Melioidosis patient serum-reactive synthetic tetrasaccharides bearing the predominant epitopes of Burkholderia pseudomallei and Burkholderia mallei O-antigens

Article abstract of DOI:10.1039/c9ob01711a

Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have been identified as attractive vaccine candidates for the development of prophylactic measures against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed of d-glucose and 6-deoxy-l-talose residues, the latter being diversely acetylated and methylated. Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-deoxy-l-talose residue. We show that these synthetic compounds strongly react with culture-confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg. Our results suggest that these tetrasaccharides could be suitable candidates for the development of vaccines and/or diagnostic tools against melioidosis and glanders.

Full text of DOI:10.1039/c9ob01711a

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Cloutier, E.
Delar, K. Muru, S. Ndong, R. R. Hoyeck, T. Kaewarpai, N. Chantratita, M. N. Burtnick, P. J. Brett and C.
Gauthier, Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB01711A.
Volume 15
Number 47
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
21
December 2017
Pages 9945-10124
Organic &
Biomolecular
Chemistry
rsc.li/obc
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
single cell transcriptome in vivo analysis (TIVA)
rsc.li/obc

Page 1 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
Melioidosis Patient Serum-Reactive Synthetic Tetrasaccharides Bearing the Predominant
Epitopes of Burkholderia pseudomallei and Burkholderia mallei O-Antigens
a
a
a
a
a
Maude Cloutier, Emmanilo Delar, Kevin Muru, Seynabou Ndong, Robert R. Hoyeck, Taniya
b
b,c
d
d
,a
Kaewarpai, Narisara Chantratita, Mary N. Burtnick, Paul J. Brett, and Charles Gauthier*
aCentre Armand-Frappier Santé Biotechnologie, Institut national de la recherche scientifique
INRS), 531, boul. des Prairies, Laval (Québec), Canada, H7V 1B7
(
E-mail: charles.gauthier@iaf.inrs.ca
bDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol
University, 420/6 Rajvithi Road, Bangkok 10400, Thailand
cMahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol
University, 420/6 Rajvithi Road, Bangkok 10400, Thailand
dDepartment of Microbiology and Immunology, University of Nevada, Reno School of Medicine,
1
664, N. Virginia Street, Reno, Nevada, 89557, United States of America
1

Organic & Biomolecular Chemistry
Page 2 of 70
View Article Online
DOI: 10.1039/C9OB01711A
ABSTRACT:
Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia
pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in
developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have
been identified as attractive vaccine candidates for the development of prophylactic measures
against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-
antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed
of D-glucose and 6-deoxy-L-talose residues, the latter being diversely acetylated and methylated.
Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of
Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential
glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-
deoxy-L-talose residue. We show that these synthetic compounds strongly react with culture-
confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg.
Our results suggest that these tetrasaccharides could be suitable candidates for the development of
vaccines and/or diagnostic tools against melioidosis and glanders.
2

Page 3 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
INTRODUCTION
Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), two closely related facultative
intracellular Gram-negative bacteria (GNB), are the respective etiologic agents of melioidosis and
glanders. Endemic in tropical and sub-tropical regions and sporadically found worldwide,1, 2 these
two neglected bacteria are highly virulent and, if left untreated, are fatal in up to 50% of infected
patients.3-5 Notably, the urgent public health issues that these diseases represent is highlighted by
the fact that melioidosis is considered the third most common cause of infectious diseases-related
6
deaths in Northeast Thailand. The protean manifestations of melioidosis and glanders can hinder
diagnosis, and treatment of infected patients is challenging due to the intracellular nature of these
pathogens as well as their intrinsic resistance to commonly used antibiotics such as
7
aminoglycosides, polymyxins, and β-lactams. Importantly, in addition to the public health threat
that they represent, Bp and Bm are considered potential biological weapons by the U. S. Centers
for Disease Control and Prevention, as they have been classified as “Tier 1” biological select
agents.8
Notwithstanding the efforts put towards the development of prophylactic measures against
melioidosis and glanders, there are currently no licensed vaccines against these diseases. In light
9
of the many attempts to develop protective vaccines, recent studies underlined the potential of
using lipopolysaccharides (LPS) as subunit vaccines owing to their stimulating effect on the
adaptive immune system.10 LPSs are surface-exposed antigenic and virulence-associated
1
1
polysaccharides anchored in the outer membrane of GNB such as Bp and Bm. It was shown that
LPS-specific monoclonal antibodies (mAbs) have the ability to passively protect animal models
from infection,12-17 and that immunization of mice with LPS conjugates induces significant
protection against lethal challenges of Bp.10, 18
3

Organic & Biomolecular Chemistry
Page 4 of 70
View Article Online
DOI: 10.1039/C9OB01711A
1
9
The predominant serotype A (97%) O-antigens (OAgs) of Bp and Bm LPSs are of similar
composition and consist of linear heteropolymers whose repeating unit is a disaccharide of the
following structure: [3)-6-deoxy-α-L-talopyranosyl-(13)-β-D-glucopyranosyl-(1] (Figure
1
). One atypical characteristic of these OAgs lies in the non-stoichiometric methylation and
acetylation patterns of the 6-deoxy-L-talose residues, which slightly vary between species. It has
been revealed that the predominant inner disaccharide unit of both Bp and Bm is acetylated at O-2.
In addition, the terminal residue of Bm is 3-O-methylated and 2-O-acetylated whereas Bp’s is also
acetylated at O-4.20-25
terminal residues
intra-chain residues
OH
O
HO
(outer core)
O
OH
O
O
HO
O
HO
HO
O
OAc
n
O
RO
OMe
HO
OAc
B. pseudomallei (R = Ac)
B. mallei (R = H)
Figure 1. Structures of B. pseudomallei and B. mallei LPS O-antigens showing the predominant
terminal and intra-chain epitopes.
We recently described the synthesis of seven di- and trisaccharides (1–7) as the minimal structures
2
6
mimicking all of the reported substitution patterns of Bp and Bm OAgs (Figure 2A). Enzyme-
linked immunosorbent assay, saturation transfer difference-nuclear magnetic resonance, and
surface plasmon resonance assays were jointly used to assess the molecular interactions of the
synthetic oligosaccharides with LPS-specific mAbs. These biophysical studies revealed the crucial
role of the capping 3-O-methylated-6-deoxy-L-talose residues in the binding process. Moreover,
we showed that high-titer antibody responses can be raised in mice injected with a CRM197:Bm-
4

Page 5 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
like disaccharide 6 construct and that these responses were cross-reactive with Bm-like LPS. In
contrast, a CRM197:Bp-like disaccharide 7 construct was not able to induce a strong antibody
response in both BALB/c and C57BL/6 mice suggesting a gap in the mice B cell repertoire against
the OAg motif bearing an additional 4-O-acetyl group. We also showed that Thai melioidosis
patient serum samples can react with Bp-like disaccharide 7, which indicates that, unlike mice,
humans can generate antibody responses against this unique OAg epitope.
A
OH
O
OH
O
HO
O
2 5
O(CH ) NH2
HO
O
2 5
O(CH ) NH2
O
O
1
R O O
OH
RO
OMe
OH
HO
O
OR2
OAc
OH
HOOH
1
2
6 R = H
R = Ac
1
2
3
4
5
R = H; R = H
7
1
2
R = H; R = Ac
1
2
R = H; R = Me
1
2
R = Ac; R = Me
1
2
R = Ac; R = Ac
B
OH
O
HO
O
2 5
O(CH ) NH2
A
B
O
OH
O
HO
O
H
HO
O
O
C
D
O
OAc
RO
OH
OMe
8
9
R = Ac
R = H
OAc
Figure 2. (A) Structures of previously synthesized di- and trisaccharides (1–7); and (B) target
synthetic tetrasaccharides 8 and 9 related to Bp and Bm LPS OAgs, respectively.
We now show that longer oligosaccharidic fragments, such as tetrasaccharides 8 and 9 (Figure 2B),
bearing both internal and terminal epitopes of Bp and Bm OAgs are suitable candidates for the
development of diagnostics and subunit vaccines against melioidosis and glanders. We herein
describe a synthetic approach enabling the preparation of tetrasaccharides related to Bp and Bm
LPS OAg featuring the main intrachain and terminal acetylation and methylation patterns. The
reactivity profiles of synthetic oligosaccharides 1–9 were assessed against Thai melioidosis patient
5

