A Facile Synthesis of the Tetrasaccharide Repeating Unit of the O-Antigen from Cronobacter turicensis

Article abstract of DOI:10.1080/07328303.2015.1027825

(Figure Presented) The para-methoxyphenyl glycoside of a tetrasaccharide repeating unit, β-D-GlcNAc-(1→3)-β-D-GlcNAc-(1→3)-[α-D-Glc-(1→2)]-α-L-Rha-O-(C₆H₄-p-OMe), of the polysaccharide O-antigen from Cronobacter turicensis was synthe

Full text of DOI:10.1080/07328303.2015.1027825

This article was downloaded by: [FU Berlin]
On: 10 May 2015, At: 01:22
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
A Facile Synthesis of the Tetrasaccharide
Repeating Unit of the O-Antigen from
Cronobacter turicensis
Yin Qiaoab, Guofeng Gua & Zhongwu Guoa
a National Glycoengineering Research CenterShandong University,
Jinan, 250010, PR China
b School of Chemistry and Chemical EngineeringShandong University,
Jinan 250010, PR China
Published online: 08 Apr 2015.
Click for updates
To cite this article: Yin Qiao, Guofeng Gu & Zhongwu Guo (2015) A Facile Synthesis of the
Tetrasaccharide Repeating Unit of the O-Antigen from Cronobacter turicensis, Journal of
Carbohydrate Chemistry, 34:3, 121-132, DOI: 10.1080/07328303.2015.1027825
To link to this article: http://dx.doi.org/10.1080/07328303.2015.1027825
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Carbohydrate Chemistry, 34:121–132, 2015
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1027825
A Facile Synthesis of the
Tetrasaccharide Repeating
Unit of the O-Antigen from
Cronobacter turicensis
Yin Qiao,1,2 Guofeng Gu,1 and Zhongwu Guo1
1National Glycoengineering Research Center, Shandong University, Jinan 250010, PR
China
2School of Chemistry and Chemical Engineering, Shandong University, Jinan 250010,
PR China
GRAPHICAL ABSTRACT
Received February 7, 2015; accepted March 6, 2015.
Address correspondence to G. Gu or Z. Guo, National Glycoengineering Research Cen-
ter, Shandong University, Jinan 250010, PR China. E-mail: guofenggu@sdu.edu.cn;
zwguo@sdu.edu.cn
121

Y. Qiao et al.
122
The para-methoxyphenyl glycoside of a tetrasaccharide repeating unit, β-D-GlcNAc-
(1→3)-β-D-GlcNAc-(1→3)-[α-D-Glc-(1→2)]-α-L-Rha-O-(C6H4-p-OMe), of the polysac-
charide O-antigen from Cronobacter turicensis was synthesized by an efficiently and
linear assembly strategy. Consecutive glycosylation of the 3-O- and 2-O-positions of an
L-rhamnopyranoside derivative with free 2,3-hydroxyl groups using a D-glucosaminyl
and a D-glucosyl donor, respectively, accomplished a branched trisaccharide rapidly.
Selective 3ꢁ-O-deacetylation of the trisaccharide followed by glucosaminylation af-
forded the fully protected tetrasaccharide. Multistep protecting group manipulations
of the fully protected tetrasaccharide eventually completed the synthesis of the target
molecule.
Keywords Cronobacter turicensis; O-Antigen; Synthesis; Regioselective glycosylation
INTRODUCTION
Cronobacter spp., previously classified as Enterobacter sakazakii, is a genus of
gram-negative and opportunistic bacterial pathogens that can lead to various
life-threatening human diseases, including meningitis, necrotizing entero-
colitis, and septicemia.[1] Cronobacter infections have received considerable
attention on account of its high risk for neonates and infants and cause of
fatalities.[2] More recently, powdered infant formula has been identified as a
source of contamination of Cronobacter organisms in some infant infection
cases.[3] Consequently, sensitive technologies for the detection and control of
Cronobacter bacterium in powdered infant formula and safe and effective ther-
apeutic protocols for the prevention and treatment of Cronobacter infections
are needed.
Bacterial cell surface lipopolysaccharides (LPSs) are the major compo-
nents of gram-negative bacterial cell glycocalyx and play an important role in
bacterium–host interactions and regulation of the host immune response,[4–6]
thereby making them attractive antigen candidates for the development of
carbohydrate-based antibacterial vaccines.[6,7] O-polysaccharide (OPS), also
known as O-antigen, as the main component of LPS is exposed on the bac-
terial cell surface and also protects bacteria against host immune recogni-
tion, complement attack, and other related responses.[8] Recently, Czerwicka
and coworkers isolated and characterized the OPSs from three C. turicensis
sequence type 5 strains, and these OPSs have the same repeat unit: β-D-
GlcpNAc-(1→3)-β-D-GlcpNAc-(1→3)-[α-D-Glcp-(1→2)]-β-L-Rhap-(1→4).[9] We
present here the first chemical synthesis of this tetrasaccharide unit as a para-
methoxyphenyl (MP) glycoside 1 (Fig. 1). In the synthetic target, we designed
to have the MP group at the reducing end, because this group can be read-
ily selectively removed in the presence of other protecting groups to facilitate
further elaborations of the fully protected oligosaccharides at the reducing end,
such as its activation for coupling with other molecules, constructing oligomers
of this repeating unit, and so on.

