Synthesis and immunogenicity of PG-tb1 monovalent glycoconjugate

Article abstract of DOI:10.1016/j.ejmech.2017.03.058

A PG-tb1 hapten from the West Beijing strains of Mycobacterium tuberculosis cell wall has been efficiently synthesized and conjugated to CRM₁₉₇ in a simple way as linker-equipped carbohydrate by applying squaric acid chemistry for an original neoglycoprotein, creating a potent T-dependent conjugate vaccine. The intermediate monoester can be easily purified and the degree of incorporation can be monitored by MALDI-TOF mass spectrometry. After administered systemically in mice without any adjuvant, the conjugate induced high antigen-specific IgG levels in serum. Furthermore, following the third immunization, significant antibody titers frequently exceeding 0.8 million were observed in the sera of mice vaccinated with PG–CRM₁₉₇ conjugate which showed the potential for preparation of TB vaccine.

Full text of DOI:10.1016/j.ejmech.2017.03.058

Accepted Manuscript
Synthesis and immunogenicity of PG-tb1 monovalent glycoconjugate
Xin Meng, Chuanming Ji, Chao Su, Di Shen, Yaxin Li, Peijie Dong, Ding Yuan,
Mengya Yang, Song Bai, Demei Meng, Zhenchuan Fan, Yang Yang, Peng Yu, Tao
Zhu
PII:
S0223-5234(17)30224-6
10.1016/j.ejmech.2017.03.058
EJMECH 9318
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2017
Revised Date: 16 March 2017
Accepted Date: 24 March 2017
Please cite this article as: X. Meng, C. Ji, C. Su, D. Shen, Y. Li, P. Dong, D. Yuan, M. Yang, S. Bai,
D. Meng, Z. Fan, Y. Yang, P. Yu, T. Zhu, Synthesis and immunogenicity of PG-tb1 monovalent
glycoconjugate, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.058.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and immunogenicity of PGL-tb1
monovalent glycoconjugate
Leave this area blank for abstract info.
Xin Meng, Chuanming Ji, Chao Su, Di Shen, Yaxin Li, Peijie Dong, Ding Yuan, Mengya Yang, Song Bai,
Demei Meng, Zhenchuan Fan, Yang Yang, Peng Yu, Tao Zhu

ACCEPTED MANUSCRIPT
Synthesis and immunogenicity of PG-tb1 monovalent glycoconjugate
Xin Meng a, Chuanming Ji a, Chao Su a, Di Shen a, Yaxin Li a, Peijie Dong a, Ding Yuan a, Mengya Yang a,
Song Bai b, Demei Meng c, Zhenchuan Fan c, Yang Yang a, , Peng Yu a, , Tao Zhu a,
a China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Sino-French Joint Lab of Food Nutrition/Safety and Medicinal
Chemistry, Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin
University of Science and Technology, Tianjin 300457, China
b Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF)
c Key Laboratory of Food Nutrition and Safety of Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin
300457, China
ARTICLE INFO
ABSTRACT
A PG-tb1 hapten from the West Beijing strains of Mycobacterium tuberculosis cell wall has
been efficiently synthesized and conjugated to CRM197 in a simple way as linker-equipped
carbohydrate by applying squaric acid chemistry for an original neoglycoprotein, creating a
potent T-dependent conjugate vaccine. The intermediate monoester can be easily purified and
the degree of incorporation can be monitored by MALDI-TOF mass spectrometry. After
administered systemically in mice without any adjuvant, the conjugate induced high antigen-
specific IgG levels in serum. Furthermore, following the third immunization, significant
antibody titers frequently exceeding 0.8 million were observed in the sera of mice vaccinated
with PG–CRM197 conjugate which showed the potential for preparation of TB vaccine.