Organic & Biomolecular Chemistry
Page 6 of 70
View Article Online
DOI: 10.1039/C9OB01711A
serum samples highlighting that tetrasaccharides 8 and 9 act as exquisite mimics of the whole Bp
and Bm LPS OAgs.
RESULTS AND DISCUSSION
Retrosynthetic approach
The presence of acetyl groups in target tetrasaccharides 8 and 9 posed a substantial challenge in
the development of the synthetic route, which had to be designed so that their migration or cleavage
would be avoided. Retrosynthetic analysis of these oligosaccharides, as depicted in Figure 3, led
2
6
26
to the identification of synthons 13, 14, 15, and 16 as the most readily accessible functionalized
monosaccharides that could enable their assembly. Our synthetic approach would be based on a
convergent [2 + 2] glycosylation strategy in which disaccharides 11 and 12, assembled from
building blocks 13–16, would be coupled into fully protected tetrasaccharide 3, the latter being a
precursor of both Bp- and Bm-like oligosaccharides 8 and 9, respectively. We plan to introduce O-
acetyl and O-methyl groups in synthons 13 and 14 prior to the synthesis of the tetrasaccharides.
These building blocks would have to be designed in order to preserve orthogonality among the
temporary protecting groups and acetates. The acetyl groups at O-2 on rhamnosides 13 and 14 as
2
7
well as the (2-azidomethyl)benzoyl (AZMB) group of glucoside 15 would act as neighbouring
participating groups (NPG) to ensure the stereoselective formation of 1,2-trans glycosidic linkages
2
8
in the target compounds. Moreover, assisted cleavage of the AZMB group, which involves the
reduction of the primary azide into the corresponding amine followed by in situ intramolecular
cyclization into an isoindolinone, thereby releasing the C2 hydroxyl group,27-29 would be achieved
during the final hydrogenolysis step. The anomeric position of monosaccharides 13 and 14 would
be activated with a trichloroacetimidate (TCA) leaving group in an attempt to achieve high-yielding
6

Page 7 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
couplings, as previously reported for structurally similar L-rhamnose donors.30 In contrast,
glucoside 15 would be thiolated at the anomeric position to ensure its orthogonal glycosylation
with respect to donor 14. The methylphenylthio (STol) group is known to be highly stable under a
broad variety of conditions while being readily accessible from peracetylated sugars,31 and
thioglucoside 15 would be converted into other donors in the event that the coupling proves
ineffective.
OH
O
1) delevulinoylation
2) epimerization
3) hydrogenolysis
Ph
O
LevO
O
O
HO
O
O(CH ) NH
O
O(CH ) N3
O
2 5
2
O
2 5
OH
O
O
HO
OH
Ph
O
OBn
LevO
MeO
HO
O
O
O
O
O
OAc
OAc
O
O
RO
OH
OAZMB
OMe
8
R = Ac
OAc
OAc
9
R = H
10
[2 + 2] glycosylation
NH
O
CCl3
Ph
O
Ph
O
O
Ph
O
O
STol
O
O
O(CH ) N3
O
R2
2 5
O
O
O
O
LevO
HO
OAZMB
LevO
OBn
OR1
O
RO
LevO
MeO
O
OAc
1
2
15 R = AZMB; R = STol
1
1
3 R = TBS
4 R = Me
OAc
HO
OAc
12
1
2
16 R = Bn; R = O(CH ) N
5 3
2
1
1
Figure 3. Retrosynthesis analysis of target tetrasaccharides 8 and 9.
As 6-deoxy-L-talose derivatives are not commercially available, rhamnosides 13 and 14 would
instead be employed. We envisioned to conduct both C4 epimerization simultaneously at a later
stage of the synthetic route via a two-step oxidation/reduction sequence.32 To reach Bp-like
tetrasaccharide 8, we intended to take advantage of the steric hindrance surrounding the inner 6-
deoxy-L-talose residue to acetylate regioselectivity the O-4 position of the non-reducing end
residue. Finally, acceptor 16 would be equipped with an azidolinker at C1, which upon reduction
would allow the coupling of the fully assembled, unprotected oligosaccharides to activated ELISA
plates enabling antigenicity assays with serum samples and/or covalent coupling with carrier
proteins.
7

Organic & Biomolecular Chemistry
Page 8 of 70
View Article Online
DOI: 10.1039/C9OB01711A
First generation synthesis of tetrasaccharides
As depicted in Scheme 1, the synthesis of glucoside acceptor 15 was performed in three steps from
3
3
known diol 17. A para-methoxybenzyl (PMB) group was selectively introduced at O-3 through
3
4
the formation of a stannylene acetal. This step was followed by esterification of the resulting
alcohol35 with 2-(azidomethyl)benzoic acid (AZMBOH), which was prepared using Steglich
conditions.30,36 The PMB group finally underwent oxidative cleavage using 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ), furnishing acceptor 15.
A
1
) Bu SnO, tol, reflux;
2
then PMBCl, TBAI, CsF
DMF, 90 °C, 51% (82% brsm)
Ph
O
Ph
O
O
O
O
O
O
STol
STol
N3
RO
HO
2) AZMBOH, DCC, DMAP
OH
DCM, reflux, 88%
O
17
AZMB
18 R = PMB
DDQ, DCM/H O 10:1
2
rt, 77%
15 R = H
B
OAll
1) TBSCl, imidazole
OAll
THF, reflux
O
O
LevO
LevO
TBSO
HO
2) Ac2O, Py, DMAP
OH
OAc
9
2% (2 steps)
) [Ir(COD){PMe(Ph) } ] .PF
6
1
9
20
+
–
1
2
2
H , THF
2
2
) I , THF/H O
2 2
8
4% (2 steps)
NH
CCl3
O
CCl CN, Cs CO
3
OH
3
2
acetone, DCM
O
O
LevO
LevO
TBSO
98%
TBSO
OAc
OAc
13
21
1
6, TMSOTf
Ph
O
O
Et O, –10 °C
O
2
O(CH ) N
2 5 3
O
91%
OBn
O
LevO
RO
OAc
22 R = TBS
12 R = H
8

Page 9 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
Scheme 1. (A) Synthesis of 2-O-AZMB-containing glucose derivative 15; and (B) failed attempts
to cleave the TBS group in disaccharide 22.
2
6
Allyl rhamnoside 19 was used as starting material for the preparation of TCA 13. Imidazole was
used as catalyst for the selective protection of the C3 hydroxyl with a tert-butyldimethylsilyl (TBS)
group and the remaining free OH was acetylated under standard conditions, yielding rhamnoside
2
0 in 92% yield over two steps. Allyl isomerization into the corresponding 1-propenyl ether was
accomplished using an iridium-based catalyst37, 38 and was followed by its hydrolysis promoted by
3
9
iodine in a mixture of THF and water. Resulting hemiacetal 21 was successfully converted into
TCA 13 (∼9:1 α/β mixture) through a standard procedure40 involving the use of
trichloroacetonitrile and cesium carbonate. The latter was coupled with acceptor 16 under the
promotion of trimethylsilyl trifluoromethanesulfonate (TMSOTf),41-44 providing disaccharide 22
in an excellent 91% yield as the sole α-anomer. In order to lengthen the disaccharide, deprotection
of the TBS group was attempted using Et N·3HF, but acetyl cleavage was unfortunately observed.
3
Tetra-n-butylammonium fluoride (TBAF)-mediated cleavage, with and without acetic acid buffer,
also led to acetyl deprotection.
Due to the poor selectivity of the silyl ether deprotection, we prepared thiorhamnoside 25, bearing
an orthogonal PMB group at O-3 instead of a TBS group as previously reported by us (Scheme
2
6
45
2
). Alcohol 23 was therefore levulinoylated under the action of dicyclohexylcarbodiimide
(DCC) and catalytic DMAP and the isopropylidene group was cleaved in acidic media, furnishing
diol 24 in excellent yield. Tin acetal chemistry was taken advantage of for the selective alkylation
of the O-3 position and the remaining hydroxyl group was acetylated into donor 25. N-
Iodosuccinimide (NIS)/silver triflate (AgOTf)-promoted glycosidation46, 47 of the latter with
9