Synthesis of O-antigen of Cronobacter turicensis
123
Figure 1: The chemical structure of target compound (1).
RESULTS AND DISCUSSION
Retrosynthetic disconnection of the synthetic target 1 resulted in three key
monosaccharide building blocks, namely, D-glucosaminyl trichloroacetimidate
2[10] and D-glucosyl thiosglycoside 4[11,12] as glycosyl donors and a 2,3-diol
derivative 5 of L-rhamnose[13,14] as a glycosyl acceptor (Sch. 1). In this design,
we intended to take advantage of the demonstrated higher reactivity of the
rhamnosyl 3-OH group than its 2-OH group[15,16] to accomplish consecutive
3-O- and 2-O-glycosylation, which would significantly simplify the monosac-
charide building block preparation and the protection tactics, as well as the
target molecule assembly.
Scheme 1: Retrosynthetic plan for the synthetic target 1.
The thioglucoside donor 4 and the diol acceptor 5 were synthesized
according to the literature procedures.[11,13] The trichloroacetimidate donor
2,[10] predominantly in the β-form, was prepared from compound 6[17,18] in
a 52% overall yield in two steps, including removal of the MP group at the
anomeric position with cerium ammonium nitrate (CAN)[19] and trichloroace-
timidation[20] of the resulting hemiacetal with trichloroacetonitrile and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (Sch. 2). The β-configuration of 2 was

Y. Qiao et al.
124
confirmed by the large coupling constant of the H-1 signal (δ 6.72 ppm, J1,2
=
1
9.0 Hz) in its H NMR spectrum. It was then used to regioselectively glycosy-
late rhamnosyl diol 5 in the presence of a catalytic amount of trimethylsilyl
triflate (TMSOTf) at –15◦C to afford the desired β-(1→3)-linked disaccharide
7 in a good isolated yield (58%). The 1→3 linkage in 7 was confirmed by an
acetylation experiment, and we found that the H-2Rha signal (δ 5.38 ppm) in
1
the H NMR spectrum of the acetylated product 8 shifted downfield by more
than 1 ppm, as compared to that of 7 (δ 4.30 ppm). The newly formed glycosidic
linkage was proved β, judging from the coupling constant of its H-1GlcN signal
(δ 5.81 ppm, J1,2 = 8.4 Hz). Coupling of 7 with thioglucopyranoside donor 4 (1.5
eq.) in the presence of N-iodosuccinimide (NIS) and a catalytic amount of TM-
SOTf in anhydrous diethyl ether[21] at –30◦C successfully furnished the trisac-
charide as an α,β-mixture (3:1 ratio) in an 83% overall yield. The two isomers
were further separated by column chromatography, and the desired α-isomer 9
was obtained in a 62% isolated yield. The coupling constant (J1,2 = 3.0 Hz) of
1
the H-1Glc signal of 9 at δ 5.12 ppm in its H NMR spectrum confirmed the α-
configuration of the new glucosidic linkage. Selective deacetylation of 9 using
sodium methoxide in methanol produced the trisaccharide acceptor 3 in a 93%
yield. Subsequently, compound 3 was glycosylated again with trichloroacetimi-
date 2, which was catalyzed with TMSOTf (0.1 eq.) to afford the fully protected
tetrasaccharide 10 in a good yield (73%). The newly introduced glucosaminyl
residue in 10 was confirmed being β-linkage based on the coupling constant
(J1,2 = 7.8 Hz) of the H-1 signal at δ 5.51 ppm in its 1H NMR spectrum. It was
interesting to observe that some of the proton and carbon signals of the rham-
nosyl and glucosaminyl residues in the 1H and 13C spectra of compounds 8–10
were significantly broadened, in some cases too broad to be identifiable, as com-
pared to signals of other nuclei in the same structure or even the same sugar
residue. Similar results were observed for NMR spectra obtained in different
solvents. However, the MS data of these compounds were in perfect agreement
with the expected structures, and furthermore, all of the proton and carbon
signals became sharp and clear after deprotection. A potential explanation for
this phenomenon was that these fully protected and branched structures pos-
sessed enormous steric hindrance and thereby limited their conformational
freedom. As a result, the NMR signals of these compounds were broadened.
Finally, global deprotection of 10, including replacement of the Phth groups
with Ac groups on the amino group of glucosaminyl residues, was achieved in
three separate steps to cope with the different solubility of involved interme-
diates. First, 10 was treated with hydrazine hydrate in refluxing ethanol to
concomitantly remove the Ac and Phth groups.[22] Then, the exposed amino
groups were selectively acetylated with acetic anhydride in methanol, which
was followed by hydrogenolytic debenzylation in 50% aqueous acetic acid with
10% Pd/C as the catalyst to afford the desired product 1 in a 67% overall yield
after purification by size-exclusion chromatography on a Bio-Gel P-2 column.

