Concise synthesis of di- and trisaccharides related to the O-antigens from Shigella flexneri serotypes 6 and 6a, based on late stage mono-O-acetylation and/or site-selective oxidation

Article abstract of DOI:10.1016/j.tet.2013.10.011

Shigella flexneri serotypes 6 and 6a are closely related bacteria causing shigellosis in humans. Their O-antigens are {→4)-β-d-GalpA-(1→3)- β-d-GalpNAc-(1→2)-[3Ac/4Ac]-α-l-Rhap-(1→2) -α-l-Rhap-(1→}_n acidic polysaccharides ({AB _AcCD}_n), which only differ in the degree of O-acetylation. A concise synthesis of two disaccharides (BC, B_AcC) and four trisaccharides, representing portions and/or analogs of the O-antigens, is described. A protected intermediate compatible with late stage 3 _C-O-acetylation, and/or galactosyl (A) to galacturonic acid (A) conversion, was designed and assembled from trichloroacetimidate and thioglycoside donors tuned for high yielding glycosylation and excellent stereocontrol. The galacturonic moiety was efficiently introduced from galactose using a TEMPO/NaOCl/NaClO₂-based oxidation protocol optimized for full compatibility with sensitive moieties, such as allyl ethers and acetates. Final Pd/C-mediated deprotection provided the targets, including the propyl glycoside AB_AcC, its non O-acetylated counterpart ABC, and the non acidic analogs A B_AcC and A BC. The BC and ABC oligosaccharides are also portions of the O-antigen from Escherichia coli O147, which causes diarrhea in pigs.

Full text of DOI:10.1016/j.tet.2013.10.011

Tetrahedron 69 (2013) 10337e10350
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Concise synthesis of di- and trisaccharides related to the O-antigens
from Shigella flexneri serotypes 6 and 6a, based on late stage mono-
O-acetylation and/or site-selective oxidation
,
b
,
c
,
,
b
,
,b
Pierre Chassagne a
y, Laurent Raibaut a z, Catherine Guerreiro a
,
Laurence A. Mulard a
,b,
*
a
ꢀ
ꢀ
Institut Pasteur, Unite de Chimie des Biomolecules, 28 rue du Dr Roux, 75015 Paris, France
b CNRS UMR3523 Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France
c
ꢀ
ꢀ
Universite Paris Descartes Sorbonne Paris Cite, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2013
Received in revised form 1 October 2013
Accepted 4 October 2013
Available online 11 October 2013
Shigella flexneri serotypes 6 and 6a are closely related bacteria causing shigellosis in humans. Their O-
antigens are {/4)- -GalpA-(1/3)- -GalpNAc-(1/2)-[3Ac/4Ac]- -Rhap-(1/2)- -Rhap-(1/}n
b
-D
b-
D
a
-L
a-L
acidic polysaccharides ({ABAcCD}n), which only differ in the degree of O-acetylation. A concise synthesis
of two disaccharides (BC, BAcC) and four trisaccharides, representing portions and/or analogs of the O-
antigens, is described. A protected intermediate compatible with late stage 3C-O-acetylation, and/or
galactosyl (Aꢀ) to galacturonic acid (A) conversion, was designed and assembled from tri-
chloroacetimidate and thioglycoside donors tuned for high yielding glycosylation and excellent stereo-
control. The galacturonic moiety was efficiently introduced from galactose using a TEMPO/NaOCl/
NaClO2-based oxidation protocol optimized for full compatibility with sensitive moieties, such as allyl
ethers and acetates. Final Pd/C-mediated deprotection provided the targets, including the propyl gly-
coside ABAcC, its non O-acetylated counterpart ABC, and the non acidic analogs AꢀBAcC and AꢀBC. The BC
and ABC oligosaccharides are also portions of the O-antigen from Escherichia coli O147, which causes
diarrhea in pigs.
Keywords:
Carbohydrates
Glycosylation
Gram negative bacteria
Lipopolysaccharide
TEMPO
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
avoids the handling of LPS. Our approach is based on the molecular
design of synthetic oligosaccharide haptens to serve as functional
Shigella flexneri is a human Gram negative entero-invasive
bacterium that is a common cause of diarrhea worldwide.1e3 It
remains a major burden in developing countries, especially in the
pediatric population.2,4,5 All known serotypesdvarying in geo-
graphic and temporal distributionsdcan be isolated from patients,4
and epidemiological surveys indicate an increasing occurrence of S.
flexneri serotype 6 (SF6) in several settings.2,6 This serotype was
identified as one to include in a multivalent Shigella vaccine.4,7
As for other Shigellae, the polysaccharide partdor O-antigen (O-
Ag)dof the SF6 lipopolysaccharide (LPS) is thought to be a primary
target of the host’s humoral immune response generated following
natural infection. Hence, SF6 detoxified LPS-conjugate vaccine
prototypes were recently documented.8 As part of our current effort
toward a potent Shigella glycovaccine, we pursue a strategy that
mimics of the natural O-Ag of interest.9 Access to well-defined
synthetic frame-shifted fragments of the O-Ag is a pre-required
step to warrant a successful development.10,11
Recently, we have confirmed the molecular composition of the
SF6 O-Ag (Fig. 1),14 the basic RU of which is a linear acidic tetra-
saccharide made of one
D
-galacturonic acid (A), one N-acetyl-
D-
galactosamine (B) and two
L-rhamnose residues (C, D), with
rhamnose C being unevenly monoacetylated at its 3- or 4-OH.
Moreover, in a recent survey of S. flexneri O-Ag structures, Per-
epelov et al. distinguish between type SF6 (minor) and type SF6a
(major), which differ only in their degree of 3C/4C-O-acetylation.12
As part of our general concern for S. flexneri O-Ags, we initiated
a careful investigation of the role of the 3C/4C-O-acetylation pattern
on O-Ag properties in the case of SF6 and SF6a. Toward this aim, we
first reported the synthesis of a set of SF6-specific di- to tetra-
saccharides having a
-galacturonic acid (A) at the reducing end.14
D
* Corresponding author. Tel.: þ33 140613820; fax: þ33 145688404; e-mail ad-
Herein, we describe the linear synthesis of 6 frame-shifted di- to
trisaccharides having rhamnose 3AcC at their reducing end. The non
O-acetylated analogs encompassing rhamnose C were isolated as
well, together withdwhenever possibledthose having rhamnose
dress: laurence.mulard@pasteur.fr (L.A. Mulard).
y
Present address: Glycom A/S, DTU, Bld 201, 2800 Kgs Lyngby, Denmark.
Present address: UMR CNRS 8161, Universite Lille Nord de France, Institut
z
ꢀ
Pasteur de Lille, 59021 Lille, France.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.10.011

10338
P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
4AcC. Note that the 4C-O-acetylated regioisomers impersonate the
minor O-acetylation pattern of the SF6 and SF6a O-Ags.12,14 More-
over, the non O-acetylated oligosaccharides are representative of
portions of the natural SF6 and SF6a O-Ags on the one hand12,14 and
of fragments of the closely related O-Ag from Escherichia coli O147
on the other hand.13 In view of appreciating the influence of the
uronate function on the O-Ag antigenic and/or immunogenic
properties, analogs comprising a galactose residue (Aꢀ) instead of
the natural galacturonic acid (A) were also prepared by advanta-
geously exploiting selected intermediates.
Under the best conditions (ꢁ65 to ꢁ40 ꢀC, 1.2 equiv of donor in
DCM), only traces of the unwanted a-linked condensation product
were formed. The isolated yield of the required
b-(1/2)-linked
disaccharide reached 78%. Treatment of the fully protected di-
saccharide 5 with Pd/C under a hydrogen atmosphere enabled the
concomitant benzyl protecting group removal, allyl reduction, and
hydrodechlorination of the 2B-trichloroacetamide. The target BC
propyl glycoside 8 was obtained in 77% yield following RP-HPLC
purification (Scheme 1). Besides, CAN-mediated oxidative cleavage
of the PMB ether in disaccharide 5 (64%) and subsequent acetylation
of the unmasked hydroxyl group in alcohol 6 furnished the 3C-O-
acetyl analog 7 (97%). Likewise, an ethanolic solution of the latter
was treated with Pd/C under a hydrogen atmosphere to give the
B3AcC-Pr disaccharide 9 in 67% following RP-HPLC purification. The
regioisomer resulting from the possible acetyl migration onto the
vicinal hydroxyl group was not identified.
Fig. 1. Repeating unit (RU, ABAcCD) of the O-Ags from SF6 and SF6a showing the non
stoichiometric acetylation at O-3C/O-4C.12 The corresponding non O-acetylated ABCD
tetrasaccharide defines the O-Ag from Escherichia coli O147.13
2. Results and discussion
All target oligosaccharides were prepared as a-propyl glycosides
in order to block their reducing end in a form mimicking the natural
linkages found in the O-Ag. The choice of propyl glycosides is in line
with our previous work on this series,14 as well as on other S.
flexneri serotypes.15 It is meant at facilitating the synthesis, owing
to the easy access to allyl glycoside building blocks, in the absence
of any anticipated major influence on antigenicity.
The strategy was designed in order to warrant the isolation of
six oligosaccharides, namely the non O-acetylated targets BC-Pr (8),
AꢀBC-Pr (29), and ABC-Pr (32) as well as their O-acetylated coun-
terparts B3AcC-Pr (9), AꢀB3AcC-Pr (27), and AB3AcC-Pr (30). The 3C-
O-acetyl moiety (Ac) was introduced at an intermediate stage of the
synthesis, to serve either as a required substituent or as a protecting
group. The corresponding hydroxyl group was initially masked by
use of a para-methoxybenzyl (PMB) ether. Thus, stepwise chain
extension starting from a 3-O-PMB-rhamnoside acceptor serving as
a common precursor to residue C or 3AcC, enabled a careful in-
vestigation and optimization of each glycosylation step. Among the
numerous N-protecting groups ensuring anchimeric assistance in
the formation of 1,2-trans glycosidic linkages involving 2-
acetamido-glycosyl donors,16 such as that required in the [BþC]
glycosylation, we favored the trichloroacetamide moiety owing to
Scheme 1. Synthesis of the BC-Pr (8) and B3AcC-Pr (9) disaccharides. Reagents and
conditions: (a) (i) Bu2SnO, toluene, reflux, 5 h; (ii) PMBCl, TBAI, CsF, reflux, 5 h (81% for
2, 8% for 3); (b) NIS/TMSOTf, DCM, ꢁ65 to ꢁ40 ꢀC, 1 h (78%); (c) H2, Pd/C 10%, EtOH, rt,
48 h (77% for 8, 67% for 9); (d) CAN, MeCN/H2O, rt, 1 h (64%); (e) Ac2O, pyridine, rt, 24 h
(97%).
2.2. Synthesis of trisaccharides AꢀB3AcC-Pr (27), AꢀBC-Pr (29),
AB3AcC-Pr (30), and ABC-Pr (32)
In contrast to the above synthesis, chain elongation at the 3B-OH
is required to reach any of the trisaccharide targets. Thus, starting
from the same rhamnoside acceptor 2, two routes to the di-
saccharide acceptor 14 were investigated (Scheme 2). The simplest
one (Route A) involved the readily accessible peracetylated thio-
glycoside 12,14 and functionalization post glycosylation. Several
attractive odorless thioglycoside donors have been proposed in
oligosaccharide synthesis.20e22 Along this line, route A was also
investigated using thioglycoside 11, a closely related analog of do-
nor 12, which was easily obtained from commercially available 2-
methyl-5-tert-butylthiophenol.22 In the second route (Route B),
the galactosamine residue was introduced using a novel orthogo-
nally protected donor 17.
its powerful b-directing effect and possible conversion to an acet-
amide in the presence of an acetate.17
2.1. Synthesis of disaccharides BC-Pr (8) and B3AcC-Pr (9)
Tin-mediated introduction of a PMB moiety at O-3 of diol 118 was
achieved in a high 81% yield on a multigram scale providing the
known rhamnoside acceptor 219 together with the yet unreported
regioisomer 3 (8%). Several 2-deoxy-2-N-trichloroacetyl D-galacto-
syl donors acting as chain terminators could be envisioned as pre-
cursors to residue B. We reasoned that, to prevent extensive side
reactions at the 3C-acetate site, the trichloroacetamide to acetamide
conversion would be best performed under Pd/C hydrogen-
mediated hydrodechlorination.17 Therefore, a 3,4,6-tri-O-benzyl
donor was selected as precursor to residue B. When a DCM solution
Donors 12 and 11 were obtained in three steps from D-galac-
tosamine hydrochloride in 64% and 56% yield, respectively. The NIS/
TMSOTf-promoted condensation of allyl glycoside 2 and thio-
of the known thiophenyl
b
-
D
-glycoside14 4 and rhamnoside 2 was
glycoside 12 or 11, run in DCM at 0 ꢀC, provided the desired
b
-
1
treated with the NIS/TMSOTf activator system at 0 ꢀC, the
b-con-
(1/2)-linked disaccharide 13 (NMR data for C-1B: JCH 161.5 Hz,
and H-1B: 1J1,2 8.5 Hz) in a disappointing yield (Table 1, entries 1 &
2). Identification of the known oxazoline23 15 (NMR data for H-1:
1
densation product 5 (NMR data for C-1B: JCH 163.5 Hz, and H-1B:
1J1,2 8.4 Hz) was isolated as the major product (69%). However, the
product of
a
-glycosylation 10, identified by mass spectrometry and
d
6.24 ppm, 1J1,2 7.0 Hz) in admixture with unreacted 2 encouraged
1
1
NMR analysis (NMR data for C-1B: JCH 174.5 Hz, and H-1B: J1,2
3.5 Hz), was isolated in a significant 13% yield. To our satisfaction,
additional investigation toward improved reaction conditions.
Changing the promoter system from NIS/TMSOTf to NIS/TfOH had
no influence (Table 1, entries 1 & 3). In contrast, increasing the
lowering the reaction temperature improved the
a/b selectivity.

P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
10339
Scheme 2. Synthesis of disaccharides 14 and 17 from rhamnoside 2. Reagents and conditions: (a) 2, DCM, see Table 1; (b) (i) NaOMe, MeOH, rt; (ii) PhCH(OMe)2, CSA, MeCN, rt (84%
for 14, 93% for 16); (c) LevOH, DCC, DMAP, DCM, rt, 1 h (90%); (d) H2NNH2$H2O, AcOH/pyridine, 0 ꢀC to rt, 1 h (86%).
Table 1
a side-product during protocol optimization. Alike in the synthesis
of disaccharide 13, the 1,2-cis linked isomer was not observed. This
outcome diverges from previous reports involving donors closely
Conditions for the synthesis of disaccharides 13 and 18 from acceptor 2
Entry Donor (equiv) Promoter Temperature Time
Product/
Yield %
related to thioglycoside 17.24 It substantiates the powerful
b-
1
2
3
4
5
6
7
8
9
12 (1.2)
11 (1.2)
12 (1.2)
12 (1.2)
11 (1.2)
12 (1.5)
17 (1.1)
17 (1.2)
17 (1.2)
NIS (1.5)/TMSOTf (0.1)
NIS (1.5)/TMSOTf (0.1)
NIS (1.5)/TfOH (0.1)
NIS (1.5)/TfOH (0.1)
NIS (1.5)/TfOH (0.1)
NIS (1.5)/TfOH (0.1)
NIS (1.5)/TMSOTf (0.1)
NIS (1.5)/TfOH (0.1)
0
0
0
0
0
ꢀC to rt
ꢀC to rt
ꢀC to rt
ꢀC to rt
ꢀC to rt
1 h
1 h
1 h
2 h
13/56
13/54
13/54
13/63
13/62
13/73
directing effect of the trichloroacetamido moiety in 4,6-O-benzyli-
dene protected galactosamine donors, and confirms the hypothesis
that despite its rigidity, the 1,3-dioxane ring in galacto-derivatives
1.5 h
does not interfere with a b-coupling transition state in the absence
ꢁ60 ꢀC to rt 3 h
of any donor/acceptor mismatched pairing.24 Furthermore, excess
of donor 17, longer reaction time, and lower reaction temperature
resulted in a higher yield of disaccharide 18 (Table 1, entries 7e10),
whereas changing the promoter system from NIS/TfOH to BSP/
TTBP/Tf2O25 impaired the glycosylation outcome (Table 1, entries 11
& 12). Under the best identified conditions (ꢁ65 to 40 ꢀC, 1.5 equiv
of donor 17 in DCM), the fully protected BC disaccharide 18 was
isolated in 82% yield (Scheme 2 and Table 1, entry 10). Conventional
hydrazinolysis of the 3B-levulinoyl ester gave alcohol 14 (86% from
18). This second route provided acceptor 14, serving as precursor to
trisaccharides 27, 29, 30, and 32, in two steps and 71% yield from
allyl rhamnoside 2. When referring to acceptor consumption,
comparison of the two routes to the BC intermediate 14 supports
our preference for the pre-functionalized donor 17. However, since
route B involves two extra functionalization steps, it may not
necessarily be favored when engaging easily accessible acceptors,
such as rhamnoside 2. Interestingly, the conditions and outcome of
the [17þ2] condensation resemble that of the [12þ2] coupling,
which suggests that the protecting pattern of donor B does not
interfere.
0
0
ꢀC to rt
ꢀC to rt
45 min 18/64
1.5 h 18/62
20 min 18/69
NIS (1.5)/TMSOTf (0.1) ꢁ65 ꢀC to
ꢁ40 ꢀC
10
17 (1.5)
17 (1.2)
17 (1.2)
NIS (1.5)/TfOH (0.1)
ꢁ65 ꢀC to
ꢁ40 ꢀC
1.5 h
18/82
11a
12a
BSP (1.3)/TTBP (2.0)/
Tf2O (1.2)
BSP (1.5)/TTBP (1.8)/
Tf2O (1.5)
ꢁ65 ꢀC to
ꢁ40 ꢀC
50 min 18/47
ꢁ60 ꢀC to rt 2 h
18/38
a
Pre-activation system, inverse procedure.
reaction time resulted in a slightly higher yield (Table 1, entries 1 &
4, 2 & 5). Under the conditions used, the choice of thioglycoside
donor 11 versus 12 had no noticeable impact on the outcome of the
glycosylation reaction. Finally, lowering the temperature at which
the promoter was added favored glycosylation over oxazoline for-
mation. Under the best identified conditions (Table 1, entry 6), di-
saccharide 13 was isolated in an acceptable 73% yield (Scheme 2).
To our satisfaction, the corresponding
a
-(1/2)-linked condensa-
ꢀ
tion product was not detected. Zemplen deacetylation of in-
termediate 13 gave the corresponding triol, which was readily
converted into acceptor 14 by regioselective 4,6-O-benzylidenation
(84% over two steps). Following this route, compound 14 was
prepared in 61% yield over three steps starting from rhamnoside 2.
In route B, phenyl thioglycoside 17 served as a pre-functionalized
galactosaminyl donor, which is easily accessible from triacetate 12.
The introduction of the galacturonic acid residue could be envi-
sioned according to either the ‘post glycosylation oxidation’ or the
‘pre glycosylation oxidation’ strategies.26 The latter route remains
poorly exemplified for the formation of b-D
-galacturonate linkages.27
In contrast, several reports attest of the efficiency of the former,
which advantageously allows the synthesis of both the uronic
targets and their non oxidized analogs. Interest in this strategy has
increased following the introduction of the 2,2,6,6-tetramethyl-1-
piperidinyloxy reagent (TEMPO). In combination with an appropri-
ate co-oxydant, TEMPO was successfully used for the careful oxida-
tion of either rather heavily protected oligosaccharides28 or lightly
protected ones.29 In view of its selectivity for primary hydroxyl
groups, TEMPO was herein selected as an attractive agent to oxidize
ꢀ
Zemplen deacetylation of the latter followed by acetylation gave
benzylidene acetal24 16 (93% over two steps). Next, reaction of the
latter with levulinic acid and DCC in the presence of DMAP gave the
fully protected 17 (90%). Condensation of donor 17 and acceptor 2
under conditions applied to donors 11 or 12 led to similar obser-
vations (Table 1). The glycosylation product had the desired 1,2-
trans stereochemistry (NMR data for C-1B: 1JCH 162.4 Hz, and H-1B:
1J1,2 8.4 Hz). Despite being present in diminished amounts owing to
extended reaction time, oxazoline 19 was identified repeatedly as
the 6-OH of a
b-linked terminal D-galactopyranosyl residue within
a partially protected AꢀBC trisaccharide. Accordingly, the known