Organic & Biomolecular Chemistry
Page 10 of 70
View Article Online
DOI: 10.1039/C9OB01711A
acceptor 16 at –10 °C in DCE provided disaccharide 26 in 90% yield. Satisfyingly, only the α-
anomer was formed, which was then successfully transformed into acceptor 12 following PMB
deprotection without noticeable acetyl migration.
STol
STol
1
) LevOH, DCC, DMAP
DCM, 50 °C, 97%
O
O
HO
LevO
O
HO
O
2) 80% aq. HOAc
OH
2
4
60 °C, 90 %
23
1) Bu SnO, tol, reflux;
2
then PMBCl, TBAI, CsF
tol, 40 °C, 88%
2
) Ac O, Py, DMAP
2
97%
Ph
O
STol
O
O
O(CH ) N 16, NIS, AgOTf, 4 Å MS
2
5 3
O
O
DCE, –10 °C
90%
OBn
LevO
PMBO
25
O
LevO
OAc
RO
OAc
2
6 R = PMB
2 R = H
DDQ, DCM/H O
2
10:1, 87%
1
Scheme 2. Synthesis of disaccharide acceptor 12.
Planning on employing a [2 + 2] glycosylation strategy (Scheme 3), disaccharide 11 was then
prepared through TMSOTf-promoted glycosylation of TCA 726 and acceptor 15. However,
complete conversion could not be achieved and disaccharide 11 was contaminated with donor 14
even upon extensive column chromatography purifications. Synthesis of fully protected
tetrasaccharide 10 was nonetheless attempted using NIS/AgOTf but proved unsuccessful, as NMR
and HRMS analyses showed no sign of the expected glycosidic bond.
1
0

Page 11 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
NH
CCl3 15, TMSOTf, Et O/DCE
Ph
O
O
O
O
STol
OAZMB
2
O
–10 °C, ~50%
O
LevO
MeO
O
LevO
MeO
OAc
OAc
1
4
11
(
contaminated with 14)
Ph
O
LevO
O
O
O
O(CH ) N3
2 5
O
O
Ph
O
OBn
LevO
MeO
O
O
O
OAc
O
OAZMB
12, NIS, AgOTf
Å MS, DCM
OAc
4
10
Scheme 3. Attempt to synthesize tetrasaccharide 10 via a [2 + 2] glycosylation.
Second generation synthesis of tetrasaccharides
As our previously envisioned methodology appeared ineffective, we chose to focus our attention
on an alternative sequential [1 + 1 + 1 + 1] glycosylation pathway (Scheme 4). First, disaccharide
acceptor 12 was glycosylated with donor 18 using the aforementioned conditions, yielding
trisaccharide 27. The AZMB group appeared to be an efficient NPG, as the β-anomer was
exclusively formed. DDQ-mediated dealkylation of trisaccharide 27 enabled its coupling with
thiorhamnoside 29, which was prepared from intermediate 24 through a methodology similar as
the one used for donor 25. In the course of this reaction sequence, O-methylation of diol 24 partially
occurred at O-2, resulting in a mixture of regioisomers. Upon glycosylation, tetrasaccharide 10 was
produced in a 65% yield, along with an inseparable unknown impurity. To achieve the desired
6
dTalp configuration, the levulinoyl groups in compound 10 were selectively cleaved under the
action of hydrazine and epimerization of both free hydroxyl groups in resulting tetrasaccharide 30
was attempted.
1
1

Organic & Biomolecular Chemistry
Page 12 of 70
View Article Online
DOI: 10.1039/C9OB01711A
Ph
O
Ph
O
O
LevO
O
O
O(CH ) N 18, NIS, AgOTf, 4 Å MS
O
2
5
3
O(CH2)5N3
O
O
DCE, –10 °C, 78%
O
OBn
Ph
O
OBn
O
O
O
O
LevO
RO
OAc
HO
OAZMB
OAc 12
2
7 R = PMB
8 R = H
DDQ, DCM/H O 10:1
2
rt, 75%
2
1
) Bu SnO, tol, reflux;
2
STol
then MeI, CsF, 80 °C, 93%
) Ac O, Py, DMAP, 96%
2
2
O
NIS, AgOTf, 4 Å MS
DCE, –10 °C, 77%
LevO
24
MeO
OAc
29
Ph
O
Ph
O
O
O
RO
O
O(CH ) N3
O
2 5
O(CH ) N
2 5 3
O
O
O
O
Ph
O
HO
OBn
Ph
O
OBn
O
RO
O
O
O
O
O
O
O
OAc
OAc
O
O
HO
OAZMB
OAZMB
10 R = Lev
0 R = H
3
1
OMe
OAc
MeO
OAc
Table S1
epimerization failed)
H NNH , Py
(
2
2
HOAc, 86%
3
Scheme 4. Synthesis of rhamnose-containing tetrasaccharide 10 via a [1 + 1 + 1 + 1] glycosylation
sequence.
Several approaches have been developed for the epimerization of rhamnose glycosides. For
instance, Kovác reported the synthesis of the C-2 rhamnose epimer L-quinovose through the
formation of 1,2-O-stannylene acetals.48 Chloral/DCC-induced inversion, which requires the
4
9
50,51
presence of a 2,3,4-triol, resulted in C-3 epimerization of rhamnose. The Mitsunobu reaction
has been shown to lead to an elimination reaction in rhamnosides due to the axial H-5.32
Intramolecular displacement of an O-4 triflate has also been reported for the synthesis of 6-deoxy-
taloside from rhamnose.52,53 However, this approach has been shown to be less reliable compared
to the oxidation-reduction sequence in which the stereochemical outcome can be consistently
predicted using the Cram chelate model.32, 54-56
Therefore, our strategy was based on an oxidation step (see Table S1) followed by a stereoselective
5
7
reduction using NaBH in a mixture of MeOH and DCM. Pfitzner-Moffatt oxidation (entry 1)
4
1
2

Page 13 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
was first tried as we previously showed that this condition was high-yielding for similar
2
6
substrates, nevertheless complete degradation of the starting material was observed. The same
reaction was conducted at –78 °C (entry 2) with the hope of stabilizing the reactive intermediate
formed between phenyl dichlorophosphate (PDCP) and DMSO, but without success. Substituting
the PDCP reagent with oxalyl chloride32 also gave no conversion (entry 3). Other oxidation
5
8
reactions were carried out using DMSO and Ac O at room temperature (entry 4) or Dess-Martin
2
2
6
periodinane in refluxing DCE (entry 5), but both failed to provide clean oxidation products.
Third generation synthesis of tetrasaccharides
As the previous double epimerization step could not be carried out successfully, we hypothesized
that the simultaneous presence of two hydroxyl groups could lead to macromolecular interactions
preventing the oxidation step to occur. Therefore, we envisioned that the introduction of a terminal
6
-deoxy-talose residue would restrict these interactions and consequently enable the epimerization
of the inner rhamnose unit. As depicted in Scheme 5, derivatives 34 and 35 were prepared from
building block 29. Delevulinoylation of the latter was performed using standard conditions.
Pfitzner-Moffatt oxidation followed by reduction of the crude ketone with NaBH furnished
4
thiotaloside 33 in 65% yield over three steps. Acetylation of the remaining hydroxyl group using
Ac O and catalytic DMAP in refluxing EtOAc led to building block 34, which mimics Bp LPS
2
OAg terminal residue. As for Bm-like LPS OAg terminal residue, a chloroacetyl group was used
as a temporary protecting group, which could be further transformed into an acetyl group following
its orthogonal deprotection. This reaction was performed using chloroacetic anhydride and catalytic
DMAP in refluxing EtOAc providing building block 35 in an almost quantitative yield.
1
3