Synthesis of O-antigen of Cronobacter turicensis
125
The structures of 1 and all synthetic intermediates were fully characterized by
1H NMR, 13C NMR, and ESI-HRMS spectra.
Scheme 2: Synthesis of the target molecule 1.
CONCLUSION
In summary, we have described herein a concise and efficient synthesis of the
tetrasaccharide repeating unit of the O-antigen from C. turicensis LPS. This
synthesis is highlighted with the regioselective 3-O-glycosyltion of a 2,3-diol
glycosyl acceptor 5, which was followed by 2-O-glycosyltion, based on the ob-
servation that in rhamnose derivatives carrying free 2,3-OH groups, the 3-OH
group is more nucleophilic than the 2-OH group. This has significantly simpli-
fied the target synthesis in terms of both monosaccharide building block prepa-
ration and protecting group design. It is also worth pointing out that the MP
group in the structure of fully protected tetrasaccharide 10 can be selectively
removed to facilitate further elaborations at the reducing end, which should

Y. Qiao et al.
126
enable its application to conjugating with other biomolecules useful for studies
such as vaccine development. We are currently pursuing these directions.
EXPERIMENTAL SECTION
General Methods
Optical rotations were determined at 20◦C with a Rudolph Autopol I au-
1
tomatic polarimeter. H and 13C NMR spectra were recorded with an Agilent
600 MHz spectrometer for solutions in CDCl3 or D2O. Chemical shifts (δ) are
given in ppm downfield from internal Me4Si or with DHO signal as a ref-
erence, where D2O was used as the solvent. Positive-mode electrospray ion-
ization (ESI) high-resolution mass spectroscopy (HRMS) was recorded on a
JEOL JMS-DX-303HF spectrometer. Thin layer chromatography (TLC) was
performed on silica gel HF254 plates with detection by charring using 30% (v/v)
H2SO4 in MeOH or by a UV detector. Silica gel column chromatography was
conducted with mixtures of ethyl acetate and petroleum ether (b.p. 60–90◦C)
as the eluents. Solution concentrations were performed at <60◦C under dimin-
ished pressure.
3-O-Acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranosyl tric-
hloroacetimidate (2)[10]
To a solution of para-methoxyphenyl 3-O-acetyl-4,6-O-benzylidene-2-
deoxy-2-phthalimido- β-D-glucopyranoside 6[17,18] (5.45 g, 10.0 mmol) in
CH3CN and H2O (60 mL, v/v 4:1) was added CAN (8.22 g, 15.0 mmol) at 0◦C.
The reaction mixture was stirred for 40 min at 0◦C, at which time TLC in-
dicated the disappearance of the starting material. The reaction mixture was
then diluted with EtOAc (200 mL) and washed with saturated aq. NaHCO3
solution and water. The organic layer was dried over Na2SO4, filtered, and con-
centrated. The resulting residue was crystallized from hexane-EtOAc (2:1) to
yield the crude hemiacetal (3.2 g) as a pale yellow solid. This above compound
was dissolved in CH2Cl2 (20 mL), and trichloroacetonitrile (3.6 mL, 36.5 mmol)
was added. The reaction mixture was stirred with a catalytic amount of DBU
(0.4 mL, 2.67 mmol) at 0◦C for 2 h. After concentration, the residue was puri-
fied by silica gel flash column chromatography with 2:1 petroleum ether-ethyl
20
acetate as the eluent to afford 2 (3.03 g, 52%) as a white foamy solid. [α]D
1
32.2 (c 0.6, CHCl3); H NMR (600 MHz, CDCl3): δ 8.65 (s, 1H, NH), 7.87–7.55
(m, 9H, Ph), 6.72 (d, 1H, J = 9.0 Hz, H-1), 6.00 (dd, 1H, J = 10.2, 9.6 Hz, H-3),
5.56 (s, 1H, PhCH), 4.60 (dd, 1H, J = 10.2, 9.0 Hz, H-2), 4.50 (dd, 1H, J = 10.2,
4.8 Hz, H-6a), 3.96 (m, 1H, H-5), 3.89 (t, 1H, J = 10.2 Hz, H-6b), 3.88 (t, 1H, J
= 9.6 Hz, H-4); 13C NMR (150 MHz, CDCl3): δ 170.1, 160.6, 136.7, 134.4, 131.2,
129.3, 128.3, 126.3, 123.7, 101.8, 94.0, 90.1, 78.9, 69.4, 68.4, 67.0, 54.3, 20.6.

