Synthesis and immunological study of a wall teichoic acid-based vaccine against E. faecium U0317

Article abstract of DOI:10.1080/07328303.2017.1390576

A repeat unit of cell wall teichoic acids (WTA) isolated from E. faecium U0317 was chemically synthesized efficiently by a stepwise strategy. It was derivatized with a 5-aminopentanyl linker to facilitate conjugation with carrier proteins KLH and HSA. Immunological studies of the KLH conjugate 1 demonstrated that it could provoke robust immune responses and high titers of IgG antibodies, which could successfully recognize the synthesized WTA repeat unit 3. This result suggested that synthetic glycoconjugate 1 could be a promising vaccine candidate against E. faecium for further studies.

Full text of DOI:10.1080/07328303.2017.1390576

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Synthesis and immunological study of a wall
teichoic acid-based vaccine against E. faecium
U0317
Zhifang Zhou, Wenzhang Ding, Chen Li & Zhimeng Wu
To cite this article: Zhifang Zhou, Wenzhang Ding, Chen Li & Zhimeng Wu (2017): Synthesis and
immunological study of a wall teichoic acid-based vaccine against E. faecium U0317, Journal of
Carbohydrate Chemistry, DOI: 10.1080/07328303.2017.1390576
To link to this article: http://dx.doi.org/10.1080/07328303.2017.1390576
Published online: 07 Nov 2017.
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Date: 11 November 2017, At: 09:08

JOURNAL OF CARBOHYDRATE CHEMISTRY
, VOL. , NO. , –
https://doi.org/./..
Synthesis and immunological study of a wall teichoic
acid-based vaccine against E. faecium U
Zhifang Zhou, Wenzhang Ding, Chen Li, and Zhimeng Wu
Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of
Biotechnology, Jiangnan University, Wuxi, China
ABSTRACT
ARTICLE HISTORY
Received  July 
Accepted  October 
A repeat unit of cell wall teichoic acids (WTA) isolated from
E. faecium U0317 was chemically synthesized efficiently by a
stepwise strategy. It was derivatized with a 5-aminopentanyl
linker to facilitate conjugation with carrier proteins KLH and HSA.
Immunological studies of the KLH conjugate 1 demonstrated
that it could provoke robust immune responses and high titers
of IgG antibodies, which could successfully recognize the syn-
thesized WTA repeat unit 3. This result suggested that synthetic
glycoconjugate 1 could be a promising vaccine candidate against
E. faecium for further studies.
KEYWORDS
E. feacium; wall teichoic acid;
vaccine; glycoconjugate
GRAPHICAL ABSTRACT
2 0 0 0 0 0
1 5 0 0 0 0
1 0 0 0 0 0
5 0 0 0 0
0
7
d
1
8
1
4
d
0
d
d
2
d 2
A n tis e ra o n d iffe re n t d a y s
Introduction
The multi-drug resistant gram-positive bacteria enterococcal species have become
the third most threatening pathogens, which have caused serious nosocomial infec-
tions in the intensive care unit (ICU), such as bloodstream infections and surgical
site infections.[1] For example, Enterococcus faecium was found to be one of the most
common pathogens among the enterococci species in the hospital environment.[2]
The treatment of infections caused by E. faecium, especially the vancomycin-
resistant Enterococcus (VRE) strains, faces increasing difficulties because of the
endogenous and acquired resistance to many antibiotics.[3] For the control of multi-
drug resistant bacterial infections, vaccination can be an effective strategy.[4–5]
Therefore, it is highly urgent to develop vaccine strategies to combat against E. fae-
cium infections.
CONTACT Prof. Zhimeng Wu
zwu@jiangnan.edu.cn
The Key Laboratory of Carbohydrate Chemistry &
Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , China.
©  Taylor & Francis Group, LLC