.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Carbohydrate
Synthesis
Bioconjugation
Glycoconjugate
Vaccine
1. Introduction
mediating cytokines including tumor necrosis factor alpha and
interleukin 12 and subsequent evidence demonstrated this was
Tuberculosis (TB) is a worldwide infectious disease caused by
the bacterium Mycobacterium tuberculosis (Mtb). The best
estimate is that there were 1.4 million TB deaths in 2015, and an
additional 0.4 million deaths resulting from TB disease among
HIV-positive people [1]. A vaccine (BCG) is still commonly
used in human anti-tuberculosis, but their immune preventive
effect is not ideal, the overall effective rate of prevention and
cure of not more than 50% [2,3]. With the emergence of multi-
drug resistant strains and hypervirulent strains, prevention and
treatment of tuberculosis is crucial.
Several Mtb strains from Asia, Africa, and India were found to
synthesize closely related phenolic glycolipids (PGL-tb1) in
which the phthiocerol is connected to a glycosylated phenol.
PGL-tb1 is suspected to be involved in hypervirulence of specific
Mtb strains [4,5]. The interplay of Mtb with the human host is
very complex, with PGL-tb1 as one of the most unusual
virulence factors modulating its defense systems and causing
disease. The West Beijing strains of Mtb, HN878, W4, and
W451, contain PGL as part of their cellular envelope [6,7]. These
due to PGL [8-12]. Thus, it is believed that PGL works in concert
with other factors to enhance the virulence of Mtb. The
disclosure herein focuses on phenolic glycosyl residues (PG-tb1),
excluding the phthiocerol dimycocerosates part of PGL.
As is well known, pure oligosaccharides are poor immunogens
and most of them are TI antigens. Synthesis of oligosaccharide
neoglycoconjugates as vaccines could be a way to convert a TI to
TD antigen [13,14]. Glycoconjugate vaccines obtained by
covalent linkage of poorly immunogenic sugar antigens to a
protein carrier play a crucial role in the prevention of many
deadly infectious diseases [15,16]. Proteins and peptides are
usually TD antigens since they require stimulation from helper T
lymphocytes in order to elicit an immune response [17,18].
Conjugate vaccines have been developed using several protein
carriers, one of which is CRM197. CRM197 is a non-toxic mutant
of diphtheria toxin and is a well defined protein and is utilized as
a carrier protein in a number of approved conjugate vaccines for
diseases such as infections and have been demonstrated excellent
efficacy and safety [19-22].
strains demonstrated reduced production of important immune-
———
*** Corresponding author
*** Corresponding author
*** Corresponding author
E-mail addresses: yyang@tust.edu.cn (Y. Yang), yupeng@tust.edu.cn (P. Yu), tao.zhu@cansinotech.com (T. Zhu)

2
removable temporary protecting group in the C3 position [26].
Precluded by the first synthesis of PGL-tb1 [23], we now
ACCEPTED MANUSCRIPT
The glycosylation reaction between thioglycoside donor 3 and
monosaccharide acceptor 4 was performed by activation with N-
iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH)
in dichloromethane at -25 °C, affording the required disaccharide
6 in an excellent 83% yield. Debenzoylation under Zemplën
conditions gave the alcohol in quantitative yield, and the
resulting alcohol was protected with a benzyl group by treatment
with benzyl bromide and sodium hydride in N, N-
Dimethylformamide. Subsequent selectively excised allyl ether
by catalytic PdCl2 in methanol afforded the disaccharide 7 in
65% yield over three steps. The obtained disaccharide acceptor 7
was welded together with fucose donor 2 under similar
glycosylation condition, yielding trisaccharide 8 in 81% yield
total chemically synthesized pure and molecularly defined
construct and attached the triglycosyl phenol to CRM197 for
original neoglycoprotein. The squaric acid chemistry of
conjugation of two amine species discovered by Tietze [24] has
been shown to be
a useful means for preparation of
neoglycoproteins in this study. Furthermore, this manuscript
describes one of our strategies to secure consistently robust
murine antibody responses to the small molecular weight PG-tb1
hapten derived from W-Beijing strains. We would go on from the
chemistry phase to evaluate the immunogenicity of the
monovalent glycoconjugate in mice. In addition, the density of
PG-tb1 epitope on the protein surface can be easily controlled by
the reaction ratio and characterized by Matrix Assisted Laser
Desorption/Ionization Time-of-Flight Mass Spectrometry
(MALDI-TOF-MS). This paper reports our initial results toward
the development of PG-tb1 conjugate vaccine.
and complete α-s
electivity. The 1H NMR and 13C NMR spectrum
of compound 8 agreed with the reported data [25]. After
deacetylation and finally methylated by treatment with an excess
of sodium hydride and methyl iodide to furnish compound 9 in
good yield.