Synthesis of O-antigen of Cronobacter turicensis
127
para-Methoxyphenyl 3-O-Acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→3)-4-O-benzyl-α-L-rhamnopyranoside (7)
To a solution of 5 (450 mg, 1.25 mmol) and 2 (760 mg, 1.30 mmol) in anhy-
drous CH2Cl2 (10 mL) was added TMSOTf (24 μL, 0.13 mmol) at –15◦C under
a N2 atmosphere. After the reaction mixture was stirred for 1 h, it was neu-
tralized with Et3N and then concentrated. The resulting residue was purified
by flash column chromatography (2:1 petroleum ether-ethyl acetate) to yield
20
1
7 (566 mg, 58%) as a white foamy solid. [α]D –44.6 (c 1.2, CHCl3); H NMR
(600 MHz, CDCl3): δ 7.75–7.35 (m, 9H, Ph), 7.15–7.06 (m, 3H, Ph), 6.98 (d,
2H, J = 9.0 Hz, Ph), 6.89 (d, 2H, J = 6.6 Hz, Ph), 6.81 (d, 2H, J = 9.0 Hz,
Ph), 5.87 (dd, 1H, J = 10.2, 9.0 Hz, H-3GlcN), 5.81 (d, 1H, J = 8.4 Hz, H-1GlcN),
5.56 (s, 1H, PhCH), 5.42 (d, 1H, J = 1.2 Hz, H-1Rha), 4.45 (dd, 1H, J = 10.2,
8.4 Hz, H-2GlcN), 4.43 (dd, 1H, J = 10.2, 4.2 Hz, H-6aGlcN), 4.40 (d, 1H, J =
11.4 Hz, PhCH2), 4.30–4.24 (m, 2H, H-2Rha and PhCH2), 4.10 (dd, 1H, J = 9.6,
3.6 Hz, H-3Rha), 3.89–3.80 (m, 3H, H-4,5,6bGlcN), 3.76 (s, 3H, -OCH3), 3.74 (m,
1H, H-5Rha), 3.41 (t, 1H, J = 9.6 Hz, H-4Rha), 2.83 (d, 1H, J = 1.8 Hz, -OH),
1.87 (s, 3H, Ac), 1.07 (d, 3H, J = 6.6 Hz, H-6Rha); 13C NMR (150 MHz, CDCl3):
δ 170.1, 154.8, 150.0, 138.0, 136.7, 117.5, 114.6, 101.8 (PhCH), 99.2 (C-1GlcN),
97.6 (C-1Rha), 82.9 (C-3Rha), 78.8 (C-4GlcN), 78.6 (C-4Rha), 74.6 (PhCH2), 70.0 (C-
2Rha), 69.7 (C-3GlcN), 68.5 (C-6GlcN), 67.7 (C-5Rha), 66.4 (C-5GlcN), 55.6 (-OCH3),
55.4 (C-2GlcN), 20.5 (-COCH3), 17.6 (C-6Rha); ESI-HRMS (positive ion): calcd for
(C43H43NO13+NH4+): 799.3073; found m/z: 799.3079.
para-Methoxyphenyl 3-O-Acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→3)-2-O-acetyl-4-O-benzyl-α-L-rhamnopyranoside (8)
A solution of 7 (15 mg, 0.019 mmol) in pyridine (3 mL) and acetic anhy-
dride (2 mL) was stirred at rt overnight. The reaction mixture was then co-
evaporated with toluene (2 × 10 mL), and the resulting residue was purified
by flash column chromatography (2:1 petroleum ether-ethyl acetate) to yield
20
1
8 (15 mg, 95%) as a white foamy solid. [α]D –18.8 (c 0.6, CHCl3); H NMR
(600 MHz, CDCl3): δ 7.70–7.09 (m, 12H, Ph), 6.96 (d, 2H, J = 9.0 Hz, Ph), 6.90
(d, 2H, J = 6.6 Hz, Ph), 6.80 (d, 2H, J = 9.0 Hz, Ph), 5.85 (dd, 1H, J = 10.2,
9.0 Hz, H-3GlcN), 5.74 (d, 1H, J = 8.4 Hz, H-1GlcN), 5.53 (s, 1H, PhCH), 5.38 (dd,
1H, J = 3.0, 1.8 Hz, H-2Rha), 5.28 (d, 1H, J = 1.8 Hz, H-1Rha), 4.44 (d, 1H, J
= 12.0 Hz, PhCH2), 4.41 (dd, 1H, J = 9.0, 3.0 Hz, H-6aGlcN), 4.39 (dd, 1H, J
= 10.2, 8.4 Hz, H-2GlcN), 4.27 (d, 1H, J = 12.0 Hz, PhCH2), 4.24 (dd, 1H, J =
9.6, 3.6 Hz, H-3Rha), 3.80–3.72 (m, 7H, H-4,5,6bGlcN, H-5Rha, and -OCH3), 3.40
(t, 1H, J = 9.6 Hz, H-4Rha), 2.18 (s, 3H, Ac), 1.86 (s, 3H, Ac), 1.10 (d, 3H, J
= 6.0 Hz, H-6Rha); 13C NMR (150 MHz, CDCl3): δ 170.2, 170.1, 155.0, 149.9,
137.9, 136.9, 117.7, 114.5, 101.7, 99.0, 96.0, 79.2, 79.1, 79.0, 74.7, 71.3, 69.9,
68.5, 67.9, 66.0, 55.6, 55.5, 21.1, 20.5, 17.7; ESI-HRMS (positive ion): calcd for
(C45H45NO14+NH4+): 841.3178; found m/z: 841.3189.