10340
P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
peracetylated trichloroacetimidate donor30 20, was thought to suit
the requirements for both protecting group orthogonality and syn-
thetic efficacy. Indeed, the ‘anomalous’ difference in stability of vic-
In view of the possible sensitivity of allyl ether and/or acetates to
oxidative conditions,36,37 such as in the ‘Anelli’s method’,38,39 and
of the propensity of
b-galactopyranosides to form 3,6-lactones
inal versus isolated O-acetyl protecting groups under Zemplen
upon TEMPO-mediated oxidation,40 the conversion of mono-
acetate 23 to the corresponding uronic acid 25 was investigated
under various conditions (Table 2). The poor solubility of in-
termediate 23 both in DCM and in water was incompatible with the
biphasic system recommended as being critical for a high yielding
oxidation step. Hence, all reactions were run in a homogenous
MeCN/water (or MeCN/aqueous buffer) system. The use of TEMPO
in combination with 1,3-dibromo-5,5-dimethylhydantoin was fa-
vored owing to its compatibility with O-acetyl groups.41 Yet, this
reagent system was rapidly abandoned since in our hands the de-
sired oxidation product 25 was formed within complex mixtures
(not shown). Under Anelli’s conditions, modified according to J.
Xie42 for compatibility with an allyl aglycone, the reaction was
slow, while degradation gradually increased. Nevertheless, re-
peated addition of TEMPO improved the conversion and the target
trisaccharide 25 was isolated in an acceptable 49% yield (Table 2,
entry 1). In an attempt to increase the conversion, trisaccharide 23
was engaged in a two-step oxidation process according to Huang
and collaborators.43 However, the remaining unreacted alcohol 23
suggested lack of completion, possibly due to solvent limitation,
and the procedure was not investigated further (Table 2, entry 2).
The orthogonality of TEMPO/BAIB ([bis(acetoxy)iodo]benzene)44 to
alkenes was exemplified previously.14,45,46 Herein, tetraol 23 was
reacted with excess TEMPO and BAIB providing oxidation product
25, albeit in a rather low and poorly reproducible 39% yield (not
shown). Therefore, inspired by the work of Zhao and collabora-
tors36 and of Kovensky’s team,47 a combination of TEMPO, NaOCl,
and NaOCl2 in buffer was investigated. However, in view of the
substantial timelength of the reported oxidation procedure
(4e14 days),47 the amount of TEMPO was increasedd0.3 versus
0.05 equivdto speed up the transformation and prevent acetate
cleavage. The initial transformation was clean though far from
completion, reaching a moderate 35% yield of trisaccharide 25
(Table 2, entry 3). The presence of unreacted tetraol 23 suggested
a rupture in the catalytic cycle. In the absence of any isolated
chlorinated side-product, the amount of TEMPO was thought to
interfere. This assumption along with the possible visual follow-up
of the reaction progress36 encouraged the implementation of a se-
quential procedure. The new proposed protocol involved the re-
peated addition of small portions of NaOCl and a slightly increased
reaction temperature. The slow 23/25 conversion was kept under
control, and under optimized conditions (Table 2, entry 4), isolation
ꢀ
conditions31,32 was occasionally found attractive,33,34 and even
valuable for the preparation of site-specifically O-acetylated oligo-
saccharides.17,35 In designing a precursor to residue A, we rational-
ized that the strategy primarily developed for the synthesis of S.
flexneri 3a oligosaccharides17,35 could be adapted to a suitable AꢀBC
intermediate without affecting a 3C-acetate.
Conventional reaction of trichloroacetimidate 20 and acceptor
14 in the presence of catalytic TMSOTf gave the fully protected AꢀBC
(21, 72%). CAN-mediated oxidative cleavage of the PMB ether and
consecutive O-acetylation readily furnished pentaacetate 22 (82%
over two steps). Upon scale-up, the [20þ14] glycosylation, PMB
removal, and O-acetylation steps provided pentaacetate 22 in
a good 74% overall yield (Scheme 3). Chemoselective deacetylation
of trisaccharide 22 was attempted using either potassium carbon-
ate or methanolic sodium methoxide. The latter was the most se-
lective. Performing the transesterification at 0 ꢀC in the presence of
0.15 equiv of NaOMe gave the key tetraol 23 in a satisfactory 70%
yield together with pentaol 24 (14%). Otherwise, treatment of the
fully protected 22 under harsher conditions (0.5 equiv NaOMe, rt)
enabled its efficient conversion into the same 24 (92%), which is the
key intermediate to trisaccharides AꢀBC-Pr and ABC-Pr (Scheme 4).
1H NMR analysis, especially of the chemical shift of H-3C in the two
products of transesterificationddH at 5.05 and 3.93 for 23 and 24,
respectivelydascertained the site of O-acetylation in tetraol 23.
Scheme 3. Synthesis of trisaccharide 22 from disaccharide 14. Reagents and condi-
tions: (a) TMSOTf, toluene, ꢁ10 ꢀC, 20 min (72%); (b) (i) CAN, MeCN/H2O, rt, 30 min; (ii)
Ac2O, pyridine, DMAP, rt, 2 h (82% from 21 over two steps, 74% from 14 over three
steps).
Scheme 4. Synthesis of the propyl glycosides 27, 29, 30, and 32 from a common trisaccharide precursor 22. Reagents and conditions: (a) NaOMe, MeOH, 0 ꢀC, 2.5 h (70% for 23); (b) NaOMe,
MeOH, rt,2h(92%for24);(c)TEMPO,NaOCl,NaClO2,MeCN/phosphatebuffer,45ꢀC,24h(90%for25,86%for26);(d)Pd/C,MeOH, 30ꢀC,H-CubeÔ‘fullH2’mode,73%for27/28(5:1);(e)(i)Pd/
C, H2, MeOH, rt, 1 day, (ii) Pd/C, H2, Et3N, MeOH/H2O, rt, 2 days (51% for 29, 70% for 32); (f) (i) Pd/C, H2, THF/H2O, rt, 1 day, (ii) Pd/C, H2, Et3N, THF/H2O, rt, 2 days (47%/16% for 30/31).

P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
10341
Table 2
Oxidation of tetraol 23 into uronic acid 25
Entry
Oxidation system: reagent (equiv)
Medium
Duration
Temperature
Yield %
1
2
3
4
TEMPO (1.1), NaOCl, NaBr, NaHCO3 then TEMPO (0.5) twice
TEMPO (0.3), NaBr, nBu4NBr, NaOCl, NaHCO3 then NaClO2, 2-methyl-2-butene
TEMPO (1.1), NaOCl, NaBr, NaHCO3 then TEMPO (0.5) twice
TEMPO (0.3), NaOCl, NaClO2
MeCN/H2O (2:1)
MeCN/H2O (1:1)
MeCN/Phosphate buffer pH 6.7 (1:1)
MeCN/Phosphate buffer pH 6.7 (1:1)
6 h
1 h
4 days
24 h
rt
rt
35 ꢀC
45 ꢀC
49
n.d.
37
90
n.d. not determined.
of the oxidized 25 reached an excellent reproducible 90% yield.
When pentaol 24 was reacted with TEMPO/NaOCl/NaOCl2 under
the same conditions, the corresponding uronic acid derivative 26
was isolated in a similar yield (86%), which validated the process in
our hands (Scheme 4).
3. Conclusion
Seven oligosaccharides representing portions of the O-Ags of
the human enteric pathogens SF6 and SF6a or of E. coli O147, were
synthesized by appropriate combination of thioglycoside and tri-
chloroacetimidate donors tuned for high yielding glycosylation and
optimal stereocontrol of the newly installed glycosidic linkages.
Fine adjustment of an oxidation protocol involving the TEMPO/
NaOCl/NaClO2 system, enabled the efficient chemoselective Galp to
GalpA conversion within trisaccharide substrates comprising sec-
ondary hydroxyl groups, in addition to allyl and O-acetyl moieties,
known for their sensitivity to standard oxidation medium. Com-
bined with an efficient PMB/Ac replacement protocol, this newly
established oxidation procedure enabled the site-directed trans-
formation of a fully protected trisaccharide intermediate into seven
functionalized targets. All RP-HPLC purified oligosaccharides will
serve at investigating the molecular network of O-Ag/antibody
interactions governing the serotyping for these enteric pathogens.
Having all four trisaccharides 23e26 in hands, we next focused
on their final conversion into the propyl glycosides 27, 29, 30, and
32, respectively. In a first attempt, Pd/C-catalyzed hydrogenolysis of
the benzyl ether and benzylidene acetal of the 3C-O-acetyl in-
termediate 23, along with concomitant hydrodechlorination of the
2B-protecting group into an acetamide and allyl to propyl conver-
sion, was performed in a microfluidic flow reactor (H-CubeÔ) using
a diluted methanolic solution (60 mL/mmol) and a hydrogen
pressure of 10 bar. Under these conditions, adding Et3N to the re-
action mixture was found to be crucial in order to avoid extensive
repeated turnovers. Finally, the required trisaccharide 27 (AꢀB3AcC-
Pr) was obtained in admixture with its regioisomer 28 (AꢀB4AcC-Pr)
following RP column chromatography (73%, 5:1 ratio). Although an
additional round of RP purification had enabled the isolation of
each one of the pure monoacetates, the 4-O-acetylated 28 was not
stable in solution. It rapidly evolved into a 2:1 mixture of tri-
saccharides 27 and 28. Alternatively, the corresponding uronic acid
25 was subjected to the same transformation, albeit in THF/H2O at
ambient hydrogen pressure.14 The 4C-O-acetyl trisaccharide
AB4AcC-Pr (31) was isolated in a meaningful 16% yield in addition to
the AB3AcC-Pr (30) derivative (47%), following RP-HPLC purification.
We have previously reported the successful obtaining of the closely
related O-acetylated SF6 tetrasaccharide B3AcCDA-Pr following
catalytic hydrogenation under neutral conditions of a precursor
having the same set of protecting groups as allyl glycosides 23 and
25.14 In the present case, the hydrodechlorination under neutral
conditions of trichloroacetamides 23 and 25 stopped at the chlor-
oacetamide stage as indicated from LCeMS monitoring of the re-
action (not shown). The acetamide targets were present as traces
only and basic conditions were needed to reach completion of the
2B-NHCl3Ac/2B-NHAc conversion. This different behavior may
4. Experimental section
4.1. General
Anhydrous (anhyd) solventsdincluding toluene (Tol), dichloro-
methane (DCM), tetrahydrofuran (THF), N,N-dimethylformamide
(DMF), methanol (MeOH), and pyridinedwere delivered on mo-
lecular sieves and used as received. Additional solvents cited in the
text are abbreviated as Chex (cyclohexane), MeCN (acetonitrile), and
EtOAc (ethyl acetate), in addition to acetone. Reactions requiring
anhyd conditions were run under an argon (Ar) atmosphere, using
ꢁ
dried glassware. 4 A Molecular sieves were activated before use by
heating under high vacuum. Analytical thin-layer chromatography
(TLC) was performed with silica gel 60F254, 0.25 mm pre-coated TLC
aluminum foil plates. Compounds were visualized using UV254 and/
or orcinol (1 mg mLꢁ1) in 10% aq H2SO4 with charring. Flash column
chromatography was carried out using silica gel (particle size
originate from
a
more hindered environment of the tri-
40e63
carried out using a Kromasil 5
preparative column. Unless stated otherwise, analytical RP-HPLC
of the final compounds ( m C18
300 A 2.1ꢂ100 mm analytical column eluting with a 0e35% linear
gradient of MeCN in 0.01 N aq TFA over 20 min at a flow rate of
0.35 mL minꢁ1. NMR spectra were recorded at 303 K on a Bruker
Avance spectrometer equipped with a BBO probe at 400 MHz (1H)
and 100 MHz (13C). Spectra were recorded in deuterated chloroform
(CDCl3), deuterated methanol (MeOD), and deuterated water (D2O).
mm, unless indicated otherwise). RP-HPLC purification was
ꢁ
chloroacetamide moiety within trisaccharides 23 and 25 in com-
parison to its location in tetrasaccharide B3AcCDA-Pr. In fact, O-
acetyl migration at the very last step of a synthesis is not without
precedent. In one exemple, although performing the hydro-
genolysis of O-acetylated oligosaccharides protected in the form of
benzylidene acetal, benzyl, and p-methoxybenzyl ether, under
slightly acidic conditions could prevent migration, a mixture of
regioisomers was still isolated following purification.48 Herein, the
basic conditions required for an efficient reduction of the tri-
chloroacetamide in 23 and 25 favored the 3C-O-acetyl migration to
the vicinal 4C-OH. Indeed, O-acetyl intramolecular migration was
shown to already occur at pH 6.8.49 Interestingly, we recently
revealed the structure of the O-Ag from SF6 strain 2924-71,
showing that acetylationdwhen presentdoccurred either at OH-
4C (minor form) or at OH-3C (major form).14 The 4C-O-acetylated
trisaccharides 28 and 31 are therefore of high interest toward the
determination of the SF6 immunodominant epitopes. Toward the
same goal, the non O-acetylated trisaccharides 24 and 26 were
transformed into the corresponding propyl glycosides AꢀBC-Pr (29)
and ABC-Pr (32) in 51% and 70% yield, respectively.
mm C18 100 A 10ꢂ250 mm semi-
l¼215 nm) used a Symmetry 3.5
m
ꢁ
Chemical shifts (d) are reported in parts per million (ppm) relative to
residual solvent peak (CHCl3, MeOH), and DSS (4,4-dimethyl-4-
silapentane-1-sulfonic acid) in the case of D2O at 7.28/77.0, 2.50/
39.5 and 0.00/0.00 ppm for the 1H and 13C spectra, respectively.
Coupling constants are reported in hertz (Hz). Elucidations of
chemical structures were based on 1H, COSY, DEPT-135, HSQC,
decoupled HSQC,13C, decoupled 13C, and HMBC. Signals are reported
as s (singlet), d (doublet), t (triplet), dd (doublet of doublet), q
(quadruplet), qt (quintuplet), dt (doublet of triplet), dq (doublet of
quadruplet), ddd (doublet of doublet of doublet), m (multiplet). The
signals can also be described as broad (prefix b), pseudo (prefix p),

