyl 3-O-
benzyl-4-(3-hydroxy-3-methyl-butanamido)-4,6-dideoxy-2-O-
H-3D,
A part of an AB system, OCHHCH2NH), 3.64 (t, 1H,
J3B,4B = J4B,5B = 9.4 Hz, H-4B), 3.53 (br t, 1H, J1A,2A = 7.8 Hz,
J2A,3A = 8.9 Hz, H-2A), 3.58–3.48 (m, 3H, H-4C, H-5D, B part of an
AB system, OCHHCH2N), 3.43–3.37 (m, 3H, H-4D, OCH2CH2NH),
3.35 (br t, 1H, J2A,3A = J3A,4A = 8.9 Hz, H-3A), 3.08–3.06 (m, 2H, H-
4A, H-5A), 1.28–1.25 (m, 9H, H-6B, H-6C, H-6D), 1.06 (d, 3H,
J5A,6A = 5.4 Hz, H-6A); 13C NMR (75 MHz, CDCl3) d 156.7 and
156.4 (NCOOCH2C6H5), 138.8, 138.6, 138.5, 138.3, 137.9, 128.8,
128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9 (NCO-
OCH2C6H5, 7 ꢂ OCH2C6H5, NCH2C6H5), 104.0 (C-1A), 100.3 (C-1B),
methyl-b-
pyranosyl-(1?3)-2,4-di-O-benzyl-
3,4-di-O-benzyl- -rhamnopyranoside (43)
To a solution of 42 (29 mg, 18.2 mol) in a mixture of CH2Cl2
(250 L) and EtOH (1 mL) was added sodium borohydride
(1.4 mg, 36.4
reaction mixture was stirred at rt for 1 h and then was concen-
trated in vacuo. The residue was dissolved in CH2Cl2. The organic
layer was washed with water and brine, dried over Na2SO4, filtered,
D
-glucopyranosyl-(1?3)-2,4-di-O-benzyl-
a-L-rhamno
a
-L
-rhamnopyranosyl-(1?2)-
a
-L
l
l
lmol) and a catalytic amount of NiCl2ꢀ6H2O. The

130
O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
and concentrated in vacuo to provide 2-[(N,N0-benzyl-benzyloxy-
carbonyl)amino]ethyl 4-amino-3-O-benzyl-4,6-dideoxy-2-O-
methyl-b-glucopyranosyl-(1?3)-2,4-di-O-benzyl- -rhamnopyr-
anosyl-(1?3)-2,4-di-O-benzyl- -rhamnopyranosyl-(1?2)-3,4-
di-O-benzyl- -rhamnopyranoside which was used in the next
tion. [
a
]
D ꢁ13 (CHCl3, c 1.2); 1H NMR (600 MHz, CDCl3) d 5.07 (br s,
1H, H-1B), 4.98 (br s, 1H, H-1C), 4.96 (br s, 1H, H-1D), 4.75 (m, 1H,
H-1A (+D2O)), 4.30 (br s, 1H, H-2B), 4.17 (br s, 1H, H-2C), 4.02–3.98
(m, 3H, H-2D, H-3B, A part of an AB system, OCHHCH2NH3OOCCF3),
3.93–3.92 (m, 1H, H-3D), 3.89–3.81 (m, 3H, H-5B, H-3C, H-5C),
3.75–3.67 (m, 2H, H-5D, B part of an AB system, OCHHCH2N-
H3OOCCF3), 3.67–3.62 (m, 2H, H-4A, H-4D), 3.65 (s, 3H, OCH3),
3.59–3.51 (m, 4H, H-3A, H-4B, H-4C, H-5A), 3.28 (br s, 2H,
OCH2CH2NH3OOCCF3), 3.16 (br t, J = 8.4 Hz, H-2A), 2.48 (br s, 2H,
NHCOCH2C(CH3)2OH), 1.36–1.30 (m, 15H, NHCOCH2C(CH3)2OH,
H-6B, H-6C, H-6D), 1.25 (d, 3H, J5A,6A = 5.7 Hz, H-6A); 13C NMR
a-L
a
-L
a
-L
step without purification. To a solution of crude amine in dry
DMF (1 mL) was added dropwise 3-hydroxy-3-methylbutanoic
acid (3
lL, 21.8
lmol), followed by HATU (8.8 mg, 21.8
lmol),
and then DIPEA (4
l
L, 21.8 mol). The reaction mixture was stirred
l
under argon at rt for 18 h and was then diluted with CH2Cl2,
washed with saturated aqueous NaHCO3, and water. The organic
phase was dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography on silica gel
using cyclohexane/EtOAc (6:4) as eluent to provide 43 (12 mg,
40% over 2 steps) as a colorless oil (mixture of conformers). Rf
(75 MHz, CDCl3)
d
174.1 (NHCOCH2C(CH3)2OH), 163.0 (q,
2JC,F = 36 Hz, CF3COO), 116.3 (q, JC,F = 292 Hz, CF3COO), 103.6 (C-
1A), 102.1, 102.0 (C-1B and C-1C), 98.5 (C-1D), 83.3 (C-2A), 79.6
(C-2D), 78.4 (C-3B), 77.9 (C-3C), 72.8 (C-3A), 72.0 (C-4D), 71.3
and 71.1 (C-4B and C-4C), 70.8 (C-5A), 70.2 (NHCOCH2C(CH3)2OH),
69.8 (C-2C, C-2B, C-3D), 69.4, 69.3 (C-5B and C-5C), 69.1 (C-5D),
63.5 (OCH2CH2NH3OOCCF3), 60.0 (OCH3), 56.6 (C-4A), 48.9
(NHCOCH2C(CH3)2OH), 39.0 (OCH2CH2NH3OOCCF3), 28.3, 28.1
(NHCOCH2C(CH3)2OH), 17.0 (C-6A), 16.7, 16.6 (C-6B, C-6C and C-
6D); HR-ESI-MS m/z calcd for C32H58N2O18 759.3763 [M+H]+, found
759.3745.