Y. Qiao et al.
128
para-Methoxyphenyl 3-O-Acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→2)]
-4-O-benzyl-α-L-rhamnopyranoside (9)
To a solution of 7 (482 mg, 0.616 mmol) and 4 (600 mg, 0.927 mmol) in
anhydrous Et2O (20 mL) was added NIS (312 mg, 1.39 mmol) and TMSOTf
(17 μL, 0.093 mmol) at –30◦C under a N2 atmosphere. The reaction mixture
was stirred for 30 min, and TLC indicated the complete disappearance of 7. The
reaction mixture was then neutralized with Et3N and concentrated, and the
resulting residue was purified by flash column chromatography (2:1 hexane-
ethyl acetate) to furnish the trisaccharide product as an α,β-mixture (665 mg).
Then, the above mixture was further purified by flash column chromatography
with 15:1 toluene-ethyl acetate as the eluents to yield 9 (388 mg, 62%) as a
20
1
white foamy solid. [α]D –5.0 (c 1.0, CHCl3); H NMR (600 MHz, CDCl3): δ
7.65–7.23 (m, 29H, Ph), 7.14–7.08 (m, 3H, Ph), 6.90 (d, 2H, J = 9.0 Hz, Ph),
6.89 (br s, 2H, Ph), 6.78 (d, 2H, J = 9.0 Hz, Ph), 5.85 (t, 1H, J = 10.2 Hz, H-
3GlcN), 5.81 (br d, 1H, J = 7.2 Hz, H-1GlcN), 5.42 (d, 1H, J = 1.8 Hz, H-1Rha),
5.28 (s, 1H, PhCH), 5.12 (d, 1H, J = 3.0 Hz, H-1Glc), 5.00, 4.92 (2 d, 2 × 1H, J
= 11.2 Hz, PhCH2), 4.97, 4.64 (2 d, 2 × 1H, J = 11.4 Hz, PhCH2), 4.72, 4.63
(2 d, 2 × 1H, J = 10.8 Hz, PhCH2), 4.71, 4.60 (2 d, 2 × 1H, J = 11.4 Hz,
PhCH2), 4.42 (dd, 1H, J = 10.2, 8.4 Hz, H-2GlcN), 4.27 (dd, 1H, J = 10.2, 4.8 Hz,
H-6aGlcN), 4.23 (d, 1H, J = 10.8 Hz, PhCH2), 4.21 (dd, 1H, J = 3.0, 1.8 Hz, H-
2Rha), 4.18 (dd, 1H, J = 8.4, 3.0 Hz, H-3Rha), 4.17–4.13 (m, 2H, H-5Glc, PhCH2),
4.12 (t, 1H, J = 9.6 Hz, H-3Glc), 3.90, 3.84 (br d, 2 × 1H, J = 10.2 Hz, H-6a,bGlc),
3.80 (t, 1H, J = 9.6 Hz, H-4Glc), 3.76 (s, 3H, -OCH3), 3.75–3.70 (m, 2H, H-5Rha
,
H-5GlcN), 3.64 (t, 1H, J = 9.0 Hz, H-4GlcN), 3.62 (dd, 1H, J = 9.6, 3.6 Hz, H-2Glc),
3.58 (t, 1H, J = 10.2 Hz, H-6bGlcN), 3.38 (br s, 1H, H-4Rha), 1.86 (s, 3H, Ac), 0.98
(br s, 3H, H-6Rha); 13C NMR (150 MHz, CDCl3): δ 170.1, 154.8, 150.3, 139.0,
138.8, 138.4, 138.1, 137.9, 136.9, 117.8, 114.5, 101.4 (PhCH), 100.0 (C-1GlcN),
96.4 (C-1Glc), 96.2 (C-1Rha), 81.7 (C-3Glc), 80.4 (C-2Glc), 80.1 (C-3Rha), 79.1 (C-
4Rha), 78.8 (C-4GlcN), 77.7 (C-4Glc), 76.6 (C-2Rha), 75.6, 74.6, 73.6, 72.7 (5C, 5 ×
PhCH2), 70.8 (C-5Glc), 69.9 (C-3GlcN), 68.6 (C-6Glc), 68.5 (2C, C-5Rha and C-6GlcN),
55.9 (C-5GlcN), 55.6 (2C, C-2GlcN and -OCH3), 20.6 (-OCCH3), 17.9 (C-6Rha); ESI-
HRMS (positive ion): calcd for (C77H77NO18+NH4+): 1321.5479; found m/z:
1321.5493.
para-Methoxyphenyl 4,6-O-Benzylidene-2-deoxy-2-phthalimido-β-D-glucopyra-
nosyl-(1→3)-[2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-(1→2)]-4-O-benz-
yl-α-L-rhamnopyranoside (3)
To a solution of 9 (326 mg, 0.25 mmol) in methanol (10 mL) was added
1 M NaOMe in MeOH solution dropwise until pH = 10 was reached. The reac-
tion mixture was stirred at rt for 2 h and then neutralized with Amberlite IR
120 (H+). The reaction solution was filtered and concentrated. The resulting