10342
P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
overlapped (suffix o) or partially overlapped (suffix po). Of the two
magnetically non-equivalent geminal protons at C-6, the one reso-
nating at lower field is denoted H-6a, and the one at higher field is
denoted H-6b. Interchangeable assignments are marked with an
asterisk. Sugar residues are lettered according to the lettering of the
RU of the SF6 O-Ag and identified by a subscript in the listing of
signal assignments. LCeMS analysis was performed on a Waters Q-
TOFmicro mass spectrometer equipped with an electrospray ion
4.3. Allyl (3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido-
-galactopyranosyl)-(1/2)-4-O-benzyl-3-O-para-methox-
ybenzyl- -rhamnopyranoside (5)
b-D
a-L
A mixture of acceptor 2 (300 mg, 0.72 mmol), donor 4 (597 mg,
ꢁ
0.87 mmol, 1.2 equiv), and powdered 4 A MS (750 mg) in
anhyd DCM (8.3 mL) was stirred at rt under an Ar atmosphere for
1 h. The reaction mixture was cooled to ꢁ65 ꢀC, then NIS (244 mg,
source (ESI) coupled to an Alliance HPLC equipped with a Waters
1.09 mmol, 1.5 equiv) and TMSOTf (13 mL, 72 mmol, 0.1 equiv) were
ꢁ
Symmetry 3.5
m
m C18 100 A 2.1ꢂ100 mm column. HRMS spectra
added. The reaction mixture was stirred for 1 h, while the bath
temperature was allowed to reach ꢁ40 ꢀC. Since a TLC control (Tol/
EtOAc 9:1) indicated that no acceptor (Rf 0.10) nor donor (Rf 0.42)
remained, and that a new product (Rf 0.22) was present, the re-
action was quenched with Et3N. Solids were filtered, and the filtrate
was concentrated to dryness. The residue was purified by flash
chromatography (Tol/EtOAc 95:5 to 9:1) to give disaccharide 5
were recorded on a WATERS QTOF Micromass instrument in the
positive-ion electrospray ionization (ESIþ) mode. Solutions were
prepared using 1:1 MeCN/H2O containing 0.1% formic acid. Optical
rotations were measured at 25 ꢀC, in CHCl3 solutions, except where
indicated otherwise, with a BS Bellingham automatic polarimeter,
Model ADP220.
(568 mg, 78%) as a light yellow solid. The
b
-condensation product 5
25
had [
a
]
ꢁ10 (c 1.0). 1H NMR (CDCl3),
d 7.41e7.26 (m, 22H, HAr),
D
4.2. Allyl 4-O-benzyl-3-O-para-methoxybenzyl-
pyranoside (2) and allyl 4-O-benzyl-2-O-para-methoxybenzyl-
a
-
L
-rhamno-
6.91 (d, 1H, JNH,2¼7.1 Hz, NH), 6.82 (m, 2H, J¼8.8 Hz, HArPMB), 5.85
(m, 1H, CH]All), 5.23 (m, 1H, Jtrans¼17.2 Hz, Jgem¼1.6 Hz, ]CH2All),
5.14 (m, 1H, Jcis¼10.4 Hz, ]CH2All), 4.96 (dpo, 1H, J1,2¼8.4 Hz, H-1B),
4.93 (dpo, 1H, J¼11.7 Hz, HBn), 4.92 (d, 1H, J¼10.9 Hz, HBn), 4.82 (d,
1H, J1,2¼1.6 Hz, H-1C), 4.68 (d, 1H, J¼11.4 Hz, HBn), 4.63 (d, 1H,
J¼11.5 Hz, HBn), 4.60e4.55 (m, 4H, HBn), 4.50 (d, 1H, J¼11.8 Hz, HBn),
a
-L-rhamnopyranoside (3)
Bu2SnO (37.2 g, 149.5 mmol,1.1 equiv) was added to a solution of
diol 1 (40.0 g, 135.8 mmol) in anhyd Tol (1.1 L). The mixture was
stirred for 5 h at reflux using a ‘DeaneStark’ apparatus and sub-
sequently concentrated under reduced pressure to give a 0.4 M
solution. After cooling to rt, dry CsF (21.05 g, 138.6 mmol, 1.0 equiv),
dry TBAI (65.25 g, 175.6 mmol, 1.3 equiv), and 4-methoxybenzyl
chloride (20.3 mL, 149.5 mmol, 1.1 equiv) were added. The re-
action mixture was stirred for 5 h at reflux, at which time moni-
toring by TLC (Chex/EtOAc 6:4) showed the conversion of the
starting material (Rf 0.22) into a major less polar product (Rf 0.51).
The temperature was lowered to 0 ꢀC, salts were removed by fil-
tration (Tol wash), and volatiles were evaporated under reduced
pressure. The residue was purified by flash chromatography (Chex/
EtOAc 9:1 to 7:3) to give by order of elution, the yet undescribed
regioisomer 3 (4.1 g, 8%) and acceptor 2 (48.1 g, 81%), both as
4.46 (d,1H, J¼11.8 Hz, HBn), 4.13e4.02 (m, 2H, H-2B, HAll), 4.00 (ddpo
,
1H, J2,3¼10.8 Hz, J3,4¼2.4 Hz, H-3B), 3.99e3.96 (m, 2H, H-4B, H-2C),
3.90 (m, 1H, HAll), 3.84 (dd, 1H, J2,3¼3.1 Hz, J3,4¼9.4 Hz, H-3C), 3.78
(s, 3H, CH3PMB), 3.68 (dqpo, 1H, J4,5¼9.4 Hz, J5,6¼6.2 Hz, H-5C), 3.66
(ddpo, 1H, J5,6a¼7.0 Hz, J6a,6b¼8.8 Hz, H-6aB), 3.58 (ddpo, 1H,
J5,6b¼5.4 Hz, H-6bB), 5.55 (ptpo, 1H, H-5B), 3.45 (t, 1H, H-4C), 1.31 (d,
3H, H-6C). 13C NMR (CDCl3),
d
161.6 (NHCO), 159.3 (CIVPMB), 138.8,
138.4, 138.0, 137.7 (4C,
CIVAr), 134.0 (CH]All), 130.7 (CIVAr),
129.7e127.5 (22C, CAr), 116.8 (]CH2All), 113.9 (2C, CArPMB), 100.5 (C-
1B, 1JCH¼163.5 Hz), 98.6 (C-1C, 1JCH¼175.4 Hz), 92.9 (CCl3), 80.8 (C-
4C), 79.6 (C-3C), 78.7 (C-3B), 75.5 (CBn), 74.9 (C-2C), 74.6 (CBn), 73.7
(C-5B), 73.6 (CBn), 72.3 (3C, 2CBn, C-4B), 68.7 (C-6B), 67.9 (C-5C), 67.6
(CH2All), 55.5 (C-2B), 55.2 (CH3PMB), 17.9 (C-6C). HRMS (ESIþ): m/z
1012.2973 (calcd for C53H58Cl3NO11Na [MþNa]þ: m/z 1012.2949).
25
25
orange-yellow oils. Alcohol 2 had [
a]
D ꢁ41 (c 1.0), lit.19
[
a]
D ꢁ22
(c 1.0). 1H NMR (CDCl3),
d
7.43e7.29 (m, 7H, HAr), 6.95e6.89 (m, 2H,
H
ArPMB), 5.96 (m, 1H, CH]All), 5.34 (m, 1H, Jtrans¼17.2 Hz,
4.4. Allyl (3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido-
Jgem¼1.7 Hz, ]CH2All), 5.24 (m, 1H, Jcis¼10.4 Hz, ]CH2All), 4.96 (d,
1H, J¼11.1 Hz, HBn), 4.91 (d, 1H, J1,2¼1.5 Hz, H-1), 4.69 (d, 1H, HBn),
4.68 (d, 1H, J¼11.2 Hz, HPMB), 4.64 (d, 1H, HPMB), 4.21 (m, 1H, HAll),
4.08 (br dd, 1H, H-2), 4.01 (m, 1H, HAll), 3.93 (dd, 1H, J2,3¼3.2 Hz,
J3,4¼9.2 Hz, H-3), 3.48 (dq, 1H, J4,5¼9.4 Hz, J5,6¼6.1 Hz, H-5), 3.78 (s,
3H, CH3PMB), 3.58 (pt, 1H, H-4), 2.23 (br s, 1H, OH-2), 1.41 (d, 3H, H-
b-D
-galactopyranosyl)-(1/2)-4-O-benzyl-a-L-rhamnopyrano-
side (6)
Water (3.7 mL) and CAN (1.61 g, 2.94 mmol, 4.0 equiv) were
added to a solution of disaccharide 5 (727 mg, 0.74 mmol) in MeCN
(37 mL). After stirring for 1 h at rt, a TLC follow up (Chex/EtOAc 8:2)
indicated the complete conversion of the p-methoxybenzyl de-
rivative 5 (Rf 0.44) into a more polar product (Rf 0.25). The reaction
was quenched by adding satd aq NaHCO3. The reaction mixture was
diluted with water and the aq phase was extracted three times with
DCM. The combined extracts were washed with brine, dried by
passing through a phase separator filter, and evaporated. The res-
idue was purified by flash chromatography (Chex/EtOAc 8:2 to 7:3)
6). 13C NMR (CDCl3),
130.6 (CIVAr), 130.3e127.7 (7C, CAr), 117.2 (]CH2All), 114.0 (2C,
ArPMB), 98.6 (C-1), 80.1, 79.9 (2C, C-3, C-4), 75.3 (CBn), 71.7 (CPMB),
d 159.5 (CIVPMB), 138.8 (CIVAr), 134.1 (CH]All),
C
68.6 (C-2), 67.8 (CH2All), 67.7 (C-5), 55.2 (CH3PMB), 18.0 (C-6). HRMS
(ESIþ): m/z 437.1929 (calcd for C24H30O6Na [MþNa]þ: m/z
437.1940).
25
Alcohol 3 had [
a
]
D ꢁ18 (c 1.0). 1H NMR (CDCl3),
d 7.41e7.27 (m,
7H, HAr), 6.95e6.90 (m, 2H, HArPMB), 5.87 (m, 1H, CH]All), 5.27 (m,
1H, Jtrans¼17.2 Hz, Jgem¼1.7 Hz, ]CH2All), 5.19 (m, 1H, Jcis¼10.4 Hz,
]CH2All), 4.93 (d, 1H, J¼11.1 Hz, HBn), 486 (d, 1H, J1,2¼1.4 Hz, H-1),
4.70 (d, 1H, J¼11.5 Hz, HBn), 4.67 (d, 1H, HBn), 4.53 (d, 1H, HBn), 4.16
(m, 1H, HAll), 4.01e3.92 (m, 2H, H-3, HAll), 3.84 (s, 3H, CH3PMB), 3.75
(dd,1H, J2,3¼3.9 Hz, H-2), 3.71 (dq, 1H, J4,5¼9.4 Hz, J5,6¼6.3 Hz, H-5),
3.32 (pt, 1H, J3,4¼9.3 Hz, H-4), 2.23 (d, 1H, JOH,3¼9.5 Hz, OH-3), 1.35
to give alcohol 6 (552 mg, 64%) as a white solid. The latter had
25
[
a
]
D ꢁ3 (c 1.0). 1H NMR (CDCl3),
d 7.40e7.27 (m, 20H, HAr), 6.97 (d,
1H, JNH,2¼6.9 Hz, NH), 5.85 (m, 1H, CH]All), 5.24 (m, 1H,
Jtrans¼17.2 Hz, Jgem¼1.6 Hz, ]CH2All), 5.13 (m, 1H, Jcis¼10.4 Hz, ]
CH2All), 5.07 (dpo, 1H, J1,2¼7.9 Hz, H-1B), 4.92 (d, 1H, J¼11.3 Hz, HBn),
4.91 (br s, 1H, H-1C), 4.84 (d, 1H, J¼11.1 Hz, HBn), 4.70 (d, 1H,
J¼11.4 Hz, HBn), 4.66 (d, 1H, J¼11.0 Hz, HBn), 4.59 (d, 1H, J¼11.7 Hz,
(d, 3H, H-6). 13C NMR (CDCl3),
d
159.5 (CIVPMB), 138.6 (CIVAr), 133.8
H
Bn), 4.56 (d, 1H, J¼11.7 Hz, HBn), 4.51 (d, 1H, J¼11.8 Hz, HBn), 4.48
(CH]All), 129.8 (CIVAr), 129.7e127.7 (7C, CAr), 117.2 (]CH2All), 114.0
(2C, CArPMB), 96.1 (C-1), 82.4 (C-4), 78.3 (C-2), 75.1, 72.7 (2C, CBn),
71.6 (C-3), 67.7 (CH2All), 67.2 (C-5), 55.3 (CH3PMB), 18.0 (C-6). HRMS
(ESIþ): m/z 437.1835 (calcd for C24H30O6Na [MþNa]þ: m/z
437.1940).
(d, 1H, J¼11.8 Hz, HBn), 4.14e4.01 (m, 4H, H-2B, HAll, H-3B, H-4B),
4.01e3.96 (m, 2H, H-2C, H-3C), 3.91 (m, 1H, HAll), 3.69 (dqpo, 1H,
J4,5¼9.5 Hz, J5,6¼6.2 Hz, H-5C), 3.68e3.60 (m, 3H, H-6aB, H-5B, H-
6bB), 3.32 (pt, 1H, J3,4¼9.2 Hz, H-4C), 2.37 (d, 1H, JOH,3¼7.3 Hz, OH-
3C), 1.32 (d, 3H, H-6C). 13C NMR (CDCl3),
d 162.0 (NHCO), 138.5,