Organic & Biomolecular Chemistry
Page 14 of 70
View Article Online
DOI: 10.1039/C9OB01711A
STol
O
RO
MeO
OAc
2
9 R = Lev
2 R = H
H NNH , Py
HOAc, 91%
2
2
3
1
) PDCP, DMSO, Et N
3
DCM, –10 °C
) NaBH , DCM, MeOH
2
4
71% (2 steps)
STol
O
OMe
RO OAc
3
3
3
3 R = H
Ac O, DMAP
EtOAc, 90%
2
(
ClAc) O, DMAP
EtOAc, 95%
4 R = Ac
5 R = AcCl
2
Scheme 5. Synthesis of 6-deoxy-L-talopyranoside derivatives 34 and 35.
To prepare Bp and Bm LPS OAg-like tetrasaccharides, trisaccharide acceptor 28 was coupled with
thiotaloside 34 and 35, respectively, under previously mentioned conditions, affording
tetrasaccharides 36 and 37 exclusively as the α-anomers (Scheme 6). Upon delevulinoylation of
these latter, either Dess-Martin periodinane reagent or Pfitzner-Moffatt conditions were
successfully used for their oxidations. In both cases, the resulting ketone was filtered over silica
gel and then reduced with complete stereoselectivity into its talose configuration under the action
of NaBH . This two-step sequence enabled the isolation of tetrasaccharide 40 in a moderate 52%
4
yield due to its partial degradation during the oxidation step. Derivative 31 was ultimately reached
in a 58% yield over three steps following chloroacetyl cleavage using thiourea in a mixture of
pyridine and methanol.
1
4

Page 15 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
Ph
O
LevO
O
O
O
O(CH ) N3
2 5
O
O
Ph
O
OBn
O
O
HO
OAc
OAZMB
2
8
34 or 35, NIS, AgOTf
4
A MS, DCE, –10 °C
quant. (for 36), 82% (for 37)
2
Ph
O
R O
O
O
O(CH ) N3
2 5
O
O
Ph
O
OBn
O
O
O
O
OAc
1
O
R O
OAZMB
OMe
1
2
OAc
36 R = Ac; R = Lev
1
2
H NNH , Py
37 R = AcCl; R = Lev
2
2
HOAc
8
5% (for 38), 65% (for 39)
1
2
3
8 R = Ac; R = H
1
2
3
9 R = AcCl; R = H
for 40
for 31
1
. Dess-Martin periodinane 1. DMSO, PDCP, Et N, –10 °C
3
DCE, 70 °C
2. NaBH , DCM, MeOH
4
2
. NaBH , DCM, MeOH
3. thiourea, Py/MeOH 1:1
60 °C, 58% (3 steps)
4
52% (2 steps)
Ph
O
O
O
O(CH ) N3
2 5
O
O
Ph
O
HO
OBn
O
O
O
O
OAc
O
RO
OAZMB
OMe
OAc
4
0 R = Ac
31 R = H
Scheme 6. Final approach for the synthesis of protected tetrasaccharides 31 and 40.
Global deprotection of tetrasaccharides
Two alternative pathways were studied for the global deprotection of tetrasaccharides 40 and 31 in
order to reach target compounds 8 and 9, respectively. The first pathway (route A) consisted in the
selective reduction of both azides followed by hydrogenolysis of the remaining benzyl and
benzylidene groups, whereas the second route (route B) only involved the hydrogenolysis step. We
therefore investigated a series of conditions for the selective reduction of both azides via the
Staudinger reaction, which would result in the assisted cleavage of the AZMB group. We first
planned to use trisaccharide 27 as a model compound, as we hypothesized that if difficulties had
1
5

Organic & Biomolecular Chemistry
Page 16 of 70
View Article Online
DOI: 10.1039/C9OB01711A
to arise during AZMB deprotection, it would be due to the electrophilic acetyl group at the inner
talose residue. Intramolecular rearrangement of iminophosphorane intermediates during the
Staudinger reduction can indeed occur in compounds containing a neighbouring ester.28
Preliminary reduction tests were first conducted with trisaccharide 27, but we noticed that the
reduction of the azidolinker reduction required harsher conditions than the AZMB group itself. Our
hypothesis was that the formation of the isoindolinone by-product acted as the driving force for the
AZMB cleavage. Our attention therefore shifted toward disaccharide 26 and conditions were
optimized for the sole reduction of the azidolinker (Table 1). Triphenylphosphine was first used in
3
0
a mixture of THF/H O in the presence of SiO (entry 1). After 20 h, the starting material was
2
2
totally consumed but, as shown by TLC, the intermediates were not fully hydrolysed. Following
work-up with aqueous NaHCO and purification, target amine 41 was isolated in low yield (21%).
3
We then attempted to conduct the reaction through a two-step procedure, i.e., 1) formation of the
iminophosphorane and 2) its hydrolysis, while heating the reaction mixture to 60 °C, but a lower
yield of amine 41 was obtained (entry 2). Switching the solvent for DMF and avoiding the work-
up procedure allowed to improve the yield to 38% (entry 3). Satisfyingly, using the same reaction
conditions than in entry 3 while avoiding the work-up provided amine 41 in excellent yield (88%,
entry 4). Tris(4-methoxyphenyl)phosphine [P(PMP) ] was also tested but without yield
3
improvement (entry 5). The optimized reduction conditions were then applied to trisaccharide 27
bearing both the azidolinker and the AZMB group, providing alcohol 42 in good yield (76%, entry
6
). These conditions finally successfully furnished tetrasaccharide 43 from diol 31 (63%, entry 7),
which was then deprotected into target compound 9 through a Pd-catalyzed hydrogenolysis, as
shown in Scheme 7.
1
6

Page 17 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
Table 1. Optimization of the Staudinger reaction for azide reduction
entry
substrate
conditions
work-up
product
yield (%)a
Ph
O
O
LevO
O
O(CH ) NH2
O
2 5
PPh SiO
3
,
2
O
NaHCO3
H O, DCM
2
PMBO
OBn
1
26
THF/H O, rt
21
2
OAc
2
0 h
4
1
PPh , THF
60 °C, 2 h then
H O, 60 °C, 4 h
2
3
NaHCO3
H O, DCM
2
2
3
26
26
41
41
41
13
38
PPh , DMF, 0 °C
3
to rt
toluene co-
2
h, then H O rt, concentration
2
2
6 h
PPh THF, 60 °C
3
,
4
5
26
26
2 h then H O
concentration
concentration
88
63
2
6
0 °C, 4 h
P(PMP) THF
3
60 °C, 2 h then
41
H O, 60 °C, 4 h
2
Ph
O
LevO
O
O
O
O(CH ) NH2
2 5
O
PPh THF, 60 °C
O
3
,
Ph
O
O
OBn
O
6
7
27
2 h then H O
concentration
PMBO
76
63
2
OAc
OH
6
0 °C, 4 h
4
2
PPh THF, 60 °C
3
,
31
2 h then H O, 60 concentration
43
2
°
C, 4 h
aIsolated yield.
1
7