2. Results and discussion
2.1. Chemistry
2.1.1 Synthesis of PG-tb1 triglycosyl phenol monomer
The synthetic strategy of hapten 1 was outlined in Scheme 1,
with an amine function as the linker, for use in conjugation.
Retrosynthetic analysis of the glycosylated phenol with a hexyl-
amine linker indicates that it can be chemically assembled from
thiofucosyl donor 2, thiorhamnosyl donor 3, p-iodophenol
rhamnoside acceptor 4 and commercially available 6-chloro-1-
hexyne 5. The building blocks 2, 3 and 4 were synthesized as
described previously [25].
Scheme 3. Synthesis of activated PG-tb1 monomer. Reagents and
conditions: a) [PdCl2(PPh3)2], PPh3, CuI, Et3N, 40 , 88%; b) NaN3,
DMF, TBAI, 60 , 82%; c) Pd(OH)2/C, H2, EtOH-EtOAc, rt, 78%.
Another key step, Sonogashira cross-coupling reaction
between the terminal alkyne 5 and iodophenyl trisaccharide 9,
gave the desired 10 in 88% yield. The strategy avoided a
stereoselective glycosylation step with an activated trisaccharide
in a late stage of the synthesis. It will be noted that the alkyne
function could be fully reduced to the corresponding aliphatic
fragment together with the removal of the benzyl protecting
groups and azido group reduction by only one step. An additional
advantage is that the amine group in the handle on the reducing
terminus could be utilized to install a bifunctional “spacer” of the
required length that render them amenable to conjugation.
Scheme 1. Retrosynthetic analysis of PG-tb1 monomer 1.
2.1.2 Preparation of PG-tb1 squarate derivative
The conjugation strategy whereby oligosaccharide is
covalently linked to protein to yield a conjugate vaccine is a
major factor that influences the synthetic strategy of
oligosaccharide assembly and deprotection. Furthermore, the
chemistry of conjugation may further impart undesirable
immunological properties to the vaccine. In this study, we used
the homobifunctional reagent, diethyl squarate [27], which
afforded reproducible conjugation in high yields under mild
conditions with small amounts of oligosaccharide and protein at
low concentration. Meanwhile, introduction of spacers (linkers)
to either PG antigen or protein carriers, which is involved in
commonly applied protocols, is not required. This strategy
involves a two-step, pH-dependent conjugation of two amines.
First, the alkyne-azide compound 11 was reduced with
Pd(OH)2/C under hydrogen atmosphere. Then, the resulting
amine was reacted with 5 equiv of the squaric acid diethyl ester
in ethanol/water solution followed by adding saturated sodium
Scheme 2. Reagents and conditions: a) NIS, TfOH, 4 Å molecular sieves,
CH2Cl2, -25
to rt, 2h, 83%; b) (i) MeONa, MeOH, rt, 4h; (ii) BnBr,
NaH, DMF, 0 to rt, 2 h; (iii) PdCl2 (5 mol%), MeOH, 20 , 16 h, 65%,
over three steps; c) NIS, TfOH, CH2Cl2, 4 Å molecular sieves, -25 to
rt, 2h, 81%; d) (i) MeONa, MeOH, 20 , 4 h; (ii) MeI, NaH, DMF, 0
to rt, 16 h, 83%.
With the required building blocks 2, 3, and 4 in hand, we could
enter the convergent part of the synthesis, depicted in Scheme 2.