Synthesis of O-antigen of Cronobacter turicensis
129
residue was purified by flash column chromatography (3:2 petroleum ether-
20
ethyl acetate) to afford 3 (294 mg, 93%) as a white foamy solid. [α]D 1.6 (c
1
1.2, CHCl3); H NMR (600 MHz, CDCl3): δ 7.55–7.20 (m, 29H, Ph), 7.16–7.08
(m, 3H, Ph), 6.90 (d, 2H, J = 9.0 Hz, Ph), 6.87 (br d, 2H, J = 5.4 Hz, Ph), 6.78
(d, 2H, J = 9.0 Hz, Ph), 5.58 (br d, 1H, J = 8.4 Hz, H-1GlcN), 5.41 (d, 1H, J
= 1.8 Hz, H-1Rha), 5.28 (s, 1H, PhCH), 5.10 (d, 1H, J = 3.6 Hz, H-1Glc), 5.02,
4.97 (2 d, 2 × 1H, J = 10.8 Hz, PhCH2), 4.98, 4.62 (2 d, 2 × 1H, J = 10.8 Hz,
PhCH2), 4.74, 4.65 (2 d, 2 × 1H, J = 11.4 Hz, PhCH2), 4.71, 4.60 (2 d, 2 ×
1H, J = 10.8 Hz, PhCH2), 4.59 (ddd, 1H, J = 10.2, 9.0, 4.0 Hz, H-3GlcN), 4.32
(dd, 1H, J = 10.2, 8.4 Hz, H-2GlcN), 4.26 (dd, 1H, J = 10.2, 4.8 Hz, H-6aGlcN),
4.24–4.14 (m, 6H, H-2,3Rha, H-3,5Glc, PhCH2), 3.93, 3.84 (br d, 2 × 1H, J =
9.0 Hz, H-6a,bGlc), 3.83 (t, 1H, J = 9.0 Hz, H-4Glc), 3.77 (s, 3H, -OCH3), 3.74
(m, 1H, H-5Rha), 3.63 (m, 1H, H-5GlcN), 3.62 (dd, 1H, J = 9.0, 3.0 Hz, H-2Glc),
3.57 (t, 1H, J = 10.2 Hz, H-6bGlcN), 3.40 (br t, 1H, J = 9.0 Hz, H-4Rha), 3.37
(t, 1H, J = 9.0 Hz, H-4GlcN), 2.01 (br d, 1H, J = 4.0 Hz, -OH), 1.00 (br d, 3H,
J = 6.0 Hz, H-6Rha); 13C NMR (150 MHz, CDCl3): δ 154.8, 150.3, 139.1, 138.9,
138.5, 138.1, 137.9, 137.0, 117.8, 114.5, 101.7 (PhCH), 100.5 (C-1GlcN), 96.1 (2C,
C-1Glc, C-1Rha), 81.72 (C-4GlcN), 81.67 (C-3Glc), 80.5 (C-2Glc), 79.8 (C-3Rha), 79.2
(C-4Rha), 77.7 (C-4Glc), 76.2 (C-2Rha), 75.5, 74.8, 74.0, 73.6, 72.6 (5C, 5 × PhCH2),
70.8 (C-5Glc), 68.63 (C-6Glc), 68.55 (C-5Rha), 68.5 (2C, C-3,6GlcN), 55.9 (C-5GlcN),
55.9 (C-2GlcN), 55.6 (-OCH3), 18.0 (C-6Rha); ESI-HRMS (positive ion): calcd for
(C75H75NO17+NH4+): 1279.5373; found m/z: 1279.5372.
para-Methoxyphenyl 3-O-Acetyl-4ꢁ,6ꢁ-O-benzylidene-2-deoxy-2-phthalimido-β-
D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-
glucopyranosyl-(1→3)-[2,3,4, 6-tetra-O-benzyl-α-D-glucopyranosyl-(1→2)]-
4-O-benzyl-α-L-rhamnopyranoside (10)
To a solution of 3 (185 mg, 0.146 mmol) and 2 (130 mg, 0.22 mmol) in an-
hydrous CH2Cl2 (5 mL) was added TMSOTf (6 μL, 0.03 mmol) at 0◦C under a
N2 atmosphere. After the mixture was stirred for 50 min, it was neutralized
with Et3N and concentrated. The resulting residue was purified by flash col-
umn chromatography (3:2 petroleum ether-ethyl acetate) to yield 10 (180 mg,
20
1
73%) as a white foamy solid. [α]D –22.5 (c 1.0, CHCl3); H NMR (600 MHz,
CD3COCD3): δ 7.80–7.10 (m, 43H, Ph), 6.90 (br d, 2H, J = 9.0 Hz, Ph), 6.80
ꢁ
(d, 2H, J = 9.0 Hz, Ph), 5.64 (d, 1H, J = 8.4 Hz, H-1GlcN ), 5.57 (s, 1H, PhCH),
ꢁ
5.52 (t, 1H, J = 9.6 Hz, H-3GlcN ), 5.43 (s, 1H, PhCH), 5.40–5.32 (br s, 1H),
5.30 (d, 1H, J = 3.6 Hz, H-1Glc), 4.96 (d, 1H, J = 11.4 Hz, PhCH2), 4.93 (d,
1H, J = 10.8 Hz, PhCH2), 4.86 (d, 1H, J = 11.4 Hz, PhCH2), 4.82 (dd, 1H, J
= 10.2, 8.4 Hz), 4.79–4.60 (m, 5H, PhCH2), 4.38–4.29 (m, 1H), 4.28–4.20 (m,
3H), 4.17 (dd, 1H, J = 6.6, 3.0 Hz), 4.08–4.01 (m, 3H), 3.96–3.88 (m, 1H, H-6),
3.87–3.80 (m, 1H, H-6), 3.79–3.63 (m, 6H), 3.71 (s, 3H, -OCH3), 3.61–3.55 (m,
1H), 3.54 (dd, 1H, J = 9.6, 3.0 Hz, H-2Glc), 3.38–3.30 (m, 1H), 1.64 (s, 3H, Ac),
0.90–0.60 (br s, 3H, H-6Rha); 13C NMR (150 MHz, CD3COCD3): δ 154.8, 150.3,