Organic & Biomolecular Chemistry
Page 18 of 70
View Article Online
DOI: 10.1039/C9OB01711A
Ph
O
PPh3, THF, 60 °C
O
O
O(CH ) NH
2 5 2
O
then H O, 60 °C
2
O
Ph
O
HO
OBn
63%
O
Route A
31
O
O
O
OAc
O
HO
OH
OMe
43
OAc
For 9
Pd black, H , HCl
2
MeOH, 40 °C
91%
For 8
Pd black, H , HCl
MeOH, 40 °C
OH
O
2
O(CH ) NH +Cl–
2 5 3
HO
O
O
OH
O
60%
HO
OH
Route B
40
HO
O
O
O
OAc
RO
OMe
OH
8
9
R = Ac
R = H
OAc
Scheme 7. Alternative pathways for the final deprotection of tetrasaccharides 31 and 40.
Alternatively, reduction of the azides into the corresponding amines and cleavage of the permanent
protecting groups were carried out via a one-step hydrogenolysis procedure, enabling the
conversion of compound 40 into Bp-like tetrasaccharide 8. Noteworthy, the presence of the
azidolinker required the addition of concentrated HCl (1.0 equiv) in the reaction mixture in order
to protonate the amine formed upon its reduction, as primary amines are known to poison transition
2
6
metal catalysts. Partial protonation of the 2-(aminomethyl)benzoyl group therefore also occurred
prior to completion of the intramolecular cyclization, preventing the complete release of the
corresponding hydroxyl group. The partial cleavage of the AZMB group not only diminished the
isolated yield, but also complicated the purification of the target compound. First, exclusion size
chromatography using LH-20 resin was employed to purify the tetrasaccharides from the
isoindolinone, which was released following AZMB cleavage. Then, reverse phase
chromatography was required to isolate tetrasaccharide 8 from the derivative still bearing the
protonated 2-(aminomethyl)benzoyl group. Despite this, direct hydrogenolysis of compound 40
enabled the isolation of pure tetrasaccharide 8 in good yield (60%), as shown in Scheme 7.
1
8

Page 19 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
Reactivity of the synthetic oligosaccharides with Bm LPS-specific mAbs.
Studies from our laboratories and others have shown that LPS-specific mAbs differentially
recognize Bm and Bp OAgs as well as disaccharides 6 and 7.23, 26, 59 To determine whether
tetrasaccharides 8 and 9 synthesized in this study (oligosaccharides 8 and 9) were recognized by
Bm LPS-specific mAbs 3D11 and 9C1-2, ELISAs were conducted using oligosaccharides 6-9
along with Bp LPS controls. As shown in Figure 4, mAbs 3D11 and 9C1-2 reacted strongly with
RR4744 LPS (4-O-acetyl mutant), oligosaccharides 6 and 9, but only weakly with RR2808 LPS
(
wild type), and oligosaccharides 7 and 8. These findings are consistent with our previous work
and also show that mAb 3D11 appears to recognize the newly synthesized tetrasaccharide
oligosaccharide 9), which represents both intrachain and terminal residues associated with Bm
(
OAg, more strongly than the previously synthesized disaccharide (oligosaccharide 6). The
reactivity levels of mAb 9C1-2 with RR2808 LPS and oligosaccharides 6 and 9 were similar to one
another. Taken together, these results suggest that the epitopes on Bm OAg that are recognized by
these two mAbs are slightly different from one another. Importantly, since 9C1-2 has been shown
1
5
to be passively protective in a mouse model of glanders, these data support the potential use of
oligosaccharide 9 in the development of novel glanders vaccine candidates.
1
9

Organic & Biomolecular Chemistry
Page 20 of 70
View Article Online
DOI: 10.1039/C9OB01711A
mAb 3D11 (IgG1)
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
S
S
6
7
8
9
P
P
o
o
o
o
L
L
g
g
g
g
i
i
i
i
4
8
l
l
l
l
4
0
O
O
O
O
7
4
8
2
R
R
R
R
Target Antigen
mAb 9C1-2 (IgG2b)
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
S
S
6
7
8
9
P
P
o
o
o
o
L
L
g
g
g
g
i
i
i
i
4
8
l
l
l
l
4
0
O
O
O
O
7
4
8
2
R
R
R
R
Target Antigen
Figure 4. Interactions of Bm LPS-specific mAbs with Bp LPS antigens and synthetic
oligosaccharides. Reactivity profiles of mAbs 3D11 and 9C1-2 with LPS antigens purified from
Bp strains RR2808 (wild type) and RR4744 (4-O-acetyl mutant) and synthetic oligosaccharides
6
-9 as determined by ELISA. Values represent the means ± SD of assays conducted in triplicate.
Reactivity of the synthetic oligosaccharides with melioidosis patient serum.
We have previously demonstrated that both Bp OAg and oligosaccharide 7 are recognized more
strongly by serum from culture-confirmed Thai melioidosis patients compared to serum from
2
6
59
healthy donors. Extending upon these findings, ELISAs were used to assess the reactivity of an
2
0

Page 21 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
expanded set of culture-confirmed Thai melioidosis patient and Thai healthy donor serum samples
with oligosaccharides 1-9 along with a Bp LPS control. Results demonstrated strong reactivity of
immune serum samples with Bp LPS, oligosaccharides 8 and 9, moderate reactivity with
oligosaccharide 2, and low to moderate reactivity with oligosaccharides 1, 3, 4, 5, 6 and 7 (based
upon mean reactivities; Figure 5). In contrast, the healthy donor serum samples showed relatively
low reactivity with all of the tested target antigens (based upon mean reactivities; Figure 5). These
results agree with our previous studies and indicate that oligosaccharides 8 and 9, which consist of
a combination of epitopes associated with oligosaccharides [2 + 7] and [2 + 6], respectively, closely
mimic the antigenicity of native Bp OAg. Additionally, these results demonstrate that the lack of
the C4 O-acetyl group in Bm-like tetrasaccharide 9 does not significantly impact its recognition by
melioidosis patient serum. This is not entirely surprising since human immune responses against
OAgs following infection with Bp would be expected to be polyclonal and thus recognize multiple
epitopes at the non-reducing end of the polysaccharide. Based on these observations, we anticipate
that glycoconjugates synthesized using either oligosaccharide 8 or 9 will be able to stimulate the
production of carbohydrate-specific polyclonal responses capable of recognizing both native Bp
and Bm OAgs. Collectively, our findings suggest that oligosaccharides 8 and 9 may be valuable
tools for developing countermeasures to combat diseases caused by Bm and Bp.
2
1

Organic & Biomolecular Chemistry
Page 22 of 70
View Article Online
DOI: 10.1039/C9OB01711A
Healthy Donor Serum
4
3
2
1
0
S
1
2
3
4
5
6
7
8
9
P
o
o
o
o
o
o
o
o
o
L
g
g
g
g
g
g
g
g
g
i
i
i
i
i
i
i
i
i
8
l
l
l
l
l
l
l
l
l
0
O
O
O
O
O
O
O
O
O
8
2
R
R
Target Antigen
Melioidosis Patient Serum
4
3
2
1
0
S
1
2
3
4
5
6
7
8
9
P
o
o
o
o
o
o
o
o
o
L
g
g
g
g
g
g
g
g
g
i
i
i
i
i
i
i
i
i
8
l
l
l
l
l
l
l
l
l
0
O
O
O
O
O
O
O
O
O
8
2
R
R
Target Antigen
Figure 5. Reactivity of human serum samples with Bp LPS and synthetic oligosaccharides. Serum
samples from culture confirmed Thai melioidosis patients (n = 42) and Thai healthy donors (n =
4
2) were assayed for reactivity with Bp RR2808 LPS and synthetic oligosaccharides 1-9 using
single-dilution ELISAs (1:2,000). Black dots represent the means of assays conducted in duplicate
for individual samples. Black bars represent mean reactivity for each target antigen.
2
2