1
0.56 (cyclohexane/EtOAc 5:5); [
(600 MHz, CDCl3)
OCH2C6H5, NCH2C6H5), 5.32 (br s, 1H, NH), 5.18 (br s, 2H,
NCOOCH2C6H5), 5.10 (br s, 1H, H-1B), 5.05 (d, 1H, J1C,2C = 1.8 Hz,
a
]
ꢁ32 (CHCl3, c 1.2); 1H NMR
D
d
7.42–7.12 (m, 45H, NCOOCH2C6H5, 7 ꢂ
H-1C), 5.00 (d, 1H, JA,B = 10.6 Hz,
A part of an AB system,
OCHHC6H5), 4.86 (d, 1H, JA,B = 11.4 Hz, A part of an AB system,
OCHHC6H5), 4.68 (d, 1H, JA,B = 11.4 Hz, A part of an AB system,
OCHHC6H5), 4.65–4.46 (m, 14H, H-1A, H-1D, 4 ꢂ B part of an
4 ꢂ AB system, 4 ꢂ OCHHC6H5, 3 ꢂ OCH2C6H5, NCH2C6H5), 4.13–
4.11 (m, 2H, H-3B, H-3C), 3.92–3.91 (m, 2H, H-2B, H-2D), 3.83–
3.79 (m, 2H, H-5B, H-2C), 3.79–3.71 (m, 3H, H-5C, H-3D, A part
of an AB system, OCHHCH2NH), 3.67–3.62 (m, 3H, H-4A, H-5A,
H-4B), 3.64 (s, 3H, OCH3), 3.58–3.47 (m, 3H, H-4C, H-5D, B part
of an AB system, OCHHCH2N), 3.47–3.34 (m, 3H, H-4D,
OCH2CH2NH), 3.33 (br t, J = 9.2 Hz, H-3A), 3.18 (dd, 1H,
J1A,2A = 7.8 Hz, J2A,3A = 8.7 Hz, H-2A), 2.22–2.18 (m, 1H, A part of
an AB system, NHCOCHHC(CH3)2OH), 2.11–2.04 (m, 1H, B part of
an AB system, NHCOCHHC(CH3)2OH), 1.26 (br s, 6H, NHC-
OCH2C(CH3)2OH), 1.35–1.19 (m, 9H, H-6B, H-6C, H-6D), 1.04 (d,
3H, J5A,6A = 6.0 Hz, H-6A); 13C NMR (75 MHz, CDCl3) d 172.5
(NHCOCH2C(CH3)2OH), 156.9 and 156.4 (NCOOCH2C6H5), 139.1,
138.9, 138.6, 138.4, 137.9, 137.8, 128.7, 128.6, 128.4, 128.3,
128.2, 127.8, 127.5 (NCOOCH2C6H5, 7 ꢂ OCH2C6H5, NCH2C6H5),
103.9 (C-1A), 100.5 (C-1B), 99.3 (C-1C and C-1D), 85.1 (C-2A),
81.1 (C-4B), 80.7 (C-4C), 80.7 (C-4D), 80.3 (C-3A), 79.9 (C-3D),
79.4 (C-2B), 79.0 (C-3B), 78.1 (C-2C), 77.3 (C-3C), 75.6 (OCH2C6H5),
75.0 (C-2D), 74.8 (2 ꢂ OCH2C6H5), 74.7 (OCH2C6H5), 73.9
(OCH2C6H5), 72.4 (OCH2C6H5), 72.3 (OCH2C6H5), 71.1 (C-5A), 69.6
(NHCOCH2C(CH3)2OH), 68.9 (C-5C), 68.6 (C-5B), 68.3 (C-5D), 67.5
(NCOOCH2C6H5), 65.7 (OCH2CH2N), 60.7 (OCH3), 55.9 (C-4A), 51.9
and 51.8 (NCH2C6H5), 47.9 (NHCOCH2C(CH3)2OH), 47.0 and 46.0
(OCH2CH2N), 18.3, 18.2, 18.1 (C-6A, C-6B, C-6C and C-6D); HR-
ESI-MS m/z calcd for C96H112N2O20 1635.7706 [M+Na]+, found
1635.7695.