2
Z. ZHOU ET AL.
Figure . The chemical structure of WAT isolated from E. feacium (A) and the target glycoprotein con-
jugates 1 and 2 of this study (B). CP: carrier protein; KLH: keyhole limpet hemocyanin; HAS: human
serum albumin.
Teichoic acids (TAs), a kind of glycopolymer with poly-alditol phosphate struc-
tures, are the major cell wall components of Gram-positive bacteria.[5] There
are two types of TAs, i.e., lipotechoic acid (LTA) and wall teichoic acid (WTA),
which anchors onto the membrane and couples to the peptidoglycan (PG) layer,
respectively.[5] LTAs are promising carbohydrate antigenic epitopes which could
be recognized by antibodies, and some of the anti-LTA antibodies were developed
as immunotherapeutic agents to treat or prevent S. aureus diseases.[6] It was also
demonstrated that the opsonic antibody against LTA from E. faecalis could cross-
bind with LTA from other gram-positive species, such as S. epidermidis, S. aureus
and group B Streptococcus (GBS).[7] However, one major concern of LTA-based
vaccines is that LTA may not easily accessed by antibody due to its subcapsular
location.[6] In contrast, WTAs are another type of potential carbohydrate anti-
gens for vaccine design because they have longer glycopolymer chains to form a
dense glycan layer at the cell surface and are extending beyond the cell wall.[8–9]
Recently, the chemical structure of WTA isolated from E. faecium U0317, which is
a strain isolated from human urinary tract infection and resistant to ampicillin and
ciprofloxacin, was analyzed and elucidated.[10–11] The repeating unit of this WTA
contains two 2-acetamido-2-deoxy-D-galactose residues, one glycerol (Gro), and a
phosphate (Figure 1A). Based on this structure, the defined WTA repeat unit could
be chemically synthesized and used for the development of semi-synthetic conjugate
vaccines and related immunological studies. Previously, Pozsgay’s group reported
the synthesis of ribitol phosphates oligomers in a stepwise fashion and their conjuga-
tion with bovine serum albumin.[12] Most recently, Codee’s group developed an ele-
gant approach for the synthesis of mono- to trimeric repeat units of E. faecium WTA
fragments without further immunological studies.[13] In this paper, we developed
an efficient synthetic strategy for the repeat unit of E. faecium WTA with a linker
that contained an amino group to facilitate its conjugation with other molecules.
This repeating unit was then conjugated with keyhole limpet hemocyanin (KLH) to
form glycoconjugate 1 (Figure 1B), which was applied to immunological studies in
mice. In the meantime, its human serum albumin (HSA) conjugate 2 was used as
capture reagent for enzyme-linked immunosorbent assays (ELISA).
Results and discussion
The repeating unit structure of WTA isolated from E. faecium U0317 is comprised
of a disaccharide α-GalNHAc(1→3)-GalNHAc, which is β-linked to the 2-position

JOURNAL OF CARBOHYDRATE CHEMISTRY
3
Figure . Synthetic target and the retrosynthetic analysis.
of a glycerol phosphate. Thus, the synthetic target 3 was designed to contain this
structural epitope. In the meantime, the glycerol phosphate was further derivatized
with the 5-aminopentanyl group to facilitate conjugation with carrier proteins.
The retrosynthetic analysis was shown in Figure 2, leading to a key disaccharide
building block 5, glycerol compound 6 and H-phosphate linker 7. Disaccharide 5
could be assembled from peracetylated 2-azido-2-deoxy-galactosyl imidate donor 8
and acceptor 9. Non-participating azido group in donor 8 would be used to prepare
α-(1→3)-linkage with the aid of diethyl ether as co-solvents in glycosylation, while
amino group in acceptor 9 was protected with 2,2,2-trichloroethoxycarbonyl (Troc)
as a participating group for the construction of β linkage with glycerol derivative 6.
Our synthesis started with preparing building blocks 8, 9 and 6 as shown in
Scheme 1. Reaction of D-galactosamine with triflyl azide in the presence of a cat-
alytic amount of copper sulfate was followed by acetylation to give peracetylated
2-azido-2-deoxy-galactose 11. Selective removal of the anomeric acetyl group in 11
afforded hemiacetal 12 in a 78% yield, which was then reacted with trichloroace-
tonitrile to give imidate donor 8 in a good yield.[14] 2-N-Troc-4,6-benzylidene
acetal-protected acceptor 9 was prepared according to a slightly modified literature
Scheme . Synthesis of building blocks 8, 9 and 6. Reagents and conditions: (a) ()NaN, HO, DCM,
TfO, KCO, CuSO, MeOH; () AcO, Py, DMAP, %; (b) (NH)CO, MeOH:THF = :, %; (c) TCAN,
DBU, DCM, %; (d)  M NaOH, reflux, o/n; (e) TrocCl, EtN, MeOH; (f) PhCH(OMe), TsOH, DMF; %
yield over (d) (e) and (f); (g) ,-DMP, p-TSA, Acetone, %; (h) NaH, BnBr, DMF, %; (i) THF:HO = .:
, p-TSA, %; (j) Py, TBDMSCl, %.