Page 23 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
EXPERIMENTAL SECTION
General methods
All starting materials and reagents were purchased from commercial sources and used as received
without further purification. Air and water sensitive reactions were performed in oven-dried
glassware under Ar atmosphere. Moisture sensitive reagents were introduced via a dry syringe.
Anhydrous solvents were either prepared from commercial solvents and dried over heat-gun
activated 4 Å molecular sieves or supplied over molecular sieves and used as received. Powdered
4
Å molecular sieves were activated before use by heating with a heat gun for ~5 min under high
vacuum. Reactions were monitored by thin-layer chromatography (TLC) with silica gel 60 F254
.25 mm pre-coated aluminium foil plates. Compounds were visualized by using UV254 and/or
0
-1
orcinol (1 mg·mL ) in 10% aq H SO solution with heating. Normal-phase flash column
2
4
chromatography was performed on silica gel 60 Å (15-40 μm). Reversed-phase flash column
chromatography was performed on C18 silica gel (fully capped, 25-40 μm). Size exclusion
chromatography was performed on GE Healthcare Sephadex LH-20 resin (70 μm). NMR spectra
were recorded at 297 K in the indicated solvent (CDCl , py-d or D O) with a 600 MHz instrument,
3
5
2
1
13
employing standard softwares given by the manufacturer. H and C NMR spectra were referenced
to tetramethylsilane (TMS, δ = δ = 0.00 ppm) as internal reference for spectra in CDCl and py-
H
C
3
d , or to internal acetone (δ = 2.218 ppm; δ = 30.9 ppm) for spectra in D O. Assignments were
5
H
C
2
based on H, 13C, COSY, HSQC and undecoupled HSQC experiments. Interchangeable
1
assignments are marked with an asterisk (*). Sugar moieties are identified from the reducing end
(sugar A) to the non-reducing end (sugar D). High-resolution mass spectra (HRMS) were recorded
on an ESI-Q-TOF mass spectrometer. Optical rotations [α]20 were measured on an Anton Paar
D
polarimeter. The retention factors (R ) were calculated from silica gel 60 F 0.25 mm pre-coated
f
254
2
3

Organic & Biomolecular Chemistry
Page 24 of 70
View Article Online
DOI: 10.1039/C9OB01711A
glass TLC plates. Di- and trisaccharides 1-7 have been synthesized according to our previous
work.26
ELISAs
Reactivity with Bm LPS-specific mAbs. Reactivity of the Bm LPS-specific mAbs (3D11 and 9C1-
2
) with synthetic oligosaccharides 6-9 and Bp LPS antigens was assessed by ELISA essentially as
2
6
previously described. In brief, maleic anhydride 96-well plates (Pierce) were coated overnight at
4
ꢀ°C with oligosaccharides 6-9 (5ꢀμg/mL) or purified LPS antigens (10ꢀμg/mL) solubilized in
carbonate buffer (pH 9.6). The LPS antigens used in this study were purified from Bp strains
RR2808 (∆wcbB; Bp LPS) and RR4744 (∆wcbB∆oacA; Bm-like LPS) as previously described.60
The coated plates were blocked at room temperature for 30ꢀmin with StartingBlock T20 (TBS)
Blocking Buffer (SB; Pierce), washed with Tris-buffered salineꢀ+ꢀ0.05% Tween 20 (TBS-T),
incubated for 1ꢀh at 37ꢀ°C with the mAbs diluted 1/2000 in TBS-T + 10% SB, washed with TBS-
T, and then incubated for 1ꢀh at 37ꢀ°C with a 1/2000 dilution of goat anti-mouse IgG-horse radish
peroxidase (HRP) conjugate (Southern Biotech). Following a final wash with TBS-T, the plates
were developed with TMB substrate (KPL) and read at 620ꢀnm. The data were plotted and analyzed
using GraphPad Prism 8 (GraphPad Software Inc.).
Reactivity with serum samples from Thai melioidosis patients and Thai healthy donors.
Reactivity of serum samples from culture-confirmed Thai melioidosis patients (nꢀ=ꢀ42) and Thai
healthy donors (n = 42) with oligosaccharides 1-9 and Bp LPS was assessed by ELISA. Plates were
coated with the oligosaccharides and LPS as described above and ELISAs were performed
5
9
essentially as previously described. In brief, the coated plates were blocked at 37ꢀ°C for 2 h with
TBS-T + 5% skim milk, washed with TBS-T, incubated for 30 min at 37ꢀ°C with human serum
samples diluted 1/2000 in TBS-T + 1% bovine serum albumin (BSA), washed with TBS-T, and
then incubated for 30 min at 37ꢀ°C with a 1/2000 dilution of rabbit anti-human IgG- HRP conjugate
2
4

Page 25 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
(Dako). Following a final wash with TBS-T, the plates were developed with TMB substrate
(Invitrogen), after 15 min 1 N aq HCl was added to stop the reaction and plates were read at 450ꢀnm.
The study was approved by the Ethics Committee of Faculty of Tropical Medicine, Mahidol
University (approval number MUTM 2014-079-02). Written informed consent was obtained from
all subjects.
para-Methylphenyl
2-O-ortho-(azidomethyl)benzoyl-4,6-O-benzylidene-3-O-para-
methoxybenzyl-1-thio-β-D-glucopyranoside (18). Bu SnO (1902 mg, 7.640 mmol, 1.1 equiv)
2
3
3
was added to a solution of diol 17 (2411 mg, 6.946 mmol, 1.0 equiv) in toluene (83 mL). The
mixture was refluxed with a Dean-Stark trap for 3 h, after which the solvents were evaporated
under reduced pressure. The residue was solubilized in anhydrous DMF (83 mL) and CsF (1076
mg, 7.085 mmol, 1.02 equiv), TBAI (2617 mg, 7.085 mmol, 1.02 equiv), and PMBCl (1.13 mL,
8
.34 mmol, 1.2 equiv) were successively added. The mixture was stirred at 90 °C for 16 h and the
resulting suspension was cooled at 0 °C and filtered over Celite. The solution was co-evaporated
with toluene and the residue was purified by silica gel flash chromatography (Hex/EtOAc 9:1 to
3
:7) to give para-methylphenyl 4,6-O-benzylidene-3-O-para-methoxybenzyl-1-thio-β-D-
3
5
glucopyranoside (1738 mg, 51%, 82% brsm) as a white amorphous solid. DMAP (144 mg, 1.18
mmol, 1.0 equiv), DCC (486 mg, 2.36 mmol, 2.0 equiv), and AZMBOH (313 mg, 1.77 mmol, 1.5
equiv) were successively added to a solution of the previously prepared alcohol (582 mg, 1.18
mmol, 1.0 equiv) in anhydrous DCM (12 mL). The mixture was refluxed for 4 h under Ar and the
resulting suspension was filtered over Celite. The solvent was evaporated under reduced pressure
and the residue was purified by silica gel flash chromatography (Hex/EtOAc 9:1 to 2:8), furnishing
2
0
glucoside 18 (751 mg, 98%) as a white amorphous solid: R 0.6 (Hex/EtOAc 7:3); [α] +36 (c
f
D
1
0
.7, CHCl ); H NMR (600 MHz, CDCl ) δ (ppm) 7.87–7.86 (m, 1H, CHAZMB), 7.61–7.59 (m, 1H,
3 3
CH ), 7.56–7.55 (m, 1H, CH ), 7.50–7.49 (m, 2H, 2  CH ), 7.41–7.37 (m, 4H, 4  CH ), 7.35–
Ar
Ar
Ar
Ar
2
5