Y. Qiao et al.
130
140.6, 138.9, 138.8, 138.6, 135.4 (3C), 119.0, 115.3, 102.2, 101.9, 99.6, 82.4, 81.4
(2C), 79.5, 78.8, 77.0, 76.4, 75.8, 75.2, 74.0, 72.7, 71.8, 70,7, 70.3, 69.25, 69.17,
67.3 (2C), 67.2, 56.6, 56.5, 55.9, 19.3, 18.6; ESI-HRMS (positive ion): calcd for
(C98H94N2O24+NH4+): 1700.6535; found m/z: 1700.6553.
para-Methoxyphenyl 2-Acetamido-2-deoxy-β-D-glucopyranosyl-(1→3)-2-deoxy-
2-acetamido-β-D-glucopyranosyl-(1→3)-[α-D-glucopyranosyl-(1→2)]-α-L-
rhamnopyranoside (1)
A solution of 10 (50 mg, 0.03 mmol) in ethanol (10 mL) and 80% hydrazine
hydrate (1 mL) was refluxed under an argon atmosphere for 20 h. The reaction
mixture was then concentrated under dismissed pressure and purified by flash
column chromatography (85:10:5 EtOAc-MeOH-H2O) to get a white solid. The
product was dissolved in methanol (10 mL) containing acetic anhydride (4 mL).
The reaction mixture was stirred at rt overnight and then concentrated to give
a pale yellow solid, which was then dissolved in 50% aqueous acetic acid (5 mL)
and vigorously stirred with 10% Pd/C (20 mg) under a hydrogen atmosphere
at rt for 20 h. The reaction mixture was filtered through a Celite pad, and the
filtrate was concentrated. The resulting residue was purified by size-exclusion
chromatography on a Bio-Gel P-2 column with distilled water as the eluent
20
and then lyophilized to give 1 (16.8 mg, 67% for 3 steps) as a white solid. [α]D
–6.7 (c 0.2, H2O); 1H NMR (600 MHz, D2O): δ 6.93 (d, 2H, J = 9.0 Hz, Ph), 6.80
(d, 2H, J = 9.0 Hz, Ph), 5.33 (s, 1H, H-1Rha), 4.89 (d, 1H, J = 3.0 Hz, H-1Glc),
4.52 (d, 1H, J = 9.0 Hz, H-1GlcN), 4.40 (d, 1H, J = 8.4 Hz, H-1GlcN), 4.17 (br s,
1H, H-2Rha), 4.04 (br d, 1H, J = 11.2 Hz, H-5), 3.90 (dd, 1H, J = 9.6, 3.0 Hz,
H-3Rha), 3.77–3.63 (m, 5H, 4 × H-6, H-5Rha), 3.62 (s, 3H, -OCH3), 3.61–3.51
ꢁ
(m, 5H, H-2GlcN, 2 × H-3, 2 × H-6), 3.48 (t, 1H, J = 9.6 Hz, H-2GlcN ), 3.44 (t,
1H, J = 9.6 Hz, H-4Rha), 3.40 (m, 1H, H-5), 3.36–3.24 (m, 6H, H-2Glc, H-3, 3 ×
H-4, H-5), 1.87, 1.83 (2 s, 2 × 3H, 2Ac), 1.06 (d, 3H, J = 6.0 Hz, H-6Rha); 13C
NMR (150 MHz, D2O): δ 174.2, 173.5, 154.6, 149.1, 118.6, 114.9, 103.1 (C-1GlcN),
ꢁ
100.9 (C-1GlcN ), 97.2 (C-1Glc), 96.4 (C-1Rha), 81.1, 77.8, 75.6, 75.2, 74.9, 73.0,
72.6, 71.31, 71.25, 71.18, 69.9, 69.5, 69.2, 68.5, 60.6, 60.3, 59.8, 55.55, 55.52,
54.7, 22.2, 22.1, 16.2; ESI-HRMS (positive ion): Calcd for (C35H54N2O21+H+):
839.3292; found m/z: 839.3302; calcd for (C35H54N2O21+Na+): 861.3117; found
m/z: 861.3109.
FUNDING
This work was supported by research grants from the National High
Technology Research and Development Program of China (863 Program)
(2012AA021504) and the National Major Scientific and Technological Special
Project for Significant New Drugs Development (No. 2012ZX09502001-005).