4
Z. ZHOU ET AL.
Scheme . Synthesis of target compound 3. Reagents and conditions: (a) DCM/diethyl ether (:),
TMSOTf, A MS, −°C, % (α:β = :); (b) DCM, TfOH, NIS, A MS, −°C, %; (c) TBAF:AcOH = :,
THF, %; (d) Py, pivloyl chloride; (e) Iodine, Py:HO = :, %; (f) Zn/HOAc/AcO; (g) MeONa, MeOH;
(h) H, Pd(OH)/C.
procedure.[15] Known peracetylated monosaccharide 13 was refluxed in 1 M NaOH
to remove all the acetyl groups to give 14 and the amine group was selectively pro-
tected with TrocCl under alkaline condition to obtain 15. Without further purifica-
tion, 15 was treated with benzaldehyde dimethyl acetal in DMF to afford acceptor 9
in a 63% yield over three steps. Glycerol compound 6 was prepared according to a
reported procedure in four steps.[16–18]
With these building blocks in hand, target compound 3 was assembled by a step-
wise strategy as shown in Scheme 2. Glycosylation of acceptor 9 with donor 8 pro-
moted by TMSOTf in DCM proceeded smoothly to afford 20 in a 70% yield but poor
stereoselectivity (α:β = 3:2). Performing the glycosylation in a mixture of DCM and
diethyl ether (2:1) improved the stereoselectivity to α:β = 5:1 ratio in a 78% yield.
The α-isomer of 20 was purified and the stereochemistry of the newly formed glyo-
sidic bond was confirmed by the small coupling constant (J1’, 2’ = 3.8 Hz) of the
anomeric proton at 4.9 ppm. Then, 20 was directly utilized as glycosyl donor to
couple with 6 under promotion of NIS and TfOH to obtain 21 in a moderate yield.
After removal of the TDBMS group in 21, phosphorylation of 22 was achieved by H-
phosphate reagent 7. The condensation reaction catalyzed by pivaloyl chloride and
subsequent in situ oxidation by iodine were performed in one pot to provide phos-
phorylated compound 23 in a 75% yield over two steps. To simultaneously trans-
fer both azido and Troc group to NHAc in a single step, zinc powder in a mixture
of acetic acid and acetic anhydride was applied, and the reaction proceeded very
well to give 4 in a 67% yield. Finally, global deprotection was achieved by Zemplén
transesterification reaction to remove the acetyl protecting group and subsequent
hydrogenation to remove the benzyl ether, Cbz and benzylidene acetal groups. The
final product was further desalted with a Sephadex LH 20 column to give 3 in a
41% yield after lyophilization. Its structure was fully characterized by 1H, 13C, 31
P

JOURNAL OF CARBOHYDRATE CHEMISTRY
5
1
NMR and MS data. In addition, the H-NMR spectrum of 3 was compared with
that of isolated WTA from E. faecium, which displayed the anomeric α- and β-
proton signal at δ 5.06 and 4.69 ppm with coupling constants of 3.5 and 8.5 Hz,
respectively.[10] Our synthetic sample 3 displayed the corresponding anomeric
proton signal at 4.98 (J = 3.6 Hz) and 4.60 (J = 8.4 Hz) ppm with very similar cou-
1
pling constants. Moreover, the overall H NMR spectra of synthetic and isolated
samples were very similar, which further demonstrated the successful synthesis.
Recently, Codee’s group described the synthesis of E. faecium WTA repeat unit and
its oligomers by using a common glycerol-disaccharide intermediate and phospho-
ramidite chemistry to install the phosphate group.[13] In our synthesis, we applied
a stepwise approach to synthesize the disaccharide and H-phosphonate chemistry,
instead of phosphoramidite chemistry, was utilized to install the phosphate group.
H-phosphonate chemistry, which involves more stable intermediates, was more con-
venient than the phosphoramidite method in our experience.
Free disaccharide 3 was conjugated with carrier proteins KLH and HSA through
the bifunctional glutaryl ester method as reported in the literature.[19–23] This link-
age has been widely used in other vaccine conjugate because of the simplicity of
reaction manipulation and the negligible immunogenecity.[20,22] The reaction of
3 with a large excess of active ester disuccinimidyl glutarate (DSG) 24 was per-
formed in DMF and PBS buffer at room temperature for 4 hours to generate acti-
vated monoester 25 in quantitative yield and then conjugated with KLH or HSA
carrier protein in phosphate buffered saline (PBS, pH 7.8) for three days to give
carbohydrate-protein conjugates 1 and 2 (Scheme 3). The sugar content of HSA
conjugate 2 was characterized by MALDI-TOF mass spectrometry, while that of
KLH conjugate 1 was analyzed by Morgan-Elson colorimetric method.[24] The sugar
loading of 1 and 2 was 11.5% and 11.3%, respectively.
Immunological activities of KLH conjugate 1 were evaluated in a group of six
C57BL/6J mice (6–8 weeks, female). For the immunization, the KLH conjugate
(3 µg saccharide moiety per mouse each time) was dissolved in 1 × PBS buffer
and mixed with Freund’s complete adjuvant (CFA), and the resulting emulsion was
injected intramuscularly (i.m.) into mice. After the initial immunization, the mice
were immunized three more times on days 7, 14 and 21 by subcutaneous (s.c.)
injection of the same glycoconjugate emulsion. The background blood samples
of each mouse were collected from mouse’s leg veins on day 0 before the initial
immunization. The antisera were prepared from the blood samples collected from
each mouse on days 7, 14 and 28 by the same method as the literature.[21–23] ELISA
Scheme . Preparation of disaccharide-protein conjugates 1 and 2. Reagents and conditions: (a) DSG
24, DMF and PBS buffer (:), rt, h; (b) KLH or HSA, PBS buffer, rt,  days.