P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
10343
138.3, 137.8, 137.3 (4C, CIVAr), 133.9 (CH]All), 128.6e127.7 (20C, CAr),
(C-5C), 70.3 (C-4B), 63.5 (C-6B), 55.2 (C-2B), 24.7 (CH3NHAc), 24.5
(CH2Pr), 19.1 (C-6C), 12.3 (CH3Pr). HRMS (ESIþ): m/z 432.1834 (calcd
for C17H31NO10Na [MþNa]þ: m/z 432.1846). RP-HPLC (215 nm):
tR¼10.4 min.
1
116.9 (]CH2All), 101.4 (C-1B, JCH¼164.9 Hz), 98.4 (C-1C,
1JCH¼171.2 Hz), 92.7 (CCl3), 82.4 (C-4C), 79.0 (C-3C), 78.2 (C-3B), 75.1,
74.7 (2C, CBn), 73.8 (C-5B), 73.6 (CBn), 72.2 (2C, CBn, C-4B), 71.5 (C-2C),
68.5 (C-6B), 67.7 (CH2All), 67.3 (C-5C), 55.7 (C-2B), 18.0 (C-6C). HRMS
(ESIþ): m/z 892.2393 (calcd for C45H50Cl3NO10Na [MþNa]þ: m/z
892.2398).
4.7. Propyl 2-acetamido-2-deoxy-b-D-galactopyranosyl-
(1/2)-3-O-acetyl- -rhamnopyranoside (9)
a-L
4.5. Allyl (3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido-
To a stirred solution of acetate 7 (99 mg, 108 mmol) in EtOH
b
-
D
-galactopyranosyl)-(1/2)-3-O-acetyl-4-O-benzyl-
a
-
L
-
(4.4 mL), was added 10% Pd/C (100 mg). The suspension was stirred
under a hydrogen atmosphere for 48 h. After this time, MS analysis
of the crude reaction mixture indicated a single molecular weight
corresponding to that of the target disaccharide. The reaction
mixture was filtered over a pad of CeliteÒ. Evaporation of the vol-
atiles, freeze-drying, and purification of the crude material by
preparative RP-HPLC (0e35% linear gradient of MeCN in 0.08%
aq TFA over 16 min, then 35e100% linear gradient of MeCN in 0.08%
aq TFA over 4 min, at a flow rate of 5.5 mL minꢁ1) gave disaccharide
rhamnopyranoside (7)
Alcohol 6 (410 mg, 0.47 mmol) was dissolved in dry pyridine
(2.4 mL). Acetic anhydride (45 L, 4.7 mmol, 10 equiv) was added at
m
rt and the reaction mixture was stirred under an Ar atmosphere for
24 h. At this time, a TLC control (Tol/EtOAc 7:3) showed the total
conversion of disaccharide 6 (Rf 0.27) into a less polar product (Rf
0.41). Volatiles were evaporated and co-evaporated three times
with Tol. The residue was purified by flash chromatography (Chex/
9 (32.5 mg, 67%) as a white solid following freeze-drying. Com-
25
EtOAc 9:1 to 8:2) to give acetate 7 (416 mg, 97%) as a white foam.
Disaccharide 7 [
pound 9 had [
a]
D þ5 (c 1.0, water). 1H NMR (D2O),
d 4.91 (dd, 1H,
25
a
]
ꢁ8 (c 1.0). 1H NMR (CDCl3),
d
7.41e7.24 (m,
J2,3¼3.0 Hz, J3,4¼10.0 Hz, H-3C), 4.83 (d, 1H, J1,2¼1.5 Hz, H-1C), 4.28
(d, 1H, J1,2¼8.3 Hz, H-1B), 3.98 (pt, 1H, H-2C), 3.78 (dpo, 1H,
J3,4¼3.4 Hz, H-4B), 3.74 (ddpo, 1H, J2,3¼10.8 Hz, H-2B), 3.59e3.56 (m,
4H, H-5C, H-6aB, H-6bB, H-3B), 3.52 (dt, 1H, J¼7.1 Hz, J¼9.7 Hz,
OCH2Pr), 3.47 (dd, 1H, J5,6a¼4.7 Hz, J5,6b¼7.7 Hz, H-5B), 3.41e3.34
(m, 2H, H-4C, OCH2Pr), 2.05 (s, 3H, CH3Ac), 1.95 (s, 3H, CH3NHAc), 1.47
(m, 2H, CH2Pr), 1.16 (d, 3H, J5,6¼6.2 Hz, H-6C), 0.78 (t, 3H, J¼7.4 Hz,
D
20H, HAr), 7.00 (d, 1H, JNH,2¼7.0 Hz, NH), 5.86 (m, 1H, CH]All), 5.28
(ddpo, 1H, J3,4¼9.5 Hz, H-3C), 5.26 (mpo, 1H, Jtrans¼17.3 Hz,
Jgem¼1.6 Hz, ]CH2All), 5.14 (m, 1H, Jcis¼10.4 Hz, ]CH2All), 5.03 (d,
1H, J1,2¼8.2 Hz, H-1B), 4.88 (d, 1H, J¼11.3 Hz, HBn), 4.83 (d, 1H,
J1,2¼1.7 Hz, H-1C), 4.68 (dpo, 1H, J¼11.2 Hz, HBn), 4.65 (dpo, 1H,
J¼11.4 Hz, HBn), 4.58 (br d, 2H, J¼11.0 Hz, HBn), 4.56 (d, 1H,
J¼11.2 Hz, HBn), 4.51 (d, 1H, J¼11.7 Hz, HBn), 4.46 (d, 1H, J¼11.7 Hz,
CH3Pr). 13C NMR (D2O),
d 177.1 (NHCO), 176.0 (COAc), 105.8 (C-1B,
H
Bn), 4.36 (dd, 1H, J2,3¼11.0 Hz, J3,4¼2.8 Hz, H-3B), 4.16 (ddpo, 1H,
1JCH¼163.3 Hz), 101.1 (C-1C, 1JCH¼175.7 Hz), 79.1 (C-2C), 77.3 (C-5B),
75.4 (C-3C), 72.8 (C-3B), 72.6 (C-4C), 72.2 (OCH2Pr), 71.2 (C-5C), 70.2
(C-4B), 63.4 (C-6B), 55.1 (C-2B), 24.9 (CH3NHAc), 24.5 (CH2Pr), 23.1
(CH3Ac), 19.1 (C-6C), 12.4 (CH3Pr). HRMS (ESIþ): m/z 474.1945 (calcd
J2,3¼3.1 Hz, H-2C), 4.13 (mpo, 1H, HAll), 4.02 (d, 1H, H-4B), 3.95 (m,
1H, HAll), 3.83 (mpo, 1H, H-2B), 3.80 (dqpo, 1H, J4,5¼9.4 Hz,
J5,6¼6.2 Hz, H-5C), 3.68e3.63 (m, 2H, H-6aB, H-5B), 3.56 (dd, 1H,
J5,6b¼9.1 Hz, J6a,6b¼12.3 Hz, H-6bB), 3.53 (pt, 1H, H-4C), 2.10 (s, 3H,
for C19H33NO11Na [MþNa]þ: m/z 474.1951). RP-HPLC (215 nm, Aeris
CH3Ac), 1.33 (d, 3H, H-6C). 13C NMR (CDCl3),
d
170.7 (COAc), 161.5
Peptide Phenomenex 3.6
m
m C18 100 A 2.1ꢂ100 mm analytical
ꢁ
(NHCO), 138.4, 138.3, 137.9, 137.6 (4C, CIVAr), 133.8 (CH]All),
128.5e127.2 (20C, CAr), 117.0 (]CH2All), 99.6 (C-1B, 1JCH¼164.3 Hz),
98.6 (C-1C, 1JCH¼171.9 Hz), 92.9 (CCl3), 79.5 (C-4C), 76.7 (C-3B), 75.3
(C-2C), 75.1, 74.8, 73.6 (3C, CBn), 73.5 (2C, C-3C, C-5B), 72.8 (C-4B),
72.5 (CBn), 68.2 (C-6B), 67.8 (CH2All), 67.7 (C-5C), 56.3 (C-2B), 21.2
(CH3Ac), 17.9 (C-6C). HRMS (ESIþ): m/z 934.2661 (calcd for
column, 0e20% linear gradient of MeCN in 0.08% aq TFA over
20 min at 0.3 mL minꢁ1): tR¼15.3 min.
4.8. Allyl (3,4,6-tri-O-benzyl-2-deoxy-2-trichloroacetamido-
a-
D-galactopyranosyl)-(1/2)-4-O-benzyl-3-O-para-methox-
ybenzyl- -rhamnopyranoside (10)
a-L
C
47H52Cl3NO11Na [MþNa]þ: m/z 934.2504).
A mixture of acceptor 2 (51 mg, 0.12 mmol), donor 4 (99 mg,
ꢁ
4.6. Propyl 2-acetamido-2-deoxy-
b
-D-galactopyranosyl-
0.15 mmol, 1.2 equiv), and powdered 4 A MS (100 mg) in
(1/2)- -rhamnopyranoside (8)
a
-
L
anhyd DCM (1.4 mL) was stirred at rt under an Ar atmosphere for
1 h. The reaction mixture was cooled to 0 ꢀC, then NIS (41 mg,
To a stirred solution of alcohol 5 (94 mg, 94
mmol) in EtOH
0.18 mmol, 1.5 equiv) and TMSOTf (2 mL, 12 mmol, 0.1 equiv) were
(4 mL), was added 10% Pd/C (100 mg). The suspension was stirred
under a hydrogen atmosphere for 48 h. After this time, MS analysis
of the crude reaction mixture indicated a single molecular weight
corresponding to that of the target disaccharide. The reaction
mixture was filtered over a pad of CeliteÒ. Evaporation of the vol-
atiles, freeze-drying, and purification of the crude material by
preparative RP-HPLC (0e35% linear gradient of MeCN in 0.08%
aq TFA over 16 min, then 35e100% linear gradient of MeCN in 0.08%
aq TFA over 4 min, at a flow rate of 5.5 mL minꢁ1) gave disaccharide
added. The reaction mixture was stirred for 45 min, while the bath
temperature reached rt. Since a TLC control (Tol/EtOAc 9:1) in-
dicated that no acceptor (Rf 0.10) nor donor (Rf 0.42) remained, and
that a new compound (Rf 0.22) was present, the reaction was
quenched with Et3N. Solids were filtered, and the filtrate was
concentrated to dryness. The residue was purified by flash chro-
matography (Tol/EtOAc 95:5 to 9:1) to give by order of elution the
product of
(82 mg, 69%), both as light yellow solids. The
product 10 had 1H NMR (CDCl3),
7.39e7.17 (m, 22H, HAr), 6.83 (d,
a-
D-glycosylation 10 (15 mg, 13%) and disaccharide 5
a
-condensation
8 (29.3 mg, 77%) as a white solid following freeze-drying. Com-
d
25
pound 8 had [
a]
ꢁ3 (c 1.0, water). 1H NMR (D2O),
d
4.83 (d, 1H,
1H, JNH,2¼9.3 Hz, NH), 6.80 (m, 2H, J¼8.7 Hz, HArPMB), 5.84 (m, 1H,
D
J1,2¼1.5 Hz, H-1C), 4.48 (d, 1H, J1,2¼8.4 Hz, H-1B), 3.86 (dd, 1H,
J2,3¼3.1 Hz, H-2C), 3.78 (dpo, 1H, J3,4¼2.7 Hz, H-4B), 3.75 (ddpo, 1H,
J2,3¼10.8 Hz, H-2B), 3.67 (ddpo, 1H, J3,4¼9.7 Hz, H-3C), 3.66e3.56 (m,
3H, H-6aB, H-6bB, H-3B), 3.56e3.46 (m, 3H, H-5C, H-5B, OCH2Pr),
3.35 (dt, 1H, J¼6.3 Hz, J¼9.7 Hz, OCH2Pr), 3.19 (pt, 1H, J4,5¼9.6 Hz, H-
4C), 1.91 (s, 3H, CH3NHAc), 1.48 (m, 2H, CH2Pr), 1.13 (d, 3H, J5,6¼6.3 Hz,
CH]All), 5.22 (m, 1H, Jtrans¼17.2 Hz, Jgem¼1.5 Hz, ]CH2All), 5.17 (m,
1H, Jcis¼10.5 Hz, ]CH2All), 4.99 (dpo, 1H, J¼11.3 Hz, HBn), 4.97 (dpo
,
1H, J1,2¼3.5 Hz, H-1B), 4.87 (d, 1H, J¼10.9 Hz, HBn), 4.76e4.68 (m,
4H, 2HBn, H-2B, H-1C), 4.60 (d, 1H, J¼11.3 Hz, HBn), 4.56 (d, 1H,
J¼10.9 Hz, HBn), 4.54 (d, 1H, J¼12.1 Hz, HBn), 4.47 (d, 1H, J¼11.6 Hz,
HBn), 4.41e4.32 (m, 3H, 2HBn, H-5B), 4.13 (br so, 1H, H-4B), 4.11 (mo,
H-6C), 0.76 (t, 3H, J¼7.4 Hz, CH3Pr). 13C NMR (D2O),
d
177.6 (NHCO),
1H, HAll), 4.07 (pt, 1H, J1,2¼2.0 Hz, H-2C), 3.90 (mo, 1H, HAll), 3.88
(ddo, 1H, J2,3¼3.2 Hz, J3,4¼9.6 Hz, H-3C), 3.81 (dd, 1H, J2,3¼10.9 Hz,
J3,4¼2.4 Hz, H-3B), 3.76 (s, 3H, CH3PMB), 3.69e3.62 (m, 2H, H-5C, H-
105.6 (C-1B, 1JCH¼163.8 Hz), 101.1 (C-1C, 1JCH¼173.6 Hz), 81.1 (C-2C),
77.5 (C-5B), 74.9 (C-4C), 73.3 (C-3B), 72.5 (C-3C), 72.2 (OCH2Pr), 71.1

10344
P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
6aB), 3.46 (dd, 1H, J5,6b¼5.5 Hz, J6a,6b¼8.7 Hz, H-6bB), 3.21 (pt, 1H,
J4,5¼9.4 Hz, H-4C), 1.21 (d, 3H, J5,6¼6.2 Hz, H-6C). 13C NMR (CDCl3),
went on for 3 h while the cooling bath reached rt. Since an LCeMS
profile and a TLC control (DCM/EtOAc 9:1 and Chex/EtOAc 6:4)
indicated that no acceptor (Rf 0.32, 0.43) nor donor (Rf 0.25, 0.33)
remained, but that a major new product (Rf 0.32, 0.45) was present,
the reaction was quenched by adding Et3N. Solids were filtered, and
the filtrate was concentrated to dryness. The residue was purified
by flash chromatography (Tol/EtOAc 9:1 to 8:2) to give first oxa-
zoline 1523 contaminated by some remaining acceptor and then
disaccharide 13 (598 mg, 73%) as a yellow foam.
d
161.8 (NHCO), 158.8 (CIVPMB), 138.5, 138.4, 138.0, 137.6 (4C, CIVAr),
133.5 (CH]All), 130.4 (CIVAr), 129.1e127.6 (22C, CAr), 117.5 (]CH2All),
1
113.7 (2C,
C
ArPMB), 96.4 (C-1B, JCH¼174.5 Hz), 95.7 (C-1C,
1JCH¼172.6 Hz), 92.9 (CCl3), 80.4 (C-4C), 79.1 (C-3C), 78.6 (C-3B),
75.2, 74.6, 73.6 (3C, CBn), 72.9 (C-2C), 72.2 (C-4B), 71.2 (2C, CBn), 70.2
(C-5B), 68.6 (C-6B), 68.0 (CH2All), 67.8 (C-5C), 55.2 (CH3PMB), 51.1
(C-2B), 18.2 (C-6C). HRMS (ESIþ): m/z 1012.2949 (calcd for
C
53H58Cl3NO11Na [MþNa]þ: m/z 1012.2973).
4.10.2. Route 2. A mixture of acceptor 2 (243 mg, 0.59 mmol),
ꢁ
4.9. 2-Methyl-5-tert-butylphenyl 3,4,6-tri-O-acetyl-2-deoxy-
2-trichloroacetamido-1-thio- -galactopyranoside (11)
donor 11 (431 mg, 0.70 mmol, 1.2 equiv), and powdered 4 A MS
b-D
(500 mg) in anhyd DCM (6.7 mL) was stirred at rt under an Ar at-
mosphere for 1 h, and then cooled to 0 ꢀC. NIS (198 mg, 0.88 mmol,
NaOMe (25% in MeOH, 15.9 mL, 69.6 mmol, 3.0 equiv) was
slowly added to a solution of -galactosamine hydrochloride
1.5 equiv) and TfOH (6 mL, 70 mmol, 0.12 equiv) were added, and the
D
reaction mixture was stirred at that temperature for 3 h while the
cooling bath reached rt. Since a TLC control (DCM/EtOAc 9:1 and
Chex/EtOAc 6:4) indicated that no acceptor (Rf 0.32, 0.43) nor donor
(Rf 0.25, 0.33) remained, but that a major new product (Rf 0.32,
0.45) was present, the reaction was quenched by adding Et3N.
Solids were filtered and the filtrate was concentrated to dryness.
The residue was purified by flash chromatography (Tol/EtOAc 9:1 to
(5.00 g, 23.18 mmol) in anhyd MeOH (100 mL) stirred at 0 ꢀC under
an Ar atmosphere. After 15 min, trichloroacetic anhydride (6.4 mL,
34.8 mmol, 1.5 equiv) was added dropwise, keeping the reaction
temperature close to 0 ꢀC. The mixture was stirred for 2 h, until
a TLC control (iPrOH/H2O/NH3 4:1:0.5) showed the complete con-
version of the starting material (Rf 0) to a less polar product (Rf
0.51). The reaction was quenched with Dowex-Hþ resin, the resin
was filtered and volatiles were evaporated. The residue was dis-
solved in anhyd pyridine (35 mL) and acetic anhydride (35.0 mL,
247.8 mmol, 15.0 equiv) was slowly added over 30 min at 0 ꢀC. After
stirring for 16 h at rt, a TLC control (DCM/EtOAc 9:1) showed the-
conversion of the intermediate (Rf 0) into less polar compounds
(Rf 0.68, 0.56). Solvents were evaporated and co-evaporated
three times with Tol. The residue was purified by flash chroma-
tography (DCM/EtOAc 95:5 to 6:4) to give a mixture (10.30 g, 90%)
of the peracylated galactosamine. 2-Methyl-5-tert-butylthiophenol
(2.50 mL, 22.6 mmol, 3.4 equiv) and BF3$OEt2 (2.55 mL, 20.0 mmol,
3.0 equiv) were added at rt to a solution of the latter (4.00 g,
6.66 mmol) in anhyd DCM (23 mL). After being stirred for 2.5 h
under an Ar atmosphere, a TLC control (Tol/EtOAc 7:3) of the re-
action mixture showed the conversion of the peracylated galac-
tosamine isomers (Rf 0.23, 0.28) into a major more polar product (Rf
0.41). The reaction was quenched with satd aq NaHCO3. DCM was
added, and the layers were separated. The organic phase was
washed with H2O and brine, dried by passing through a phase
separator filter, and volatiles were evaporated. The residue was
purified by flash chromatography (Tol/EtOAc 95:5 to 75:15) to give
8:2) to give disaccharide 13 (308 mg, 62%) as a yellow foam. Di-
25
saccharide 13 had [
a
]
ꢁ20 (c 1.0). 1H NMR (CDCl3),
d 7.35e7.14
D
(m, 7H, HAr), 6.82 (d, 2H, J¼8.6 Hz, HArPMB), 5.60 (d,1H, JNH,2¼8.5 Hz,
NH), 5.81 (m, 1H, CH]All), 5.25 (d, 1H, J3,4¼3.2 Hz, H-4B), 5.19 (m,
1H, Jtrans¼17.2 Hz, Jgem¼1.7 Hz, ]CH2All), 5.11 (m,1H, Jcis¼10.4 Hz, ]
CH2All), 4.93 (dd, 1H, J2,3¼11.1 Hz, H-3B), 4.79 (d, 1H, J¼10.9 Hz, HBn),
4.75 (d, 1H, J1,2¼1.7 Hz, H-1C), 4.62 (d, 1H, J¼11.1 Hz, HBn), 4.57 (d,
1H, J1,2¼8.5 Hz, H-1B), 4.51 (d, 1H, HBn), 4.46 (d, 1H, HBn), 4.15 (m,
1H, H-2B), 4.12e3.97 (m, 3H, H-6aB, H-6bB, HAll), 3.87 (m, 1H, HAll),
3.85 (m, 1H, H-2C), 3.80 (dd, 1H, J2,3¼3.1 Hz, J3,4¼9.2 Hz, H-3C), 3.75
(s, 3H, CH3PMB), 3.71 (pt, 1H, J5,6a¼6.5 Hz, J5,6b¼7.0 Hz H-5B), 3.62
(dq, 1H, J4,5¼9.5 Hz, J5,6¼6.2 Hz, H-5C), 3.31 (pt, 1H, H-4C), 2.08, 1.99,
1.91 (3s, 9H, CH3Ac), 1.24 (d, 3H, H-6C). 13C NMR (CDCl3),
d 170.3,
170.2, 170.1 (3C, COAc), 161.8 (NHCO), 159.6, 138.6 (2C, CIVAr), 133.7
(CH]All), 130.4 (CIVAr),129.8,128.4,127.7,127.5,126.4 (7C, CAr),117.2
(]CH2All), 114.2 (2C, CAr), 101.7 (C-1B), 98.2 (C-1C), 92.3 (CCl3), 81.0
(C-4C), 79.9 (C-3C), 76.6 (C-2C), 75.5 (CBn), 73.4 (CBn), 71.0 (C-5B),
70.8 (C-3B), 67.9 (C-5C), 67.7 (CH2All), 66.6 (C-4B), 61.2 (C-6B), 55.3
(CH3PMB), 52.6 (C-2B), 20.6, 20.5 (3C, CH3Ac), 17.9 (C-6C). HRMS
(ESIþ): m/z 868.1815 (calcd for C38H46Cl3NO14Na [MþNa]þ: m/z
868.1882).
thioglycoside 11 (3.11 g, 56%, three steps) as a white foam. Com-
25
pound 11 had [
a]
þ5 (c 1.0). 1H NMR (CDCl3),
d
7.63 (d, 1H,
4.11. Phenyl 4,6-O-benzylidene-2-deoxy-2-
D
J¼2.1 Hz, HAr), 7.28 (dd, 1H, J¼8.0 Hz, HAr), 7.17 (d, 1H, HAr), 6.73 (d,
1H, JNH,2¼9.0 Hz, NH), 5.46 (dd, 1H, J3,4¼3.3 Hz, J4,5¼0.9 Hz, H-4),
5.31 (dd, 1H, J2,3¼10.9 Hz, H-3), 4.91 (d, 1H, J1,2¼10.4 Hz, H-1), 4.29
(m, 1H, H-2), 4.21 (ddpo, 1H, J5,6a¼6.9 Hz, J6a,6b¼11.3 Hz, H-6a), 4.18
(ddpo, 1H, J5,6b¼6.3 Hz, H-6b), 3.98 (dt, 1H, H-5), 2.40 (s, 3H, MeAr),
trichloroacetamido-1-thio-
b-D-galactopyranoside (16)
A
solution of thioglycoside 12 (9.45 g, 17.41 mmol) in
anhyd MeOH (131 mL) was treated with 0.5 M methanolic NaOMe
(7.0 mL, 3.48 mmol, 0.2 equiv). The solution was stirred for 2 h at rt
under an Ar atmosphere. A TLC control (Chex/EtOAc 6:4 and DCM/
MeOH 8:2) showed the total conversion of the starting material (Rf
0.28, 1.0) into a more polar product (Rf 0, 0.61). The reaction was
quenched with Dowex-Hþ resin, the resin was filtered and volatiles
were evaporated to give the corresponding crude triol. Benzalde-
hyde dimethylacetal (5.2 mL, 34.8 mmol, 2.0 equiv) and CSA
(404 mg, 1.74 mmol, 0.1 equiv) were successively added to a solu-
tion of the latter in anhyd MeCN (450 mL). The mixture was stirred
under an Ar atmosphere for 16 h, at which time a TLC control (Chex/
EtOAc 1:1) showed the conversion of the triol (Rf 0) into a less polar
compound (Rf 0.32). The reaction was quenched with Et3N, and
volatiles were evaporated. The residue was purified by flash chro-
matography (Chex/EtOAc 6:4 to 4:6) to give acetal 16 (8.18 g, 93%)
2.20, 2.05, 2.02 (3s, 9H, CH3Ac), 1.34 (s, 9H, Bu). 13C NMR (CDCl3),
t
d
170.4, 170.2 (3C, COAc), 161.8 (NHCO), 149.8, 137.5, 131.6 (3C, CIVAr),
130.7, 130.2, 128.4 (3C, CAr), 92.3 (CCl3), 88.0 (C-1), 74.7 (C-5), 70.7
(C-3), 66.9 (C-4), 61.7 (C-6), 51.8 (C-2), 34.5 ðCIVt BuÞ; 31.4
ð9C; CH3t BuÞ; 20.8, 20.7, 20.6, 20.5 (4C, 3CH3Ac, CMeAr). HRMS (ESIþ):
m/z 634.0815 (calcd for C25H32Cl3NO8SNa [MþNa]þ: m/z 634.0812).
4.10. Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-
-galactopyranosyl)-(1/2)-4-O-benzyl-3-O-para-methox-
ybenzyl- -rhamnopyranoside (13)
b-D
a-L
4.10.1. Route 1. A mixture of acceptor 2 (400 mg, 0.97 mmol), donor
12 (786 mg, 1.45 mmol, 1.5 equiv), and powdered 4 A MS (900 mg)
ꢁ
25
25
in anhyd DCM (11 mL) was stirred at rt under an Ar atmosphere for
as a white solid. Thioglycoside 16 had [
a
]
D ꢁ12 (c 1.0), lit.24
[a]
D
1 h, and then cooled to ꢁ60 ꢀC. NIS (326 mg, 1.45 mmol, 1.5 equiv)
ꢁ11 (c 1.0). 1H NMR (CDCl3),
d 7.72e7.29 (m, 10H, HAr), 6.79 (d, 1H,
JNH,2¼7.6 Hz, NH), 5.58 (s, 1H, CHPh), 5.12 (d, 1H, J1,2¼10.2 Hz, H-1),
and TfOH (10
m
L, 116
mmol, 0.12 equiv) were added, and stirring