Synthesis of O-antigen of Cronobacter turicensis
131
SUPPLEMENTARY DATA
1
Supplementary data including H NMR, 13C NMR, and ESI-HRMS spectra of
2–3, 7–10, and 1 are available from the corresponding authors.
REFERENCES
1. Lai, K.K. Enterobacter sakazakii infections among neonates, infants, children, and
adults: case reports and a review of the literature. Medicine 2001, 80, 113–122.
2. Caubilla-Barron, J.; Hurrell, E.; Townsend, S.; Cheetham, P.; Loc-Carrillo, C.;
Fayet, O.; Pre`re, M.-F.; Forsythe, S.J. Genotypic and phenotypic analysis of Enterobac-
ter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive
care unit in France. J. Clin. Microbiol. 2007, 45, 3979–3985.
3. FAO/WHO. Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up for-
mulae. Microbiological Risk Assessment Series No. 15. 2008, Rome.
4. Robbins, J.B.; Schneerson, R.; Egan, W.B.; Vann, W.; Liu, D.T. Virulence properties
of bacterial capsular polysaccharides-unanswered questions. Life Sci. Res. Rep. 1980,
16, 115–132.
5. Moxon, E.R.; Kroll, J.S. The role of bacterial polysaccharide capsules as virulence
factors. Curr. Top. Microbiol. Immunol. 1990, 150, 65–85.
6. Weintraub, A. Immunology of bacterial polysaccharide antigens. Carbohydr. Res.
2003, 338, 2539–2547.
7. Plotkin, S.A. Vaccines: past, present and future. Nat. Med. 2005, 11, S5–S11.
8. Raetz, C.R.H.; Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem.
2002, 71, 635–700.
9. Czerwicka, M.; Marszewska, K.; Forsythe, S.J.; Bychowska, A.; Mazgajczyk, A.;
Dziadziuszko, H.; Ossowska, K.; Stepnowski, P.; Kaczyn´ski, Z. Chemical structure of
the O-polysaccharides isolated from Cronobacter turicensis sequence type 5 strains 57,
564, and 566. Carbohydr. Res. 2013, 373, 89–92.
10. Morando, M.; Yao, Y.; Mart´ın-Santamar´ıa, S.; Zhu, Z.; Xu, T.; Can˜ada, F.J.; Zhang,
Y.; Jime´nez-Barbero, J. Mimicking chitin: chemical synthesis, conformational analysis,
and molecular recognition of the β(1→3) N-acetylchitopentaose analogue. Chem. Eur.
J. 2010, 16, 4239–4249.
11. Veleti, S.K.; Lindenberger, J.J.; Thanna, S.; Ronning, D.R.; Sucheck, S.J. Synthesis
of a poly-hydroxypyrolidine-based inhibitor of Mycobacterium tuberculosis GlgE. J. Org.
Chem. 2014, 79, 9444–9450.
12. France, R.R.; Rees, N.V.; Wadhawan, J.D.; Fairbanks, A.J.; Compton, R.G. Selec-
tive activation of glycosyl donors utilising electrochemical techniques: a study of the
thermodynamic oxidation potentials of a range of chalcoglycosides. Org. Biomol. Chem.
2004, 2, 2188–2194.
13. Werz, D.B.; Seeberger, P.H. Total synthesis of antigen Bacillus anthracis
tetrasaccharide-creation of an anthrax vaccine candidate. Angew. Chem. Int. Ed. 2005,
44, 6315–6318.
14. Roy, B.; Field, R.A.; Mukhopadhyay, B. Synthesis of a tetrasaccharide related to
the repeating unit of the O-antigen from Escherichia coli K-12. Carbohydr. Res. 2009,
344, 2311–2316.

Y. Qiao et al.
132
15. Zhang, J.; Kong, F. Synthesis of β-D-Glcp-(1→2)-[β-D-Ribf-(1→3)]-α-L-Rhap-
(1→3)- α-L-Rhap-(1→2)-α-L-Rhap, the repeating unit of the lipopolysaccharide of Ace-
tobacter diazotrophicus PAL 5. J. Carbohydr. Chem. 2002, 21, 579–589.
16. Zhang, J.; Kong, F. Synthesis of a xylosylated rhamnose pentasaccharide-the re-
peating unit of the O-specific side chain of lipopolysaccharides from the reference
strains for Stenotrophomonas maltophilia serogroup O18. J. Carbohydr. Chem. 2002,
21, 89–97.
17. Wang, P.; Wang, J.; Guo, T.T.; Li, Y. Synthesis and cytotoxic activity of the N-
acetylglucosamine-bearing triterpenoid saponins. Carbohydr. Res. 2010, 345, 607–620.
18. Yang, Y.; Li, Y.; Yu, B. Total synthesis and structural revision of TMG-
chitotriomycin, a specific inhibitor of insect and fungal β-N-acetylglucosaminidases. J.
Am. Chem. Soc. 2009, 131, 12076–12077.
19. Fukuyama, T.; Laird, A.A.; Hotchkiss, L.M. p-Anisyl group: a versatile protecting
group for primary alcohols. Tetrahedron Lett. 1985, 26, 6291–6292.
20. Schmidt, R.R.; Michel, J. Facile synthesis of α- and β-O-glycosyl imidates: prepa-
ration of glycosides and disaccharides. Angew. Chem. Int. Ed. Engl. 1980, 19, 731–732.
21. Olsson, J.D.M.; Landstro¨m, J.; Ro¨nnols, J.; Oscarson, S.; Widmalm, G. Synthesis
of and molecular dynamics simulations on a tetrasaccharide corresponding to the re-
peating unit of the capsular polysaccharide from Salmonella enteritidis. Org. Biomol.
Chem. 2009, 7, 1612–1618.
22. Maranduba, A.; Veyrieˇres, A. Glycosylation of lactose: synthesis of branched
oligosaccharides involved in the biosynthesis of glycolipids having blood-group I ac-
tivity. Carbohydr. Res. 1986, 151, 105–119.