6
Z. ZHOU ET AL.
Figure . ELISA results of the antisera collected on different days (A) and the antibody titers of IgG
and IgM on day  antisera (B). The error bar in (A) represents the standard deviations (SD) of three
parallel experiments. Each dot in (B) represents the antibody titer of individual mouse and the black
bar represents the average titer of six mice.
experiment was carried out to measure the immune responses using the HSA
conjugate 2 as capture reagent for plate coating. Generally, the ELISA plates were
treated with conjugate 2 and blocked by 1% bovine serum albumin (BSA) solution.
Then, serial-diluted pooled antisera or the antiserum of individual mouse were
added to the plates. After the antigen-specific antibody binding to the plates, a
secondary antibody solution of goat anti-mouse IgG or IgM with alkaline phos-
phatase (AP) was added to each well. Thereafter, the plates were treated with
p-nitrophenylphosphate (PNPP) buffer to develop color and the optical density
(OD) values at 415 nm were measured by microplate reader. Finally, the antibody
titers were defined as the dilution number yielding an OD (415 nm) value of 0.2.
The ELISA results shown in Figure 3 revealed that the KLH conjugate 1 can elicit a
strong immune response, as indicated by the high titers of antibodies. Even only after
three times of immunization, the antibody titer has reached a significant level (titer:
98,529). After the fourth immunization, the total antibody titer reached as high as
135,466. More importantly, in the day 28 antisera, IgG was found to be the main
antibody isotype and a very low level of IgM was observed. This result indicated
that a T cell-dependent immunity may be involved.
To further confirm the specificity of the antisera induced by conjugate 1, a
competitive ELISA experiment was performed using a free disaccharide compound
3 as the competitive reagent. In this experiment, 96 well plates were coated with
conjugate 2 at first followed by treatment with blocking buffer containing 1% BSA.
In the meantime, compound 3 in various concentrations (from 0 to 150 µM) was
mixed and incubated with 1:10000 diluted pooled antisera of day 28 at 37°C for
30 min, and then the mixture was added to the pretreated 96-well plates at 37°C
for another 2 h. Thereafter, the secondary antibody solution of goat anti-mouse
IgG and IgM with AP was added to each well and incubated at rt for 1 h. Finally,
the plates were developed with PNPP solution and the OD values at 415 nm were
measured by microplate reader. During this procedure, the free compound 3 in
solution should compete with 2 attached on the ELISA plate for antibody binding.

JOURNAL OF CARBOHYDRATE CHEMISTRY
7
Figure . Competitive ELISA result of compound 3 with the pooled antisera induced by conjugate
1 plus CFA. The OD values at  nm reflected the levels of antibodies bound to the 2-coated ELISA
plate in the presence of various concentrations of compound 3. The error bar represents the SD of
three parallel experiments.
The OD values at 415 nm represent the levels of antibody attached to the ELISA
plate coated with conjugate 2. As shown in Figure 4, the disaccharide 3 could
compete with 2 attached to the ELISA plate for antibody binding, and displayed
concentration-dependent inhibition on the antibody binding to the plate. When the
concentration of compound 3 was 150 µM, the OD value at 415 nm was reduced to
0.73 comparing to the initial value 1.22. This result proved that at least a portion of
the antibodies induced by conjugate 1 was specific to carbohydrate antigen 3.
Conclusions
In summary, a repeat unit of WTA from E. feacium U0317 with a linker was suc-
cessfully synthesized by a linear assembly strategy and then conjugated with carrier
protein KLH for immunological studies. The immunological studies revealed that
conjugate 1 could provoke very strong immune response, and mainly IgG type of
response. The elicited antibodies could successfully recognize the WTA repeat unit
conjugated on HSA protein or its free form in solution. This research demonstrated
the possibility and potential to develop synthetic WTA-based anti-E. feacium
vaccines.
Experimental section
General methods
NMR spectra were recorded at 400 MHz. Proton chemical shifts are reported in
ppm (δ) downfield from tetramethylsilane (TMS) or in reference to the solvent
DHO (δ 4.79). Carbon chemical shifts are reported in ppm (δ) in reference to
CDCl3 (δ 77.16). Coupling constants (J) are reported in hertz (Hz). Mass spectra
(ESI) were recorded on a Bruker Amazon SL. Thin layer chromatography (TLC)
was performed on silica gel GF254 detected by charring with phosphomolibdic