P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
10345
4.43 (dd, 1H, J5,6a¼1.6 Hz, J6a,6b¼12.4 Hz, H-6a), 4.27 (d, 1H,
J3,4¼3.6 Hz, J4,5¼0.7 Hz, H-4), 4.19 (ptd, 1H, J2,3¼J3,OH¼10.3 Hz, H-3),
4.08 (dd, 1H, J5,6b¼1.7 Hz, H-6b), 3.79 (ptd, 1H, H-2), 3.63 (m, 1H, H-
The residue was purified by flash chromatography (Tol/EtOAc 92:8
to 7:3) to give disaccharide 18 (10.33 g, 82%) as a yellow foam. The
25
condensation product had [
d
a
]
þ29 (c 1.0). 1H NMR (CDCl3),
D
5), 2.61 (d, 1H, OH-3). 13C NMR (CDCl3),
d
162.0 (NHCO), 137.4
7.48e7.08 (m, 12H, HAr), 6.81 (d, 2H, J¼8.6 Hz, HArPMB), 6.76 (d, 1H,
(CIVAr), 134.0 (2C, CAr), 130.7 (CIVAr), 129.4e126.5 (8C, CAr), 101.3
(CHPh), 92.4 (CCl3), 83.8 (C-1, 1JCH¼160.6 Hz), 75.0 (C-4), 70.5 (C-3),
70.2 (C-5), 69.3 (C-6), 54.2 (C-2). HRMS (ESIþ): m/z 526.0053 (calcd
for C21H20Cl3NO5SNa [MþNa]þ: m/z 526.0026).
JNH,2¼8.1 Hz, NH), 5.80 (m, 1H, CH]All), 5.43 (s, 1H, CHPh), 5.17 (m,
1H, Jtrans¼17.2 Hz, Jgem¼1.7 Hz, ]CH2All), 5.08 (m, 1H, Jcis¼10.4 Hz,
]CH2All), 4.94 (dd, 1H, J2,3¼11.1 Hz, J3,4¼3.4 Hz, H-3B), 4.83 (d, 1H,
J1,2¼1.5 Hz, H-1C), 4.80 (d, 1H, J¼10.9 Hz, HBn), 4.70 (d, 1H,
J1,2¼8.4 Hz, H-1B), 4.62 (d, 1H, J¼11.1 Hz, HBn), 4.51 (d, 1H, HBn), 4.47
(d, 1H, HBn), 4.28 (ptd, 1H, H-2B), 4.19 (dd, 1H, J5,6a¼1.2 Hz,
J6a,6b¼12.4 Hz, H-6aB), 4.14 (d, 1H, H-4B), 4.05 (m, 1H, HAll), 3.96 (dd,
1H, J5,6b¼1.5 Hz, H-6bB), 3.93 (m, 1H, H-2C), 3.85 (m, 1H, HAll), 3.80
(dd, 1H, J2,3¼3.1 Hz, J3,4¼9.3 Hz, H-3C), 3.73 (s, 3H, CH3PMB), 3.62
(dq, 1H, J4,5¼9.5 Hz, J5,6¼6.3 Hz, H-5C), 3.35 (pt, 1H, H-4C), 3.27 (br s,
1H, H-5B), 2.74e2.41 (m, 4H, CH2Lev), 1.92 (s, 3H, CH3Lev), 1.24 (d, 3H,
4.12. Phenyl 4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-2-
trichloroacetamido-1-thio-b-D-galactopyranoside (17)
Levulinic acid (1.6 mL, 15.3 mmol, 2.0 equiv), DCC (2.83 g,
13.7 mmol, 1.8 equiv), and DMAP (186 mg, 1.52 mmol, 0.2 equiv)
were added to a solution of alcohol 16 (3.85 g, 7.63 mmol) in DCM
(27 mL). The mixture was stirred for 1 h at rt under an Ar atmo-
sphere. At this time, a TLC control (Tol/EtOAc, 7:3) showed the
conversion of alcohol 16 (Rf 0.17) into a less polar product (Rf 0.31).
The reaction mixture was filtered over a pad of CeliteÒ, diluted with
DCM, and the organic phase was washed with 10% aq HCl,
satd aq NaHCO3, and brine, dried by passing through a phase sep-
arator filter, and concentrated. The residue was purified by flash
H-6C). 13C NMR (CDCl3),
d 206.6 (COLev), 172.1 (CO2Lev), 161.7
(NHCO), 159.6 (CIVPMB), 138.7, 137.5 (2C, CIVAr), 133.9 (CH]All), 130.5
(CIVAr), 129.8e126.4 (12C, CAr), 117.1 (]CH2All), 114.2 (2C, CArPMB),
101.3 (C-1B, 1JCH¼161.5 Hz), 100.9 (CHPh), 98.5 (C-1C,
1JCH¼174.4 Hz), 92.5 (CCl3), 81.1 (C-4C), 80.1 (C-3C), 75.9 (C-2C), 75.5,
73.3 (2C, CBn), 73.0 (C-4B), 71.7 (C-3B), 68.9 (C-6B), 67.8 (2C, C-5C,
CH2All), 66.6 (C-5B), 55.3 (CH3PMB), 52.4 (C-2B), 37.7 (CH2Lev), 29.7
(CH3Lev), 28.2 (CH2Lev), 17.9 (C-6C). HRMS (ESIþ): m/z 928.2211
chromatography (Tol/EtOAc, 8:2 to 7:3) to give the fully protected
25
17 (4.1 g, 90%) as a white powder. Thioglycoside 17 had [
a]
D ꢁ3 (c
(calcd for C44H50Cl3NO13Na [MþNa]þ: m/z 928.2245).
1.0). 1H NMR (CDCl3),
d
7.64e7.16 (m, 10H, HAr), 6.62 (d, 1H,
Oxazoline 19 had
[
a]
þ105 (c 1.0). 1H NMR (CDCl3),
25
D
JNH,2¼8.1 Hz, NH), 5.44 (s, 1H, CHPh), 5.33 (dd, 1H, J2,3¼10.9 Hz,
J3,4¼3.3 Hz, H-3), 5.08 (d, 1H, J1,2¼10.1 Hz, H-1), 4.32 (dd, 1H,
J5,6a¼1.4 Hz, J6a,6b¼12.4 Hz, H-6a), 4.25 (d, 1H, H-4), 4.05 (ptd, 1H,
H-2), 3.97 (dd, 1H, J5,6b¼1.5 Hz, H-6b), 3.57 (m, 1H, H-5), 2.61e2.39
d 7.52e7.50 (m, 2H, HAr), 7.43e7.39 (m, 3H, HAr), 6.60 (d, 1H,
J1,2¼6.0 Hz, H-1), 5.58 (s, 1H, CHPh), 5.00 (dd, 1H, J2,3¼7.9 Hz,
J3,4¼2.6 Hz, H-3), 4.56 (pt, 1H, J4,5¼2.4 Hz, H-4), 4.48 (dd, 1H, H-2),
4.43 (dd, 1H, J5,6a¼1.4 Hz, J6a,6b¼12.8 Hz, H-6a), 4.09 (dd, 1H,
J5,6b¼1.8 Hz, H-6b), 3.95 (m, 1H, H-5), 2.81e2.76 (m, 2H, CH2Lev),
2.73e2.68 (m, 2H, CH2Lev), 2.15 (s, 3H, CH3Lev). 13C NMR (CDCl3),
(m, 4H, CH2Lev), 1.93 (s, 3H, CH3Lev). 13C NMR (CDCl3),
d 206.4
(COLev), 172.0 (CO2Lev), 161.4 (NHCO), 137.6, 134.0, 130.8, 129.2,
129.0, 128.5, 128.2, 126.5 (12C, CAr), 100.9 (CHPh), 92.4 (CCl3), 84.3
d
206.2 (COLev), 172.3 (CO2Lev), 163.2 (CN), 137.2 (CIVAr), 129.3, 128.3,
1
(C-1, JCH¼148.9 Hz), 73.2 (C-4), 71.1 (C-3), 69.9 (C-5), 69.2 (C-6),
126.1 (5C, CAr), 105.1 (C-1, 1JCH¼186.0 Hz), 100.7 (CHPh), 77.2 (CCl3),
72.4 (C-3), 71.5 (C-4), 70.5 (C-5), 66.5 (C-6), 66.3 (C-2), 37.9 (CH2Lev),
29.7 (CH3Lev), 28.2 (CH2Lev). HRMS (ESIþ): m/z 492.0412 (calcd for
50.8 (C-2), 37.7 (CH2Lev), 29.6 (CH3Lev), 28.1 (CH2Lev). HRMS (ESIþ):
m/z 624.0410 (calcd for C26H26Cl3NO7SNa [MþNa]þ: m/z 624.0393).
C
C
20H21Cl3NO7 [MþH]þ: m/z 492.0384), m/z 514.0226 (calcd for
4.13. Allyl (4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-2-
20H20Cl3NO7Na [MþNa]þ: m/z 514.0203), and m/z 532.0306 (calcd
trichloroacetamido-
3-O-para-methoxybenzyl-
trichloromethyl-4,5-dihydro-(4,6-O-benzylidene-1,2-dideoxy-
3-O-levulinoyl- -galactopyranoso)[2,1-d]-1,3-oxazole (19)
b
-D
-galactopyranosyl)-(1/2)-4-O-benzyl-
for C20H22Cl3NO8Na [MþH2OþNa]þ: m/z 532.0309).
a-L
-rhamnopyranoside (18) and 2-
4.14. Allyl (4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-
a
-D
b-D
-galactopyranosyl)-(1/2)-4-O-benzyl-3-O-para-methoxy-
benzyl- -rhamnopyranoside (14)
a-L
4.13.1. Route 1. A mixture of acceptor 2 (100 mg, 241
m
mol), donor
ꢁ
17 (175 mg, 290
mmol, 1.2 equiv), and powdered 4 A MS (180 mg) in
4.14.1. Route 1. A solution of disaccharide 13 (4.59 g, 5.42 mmol) in
anhyd MeOH (47 mL) was treated with 0.5 M methanolic NaOMe
(0.5 mL, 1.08 mmol, 0.2 equiv). The mixture was stirred at rt under
an Ar atmosphere for 1.5 h. When TLC (Tol/EtOAc, 7:3) showed the
complete disappearance of the starting material (Rf 0.53) and the
presence of a more polar product (Rf 0), the reaction mixture was
neutralized with Dowex-Hþ resin and filtered. The filtrate was
concentrated to give the corresponding crude triol as a white solid.
Benzaldehyde dimethylacetal (3.2 mL, 21.68 mmol, 4.0 equiv) and
anhyd DCM (2.8 mL) was stirred at rt under an Ar atmosphere for
1 h, and then cooled to ꢁ60 ꢀC. NIS (81 mg, 362
mmol, 1.5 equiv) and
TfOH (5
mL, 29 mmol, 0.12 equiv) were added. The reaction was
stopped after 30 min by adding Et3N, while the temperature of the
cooling bath had reached ꢁ40 ꢀC and a TLC control (Tol/EtOAc 7:3)
had indicated the presence of a major product (Rf 0.37). Solids were
filtered, and the filtrate was concentrated to dryness. The residue
was purified by flash chromatography (Tol/EtOAc 90:10 to 7:3) to
give by order of elution, a mixture of oxazoline 19 and unreacted
acceptor 2 (36 mg, molar ratio 1:1.3) and disaccharide 18 (151 mg,
69%), both as yellow foams.
CSA (63 mg, 271 mmol, 0.05 equiv) were added to a solution of the
latter in MeCN (108 mL). The mixture was stirred for 2 h at rt under
an Ar atmosphere, at which time a TLC control (Tol/EtOAc 4:6) in-
dicated the conversion of the triol (Rf 0.06) into a major less polar
compound (Rf 0.63). The reaction mixture was neutralized by ad-
dition of Et3N and concentrated. The residue was purified by flash
chromatography (Tol/EtOAc 8:2 to 7:3) to give acceptor 14 (3.50 g,
84%) as a white solid.
4.13.2. Route 2. A mixture of acceptor 2 (5.70 g, 13.75 mmol), donor
ꢁ
17 (12.44 mg, 20.63 mmol, 1.5 equiv), and powdered 4 A MS (10 g)
in anhyd DCM (158 mL) was stirred at rt under an Ar atmosphere
for 1 h, and then cooled to ꢁ65 ꢀC. NIS (4.64 g, 20.63 mmol,
1.5 equiv) and TfOH (147 mL,1.65 mmol, 0.12 equiv) were added, and
stirring went on for 1 h while the cooling bath reached ꢁ40 ꢀC.
Since a TLC control (Tol/EtOAc 7:3) indicated the absence of any
remaining acceptor (Rf 0.40) or donor (Rf 0.30), but the presence of
a new product (Rf 0.37), the reaction was quenched by adding Et3N.
Solids were filtered, and the filtrate was concentrated to dryness.
4.14.2. Route 2. To a solution of disaccharide 18 (10.33 g,
11.37 mmol) in anhyd pyridine (95 mL) stirred at 0 ꢀC under an Ar
atmosphere were added AcOH (64 mL, dropwise) and hydrazine
monohydrate (2.8 mL, 56.93 mmol, 5 equiv). The reaction mixture
was stirred for 1 h, while the cooling bath reached rt. At this time,