Acknowledgement
The authors are grateful to the Direction Générale de l’Arme-
ment for financial support to O.M.
References
1. Borio, L.; Frank, D.; Mani, V.; Chiriboga, C.; Pollanen, M.; Ripple, M.; Ali, S.;
DiAngelo, C.; Lee, J.; Arden, J.; Titus, J.; Fowler, D.; O’Toole, T.; Masur, H.;
Bartlett, J.; Inglesby, T. JAMA, J. Am. Med. Assoc. 2001, 286, 2554–2559.
2. Cybulski, R. J., Jr.; Sanz, P.; O’Brien, A. D. Mol. Aspects Med. 2009, 30, 490–502.
3. Bouzianas, D. G. J. Med. Chem. 2010, 53, 4305–4333.
4. Hudson, M. J.; Beyer, W.; Böhm, R.; Fasanella, A.; Garofolo, G.; Golinski, R.;
Goossens, P. L.; Hahn, U.; Hallis, B.; King, A.; Mock, M.; Montecucco, C.; Ozin, A.;
Tonello, F.; Kaufmann, S. H. E. Int. J. Me Microbiol. 2008, 298, 345–364.
5. Jones, C. An. Acad. Bras. Cienc. 2005, 77, 293–324.
6. Daubenspeck, J. M.; Zeng, H.; Chen, P.; Dong, S.; Steichen, C. T.; Krishna, N. R.;
Pritchard, D. G.; Turnbough, C. L. J. Biol. Chem. 2004, 279, 30945–30953.
7. (a) Werz, D. B.; Seeberger, P. H. Angew. Chem., Int. Ed. 2005, 44, 6315–6318; (b)
Werz, D. B.; Adibekian, A.; Seeberger, P. H. Eur. J. Org. Chem. 2007, 1976–1982.
ˇ
8. (a) Adamo, R.; Saksena, R.; Kovác, P. Carbohydr. Res. 2005, 340, 2579–2582; (b)
Adamo, R.; Saksena, R.; Kovác, P. Helv. Chim. Acta 2006, 89, 1075–1089.
9. (a) Saksena, R.; Adamo, R.; Kovác, P. Bioorg. Med. Chem. 2006, 16, 615–617; (b)
ˇ
ˇ
ˇ
Saksena, R.; Adamo, R.; Kovác, P. Bioorg. Med. Chem. 2007, 15, 4283–4310.
10. Crich, D.; Vinogradova, O. J. Org. Chem. 2007, 72, 6513–6520.
11. (a) Guo, H.; O’Doherty, G. A. Angew. Chem., Int. Ed. 2007, 46, 5206–5208; (b)
Guo, H.; O’Doherty, G. A. J. Org. Chem. 2008, 73, 5211–5220.
ˇ
12. Hou, S.; Kovác, P. Synthesis 2009, 2009, 545–550.
13. Wang, Y.; Liang, X.; Wang, P. Tetrahedron Lett. 2011, 52, 3912–3915.
14. (a) Tamborrini, M.; Werz, D. B.; Frey, J.; Pluschke, G.; Seeberger, P. H. Angew.
Chem., Int. Ed. 2006, 45, 6581–6585; (b) Tamborrini, M.; Oberli, M. A.; Werz, D.
B.; Schürch, N.; Frey, J.; Seeberger, P. H.; Pluschke, G. J. Appl. Microbiol. 2009,
106, 1618–1628; (c) Oberli, M. A.; Tamborrini, M.; Tsai, Y.-H.; Werz, D. B.;
Horlacher, T.; Adibekian, A.; Gauss, D.; Möller, H. M.; Pluschke, G.; Seeberger, P.