The central rhamnose building block 3 was equipped with a C2
participating group to ensure α-selectivity and
a readily

The purity and the degree of substitution of the PG–CRM197
conjugates was analysed by sodium dodecyl sulfate
carbonate solution at room temperature affording the
corresponding squaric acid amide ester 12. The product obtained
ACCEPTED MANUSCRIPT gel
polyacrylamide
electrophoresis
(SDS-PAGE)
on
showed only moderate UV absorption at the wavelength
characteristic of squaric acid. After that, the product was easily
purified by column chromatography followed by freeze drying to
give the PG-tb1 squarate monomethyl ester as white solid in
homogeneous 12% polyacrylamide gels (Figure 1). High
molecular weight protein bands were observed for the purified
conjugate (lane 2-4) stained with CBB and no bands for CRM197
and diffuse are observed, confirming that the product was devoid
of residual unconjugated protein and suggesting homogeneity of
the glycoconjugate molecule.
1
good yield. H NMR and 13C NMR of the methyl squarate
derivative 12 showed doubling of some signals due to the
vinylogous amide character of the squarate adduct, as has been
previously observed in Scheme 4 (see the SI for details) [28]. The
product 12 owns excellent water solubility and immediately was
used for coupling purposes.
The degree of incorporation of the oligosaccharides on protein
backbone was determined by MALDI-TOF MS using sinapinic
acid (SA) as the matrix. The expected glycoconjugate average
MW of PG-CRM197 and PG-BSA were 60277.7 Da and 76503.4
Da, respectively (Figure 2). On the basis of the MW of the
hapten, the ratio hapten/CRM197 was 3.0:1 (conjugation
efficiency, 18%), the ratio hapten/BSA was 13.2:1 (conjugation
efficiency, 73%) as shown in Table 1, similar to those published
for the coupling of oligosaccharides to CRM197 or BSA [19, 27,
29]. This corresponds to the incorporation of 13.2 ligands to BSA
and 3 ligands to CRM197 with a ~18 fold molar excess of
activated oligosaccharides. The non-integer number refers to the
average degree of hapten substitution. This results could be
explained by glycoconjugates of this type are heterogeneous with
respect to the precise number of ligands attached to protein and
with regard to the specific lysine residues that are substituted.
Next, the conjugate containing PG-tb1 epitope was used to
evaluate immunogenicity by vaccination of mice.
Table 1
The conjugation of PG-tb1 with proteins.
Conjugate
Equiv haptena/
carrier protein
17:1
Number of
Conjugation
efficiency [%]
conjugated haptensb
13
14
3.0
13.2
18
73
18:1
a Hapten is defined as triglycosyl phenol with squaric acid linker
b mmol hapten/mmol protein determined by MALDI-TOF MS
Scheme 4. Synthesis of PG-tb1 squarate conjugate and conjugation to
CRM197 or BSA. Reagents and conditions: (a) squaric acid diethyl esters,
pH 8.0; (b) CRM197/BSA, pH 9.0.
2.1.3 Synthesis and analysis of PG–protein conjugate
Coupling of half ester 12 to CRM197 was performed over 72 h
in borate buffer (pH = 9.0) at ambient temperature (to form ∼3.0
mM solution with respect to the antigen; antigen/carrier= 17:1).
Dialysis against deionized water followed by lyophilization to
afford PG-CRM197 13 as white solid for use as a vaccine. In the
same way, half ester 12 was conjugated to BSA following the
protocol described above (antigen/carrier = 18:1) in 3.0 mL
borate buffer at ambient temperature (∼2.0 mM solution with
respect to hapten) affording the corresponding conjugate PG-
BSA 14. The molar ratio of activated half ester 12 to protein
and observed hapten incorporation were tabulated in Table 1.
Figure 2. MALDI-TOF-MS profiles of CRM197 (A1), PG-CRM197 (A2),
BSA (B1), PG-BSA (B2).
2.2. Biological evaluation
The immunogenicity of the vaccine was investigated using
seven groups of 10 NIH female mice which were administered
subcutaneously over the lower abdomen at intervals of two
weeks employing three doses (0.5 µg, 2.0 µg, 5.0 µg of PG-tb1 in
100 µL of formulation per mouse) without any adjuvant, and
control group was vaccinated with sterile saline. Following
completion of the immunization regimen, enzyme-linked
immunosorbent assays (ELISA) were performed to determine the
IgG serum antibody titers achieved for each group of mice (Table
2 and Figure 3). To avoid possible cross-reactivity between sera
raised against the PG-CRM197 conjugate and CRM197, we used
PG-BSA conjugate to coat plates.