8
Z. ZHOU ET AL.
acid/EtOH or 1% H2SO4/EtOH solutions. Molecular sieves were dried under high
vacuum at 170–180°C for 6 to 10 h before use. Commercial anhydrous solvents and
other reagents were used without further purification unless otherwise mentioned.
Mass spectrometry (MS) was performed with Thermo Scientific TSQ Quantum
Ultra, Bruker Daltonics Ultraflex MALDI-TOF mass spectrometer. High resolution
mass spectrum was collected by IonSpec Ultra. Freund’s complete adjuvant (CFA)
was purchased from SigmaAldrich. Alkaline phosphatase (AP) conjugated goat
anti-mouse IgG and IgM antibodies were purchased from Jackson ImmunoRe-
search. Female C57BL/6J mice of 6–8 weeks age were purchased from Shanghai
Slac Laboratory animal Co. Ltd.
3,4,6-O-acetyl-2-azido-2-deoxy-D-galactopyranoside-1-O-
trichloroacetoimidate (8)[25] and 1-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-
3-(phenylmethoxy)-2-propanol (6)[18] was prepared according to literature.
P-methylphenyl 4,6-O-benzylidene-2-deoxy-2-(2ꢀ,2ꢀ,2ꢀ-trichlorethoxycarbony
lamino)-1 thiol-β-D-galactopyranoside (9)
Peracetylated galactosamine 13[26] (10.4 g, 18.6 mmol) was dissolved in 300 mL
of 1 M NaOH solution, and refluxed overnight. After reaction was completed, the
reaction mixture was neutralized with 2 M HCl solution in ice-water bath to pH 7–
8. Then solvents were concentrated under vacuum, residue was filtered and washed
with 100 mL MeOH. The elution was concentrated several times under vacuum by
mixture solvents of CH3OH and toluene (1:4) until solid product 14 was obtained.
Without purification, 14 was dissolved in 100 mL of CH3OH and cooled to 0°C, then
2,2,2-trichloroethyl chloroformate (3 mL, 22.3 mmol) and Et3N (3.1 mL, 22.3 mmol)
were added slowly. The reaction was kept in 0°C for 5 h, and then the reaction
mixture was concentrated and dried under vacuum. The solid residue was precipi-
tated in ice-water to give 15 for next use without further purification. Benzaldehyde
dimethyl acetal (3.3 mL, 22.3 mmol) and camphorsulfonic acid (500 mg, 2.2 mmol)
were added to 20 mL of DMF solution containing compound 15, and the mix-
ture was stirred overnight at rt. After the reaction was completed, the reaction was
quenched by Et3N. The reaction mixture was diluted by 40 mL of ice-water, and
extracted with DCM three times. Organic phase was collected and washed with
water, saturated NaHCO3 and saturated NaCl solution. The organic phase was dried,
filtered and concentrated under vacuum. The product was purified by silica gel col-
umn (ethyl acetate: toluene = 3:7) to afforded compound 9 [27] (7.2 g, 63% yield). R
f = 0.67 (MeOH /CH2Cl2 = 5:95). The 1H NMR is same as reported literature.
P-methylphenyl 3,4,6-O-acetyl-2-azido-2-deoxy-α-galactopyranosyl-(1→3)-4,6-
O-benzylidene-2-deoxy-2-(2ꢀ,2ꢀ,2ꢀ-trichlorethoxycarbonylamino)-1-thiol-β-D-
galactopyranoside (20)
Compound 8 (690 mg, 1.26 mmol) and 9 (500 mg, 1.05 mmol) was co-evaporated
with toluene for three times, then dissolved in 20 mL mixture solvents of DCM