H. J. Am. Chem. Soc. 2010, 132, 10239–10241.
4.2.33. 2-Aminoethyl 4-(3-hydroxy-3-methyl-butanamido)-4,6-
dideoxy-2-O-methyl-b-
D
-glucopyranosyl]-(1?3)- -rhamno
a
-
L
ˇ
15. (a) Wang, D.; Carroll, G. T.; Turro, N. J.; Koberstein, J. T.; Kovác, P.; Saksena, R.;
pyranosyl-(1?3)-
a-
L-rhamnopyranosyl-(1?2)-a-L-rhamnopyr
Adamo, R.; Herzenberg, L. A.; Herzenberg, L. A.; Steinman, L. Proteomics 2007, 7,
180–184; (b) Kuehn, A.; Kovác, P.; Saksena, R.; Bannert, N.; Klee, S. R.; Ranisch,
anoside (2)
ˇ
H.; Grunow, R. Clin. Vaccine Immunol. 2009, 16, 1728–1737.
16. Werz, D. B.; Seeberger, P. H.; Tamborrini, M.; Pluschke, G. U.S. Patent No
7,901,687, 2011.
17. Dhénin, S. G. Y.; Moreau, V.; Morel, N.; Nevers, M.-C.; Volland, H.; Créminon, C.;
Djedaïni-Pilard, F. Carbohydr. Res. 2008, 343, 2101–2110.
18. Dhénin, S. G. Y.; Moreau, V.; Nevers, M.-C.; Créminon, C.; Djedaïni-Pilard, F. Org.
Biomol. Chem. 2009, 7, 5184–5199.
19. Mehta, A. S.; Saile, E.; Zhong, W.; Buskas, T.; Carlson, R.; Kannenberg, E.; Reed,
Y.; Quinn, C. P.; Boons, G.-J. Chem. Eur. J. 2006, 12, 9136–9149.
20. Kuhn, R.; Trischmann, H.; Löw, I. Angew. Chem. 1955, 67, 32.
21. Milhomme, O.; John, C.; Djedaïni-Pilard, F.; Grandjean, C. J. Org. Chem. 2011, 76,
5985–5998.
Compound 43 (12 mg, 7.8
lmol) in MeOH/AcOH (12 mL/
120 L) was treated with a catalytic amount of a Pd/C under 10
l
bars of hydrogen at 50 °C for 30 min. The reaction mixture was
then concentrated in vacuo. The residue was dissolved in H2O
and was washed with CH2Cl2. The organic layer was extracted by
water. The aqueous phases were combined and concentrated un-
der vacuum. The crude was then purified by RP-HPLC (0% B during
5 min, 0–100% solvent B over 25 min, 100% B, 5 min [eluent A:
0.05% TFA in H2O; eluent B: 0.05% TFA in CH3CN:H2O 60:40], Grace
Prevail C18 column, 3 mL/min, 215 nm) to give tetrasaccharide 2
(6 mg, 88%, with counterion TFA) as a white solid after lyophiliza-
22. Plettenburg, O.; Bodmer-Narkevitch, V.; Wong, C.-H. J. Org. Chem. 2002, 67,
4559–4564.

O. Milhomme et al. / Carbohydrate Research 356 (2012) 115–131
131
23. Mukhopadhyay, B.; Field, R. A. Carbohydr. Res. 2003, 338, 2149–2152.
24. Handa, V. K.; Piskorz, C. F.; Barlow, J. J.; Matta, K. L. Carbohydr. Res. 1979, 74,
C5–C7.
25. Byramova, N. É.; Ovchinnikov, M. V.; Backinowsky, L. V.; Kochetkov, N. K.
Carbohydr. Res. 1983, 124, C8–C11.
26. Sandrine Dhénin, Ph.D. Thesis, Université de Picardie Jules Verne, Amiens,
France, 2008.
27. Note that, among the classical ester protective groups orthogonal to the
benzoyl, the choice of acetate was mandatory for the preparation of donor 6.
For example, chloroacetate does not survive nucleophilic azide substitution
steps while levulinate was not compatible with the acid-catalyzed benzylation.
28. Kim, Y.-J.; Wang, P.; Navarro-Villalobos, M.; Rohde, B. D.; DerryBerry; Gin, D. Y.
J. Am. Chem. Soc. 2006, 128, 11906–11915.
29. Severn, W. B.; Richards, J. C. J. Am. Chem. Soc. 1993, 115, 1114–1120.
30. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21–123.
31. Barbier, M.; Grand, E.; Kovensky, J. Carbohydr. Res. 2007, 342, 2635–2640.
32. Alpe, M.; Oscarson, S. Carbohydr. Res. 2002, 337, 1715–1722.
33. Bebault, G.; Dutton, G. G. S.; Warfield, C. Carbohydr. Res. 1974, 34, 174–179.