Figure 1. SDS-PAGE of PG-CRM197 conjugate. The 12%
polyacrylamide gel was stained with Coomasie brilliant blue. Lanes: M =
molecular weight markers; CRM197; PG-CRM197 (1.0 µg); PG-CRM197
(0.5 µg); PG-CRM197 (0.2 µg).

4
Tetrahedron
ACCEPTED MANUSCRIPT
Table 2. Antibody titers of mice vaccinated with PG-CRM197 conjugate.
titer vs PG-CRM197
max
dose/
0
µ
g
min.
2555
median
41432
3rd inj.
2nd inj.
3rd inj.
2nd inj.
3rd inj.
2nd inj.
3rd inj.
360489
611265
400354
360603
882938
825469
1583020
0.5
0.5
2.0
2.0
5.0
5.0
49960
93914
154527
335048
176418
451464
136728
326530
197022
434427
357764
768000
without any adjuvant, the conjugate induced high antigen-
specific IgG levels in serum. As expected, this conjugate vaccine
also showed good immunogenicity in rabbits and guinea pigs
(unpublished results). These results suggest that glycoconjugates
of the type described here should be useful tools for the
generation of high titer sera or monoclonal antibodies specific for
oligosaccharide epitopes, and most likely also other low
molecular weight haptens. On the other hand, oligosaccharides of
this limited size are attractive candidates for a vaccine, since they
can be produced economically by synthetic approaches. And
also, it should be noted that increaseing the number of PG
epitope on the conjugate and permitting their diverse spatial
arrangement still requires more detailed study.
4. Experimental section
4.1. Chemistry
Figure 3. Trisaccharide specific antibody titers following vaccination of
mice with glycoconjugate. Sera were collected after the 2nd (triangles)
and 3rd (circles) injections and assayed by ELISA on PG-BSA
conjugate-coated microtiter plates. Panels represent experiments with
different vaccine formulations: (A) 0.5 µg; (B) 2.0 µg; (C) 5.0 µg; (D)
saline. Data points are titers for individual mice. Horizontal lines in
panels represent median values.
All the materials were obtained from commercial suppliers,
and were used without further purification. Air and moisture
sensitive reactions were carried out under a steam of argon.
Solvents were purified according to standard procedures, if
required. CRM197 was kindly provided by Dr. X. Yu (Tianjin
Cansino Biotechnology Inc.). BSA was purchased from Sigma.
Thin-layer chromatography (TLC) was purchased from EMD Co.
Ltd. (German). The compounds were stained with 5% H2SO4 in
ethanol and detection with UV light was employed when
possible. Flash column chromatography was performed on silica
gel 300~400 mesh. NMR spectra were recorded on Bruker
AVANCE III (400 MHz) instruments. Chemical shifts (δ) were
reported in ppm downfield from TMS, the internal standard; J
values were given in Hertz. High resolution ESI mass spectra
were obtained at a hybrid IT-TOF mass spectrometer (Shimadzu
LCMS-IT-TOF, Kyoto, Japan). The molecular weights of
glycoconjugates were confirmed by Bruker ultrafleXtreme
MALDI-TOF/TOF mass spectrometer (Bruker Daltonics,
Bremen, Germany).
The results of our experiments from the systematic
immunizations are encouraging. Even in the absence of adjuvant,
the conjugate elicited specific IgG antibodies. All mice given the
conjugate had increases in PG specific antibody titer after two
doses, which increased further after the third dose (Table 2).
Following the third immunization, significant antibody titers
frequently exceeding 0.8 million measured against the PG–BSA
conjugate were observed in the sera of mice vaccinated with PG–
CRM197 conjugate (Figure 3, panel C). Our results show that PG
conjugated to non-toxic mutant of diphtheria toxin is highly
immunogenic in mice and induce a robust immune response.
Mice given the PG-CRM197 vaccine with medium dose without
adjuvant had high IgG titers against the synthetic PG hapten after
2 injections (median ∼197 000), while the third injection resulted
in a further increase of titer by more than a factor of 2 (median
∼430000). By comparison, the IgG response after vaccination
with saline was also observed with a low median IgG titer of
41000 (Table 2). This result illustrated the nonspecific
recognition on account of the low number of PG epitope
displayed on the carrier protein. In contrast to antibody
responses, groups vaccinated with three doses showed significant
differences (P<0.05) in immunogenicity compared with saline
group after the second or third immunization.