Organic & Biomolecular Chemistry
Page 26 of 70
View Article Online
DOI: 10.1039/C9OB01711A
7
.33 (m, 2H, 2  CHSTol), 7.10–7.09 (m, 2H, 2  CHSTol), 7.05–7.03 (m, 2H, 2  CHPMB), 6.60–
.59 (m, 2H, 2  CHPMB), 5.60 (s, 1H, H-7), 5.20 (dd, J = 9.9 Hz, J = 9.0 Hz, 1H, H-2), 4.81–4.72
6
(m, 4H, H-1, CH2AZMB, CHHPMB), 4.57 (d, J = 11.6 Hz, 1H, CHHPMB), 4.41 (dd, J6a-6b = 10.5 Hz,
J6a-5 = 5.0 Hz, 1H, H-6a), 3.87–3.82 (m, 2H, H-3 and H-6b), 3.77 (t, J = 9.3 Hz, 1H, H-4), 3.69 (s,
1
3
3
H, CH3PMB), 3.55 (td, J5-4 = 9.8 Hz, J5-6a = 5.0 Hz, 1H, H-5), 2.33 (s, 3H, CH3STol); C NMR (150
MHz, CDCl ) δ (ppm) 165.0 (COOR ), 159.3 (C ), 138.8 (C ), 137.8 (C ), 137.3 (C ), 133.8
3
AZMB
Ar
Ar
Ar
Ar
(
2C, 2  CHSTol), 133.0 (CH ), 131.2 (CH
), 130.0 (C ), 129.9, 129.8 (4C, 2  CHSTol, 2 
Ar
AZMB
Ar
CHPMB), 129.5 (CH ), 129.2 (C ), 128.6 (CH ), 128.4 (3C, 3  CH ), 128.0 (C ), 126.1 (2C, 2
Ar
Ar
Ar
Ar
Ar

CH ), 113.7 (2C, 2  CHPMB), 101.4 (C-7), 87.1 (C-1), 81.7 (C-4), 79.2 (C-3), 74.1 (CH2PMB),
Ar
7
2.1 (C-2), 70.7 (C-5), 68.7 (C-6), 55.2 (CH3PMB), 53.0 (CH2AZMB), 21.3 (CH3STol); HRMS (ESI-
+
TOF) m/z [M + Na] calcd for C H N NaO S 676.2088; found 676.2067.
3
6
35
3
7
para-Methylphenyl
glucopyranoside (15). DDQ (378 mg, 1.66 mmol, 2.0 equiv) was added to a solution of glucoside
8 (544 mg, 0.832 mmol, 1.0 equiv) in DCM (18 mL) and H O (1.7 mL) and the mixture was
2-O-ortho-(azidomethyl)benzoyl-4,6-O-benzylidene-1-thio-β-D-
1
2
stirred at rt for 2 h. Saturated NaHCO (aq) (20 mL) was added to quench the solution, which was
3
then diluted in EtOAc (30 mL) and transferred into a separatory funnel. The organic layer was
washed with saturated NaHCO (aq) (30 mL) and brine (30 mL), dried over anhydrous MgSO , and
3
4
evaporated under reduced pressure. The residue was purified by silica gel flash chromatography
(
Hex/EtOAc 90:10 to 85:15) to give alcohol 15 (343 mg, 77%) as a white amorphous solid: R 0.5
f
Hex/EtOAc 7:3); [α]D20 –26 (c 0.7, CHCl ); H NMR (600 MHz, CDCl ) δ (ppm) 8.05–8.04 (m,
1
(
3
3
1
H, CHAZMB), 7.60–7.58 (m, 1H, CH ), 7.52–7.51 (m, 1H, CH ), 7.49–7.47 (m, 2H, 2  CH ),
Ar
Ar
Ar
7
.46–7.43 (m, 1H, CH ), 7.37–7.36 (m, 5H, 2  CH , 3  CH ), 7.12–7.11 (m, 2H, 2  CHSTol),
Ar
STol
Ar
5
.56 (s, 1H, H-7), 5.15 (dd, J = 9.9 Hz, J = 8.9 Hz, 1H, H-2), 4.85–4.76 (m, 3H, H-1, CH2AZMB),
2
-1
2-3
2
6

Page 27 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
4
.41 (dd, J6a-6b = 10.5 Hz, J6a-5 = 4.9 Hz, 1H, H-6a), 4.05 (t, J = 9.0 Hz, 1H, H-3), 3.82 (t, J = 10.2
Hz, 1H, H-6b), 3.61 (t, J = 9.3 Hz, 1H, H-4), 3.55 (ddd, J5-6b = 9.7 Hz, J5-4 = 9.2 Hz, J5-6a = 4.9 Hz,
1
3
1
H, H-5), 2.35 (s, 3H, CH3STol); C NMR (150 MHz, CDCl ) : δ (ppm) 166.0 (COORAZMB), 138.9
3
(
C ), 137.3 (C ), 136.9 (C ), 133.7 (2C, 2  CH ), 133.1 (CH ), 131.3 (CHAZMB), 130.0–126.4
Ar
Ar
Ar
STol
Ar
(
11C, 2  C , 9  CH ), 102.1 (C-7), 86.7 (C-1), 80.6 (C-4), 73.7 (C-3), 73.4 (C-2), 70.6 (C-5),
Ar
Ar
+
6
8.6 (C-6), 53.3 (CH2AZMB), 21.3 (CH3STol); HRMS (ESI-TOF) m/z [M + Na] calcd for
+
C H N NaO S 556.15128; found 556.15109; m/z [M+NH ] calcd for C H N O S 551.19588;
2
8
27
3
6
4
28 31
4
6
found 551.19581.
Allyl 2-O-acetyl-3-O-tert-butyldimethylsilyl-4-O-levulinoyl-α-L-rhamnopyranoside (20).
TBSCl (1989 mg, 13.23 mmol, 4.0 equiv), imidazole (674 mg, 9.92 mmol, 3.0 equiv), and DMAP
2
6
(
81 mg, 0.7 mmol, 0.2 equiv) were successively added to a solution of diol 19 (1000 mg, 3.307
mmol, 1.0 equiv) in anhydrous THF (80 mL). The mixture was refluxed for 16 h under Ar. The
suspension was filtered over Celite, rinsed, and evaporated under reduced pressure. The residue
was purified by silica gel flash chromatography (Hex/EtOAc) to give allyl 3-O-tert-
butyldimethylsilyl-4-O-levulinoyl-α-L-rhamnopyranoside (1375 mg, quantitative) as a yellow oil:
Rf 0.4 (Hex/EtOAc 7:3); [α]D20 –40 (c 1.1, CHCl ); H NMR (700 MHz, CDCl ) δ (ppm) 5.91
1
3
3
(
dddd, J2-3a = 17.2 Hz, J2-3b = 10.4 Hz, J2-1a = 6.1 Hz, J2-1b = 5.1 Hz, 1H, H-2Allyl), 5.29 (ddt, J =
7.2 Hz, 5.4 Hz, 1.6 Hz, 1H, H-3aAllyl), 5.21 (ddt, J = 10.4 Hz, 4.8 Hz, 1.3 Hz, 1H, H-3bAllyl), 4.97
t, J = 9.6 Hz, 1H, H-4), 4.87 (d, J = 1.4 Hz, 1H, H-1), 4.17 (ddt, J = 13.1 Hz, 5.1 Hz, 1.5 Hz, 1H,
H-1aAllyl), 4.04–3.98 (m, 2H, H-3, H-1bAllyl), 3.83 (dd, J2-3 = 3.6 Hz, J2-1 = 1.5 Hz, 1H, H-2), 3.76
1
(
(
dq, J = 9.8 Hz, J = 6.3 Hz, 1H, H-5), 2.89–2.81 (m, 1H, CHH ), 2.74–2.67 (m, 1H, CHH ),
5-4 5-6 Lev Lev
2
.64 (m, 1H, CHH ), 2.54–2.47 (m, 1H, CHH ), 2.20 (s, 3H, CH3Lev), 1.19 (d, J = 6.3 Hz, 3H,
Lev Lev
1
3
H-6), 0.88 (s, 9H, C(CH3)3TBS), 0.11 (s, 3H, CH3TBS), 0.08 (s, 3H, CH3TBS); C NMR (176 MHz,
2
7