JOURNAL OF CARBOHYDRATE CHEMISTRY
9
˚
and diethyl ether(2:1) followed by adding activated 4A molecular sieve (350 mg)
under argon. The mixture was stirred at rt for 1 h. The reaction mixture was cooled
to −78°C, TMSOTf (23 µL, 0.126 mmol) was added and stirred at same tempera-
ture for 2 h. When the reaction was completed, it was quenched by adding 0.1 mL
Et3N. Molecular sieve was filtered off, and reaction mixture was purified by silica
gel column (PE:EtOAc = 2:1, Rf = 0.3) to give compound 2026 (580 mg, 64% yield).
1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 8.1 Hz, 2H), 7.53 – 7.45 (m, 2H), 7.44 –
7.35 (m, 3H), 7.06 (d, J = 8.0 Hz, 2H), 5.61 – 5.49 (m, 2H, NH+PHCH), 5.39 (d, J =
2.5 Hz, 1H), 5.31 – 5.21 (m, 2H), 5.18 (d, J = 3.5 Hz, 1H, H-1ꢀ), 4.87 (d, J = 12.0 Hz,
1H), 4.62 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 10.9 Hz, 1H), 4.44 (dd, J = 12.4, 1.4 Hz,
1H), 4.40 (d, J = 3.1 Hz, 1H), 4.22 (t, J = 6.5 Hz, 1H), 4.15 – 3.99 (m, 3H), 3.63 –
3.51 (m, 3H), 2.32 (s„ 3H), 2.15 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H).
3,4,6-O-acetyl-2-azido-2-deoxy-α-galactopyranosyl-(1→3)-4,6-O-benzylidene-
2-deoxy-2-(2ꢀ,2ꢀ,2ꢀ-trichlorethoxycarbonylamino)-β-D-galactopyranosyl-(1→2)-
β-3-O-benzyl-1-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-glycerol (21)
˚
Dried donor 20 (500 mg, 0.58 mmol), acceptor 6 (340 mg, 340 mmol) and 4A molec-
ular sieve (350 mg) was dissolved in 20 mL DCM in argon atmosphere and stirred
for 1 h. Then, NIS (261 mg, 1.16 mmol) and TfOH (10 µL, 0.058 mmol) was added
at −20°C and stirring was continued for 5 h. Et3N (0.1 mL) was added to quench the
reaction. After removal of molecular sieve, the reaction mixture was washed by sat-
urated NaS2O3, water and saturated NaCl solutions. Compound 21 (365 mg, 61%
yield) was afforded after purification in silica gel column (PE:EtOAc = 3:1, R f =
0.25). 1H NMR (400 MHz, CDCl3) δ7.63 – 7.53 (m, 2H), 7.44 – 7.23 (m, 8H), 5.84
(b, 1H, NH), 5.62 (s, 1H, PhCH), 5.42–5.43 (m, 1H), 4.62(d, J = 3.6 Hz, 1H), 5.35
(d, J = 4.0 Hz, 1H), 4.58 (s, 2H), 4.47–4.54(m, 1H), 4.33–4.39(m, 3H), 4.23(m, 1H),
4.09–4.14(m, 3H), 3.00–4.02(m, 2H), 3.73 (d, J = 5.9 Hz, 1H), 3.67 (d, J = 5.0 Hz,
1H), 3.57 (dd, J = 11.2, 3.5 Hz, 1H), 3.49 (s, 1H), 2.15 (s, 3H), 2.07 (s, 3H), 2.05(s,
3H), 0.90 (s, 9H), 0.07 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 169.9, 169.5, 154.2,
138.1, 137.5, 128.8, 128.3, 128.0, 127.8, 127.6, 126.1, 100.8, 95.8, 74.5, 73.4, 70.3,
69.3, 67.5, 67.3, 66.9, 66.4, 61.1, 56.9, 53.4, 25.9, 20.7, 20.6, 20.6, 18.2; HRMS: Cal.
for C44H59Cl3N4O16Si m/z, 1032.276; found 1055.2646[M+Na]+.
3,4,6-O-acetyl-2-azido-2-deoxy-α-galactopyranosyl-(1→3)-4,6-O-benzylidene-
2-deoxy-2-(2ꢀ,2ꢀ,2ꢀ-trichlorethoxycarbonylamino)-β-D-galactopyranosyl-(1→2)-
β-3-O-benzyl-1-hydroxyl-glycerol (22)
Compound 21 (300 mg, 0.29 mmol) was dissolved in 6 mL THF in ice-water bath.
Prepared TBAF:CH3CO2H (mol:mol = 1:1) solution was added into the reaction
and reaction mixture was kept at rt overnight. After concentration, the product
was purified by silica gel column (PE:EtOAC = 1:1, R = 0.3) to achieve com-
f
pound 22 (200 mg, 75% yield). 1H NMR (400 MHz, CDCl3) δ 7.63 – 7.53 (m, 2H),