10346
P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
follow up by TLC (Tol/EtOAc 7:3) showed the total conversion of the
starting material (Rf 0.33) into a more polar product (Rf 0.21). Fol-
lowing addition of DCM and ice cold water, the two layers were
separated and the aq phase was re-extracted twice with DCM. The
combined organic phases were washed with brine, dried by passing
through a phase separator filter, and concentrated to dryness. The
residue was purified by flash chromatography (Tol/EtOAc 8:2 to 7:3)
67.7 (CH2All), 67.2 (C-4A), 66.5 (C-5B), 61.4 (C-6A), 55.5 (C-2B), 55.3
(CH3PMB), 20.9, 20.8, 20.7, 20.4 (4C, CAc), 18.1 (C-6C). HRMS (ESIþ):
m/z 1160.2833 (calcd for
C
53H62Cl3NO20Na [MþNa]þ: m/z
1160.2828).
4.16. Allyl (2,3,4,6-tetra-O-acetyl-
(1/3)-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-
-galacto-pyranosyl)-(1/2)-3-O-acetyl-4-O-benzyl- -rham-
nopyranoside (22)
b-D-galactopyranosyl)-
b
-
to give acceptor 14 (7.94 g, 86%) as a white foam. Alcohol 14 had
D
a-L
25
[
a
]
D þ6 (c 1.0). 1H NMR (CDCl3),
d 7.54e7.31 (m, 10H, HAr), 7.22 (m,
3H, 2HArPMB, NH), 6.88 (m, 2H, HArPMB), 5.92 (m, 1H, CH]All), 5.59
(s, 1H, CHPh), 5.29 (m, 1H, Jtrans¼17.2 Hz, Jgem¼1.5 Hz, ]CH2All), 5.20
(m, 1H, Jcis¼10.4 Hz, ]CH2All), 4.95 (do, 1H, H-1C), 4.93 (dpo, 1H,
J¼10.9 Hz, HBn), 4.75 (d, 1H, J¼10.9 Hz, HBn), 4.65 (d, 1H, J¼10.9 Hz,
4.16.1. Route 1. Water (1.3 mL) and CAN (733 mg, 1.33 mmol,
4.0 equiv) were added to a solution of the fully protected 21
(380 mg, 0.33 mmol) in MeCN (13.4 mL) and the reaction mixture
was stirred for 30 min at rt. A TLC control (Tol/EtOAc 6:4) indicated
the complete disappearance of the starting material (Rf 0.56) and
the presence of a major more polar product (Rf 0.26). The reaction
was quenched with satd aq NaHCO3. The reaction mixture was
diluted with water and extracted three times with DCM. The
combined extracts were washed with brine, dried by passing
through a phase separator filter, and condensed to dryness. The
crude oil was dissolved in pyridine (16.7 mL), excess acetic anhy-
dride (3.16 mL) and DMAP (41 mg, 0.33 mmol, 1.0 equiv) were
added at rt and the reaction mixture was stirred for 2 h at this
temperature. At this time, a TLC control (Tol/EtOAc 6:4) showed the
conversion of the intermediate alcohol into a less polar product (Rf
0.48). Volatiles were evaporated and co-evaporated three times
with Tol. The residue was purified by flash chromatography (Tol/
EtOAc 8:2 to 75:25) to give pentaacetate 22 (290 mg, 82%) as
a white foam.
H
Bn), 4.60 (dpo, 1H, J1,2¼8.5 Hz, H-1B), 4.59 (dpo, 1H, HBn), 4.32 (dd,
1H, J5,6a¼1.3 Hz, J6a,6b¼12.4 Hz, H-6aB), 4.22e4.14 (m, 3H, H-2B, H-
4B, HAll), 4.10 (dd, 1H, J5,6a¼1.7 Hz, H-6bB) 4.00 (m, 1H, H-2C), 3.97
(mpo, 1H, HAll), 3.93 (ddpo, 1H, J2,3¼3.1 Hz, H-3C), 3.83 (s, 3H,
CH3PMB), 3.74 (dq, 1H, J4,5¼9.3 Hz, H-5C), 3.66 (dd, 1H, J2,3¼10.6 Hz,
J3,4¼3.5 Hz, H-3B), 3.48 (pt, 1H, J3,4¼9.5 Hz, H-4C), 3.37 (br s, 1H, H-
5B), 2.38 (s, 1H, OH-3B), 1.35 (d, 3H, J5,6¼6.2 Hz, H-6C). 13C NMR
(CDCl3),
(CH]All), 130.2 (CIVAr), 129.8, 129.2, 128.5, 128.2, 127.7, 127.4, 126.3
(12C, Ar), 117.2 (]CH2All), 114.2 (2C, CArPMB), 101.7 (C-1B,
d 163.7 (NHCO), 159.7 (CIVPMB), 138.6, 137.4 (2C, CIVAr), 133.9
C
1JCH¼161.5 Hz),101.2 (CHPh), 98.4 (C-1C, 1JCH¼173.5 Hz), 92.3 (CCl3),
81.2 (C-4C), 80.2 (C-3C), 76.7 (C-2C), 75.5 (CBn), 75.1 (C-4B), 73.7
(CBn), 73.5 (C-3B), 68.9 (C-6B), 67.8 (CH2All), 67.6 (C-5C), 67.0 (C-5B),
55.6 (C-2B), 55.4 (CH3PMB), 17.9 (C-6C). HRMS (ESIþ): m/z 830.1845
(calcd for C39H44Cl3NO11Na [MþNa]þ: m/z 830.1877).
4.15. Allyl (2,3,4,6-tetra-O-acetyl-
(1/3)-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-
-galacto-pyranosyl)-(1/2)-4-O-benzyl-3-O-para-methox-
ybenzyl- -rhamnopyranoside (21)
b-D-galactopyranosyl)-
b-
4.16.2. Route 2. A mixture of acceptor 14 (895 mg, 1.11 mmol),
donor 20 (708 mg, 1.44 mmol, 1.3 equiv), and powdered MS 4 A
ꢁ
D
a
-L
(1.8 g) in anhyd Tol (11.1 mL) was stirred at rt under an Ar atmo-
sphere for 1 h. The reaction mixture was cooled to ꢁ10 ꢀC, and
A mixture of acceptor 14 (100 mg, 0.12 mmol), donor30 20
TMSOTf (10 mL, 55 mmol, 0.05 equiv) was added. After 20 min, a TLC
ꢁ
(73 mg, 0.16 mmol, 1.2 equiv), and powdered MS 4 A (200 mg) in
control (Tol/EtOAc 9:1) showed the absence of both donor (Rf 0.32)
and acceptor (Rf 0.15) and the presence of a new major product (Rf
0.26). The reaction was quenched with Et3N, and the reaction
mixture was filtered and concentrated to dryness. The residue was
purified by flash chromatography (Tol/EtOAc 98:2 to 9:1) to give
slightly contaminated trisaccharide 21 as a white foam. This con-
taminated product was dissolved in MeCN (37 mL). Water (3.8 mL)
and CAN (2.43 g, 4.42 mmol, 4.0 equiv) were added. The reaction
mixture was stirred for 30 min, at which time a TLC control (Tol/
EtOAc 6:4) showed the complete disappearance of the starting
material (Rf 0.56) and the presence of a major more polar product
(Rf 0.26). The reaction was quenched with satd aq NaHCO3. The
reaction mixture was diluted with water and extracted three times
with DCM. The combined extracts were washed with brine, dried
by passing through a phase separator filter, and condensed to
dryness. The crude oil was dissolved in pyridine (58 mL), excess
acetic anhydride (3.12 mL) and DMAP (135 mg,1.11 mmol,1.0 equiv)
were added at rt and the reaction mixture was stirred for 2 h. At this
time, a TLC control (Tol/EtOAc 6:4) showed the conversion of the
intermediate alcohol into a less polar product (Rf 0.48). Volatiles
were evaporated and co-evaporated three times with Tol. The
residue was purified by flash chromatography (Tol/EtOAc 8:2 to
anhyd Tol (1.2 mL) was stirred at rt under an Ar atmosphere for 1 h.
The reaction mixture was cooled to ꢁ10 ꢀC, then TMSOTf (2
mL,
12 mmol, 0.1 equiv) was added. After 20 min, a TLC control (Tol/
EtOAc 9:1) showed the absence of both donor (Rf 0.32) and acceptor
(Rf 0.15) and the presence of a new major product (Rf 0.26). The
reaction was quenched with Et3N, filtered, and concentrated. The
residue was purified by flash chromatography (Tol/acetone 99:1 to
95:5) to give trisaccharide 21 (102 mg, 72%), as a white foam. The
25
condensation product had [
d
a
]
þ3 (c 1.0). 1H NMR (CDCl3),
D
7.61e7.18 (m, 12H, HAr), 7.17 (dpo, 1H, JNH,2¼6.8 Hz, NH), 6.85 (d,
2H, J¼8.6 Hz, HArPMB), 5.87 (m,1H, CH]), 5.60 (s,1H, CHPh), 5.38 (d,
1H, H-4A), 5.30 (d, 1H, J1,2¼8.4 Hz, H-1B), 5.28e5.20 (m, 2H, ]
CH2All, H-2A), 5.16 (m, 1H, Jcis¼10.4 Hz, Jgem¼1.4 Hz, ]CH2All), 4.96
(ddpo, 1H, J2,3¼10.4 Hz, J3,4¼3.3 Hz, H-3A), 4.93e4.86 (m, 2H, HBn, H-
1A), 4.86 (d, 1H, J1,2¼1.5 Hz, H-1C), 4.66 (dd, 1H, J2,3¼11.2 Hz,
J3,4¼3.2 Hz, H-3B), 4.65 (d, 1H, J¼12.0 Hz, HBn), 4.61 (d, 1H,
J¼12.1 Hz, HBn), 4.57 (d, 1H, J¼10.9 Hz, HBn), 4.31 (d, 1H, H-4B), 4.27
(dpo, 1H, J6a,6b¼11.9 Hz, H-6aB), 4.24 (ddpo, 1H, J5,6a¼6.9 Hz,
J6a,6b¼11.3 Hz, H-6aA), 4.15 (ddpo, 1H, J5,6b¼6.0 Hz, H-6bA), 4.11 (mo,
1H, HAll), 4.07 (do, 1H, H-6bB), 4.04 (dd, 1H, J2,3¼2.8 Hz, H-2C),
3.95e3.83 (m, 4H, HAll, H-5A, H-2B, H-3C), 3.81 (s, 3H, CH3PMB), 3.70
(dq, 1H, J4,5¼9.6 Hz, J5,6¼6.1 Hz, H-5C), 3.46 (pt, 1H, J3,4¼9.4 Hz, H-
4C), 3.33 (br s, 1H, H-5B), 2.18e1.96 (4s, 12H, CH3Ac), 1.30 (d, 3H, H-
65:35) to give pentaacetate 22 (870 mg, 74%, three steps) as a white
25
foam. Pentaacetate 22 had [
a
]
þ14 (c 1.0). 1H NMR (CDCl3),
D
6C). 13C NMR (CDCl3),
d
170.3, 170.2, 170.1, 169.2 (4C, COAc), 161.7
d 7.60e7.15 (m, 11H, 10HAr, NH), 5.91 (m, 1H, CH]All), 5.63 (s,
(NHCO), 159.6 (CIVPMB), 138.6, 137.8 (2C, CIVAr), 134.0 (CH]All),
1H, CHPh), 5.37 (d, 1H, H-4A), 5.34e5.16 (m, 5H, H-1B, H-2A, H-3C,
2]CH2All), 5.01 (d, 1H, J1,2¼7.9 Hz, H-1A), 4.95 (dd, 1H, J2,3¼10.3 Hz,
J3,4¼3.2 Hz, H-3A), 4.91 (d, 1H, J1,2¼1.6 Hz, H-1C), 4.75 (dd, 1H,
J2,3¼11.4 Hz, J3,4¼3.3 Hz, H-3B), 4.69 (d, 1H, J¼11.2 Hz, HBn), 4.59 (d,
1H, HBn), 4.38 (d, 1H, H-4B), 4.29 (d, 1H, J6a,6b¼12.7 Hz, H-6aB), 4.27-
4.22 (m, 2H, H-6aA, H-2C), 4.18e4.07 (m, 3H, HAll, H-6bB, H-6bA),
130.8 (CIVAr), 129.6e126.1 (12C, CAr), 116.7 (]CH2All), 113.9 (2C,
1
C
ArPMB), 100.7 (C-1A, JCH¼163.6 Hz), 100.4 (CHPh), 99.0 (C-1B,
1JCH¼162.4 Hz), 98.8 (C-1C, 1JCH¼173.4 Hz), 92.7 (CCl3), 80.7 (C-4C),
79.3 (C-3C), 76.1 (C-4B), 75.5 (CBn), 74.3 (C-2C), 73.2 (C-3B), 72.0
(CBn), 71.1 (C-5A), 70.9 (C-3A), 69.0 (C-6B), 68.9 (C-2A), 67.9 (C-5C),