Organic & Biomolecular Chemistry
Page 28 of 70
View Article Online
DOI: 10.1039/C9OB01711A
CDCl ) δ (ppm) 206.5 (CO ), 172.1 (COOR ), 133.9 (C-2Allyl), 117.6 (C-3Allyl), 98.0 (C-1), 74.3
3
Lev
Lev
(C-4), 71.8 (C-2), 70.6 (C-3), 68.2 (C-1Allyl), 66.3 (C-5), 38.0 (CH2Lev), 30.0 (CH3Lev), 28.2
(CH2Lev), 25.7 (3C, C(CH3)3TBS), 18.0 (C(CH3)3TBS), 17.5 (C-6), –4.5 (CH3TBS), –4.7 (CH3TBS);
+
HRMS (ESI-TOF) m/z [M + Na] calcd for C H NaO Si 439.21225; found 439.21102. The latter
2
0
36
7
alcohol (1000 mg, 2.400 mmol, 1.0 equiv) was dissolved in anhydrous pyridine (40 mL), and Ac O
2
(
40 mL) and DMAP (59 mg, 0.48 mmol, 0.2 equiv) were added. The mixture was stirred at rt for
6 h under Ar. The solution was diluted in toluene and evaporated under reduced pressure. The
residue was purified by silica gel flash chromatography (Hex/EtOAc) to give glycoside 20 (1013
1
2
0
1
mg, 92%) as a colorless oil: R 0.6 (Hex/EtOAc 7:3); [α] –23 (c 1.0, CHCl ); H NMR (600
f
D
3
MHz, CDCl ) δ (ppm) 5.90 (dddd, J
= 16.6 Hz, J2-3b = 10.5 Hz, J2-1a = 6.0 Hz, J2-1b = 5.3 Hz,
3
2-3a
1
1
4
H, H-2Allyl), 5.29 (ddt, J = 17.1 Hz, 3.6 Hz, 1.5 Hz, 1H, H-3aAllyl), 5.22 (ddt, J = 10.4 Hz, 3.2 Hz,
.3 Hz, 1H, H-3bAllyl), 5.10 (dd, J2-3 = 3.6 Hz, J2-1 = 1.8 Hz, 1H, H-2), 4.98 (t, J = 9.7 Hz, 1H, H-
), 4.73 (d, J = 1.6 Hz, 1H, H-1), 4.16 (ddt, J = 13.0 Hz, 5.2 Hz, 1.4 Hz, 1H, H-1aAllyl), 4.08 (dd,
J3-4 = 9.5 Hz, J3-2 = 3.6 Hz, 1H, H-3), 3.99 (ddt, J = 13.0 Hz, 6.1 Hz, 1.3 Hz, 1H, H-1bAllyl), 3.78
dq, J = 9.9 Hz, J = 6.4 Hz, 1H, H-5), 2.88–2.82 (m, 1H, CHH ), 2.70–2.62 (m, 2H, CHH ,
(
5
-4
5-6
Lev
Lev
CHH ), 2.52–2.47 (m, 1H, CHH ), 2.20 (s, 3H, CH ), 2.11 (s, 3H, CH ), 1.20 (d, J = 6.3
Lev
Lev
3Lev
3Ac
1
3
Hz, 3H, H-6), 0.82 (s, 9H, C(CH3)3TBS), 0.07 (s, 3H, CH3TBS), 0.05 (s, 3H, CH3TBS); C NMR (150
MHz, CDCl ) δ (ppm) 206.5 (CO ), 172.0 (COOR ), 170.5 (COOR ), 133.7 (C-2Allyl), 117.8
3
Lev
Lev
Ac
(C-3Allyl), 96.9 (C-1), 74.5 (C-4), 72.3 (C-2), 68.4, 68.3 (2C, C-1Allyl, C-3), 66.7 (C-5), 38.0
(
CH2Lev), 30.0 (CH3Lev), 28.2 (CH2Lev), 25.6 (3C, C(CH )
), 21.1 (CH ), 17.9, 17.6 (2C, C-6,
3 3TBS
3Ac
+
C(CH3)3TBS), –4.7 (CH3TBS), –4.9 (CH3TBS); HRMS (ESI-TOF) m/z [M + NH ] calcd for
4
+
C H NO Si 476.26742; found 476.26647; m/z [M + Na] calcd for C H NaO Si 481.22282;
2
2
42
8
22 38
8
found 481.22179.
2
8

Page 29 of 70
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01711A
2
-O-Acetyl-3-O-tert-butyldimethylsilyl-4-O-levulinoyl-L-rhamnopyranose
(21).
1,5-
Cyclooctadiene-bis(methyldiphenylphosphine)-iridium(I) hexafluorophosphate (132 mg, 0.156
mmol, 0.05 equiv) was dissolved in anhydrous THF (15 mL) and the red solution was degassed
under Ar. Hydrogen was bubbled through the solution for 5 min and the resulting yellow solution
was once again degassed under argon. A solution of rhamnoside 20 (1435 mg, 3.129 mmol, 1.0
equiv) in anhydrous THF (15 mL) was added and the reaction mixture was stirred for 2 h at rt under
Ar. Then, a solution of iodine (1588 mg, 6.258 mmol, 2.0 equiv) in THF/H O (4:1, 18 mL) was
2
added to the mixture, which was stirred for 1 h at rt. The excess of iodine was quenched by adding
a freshly prepared 10% Na S O (aq) solution and stirred until the color turned bright yellow. THF
2
2
3
was evaporated under reduced pressure and the resulting aqueous phase was extracted using EtOAc
3  25 mL). The combined organic layers were washed with saturated NaHCO (aq) (50 mL) and
(
3
brine (50 mL). The organic phase was dried over anhydrous MgSO and evaporated under reduced
4
pressure. The residue was purified by silica gel flash chromatography (Hex/EtOAc) to give
2
0
hemiacetal 21 (1100 mg, 84%, ratio α/β ∼ 85:15) as a brown oil: R 0.6 (Tol/EtOAc 1:1); [α]
–
f
D
1
1
4 (c 1.1, CHCl ); H NMR (600 MHz, CDCl , α-anomer) δ (ppm) 5.11 (m, 2H, H-1, H-2), 4.98
3
3
(t, J = 9.6 Hz, 1H, H-4), 4.14 (dd, J3-4 = 9.4 Hz, J3-2 = 3.0 Hz, 1H, H-3), 4.01 (dq, J5-4 = 9.7 Hz, J5-6
=
6.3 Hz, 1H, H-5), 3.14 (br s, 1H, OH), 2.88–2.82 (m, 1H, CHH ), 2.69–2.64 (m, 2H, CHH ,
Lev
Lev
CHH ), 2.54–2.49 (m, 1H, CHH ), 2.20 (s, 3H, CH ), 2.12 (s, 3H, CH ), 1.20 (d, J = 6.3
Lev
Lev
3Lev
3Ac
1
3
Hz, 3H, H-6), 0.83 (s, 9H, (C(CH3)3TBS), 0.08 (s, 3H, CH3TBS), 0.06 (s, 3H, CH3TBS); C NMR
(150 MHz, CDCl ) δ (ppm) 206.6 (CO ), 172.1 (COOR ), 170.7 (COOR ), 92.4 (C-1), 74.5
3 Lev Lev Ac
(C-4), 72.6 (C-2), 67.9 (C-3), 66.9 (C-5), 38.0 (CH2Lev), 30.0 (CH3Lev), 28.2 (CH2Lev), 25.6 (3C,
C(CH3)3TBS), 21.1 (CH ), 17.9, 17.7 (2C, C-6, C(CH3)3TBS), –4.7 (CH3TBS), –4.9 (CH3TBS); HRMS
3Ac
2
9