10
Z. ZHOU ET AL.
7.44 – 7.23 (m, 8H), 5.67 (b, 1H, NH), 5.59 (s, 1H, PhCH), 5.41 (s, 1H), 5.33 (dd,
J = 11.2, 3.1 Hz, 1H), 5.21 (d, J = 2.9 Hz, 1H), 4.96 (d, J = 7.4 Hz, 1H), 4.47–
4.63 (m, 4H), 4.33–4.36(m, 3H), 4.26 (t, J = 6.3 Hz, 1H), 4.16–3.98 (m, 3H), 3.92
(d, J = 5.5 Hz, 1H), 3.83–3.65 (m, 2H), 3.54 (m, 5H), 1.68(b, 1H, OH), 2.13 (s, 3H),
2.05 (s, 3H), 2.03(s, 3H); 13C NMR (101 MHz, CDCl3) δ 170.5, 169.9, 169.6, 154.2,
137.7, 137.4, 128.9, 128.4, 128.1, 127.8, 126.1, 100.9, 95.4, 81.9, 74.5, 73.5, 71.4, 70.3,
69.3 67.6, 67.3, 67.0, 66.5, 63.1, 61.3, 56.9, 53.8, 30.9, 20.8, 20.6, 20.61. HRMS: Cal.
For C38H45Cl3N4O16 m/z, 918.190; found 941.1794 [M+Na]+.
3,4,6-O-acetyl-2-azido-2-deoxy-α-galactopyranosyl-(1→3)-4,6-O-benzylidene-
2-deoxy-2-(2ꢀ,2ꢀ,2ꢀ-trichlorethoxycarbonylamino)-β-D-galactopyranosyl-(1→2)-
β-3-O-benzyl-1-(1-phosphate-5-benzyloxycarbonyl amino pentanyl)-glycerol (23)
Dried compound 22 (200 mg, 0.22 mmol) and compound 7 (120 mg, 0.45 mmol)
were dissolved in 10 mL dried pyridine. Pivaloyl chloride (57 µL) was slowly added
to the reaction mixture. After 3 h of stirring at rt, 2 mL of freshly prepared iodine
solution (pyridine:water = 9:1) was added to the reaction mixture and stirring
was continued for 1 h. Then the reaction was quenched by addition of saturated
Na2S2O3 aqueous solution. The mixture was extracted with chloroform, and the
organic phase was combined, dried and concentrated. The residue was subjected to
silica gel column chromatography (PE:EtOAc = 1:2, R f = 0.3) to afford compound
23 (199 mg, 75% yield). The compound gave very bad resolution in 1H-NMR spec-
trum; it was characterized by 31P NMR (162 MHz, CDCl3): 3.28; MALDI-TOF MS:
Cal. for C51H63Cl3N5O21P m/z, 1217.282, found 1242.299[M+Na]+.
2-acetamido-2-deoxy-α-galactopyranosyl-(1→3)-2-acetamido -2-deoxy-β-
D-galactopyranosyl-(1→2)-β-1-(1-phosphate-5-amino-pentanyl) glycerol (3)
Compound 23 (100 mg, 0.08 mmol), 600 mg activated Zn (w:w = 1:3), acetic acid
(30 mL/g) and acetic anhydride (15 mL/g) was mixed in 5 mL THF in ice-water bath.
The reaction was stirred at rt for 3 h. The reaction mixture was filtered, organic phase
was washed with water and concentrated to give compound 4 (60 mg, 67% yield).
The 1H NMR of this compound had very bad of resolution, it was characterized by
HRMS: Cal. for C52H68N3O21P, m/z, 1101.408; found 1102.4152.
Compound 4 (60 mg, 0.054 mmol) in 3 mL CH3OH was added 1 drop of
CH3ONa (about 5 mol/L). After stirred at rt for 5 h, the reaction was neutralized
with acidic resin. The crude product was dissolved in 2 mL of CH3OH and added
with 20 µL of water and 1 drop of acetic acid. Then 20% Pd(OH)2/C (w:w = 1:1) was
added under hydrogen atmosphere. After stirred for 48 h, catalyst was removed and
the resulted product was desalted by Sephadex LH-20. Compound 3 (15 mg) was
achieved after lyophilization. 1H NMR (400 MHz, D2O) δ4.97 (d, J = 3.8 Hz, 1H, H-
1), 4.60 (d, J = 8.5 Hz, 0.2H, water suppression with presat, H-1ꢀ), 4.12 (dd, J = 11.0,
3.7 Hz, 1H), 4.01 (d, J = 3.0 Hz, 1H), 3.95–3.99 (m, 1H), 3.89–3.93 (m, 4H), 3.79–
3.83 (m, 3H), 3.64–3.76 (m, 6H), 3.59–3.63(m, 2H), 3.54–3.56(m, 1H), 2.91–2.94(m,

JOURNAL OF CARBOHYDRATE CHEMISTRY
11
2H, CH2 from linker), 1.96 (s, 3H, NH Ac), 1.94 (s, 3H, NH Ac), 1.57–1.66 (m, 4H,
CH2 from linker), 1.36–1.40 (m, 2H, CH2 from linker). 13C NMR (101 MHz, D2O) δ
174.68(C = O), 174.64(C = O), 101.2(C1), 93.4(C1ꢀ), 79.5(C2, glycerol), 75.1, 74.8,
71.3, 68.3, 67.7, 65.9, 65.8, 64.5, 63.6, 61.1, 60.9, 60.6, 51.0, 49.4, 49.0, 42.6, 39.4, 32.6,
29.1, 29.0, 26.3, 25.0, 23.5, 22.3(CH3), 22.0(CH3). 31P NMR (162 MHz, D2O) δ 0.83;
MALDI-TOF MS: Cal. For C24H46N3O16P, 663.26, found 685.507 [M+Na]+.
Procedure for the activation of compound 3 to prepare compound 25
Compound 3 (15 mg) and excess amount of disuccinimidyl glutarate (DSG, 15 eq)
was dissolved in DMF and 1 × PBS buffer (v/v = 4:1, 0.5 mL) and stirred at rt for
4 h. After removing the solvent, the activated oligosaccharide 25 was separated from
the excessive reagents by precipitation with 5 mL EtOAc. Followed by washing the
precipitates 10 times with EtOAc, the white solid was dried and used in the next step
without further purification.
Procedure for the preparation of conjugate 1 and 2
The activated oligosaccharides 25 were mixed with HSA or KLH at a molar ratio
of 30:1 in 1 × PBS buffer (0.4 mL). The solution was shaken at rt for 3 days and
then the conjugates were purified by 10 kDa (for HSA conjugate) and 30 kDa (for
KLH conjugate) centrifugal filter devices to remove free sugars. The glycoconjugate
fractions were lyophilized to afford the desired glycoconjugates as white solids.
Analysis of the carbohydrate loading of glycoconjugates 1 and 2
The carbohydrate loading of conjugate 1 was measured by Morgan-Elson colori-
metric method.[24,28] Generally, dried galactosamine was weighted and dissolved
in water as solution with a series of concentrations. 3 g of potassium tetraborate
was dissolved in 50 mL as solution A, and 10 g of p-dimethylaminobenzaldehyde
(DMAB) was dissolved in 100 mL HCl:CH3CO2H solution (v:v = 12.5:87.5) as solu-
tion B. The standard samples were stirred with 50 µL of solution A and boiled for
5 min in water bath. After cooled to rt, each sample was added 1.5 mL solution B and
incubated at 37°C for 20 min. The colorimetric readout was measured at 595 nm by
spectrophotometer. To detect the carbohydrate loading in conjugate 1, the KLH pro-
tein and conjugate 1 were both hydrolyzed in 2 M HCl solution in boil water bath for
2 h. Thereafter, the solvent was removed and the lyophilized resulted samples were
determined by the same method. The carbohydrate loading was calculated by the
work-curve obtained from the standard samples. Finally, the carbohydrate loading
was defined as:
ꢀ
ꢁ
carbohydrate content mg in the sample
carbohydrate loading% =
× 100%
weight of conjugate(mg)
The loading of conjugate 1 was 11.5%.