P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
10347
3.99 (m, 1H, HAll), 3.86e3.77 (m, 3H, H-5A, H-5C, H-2B), 3.54 (pt, 1H,
J3,4¼J4,5¼9.6 Hz, H-4C), 3.47 (br s, 1H, H-5B), 2.20e1.98 (5s, 15H,
J¼11.2 Hz, HBn), 4.61 (d,1H, HBn), 4.49 (d,1H, J3,4¼3.2 Hz, H-4B), 4.44
(d, 1H, J1,2¼7.6 Hz, H-1A), 4.31 (dd, 1H, J2,3¼11.0 Hz, H-3B), 4.27e4.13
(m, 4H, H-6aB, H-6bB, H-2B, HAll), 4.03e3.97 (m, 2H, HAll, H-2C), 3.96
(ddpo,1H, J2,3¼3.3 Hz, J3,4¼9.3 Hz, H-3C), 3.82 (ddpo, 1H, J5,6a¼7.2 Hz,
J6a,6b¼11.4 Hz, H-6aA), 3.81 (ddo, 1H, J4,5¼0.8 Hz, H-4A), 3.76 (dd, 1H,
J5,6b¼4.7 Hz, H-6bA), 3.64 (mo, 1H, J4,5¼9.5 Hz, J5,6¼6.2 Hz, H-5C),
3.64 (br so, 1H, H-5B), 3.56 (ddpo, 1H, J2,3¼9.8 Hz, H-2A), 3.53 (dddo,
1H, H-5A), 3.43 (ddpo, 1H, J3,4¼3.2 Hz, H-3A), 3.42 (ptpo, 1H, H-4C),
H
Ac), 1.33 (d, 3H, J5,6¼6.2 Hz, H-6C). 13C NMR (CDCl3),
d 171.0, 170.5,
170.2, 170.0, 169.1 (5C, COAc), 161.6 (NHCO), 138.3, 137.8 (2C, CIVAr),
133.9 (CH]All), 129.7e125.9 (10C, CAr), 117.1 (]CH2All), 100.4
(CHPh), 100.3 (C-1A), 98.9 (C-1B), 98.8 (C-1C), 92.7 (CCl3), 79.5 (C-
4C), 76.4 (C-4B), 75.2 (CBn), 75.0 (C-2C), 73.6 (C-3C), 72.3 (C-3B), 71.3
(C-5A), 71.0 (C-3A), 69.1 (C-6B), 69.0 (C-2A), 68.0 (CH2All), 67.2 (C-5C),
67.1 (C-4A), 66.6 (C-5B), 61.5 (C-6A), 55.7 (C-2B), 21.2, 20.9, 20.8, 20.7,
20.5 (5C, CAc), 18.0 (C-6C). HRMS (ESIþ): m/z 1082.2361 (calcd for
1.25 (d, 3H, H-6C). 13C NMR (MeOD),
d 163.0 (NHCO), 138.8, 138.3
(2C, CIVAr), 134.1 (CH]All), 128.6e126.1 (10C, CAr), 115.8 (]CH2All),
104.8 (C-1A), 101.5 (C-1B), 101.0 (CHPh), 98.7 (C-1C), 92.8 (CCl3), 81.8
(C-4C), 78.5 (C-2C), 75.9 (C-4B), 75.7 (2C, C-3B, C-5A), 75.0 (CBn), 73.2
(C-3A), 71.1 (C-3C), 71.0 (C-2A), 69.0 (C-4A), 68.8 (C-6B), 67.6 (2C, C-
5C, CH2All), 66.8 (C-5B), 61.4 (C-6A), 54.1 (C-2B), 16.9 (C-6C). HRMS
(ESIþ): m/z 872.1804 (calcd for C37H46Cl3NO15Na [MþNa]þ: m/z
872.1830).
C
47H56Cl3NO20Na [MþNa]þ: m/z 1082.2358).
4.17. Allyl
deoxy-2-trichloroacetamido-
O-acetyl-4-O-benzyl- -rhamnopyranoside (23)
b-D
-galactopyranosyl-(1/3)-(4,6-O-benzylidene-2-
b-D-galactopyranosyl)-(1/2)-3-
a-L
A
solution of trisaccharide 22 (179 mg, 0.17 mmol) in
anhyd MeOH (10 mL) was treated by 0.5 M methanolic NaOMe
(51 L, 25
mol, 0.15 equiv) at 0 ꢀC. The mixture was stirred under
an Ar atmosphere at this temperature for 2.5 h. Follow up by TLC
(DCM/MeOH, 9:1) showed the conversion of the starting material
(Rf 0.97) into a major more polar product (Rf 0.35), the reaction
mixture was neutralized with Dowex-Hþ resin. The suspension was
filtered and volatiles were evaporated. The residue was purified by
flash chromatography (DCM/MeOH 98:2 to 9:1) to give first tetraol
23 (105 mg, 70%) as a white foam. Pentaol 24 (20 mg, 14%) was
4.19. Allyl
b
-
D
-galactopyranosyluronic acid-(1/3)-(4,6-O-
-galactopyr-
-rhamnopyranoside
m
m
benzylidene-2-deoxy-2-trichloroacetamido-b-D
anos-yl)-(1/2)-3-O-acetyl-4-O-benzyl-a-L
(25)
To a solution of tetraol 23 (100 mg, 112
phosphate buffer (1:1, 2.6 mL) were added successively NaClO2
(16 mg, 0.22 mmol, 2.0 equiv), TEMPO (5.2 mg, 34 mol, 0.3 equiv),
and a commercially available NaOCl solution (available chlorine 4%,
22 L, 11 mol, 0.1 equiv), resulting in a brown coloration of the
reaction mixture. The reaction mixture was stirred at 45 ꢀC for 24 h,
while more NaOCl (22 L, 11 mol, 0.1 equiv) was added after 5 h,
mmol) in MeCN/0.67 M
m
isolated as the second eluting product, resulting from over trans-
m
m
25
esterification. Tetraol 23 had [
a
]
þ17 (c 1.0, MeOH). 1H NMR
D
(MeOD),
d
7.58e7.52 (m, 2H, HAr), 7.40e7.24 (m, 8H, HAr), 5.95 (m,
m
m
1H, CH]All), 5.63 (s, 1H, CHPh), 5.32 (m, 1H, Jtrans¼17.1 Hz,
Jgem¼1.5 Hz, ]CH2All), 5.22 (ddpo, 1H, J2,3¼3.4 Hz, J3,4¼9.6 Hz, H-3C),
5.19 (mpo, 1H, Jcis¼10.5 Hz, ]CH2All), 5.05 (d, 1H, J1,2¼1.6 Hz, H-1C),
4.94 (d, 1H, J1,2¼8.2 Hz, H-1B), 4.65 (br s, 2H, HBn), 4.49 (d, 1H,
J3,4¼3.3 Hz, H-4B), 4.42 (ddpo, 1H, J2,3¼10.8 Hz, H-3B), 4.43 (dpo, 1H,
J1,2¼7.7 Hz, H-1A), 4.22e4.15 (m, 3H, H-6aB, H-6bB, HAll), 4.14 (dd,
1H, H-2C), 4.11e4.00 (m, 2H, H-2B, HAll), 3.84e3.72 (m, 4H, H-6aA,
H-6bA, H-4A, H-5C), 3.61 (br s, 1H, H-5B), 3.60 (ptpo, 1H, J4,5¼9.2 Hz,
H-4C), 3.56 (dd, 1H, H-2A), 3.52 (pt, 1H, H-5A), 3.42 (dd, 1H,
J2,3¼9.8 Hz, J3,4¼3.4 Hz, H-3A), 2.09 (s, 3H, CH3Ac), 1.29 (d, 3H,
7 h, and 10 h. A TLC control (DCM/MeOH 8:2þa drop of waterþa
drop of AcOH) indicated the total conversion of the starting 23 (Rf
0.73) into a more polar product (Rf 0.20). The reaction was
quenched by addition of few drops of EtOH and volatiles were
evaporated. The residue was suspended in MeOH, triturated, and
filtered. The filtrate was concentrated and the residue was purified
by flash chromatography (DCM/MeOH/H2O/AcOH 425:75:10:4 to
400:80:10:4) to give uronic acid 25 (92 mg, 90%) as a white solid,
following evaporation and repeated co-evaporations with MeOH
25
and Tol. The oxidation product 25 had [
a]
þ7 (c 1.0). 1H NMR
D
J5,6¼6.2 Hz, H-6C). 13C NMR (MeOD),
d
171.2 (COAc), 162.6 (NHCO),
(MeOD), d 7.62e7.54 (m, 2H, HAr), 7.42e7.24 (m, 8H, HAr), 5.96 (m,
138.5, 138.3 (2C, CIVAr), 133.9 (CH]All), 129.6e126.3 (10C, CAr), 116.0
(]CH2All), 104.7 (C-1A), 100.9 (CHPh), 100.7 (C-1B), 98.7 (C-1C), 92.8
(CCl3), 79.4 (C-4C), 75.9 (C-4B), 75.8 (C-2C), 75.7 (C-5A), 74.9 (CBn),
74.4 (C-3B), 73.3, 73.2 (2C, C-3C, C-3A), 71.0 (C-2A), 69.0 (C-4A), 68.7
(C-6B), 67.9 (C-5C), 67.7 (CH2All), 66.6 (C-5B), 61.3 (C-6A), 54.5 (C-2B),
20.1 (CH3Ac), 16.8 (C-6C). HRMS (ESIþ): m/z 914.1931 (calcd for
1H, CH]All), 5.72 (s, 1H, CHPh), 5.32 (m, 1H, Jtrans¼17.3 Hz,
Jgem¼1.5 Hz, ]CH2All), 5.22 (ddpo, 1H, J2,3¼3.2 Hz, J3,4¼9.8 Hz, H-3C),
5.19 (mpo, 1H, ]CH2All), 5.07 (d, 1H, J1,2¼1.5 Hz, H-1C), 4.85 (d, 1H,
J1,2¼8.8 Hz, H-1B), 4.66 (d,1H, J3,4¼3.1 Hz, H-4B), 4.64 (br s, 2H, HBn),
4.45 (dpo, 1H, J1,2¼7.7 Hz, H-1A), 4.43 (ddpo, 1H, J2,3¼11.0 Hz, H-3B),
4.23e4.16 (m, 4H, H-6aB, H-6bB, H-2B, HAll), 4.14 (d, 1H, J3,4¼3.2 Hz,
H-4A), 4.13 (dd, 1H, H-2C), 4.04 (m, 1H, HAll), 3.88 (s, 1H, H-5A), 3.76
(dq, 1H, J4,5¼9.4 Hz, J5,6¼6.2 Hz, H-5C), 3.63 (br s, 1H, H-5B), 3.60
(ptpo, 1H, H-4C), 3.57 (dd, 1H, H-2A), 3.42 (dd, 1H, J2,3¼9.6 Hz, H-3A),
C
39H48Cl3NO16Na [MþNa]þ: m/z 914.1937).
4.18. Allyl
deoxy-2-trichloroacetamido-
O-benzyl- -rhamnopyranoside (24)
b-
D-galactopyranosyl-(1/3)-(4,6-O-benzylidene-2-
b
-D-galactopyranosyl)-(1/2)-4-
2.08 (s, 3H, CH3Ac), 1.29 (d, 3H, H-6C). 13C RMN (MeOD),
d 173.8 (C-
a
-L
6A), 171.1 (COAc), 162.7 (NHCO), 138.5, 138.3 (2C, CIVAr), 133.9
(CH]All), 128.6e126.4 (10C, CAr), 116.0 (]CH2All), 104.0 (C-1A), 101.1
(C-1B), 100.9 (CHPh), 98.7 (C-1C), 92.8 (CCl3), 79.4 (C-4C), 76.1 (C-
5A), 76.0 (C-2C), 75.5 (C-4B), 74.9 (CBn), 74.0 (C-3B), 73.3, 73.2 (2C, C-
3C, C-3A), 70.7 (C-2A), 70.5 (C-4A), 68.7 (C-6B), 67.9 (C-5C), 67.7
(CH2All), 66.6 (C-5B), 54.3 (C-2B), 20.1 (CH3Ac), 16.8 (C-6C). HRMS
(ESIþ): m/z 928.1711 (calcd for C39H46Cl3NO17Na [MþNa]þ: m/z
928.1729).
A
solution of trisaccharide 22 (1.07 g, 1.01 mmol) in
anhyd MeOH (15 mL) was treated with 0.5 M methanolic NaOMe
(1.0 mL, 0.50 mmol, 0.5 equiv) at rt. The mixture was stirred at this
temperature under an Ar atmosphere for 2 h. Follow up by TLC
(DCM/MeOH, 9:1) showed the conversion of the starting material
(Rf 0.97) into a major more polar product (Rf 0.32), the reaction
mixture was neutralized with Dowex-Hþ resin. The suspension was
filtered and volatiles were evaporated. The residue was purified by
4.20. Allyl
benzylidene-2-deoxy-2-trichloroacetamido-
anos-yl)-(1/2)-4-O-benzyl- -rhamnopyranoside (26)
b-
D-galactopyranosyluronic acid-(1/3)-(4,6-O-
flash chromatography (DCM/MeOH 92:8 to 85:15) to give pentaol
b-D-galactopyr-
25
24 (786 mg, 92%) as a white foam. Pentaol 24 had [
a
]
D þ8 (c 1.0).
a-L
1H NMR (MeOD),
d
7.58e7.55 (m, 2H, HAr), 7.41e7.22 (m, 8H, HAr),
5.94 (m, 1H, CH]All), 5.63 (s, 1H, CHPh), 5.30 (m, 1H, Jtrans¼17.3 Hz,
Jgem¼1.6 Hz, ]CH2All), 5.18 (m, 1H, Jcis¼10.5 Hz, ]CH2All), 5.09 (d,
1H, J1,2¼8.0 Hz, H-1B), 5.01 (d, 1H, J1,2¼1.6 Hz, H-1C), 4.92 (d, 1H,
To a solution of pentaol 24 (250 mg, 0.112 mmol) in MeCN/
0.67 M phosphate buffer (1:1, 7.0 mL) were added NaClO2 (43 mg,
0.59 mmol, 2.0 equiv), TEMPO (14 mg, 88 mmol, 0.3 equiv), and