12
Z. ZHOU ET AL.
The carbohydrate loading of conjugate 2 was 11.3% which was calculated from
the molecular weight (conjugate 2: 75.3 kDa; HSA protein: 66.4 kDa) measured by
MALDI-TOF-MS.
Immunization of mice
The animal protocol (JN. No 20170314–20170510 [28]) for this study was approved
by the Laboratory Animal Center (LAC, SYXK(Su)2016–0044) of Jiangnan Univer-
sity, and all of the animal experiments were performed in compliance with the rel-
evant laws and institutional guidelines.
A group of six female C57BL/6J mice (6–8 weeks old) were purchased from
Shanghai Slac Laboratory animal, Co., Ltd. (Shanghai, China). Upon arrival, mice
were housed and kept in a Specific Pathogen Free (SPF) room (temperature: 25
2°C, humidity: 60 5%) with a 12 h light-dark cycle. Conjugate 1 (3 µg carbohy-
drate moiety per mouse each immunization) was dissolved in 1 × PBS buffer and
then mixed with CFA (1:1, v/v) to form an emulsion according to the protocol pro-
vided by manufacturer. The mice were inoculated on day 1 by intramuscularly (i.m.)
injection of 0.1 mL of the emulsion of vaccine and boosted 3 more times on day 7, 14
and 21 by subcutaneous (s.c.). Blood samples of each mouse were collected through
the leg veins on day 0 before the initial immunization and on day 14, 21 and 28 that
one week after each boost. The blood was clotted and centrifuged to obtain antisera
which were further stored at −80°C for analysis.
Protocols for ELISA
ELISA plates were coated with 100 µL solution of conjugate 2 (2 µg/mL) dissolved
in coating buffer (0.1 M bicarbonate, pH 9.6) overnight followed by 1 h incuba-
tion at 37°C. After washing 3 times by PBST (phosphate-buffered saline (PBS) con-
taining 0.05% Tween-20), the plates were incubated with blocking buffer (1% BSA
in PBST) at rt for 1 h to block the non-specific sites. After washing, a pooled or
individual mouse antiserum with serial half-log dilutions from 1:300 to 1:656100
in PBS was added to each well of plates (100 µL/well) respectively, and incubated
at 37°C for 2 h. In the competitive ELISA protocol, various concentrations of com-
pound 3 was incubated with 1:10000 diluted day 28 pooled antisera for 30 min at
37°C and then added to the 96-well plates pre-treated with conjugate 2 and block-
ing buffer for another 2 h. After the incubation with antisera, the plates were washed
and treated with 100 µL of 1:2500 diluted goat anti-mouse IgG or/and IgM with AP
enzyme for 1 h at rt. Finally, these plates were washed and developed with 100 µL
of p-nitrophenylphosphate (PNPP) solution (1.67 mg/mL in buffer) for 30 min at
rt. The colorimetric readout was collected by Bio-RAD iMarkTM microplate reader
at 415 nm. The optical density (OD) values were plotted against logarithmic anti-
serum dilution values to get a best-fit line. The antibody titers were calculated as the
dilution value at which an OD of 0.2 was achieved.

JOURNAL OF CARBOHYDRATE CHEMISTRY
13
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 21472070
and No. 21602084), the Project for Jiangsu Scientific and Technological Innovation Team, Fund
for Jiangsu Distinguished Professorship Program, Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions, the Fundamental Research
Funds for the Central Universities (No. JUSRP11729) and the fund from the Open Foundation
of Key Laboratory of Carbohydrate Chemistry & Biotechnology Ministry of Education (No.
KLCCB-KF201608).
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