10348
P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
a commercially available NaOCl solution (available chlorine 4%,
69 L, 0.029 mmol, 0.1 equiv), causing a brown coloration. The
reaction mixture was stirred at 45 ꢀC for 24 h, while more NaOCl
(44 L, 11 mol, 0.1 equiv) was added after 5 h, 6 h, and 10 h. A TLC
control (DCM/MeOH 8:2þa drop of waterþa drop of AcOH) showed
the total conversion of the starting material (Rf 0.73) into a more
polar product (Rf 0.15). The reaction was quenched by adding few
drops of EtOH and volatiles were evaporated. The residue was
suspended in MeOH, triturated, and filtered. The filtrate was con-
centrated and the residue was purified by flash chromatography
(DCM/MeOH/H2O/AcOH 425:75:10:4 to 400:80:10:4) to give uronic
acid 26 (218 mg, 86%) as a white solid, following evaporation and
(m, 3H, H-2A, OCH2Pr, H-4C), 2.18 (s, 3H, CH3Ac), 2.06 (s, 3H,
m
CH3NHAc), 1.60 (m, 2H, CH2Pr), 1.28 (d, 3H, J5,6¼6.2 Hz, H-6C), 0.90 (t,
3H, J¼7.4 Hz, CH3Pr). 13C NMR (D2O),
d 177.2 (NHCO), 176.2 (COAc),
m
m
107.3 (C-1A), 105.3 (C-1B), 101.2 (C-1C), 81.8 (C-3B), 79.1 (C-2C), 77.7
(C-5A), 77.1 (C-5B), 75.6 (C-3C), 75.1 (C-3A), 73.3 (C-2A), 72.8 (C-4C),
72.3 (OCH2Pr), 71.4, 71.3 (2C, C-5C, C-4A), 70.5 (C-4B), 63.7, 63.5 (2C,
C-6B, C-6A), 54.2 (C-2B), 25.0 (CH3NHAc), 24.6 (CH2Pr), 23.2 (CH3Ac),
19.2 (C-6C), 12.5 (CH3Pr). HRMS (ESIþ): m/z 636.2473 (calcd for
C
25H43NO16Na [MþNa]þ: m/z 636.2479). RP-HPLC (215 nm):
tR¼12.5 min, RP-HPLC (215 nm, Aeris Peptide Phenomenex 3.6
mm
ꢁ
C
18 100 A 2.1ꢂ100 mm analytical column, 0e20% linear gradient of
MeCN in 0.08% aq TFA over 20 min at 0.3 mL minꢁ1): tR¼14.9 min.
repeated co-evaporation with MeOH and Tol. The oxidation product
The 4-O-acetyl analog 28 had 1H NMR (D2O, partial),
d 4.96 (br so,
25
26 had [
a
]
D þ4 (c 1.0). 1H NMR (MeOD),
d
7.62e7.57 (m, 2H, HAr),
1H, H-1C), 4.79 (tpo, 1H, J1,2¼9.6 Hz, H-4C), 4.73 (do, 1H, H-1B), 4.43
(d, 1H, J1,2¼7.6 Hz, H-1A), 4.15 (do, 1H, H-4B), 4.08 (dd, 1H, H-2C),
4.05e3.97 (m, 2H, H-3C, H-2B), 3.85 (mpo, 1H, H-5C), 3.52 (ddo, 1H,
H-2A), 2.13 (s, 3H, CH3Ac), 2.02 (s, 3H, CH3NHAc), 1.61e1.56 (mo, 2H,
CH2Pr), 1.14 (d, 3H, J5,6¼6.2 Hz, H-6C), 0.89 (t, 3H, J¼7.4 Hz, CH3Pr).
HRMS (ESIþ): m/z 636.2474 (calcd for C25H43NO16Na [MþNa]þ: m/z
7.43e7.25 (m, 8H, HAr), 5.94 (m,1H, CH]All), 5.72 (s,1H, CHPh), 5.30
(m, 1H, Jtrans¼17.3 Hz, Jgem¼1.5 Hz, ]CH2All), 5.18 (m, 1H,
Jcis¼10.4 Hz, ]CH2All), 5.04 (d, 1H, J1,2¼7.7 Hz, H-1B), 5.02 (d, 1H,
J1,2¼1.3 Hz, H-1C), 4.92 (d, 1H, J¼11.2 Hz, HBn), 4.66 (d, 1H,
J3,4¼1.7 Hz, H-4B), 4.61 (d, 1H, HBn), 4.46 (d, 1H, J1,2¼7.6 Hz, H-1A),
4.37e4.28 (m, 2H, H-2B, H-3B), 4.26e4.12 (m, 4H, H-6aB, H-6bB, HAll
,
636.2479). RP-HPLC (215 nm): tR¼16.5 min, RP-HPLC (Aeris Peptide
ꢁ
H-4A), 4.04e3.98 (m, 2H, H-2C, HAll), 3.96 (ddpo, 1H, J2,3¼3.3 Hz,
J3,4¼9.3 Hz, H-3C), 3.90 (s, 1H, H-5A), 3.66 (br so, 1H, H-5B), 3.64
(dqpo, 1H, J4,5¼9.5 Hz, J5,6¼6.2 Hz, H-5C), 3.56 (dd, 1H, H-2A), 3.47
(dd, 1H, J2,3¼9.7 Hz, J3,4¼3.3 Hz, H-3A), 3.60 (ptpo, 1H, H-4C), 1.25
Phenomenex 3.6
m
m C18 100 A 2.1ꢂ100 mm analytical column,
0e20% linear gradient of MeCN in 0.08% aq TFA over 20 min at
0.3 mL minꢁ1): tR¼21.0 min.
(d, 3H, H-6C). 13C NMR (MeOD),
d
174.1 (C-6A), 163.1 (NHCO),
4.22. Propyl
b-D
-galactopyranosyl-(1/3)-(2-acetamido-2-
138.7, 138.3 (2C, CIVAr), 134.1 (CH]All), 128.7e126.4 (10C, CAr),
115.8 (]CH2All), 104.1 (C-1A), 101.7 (C-1B), 101.0 (CHPh), 98.7 (C-1C),
92.8 (CCl3), 81.7 (C-4C), 78.7 (C-2C), 76.1 (C-5A), 75.5 (C-3B), 75.2 (C-
4B), 75.0 (CBn), 73.3 (C-3A), 71.1 (C-4A), 70.7 (C-3C), 70.4 (C-2A), 68.8
(C-6B), 67.6 (2C, C-5C, CH2All), 66.8 (C-5B), 54.0 (C-2B), 16.9 (C-6C).
HRMS (ESIþ): m/z 886.1591 (calcd for C37H44Cl3NO16Na [MþNa]þ:
m/z 886.1624).
deoxy-
(29)
b
-
D
-galactopyranosyl)-(1/2)- -rhamnopyranoside
a-L
To a stirred solution of trisaccharide 24 (143 mg, 170 mmol) in
MeOH (4.1 mL), was added 10% Pd/C (115 mg). The suspension was
stirred under a hydrogen atmosphere for a day. Additional 10% Pd/C
(50 mg) and Et3N (117 mL, 0.84 mmol, 4.0 equiv) were added and the
suspension was stirred under a hydrogen atmosphere for 2 days.
After this time, MS analysis of the crude reaction mixture indicated
the presence of a single compound, with a molecular weight cor-
responding to that of the target 29. The reaction mixture was fil-
tered over a pad of CeliteÒ. Evaporation of the volatiles, freeze-
drying, and purification of the crude material by RP-HPLC (215 nm,
0e30% linear gradient of MeCN in 0.08% aq TFA over 20 min,
4.21. Propyl
oxy- -galactopyranosyl)-(1/2)-3-O-acetyl-
anoside (27) and propyl -galactopyranosyl-(1/3)-(2-
acetamido-deoxy- -galactopyranosyl)-(1/2)-4-O-acetyl-
-rhamno-pyranoside (28)
b
-
D
-galactopyranosyl-(1/3)-(2-acetamido-de-
b-D
a-L
-rhamnopyr-
b-D
b-D
a-
L
A solution of trisaccharide 23 (152 mg, 170
m
mol) in MeOH
5.5 mL minꢁ1) gave trisaccharide 29 (48.4 mg, 51%) as a white solid
25
(10 mL) was passed through a 10% Pd/C cartridge (CatCartÒ 30)
using an H-CubeÔ continuous flow hydrogenation system (‘full H2’
mode, 30 ꢀC, 1 mL min flow rate) until MS analysis of the crude
reaction mixture indicated the presence of essentially two com-
pounds, with molecular weights corresponding to that of the target
following repeated freeze-drying. Trisaccharide 29 had [
a
]
D ꢁ4 (c
1.0, water). 1H NMR (D2O),
d
4.85 (d, 1H, J1,2¼1.6 Hz, H-1C), 4.57 (d,
1H, J1,2¼8.4 Hz, H-1B), 4.33 (d, 1H, J1,2¼7.7 Hz, H-1A), 4.05 (d, 1H,
J3,4¼3.1 Hz, H-4B), 3.93e3.87 (m, 2H, H-2B, H-2C), 3.78 (do, 1H,
J3,4¼3.2 Hz, H-4A), 3.77 (ddpo, 1H, J2,3¼11.0 Hz, H-3B), 3.72e3.59 (m,
5H, H-3C, H-6aB, H-6bB, H-6aA, H-6bA), 3.59e3.48 (m, 5H, H-5A, H-
5B, H-5C, H-3A, OCH2Pr), 3.41 (ddpo, 1H, J2,3¼10.1 Hz, H-2A), 3.37
(dtpo, 1H, J¼6.3 Hz, J¼9.8 Hz, OCH2Pr), 3.21 (pt, 1H, J3,4¼J4,5¼9.7 Hz,
H-4C), 1.92 (s, 3H, CH3NHAc), 1.48 (m, 2H, CH2Pr), 1.15 (d, 3H,
trisaccharide 27 and of the N-chloroacetyl analog. Et3N (71
mL,
0.51 mmol, 3 equiv) was added and the reaction mixture was
passed through the same cartridge using the same parameters. As
the system did not evolve according to MS follow up, the pressure
was set up to 10 bar. Additional runs through the same cartridge
allowed full conversion as indicated by MS analysis. Purification of
the crude material by RP-MPLC (C18 2x20 cm, linear gradient of
J5,6¼6.2 Hz, H-6C), 0.79 (t, 3H, J¼7.4 Hz, CH3Pr). 13C NMR (D2O),
1
d
177.5 (NHCO), 107.4 (C-1A, JCH¼162.9 Hz), 105.3 (C-1B,
1JCH¼163.7 Hz), 101.1 (C-1C, 1JCH¼172.8 Hz), 82.1 (C-3B), 81.0 (C-2C),
77.5 (C-5B*), 77.2 (C-5A*), 75.0 (C-3A), 74.9 (C-4C), 73.1 (C-2A), 72.6
(C-3C), 72.2 (OCH2Pr), 71.1 (2C, C-5C, C-4A), 70.5 (C-4B), 63.5, 63.4
(2C, C-6B, C-6A), 54.1 (C-2B), 24.9 (CH3NHAc), 24.5 (CH2Pr), 19.1 (C-6C),
12.4 (CH3Pr). HRMS (ESIþ): m/z 594.2369 (calcd for C23H41NO15Na
[MþNa]þ: m/z 594.2374). RP-HPLC (215 nm): tR¼10.3 min.
MeOH/H2O 100:0 to 70:30, 50 min at a flow rate of 10 mL minꢁ1
)
gave a 5:1 mixture of trisaccharides 27 and 28 (76 mg, 73%) as
a white solid following repeated freeze-drying. The separation of
the two regioisomers was successful when eluting the material
(70 mg) from a C18 column (3.1ꢂ30 cm, 215 nm, 0e80% linear
gradient of MeCN/0.08% aq TFA 80:20 in 0.08% aq TFA over 60 min,
20 mL minꢁ1), which provided first compound 27 (38.5 mg), then
4.23. Propyl
acetamido-2-deoxy-
-rhamnopyranoside (30) and propyl
uronic acid-(1/3)-(2-acetamido-2-deoxy-
anos-yl)-(1/2)-4-O-acetyl- -rhamnopyranoside (31)
b-D
-galactopyranosyluronic acid-(1/3)-(2-
-galactopyranosyl)-(1/2)-3-O-acetyl-
-galactopyranosyl-
-galactopyr-
25
regioisomer 28 (4.5 mg). The 3-O-acetyl trisaccharide 27 had [
a
]
b-D
D
þ13 (c 1.0, water). 1H NMR (D2O),
d
5.03 (dd, 1H, J1,2¼3.0 Hz,
a-
L
b-D
b-D
J1,2¼10.0 Hz, H-3C), 4.96 (br s, 1H, H-1C), 4.49 (d, 1H, J1,2¼8.1 Hz, H-
1B), 4.47 (d,1H, J1,2¼7.7 Hz, H-1A), 4.15 (d,1H, J3,4¼2.6 Hz, H-4B), 4.11
(dd, 1H, H-2C), 3.98 (dd, 1H, J2,3¼10.9 Hz, H-2B), 3.90 (ddpo, 1H, H-
3B), 3.89 (do, 1H, H-4A), 3.80e3.71 (m, 5H, H-3A, H-6aB, H-6bB, H-
6aA, H-6bA), 3.70e3.57 (m, 4H, H-5A, H-5B, H-5C, OCH2Pr), 3.55e3.47
a-L
To a stirred solution of trisaccharide 25 (81 mg, 89
mmol) in THF/
H2O (2:1, 4.0 mL) was added 10% Pd/C (80 mg). The suspension was

P. Chassagne et al. / Tetrahedron 69 (2013) 10337e10350
10349
25
stirred under a hydrogen atmosphere for a day. Additional 10% Pd/C
(40 mg) and Et3N (50 L, 0.36 mmol, 4.0 equiv) were added and the
following repeated freeze-drying. Trisaccharide 32 had [
a
]
ꢁ24
D
m
(c 0.9, water). 1H NMR (D2O),
d
4.85 (d,1H, J1,2¼1.5 Hz, H-1C), 4.57 (d,
suspension was stirred under a hydrogen atmosphere for 2 days.
After this time, MS analysis of the crude reaction mixture indicated
the presence of a single compound, with a molecular weight cor-
responding to that of the target trisaccharide. The reaction mixture
was filtered over a pad of CeliteÒ. Evaporation of the volatiles,
freeze-drying, and purification of the crude material by semi-
preparative RP-HPLC (215 nm, 0e35% linear gradient of MeCN in
0.08% aq TFA over 16 min, then 35e100% linear gradient of MeCN in
0.08% aq TFA over 4 min, 5.5 mL minꢁ1) gave by order of elution
trisaccharides 30 (26.5 mg, 47%) and 31 (8.9 mg, 16%), both as
1H, J1,2¼8.4 Hz, H-1B), 4.38 (d, 1H, J1,2¼7.8 Hz, H-1A), 4.25 (d, 1H,
J4,5¼1.3 Hz, H-5A), 4.14 (dd, 1H, J3,4¼3.4 Hz, H-4A), 4.10 (d, 1H,
J3,4¼3.1 Hz, H-4B), 3.91 (ddpo, 1H, J2,3¼10.9 Hz, H-2B), 3.89 (ddpo, 1H,
J2,3¼3.2 Hz, H-2C), 3.76 (dd, 1H, H-3B), 3.70 (ddpo, 1H, J3,4¼9.8 Hz, H-
3C), 3.69e3.65 (m, 1H, H-6aB), 3.63 (dd, 1H, J5,6b¼4.4 Hz,
J6a,6b¼11.8 Hz, H-6bB), 3.60e3.48 (m, 4H, H-3A, H-5C, H-5B, OCH2Pr),
3.43 (dd, 1H, J2,3¼10.0 Hz, H-2A), 3.37 (dt, 1H, J¼6.4 Hz, J¼9.8 Hz,
OCH2Pr), 3.21 (pt, 1H, J4,5¼9.7 Hz, H-4C), 1.91 (s, 3H, CH3NHAc), 1.48
(m, 2H, CH2Pr), 1.14 (d, 3H, J5,6¼6.2 Hz, H-6C), 0.78 (t, 3H,
J¼7.4 Hz, CH3Pr). 13C NMR (D2O),
d 177.5 (NHCO), 174.5 (C-6A), 106.9
1
1
a white solids, following extensive freeze-drying. The 3C-O-acetyl
(C-1A, JCH¼160.1 Hz), 105.3 (C-1B, JCH¼162.5 Hz), 101.1 (C-1C,
1JCH¼174.3 Hz), 82.5 (C-3B), 81.1 (C-2C), 77.3 (C-5B), 76.3 (C-5A), 74.9
(C-4C), 74.5 (C-3A), 72.6 (C-3C), 72.4 (C-2A), 72.2 (OCH2Pr), 72.0 (C-
4A), 71.1 (C-5C), 70.2 (C-4B), 63.5 (C-6B), 54.1 (C-2B), 24.9 (CH3NHAc),
24.5 (CH2Pr), 19.1 (C-6C), 12.4 (CH3Pr). HRMS (ESIþ): m/z 608.2145
25
trisaccharide 30 had [
a
]
D ꢁ9 (c 0.7, water). 1H NMR (D2O),
d 4.97
(dd, 1H, J2,3¼3.0 Hz, J3,4¼10.0 Hz, H-3C), 4.90 (d, 1H, J1,2¼1.5 Hz, H-
1C), 4.46 (d, 1H, J1,2¼7.8 Hz, H-1A), 4.43 (d, 1H, J1,2¼8.2 Hz, H-1B),
4.29 (d, 1H, J4,5¼1.3 Hz, H-5A), 4.18 (dd, 1H, J3,4¼3.4 Hz, H-4A), 4.15
(d,1H, J3,4¼2.8 Hz, H-4B), 4.05 (dd,1H, J2,3¼2.9 Hz, H-2C), 3.92 (ddpo
,
(calcd for
(215 nm): tR¼10.5 min.
C
23H39NO16Na [MþNa]þ: m/z 608.2167). RP-HPLC
1H, J2,3¼10.9 Hz, H-2B), 3.84 (dd, 1H, H-3B), 3.74e3.66 (m, 3H, H-
6aB, H-6bB, H-5C), 3.64 (dd, 1H, J2,3¼10.0 Hz, H-3A), 3.61e3.52 (m,
2H, H-5B, OCH2Pr), 3.48 (ddpo, 1H, H-2A), 3.47e3.40 (m, 2H, H-4C,
OCH2Pr), 2.11 (s, 3H, CH3Ac), 1.99 (s, 3H, CH3NHAc), 1.54 (m, 2H,
J¼7.0 Hz, CH2Pr), 1.22 (d, 3H, J5,6¼6.2 Hz, H-6C), 0.84 (t, 3H, J¼7.4 Hz,
Acknowledgements
We thank C. Ganneau (UCB) for analytical RP-HPLC, Y.M. Coïc
and F. Baleux (UCB) for LCeMS analysis, and F. Bonhomme (CNRS
UMR 3523) for HRMS recording and NMR processing. This work
was supported by grants from the Institut Pasteur (Fellowship to
CH3Pr). 13C NMR (D2O),
d 177.1 (NHCO), 176.1 (COAc), 174.6 (C-6A),
106.7 (C-1A, 1JCH¼161.9 Hz), 105.3 (C-1B, 1JCH¼162.8 Hz), 101.2 (C-1C,
1JCH¼175.3 Hz), 82.1 (C-3B), 79.1 (C-2C), 77.1 (C-5B), 76.4 (C-5A), 75.5
(C-3C), 74.6 (C-3A), 72.7 (C-4C), 72.5 (C-2A), 72.3(OCH2Pr), 72.1 (C-
4A), 71.3 (C-5C), 70.1 (C-4B), 63.5 (C-6B), 54.1 (C-2B), 25.0 (CH3NHAc),
24.6 (CH2Pr), 23.1 (CH3NHAc), 19.1 (C-6C), 12.4 (CH3Pr). HRMS (ESIþ):
m/z 650.2252 (calcd for C25H41NO17Na [MþNa]þ: m/z 650.2272).
ꢂ
L.R.), the Ministere de l’Education Nationale, de la Recherche et de
la Technologie (MENRT) (PhD fellowship to P.C.). The research
leading to these results, including a fellowship to P.C., has received
funding from the European Commission Seventh Framework
Program [FP7/2007-2013] under Grant agreement nꢀ 261472-
STOPENTERICS.
RP-HPLC (215 nm): tR¼12.7 min.
25
The 4C-O-acetyl analog 31 had [
a
]
D ꢁ25 (c 0.5, water). 1H NMR
(D2O),
d
4.85 (d, 1H, J1,2¼1.6 Hz, H-1C), 4.68 (pt, 1H, J3,4¼J4,5¼9.8 Hz,
H-4C), 4.60 (d, 1H, J1,2¼8.5 Hz, H-1B), 4.37 (d, 1H, J1,2¼7.8 Hz, H-1A),
4.21 (d, 1H, J4,5¼1.3 Hz, H-5A), 4.12 (dd, 1H, J3,4¼3.4 Hz, H-4A), 4.11
(d, 1H, J3,4¼2.8 Hz, H-4B), 3.98 (dd, 1H, J2,3¼3.0 Hz, H-2C), 3.95e3.89
(m, 2H, H-2B, H-3C), 3.78e3.61 (m, 4H, H-3B, H-5C, H-6aB, H-6bB),
3.60e3.49 (m, 3H, H-3A, H-5B, OCH2Pr), 3.43 (dd,1H, J2,3¼10.0 Hz, H-
2A), 3.38 (dt,1H, J¼6.4 Hz, J¼9.7 Hz, OCH2Pr), 2.02 (s, 3H, CH3Ac),1.90
(s, 3H, CH3NHAc), 1.48 (m, 2H, CH2Pr), 1.04 (d, 3H, J5,6¼6.2 Hz, H-6C),
Supplementary data
1D and 2D NMR spectra, including 1H, 13C, DEPT, COSY, and
HSQC of all new compounds are provided. Supplementary data
related to this article can be found at http://dx.doi.org/10.1016/
j.tet.2013.10.011.
0.78 (t, 3H, J¼7.4 Hz, CH3Pr). 13C NMR (D2O),
d 177.6 (NHCO), 176.3
1
(COAc), 174.8 (C-6A), 106.9 (C-1A, JCH¼161.9 Hz), 105.0 (C-1B,
1JCH¼163.7 Hz), 101.3 (C-1C, 1JCH¼174.4 Hz), 82.5 (C-3B), 80.2 (C-2C),
77.4 (C-5B), 76.9 (C-4C), 76.5 (C-5A), 74.6 (C-3A), 72.6 (C-2A), 72.4
(OCH2Pr), 72.2 (C-4A), 71.0 (C-3C), 70.4 (C-4B), 69.1 (C-5C), 63.6
(C-6B), 54.2 (C-2B), 24.9 (CH3NHAc), 24.6 (CH2Pr), 23.1 (CH3NHAc),
19.1 (C-6C), 12.4 (CH3Pr). HRMS (ESIþ): m/z 650.2305 (calcd for
References and notes
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