4.1.1 Synthesis of compound 6. A mixture of compound donor 3
(804 mg, 1.60 mmol), acceptor 4 (500 mg, 1.06 mmol), and 4 Å
molecular sieve (500 mg) in dry 5 mL CH2Cl2 was stirred for 20
min at room temperature. The mixture was cooled down to -25
°C and N-iodosuccinimide (NIS, 710 mg, 3.16 mmol) and triflic
acid (27.8 µL, 0.31 mmol) were added. After stirring for 15 min
at -25 °C, the reaction mixture was warmed to room temperature
and stirred for another 30 min when TLC indicated the
completion of the reaction. The reaction mixture was neutralized
by Et3N (0.1 mL), filtered and concentrated under reduce
pressure. Chromatography (petroleum ether/ethyl acetate 5:1)
afforded the title compound 6.
3. Conclusions
A PG-tb1 hapten from the West Beijing strains of Mtb cell
wall has been synthesized and conjugated to CRM197 by applying
squaric acid chemistry for original neoglycoprotein, creating a
potent T-dependent conjugate vaccine. The conjugation
described herein is much simpler and less laborious, and afforded
conjugates in good yield. After administered systemically in mice
4.1.1.1 p iodophenyl (3-O-allyl-2-O-benzoyl-4-O-benzyl-α-L-
rhamnopyranosyl)‐ (1→3)-(4-O-benzyl-2-O-methyl)-α-
L‐ rhamnopyranoside (6). Obtained from compound donor 3 and
acceptor 4 following the procedure described above. Column
chromatography gave title compound as colorless syrup (752 mg,
83%). 1H NMR (400 MHz, CDCl3) δ 8.01 – 7.95 (m, 2H), 7.55 –

compound 9 and commercially available 6-chloro-1-hexyne 5
7.44 (m, 3H), 7.40 (t, J = 8.2 Hz, 2H), 7.32 – 7A.10C(mC,E10PHT),E6.D70 MANUSCRIPT
– 6.76 (m, 2H), 5.78 (ddd, J = 22.5, 10.8, 5.6 Hz, 1H), 5.60 (s,
1H), 5.41 (s, 1H), 5.18 – 5.09 (m, 2H), 4.97 (d, J = 10.4 Hz, 1H),
4.84 (dd, J = 21.2, 10.8 Hz, 2H), 4.57 (dd, J = 19.6, 10.8 Hz,
2H), 4.15 – 4.06 (m, 2H), 3.95 – 3.90 (m, 3H), 3.64 – 3.58 (m,
2H), 3.49 – 3.42 (m, 5H), 1.32 (d, J = 6.0 Hz, 3H), 1.17 (d, J =
6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 165.63, 156.27,
138.55, 138.49, 138.00, 134.80, 133.26, 130.11, 129.98,
128.57, 128.51, 128.48, 128.35, 128.10, 127.92, 127.88,
118.70, 117.13, 99.93, 94.88, 84.89, 80.17, 80.05, 80.01,
78.86, 77.78, 75.66, 75.54, 70.70, 69.92, 69.09, 68.60, 59.05,
18.49, 18.09. The 1H and 13C NMR spectra were consistent with
those reported [25].
following
the
procedure
described
above.
Column
chromatography gave title compound as yellow syrup (435 mg,
1
88%). H NMR (400 MHz, CDCl3) δ 7.30 – 7.18 (m, 20H), 6.90
(d, J = 8.8 Hz, 2H), 5.44 (s, 1H), 5.21– 5.09 (m, 3H), 4.78 (d, J =
11.6 Hz, 1H), 4.60 – 4.47 (m, 3H), 4.20 – 4.12 (m, 2H), 4.00 (dd,
J= 9.4, 2.8 Hz, 1H), 3.87 (dq, J = 15.2, 6.4 Hz, 1H), 3.78 – 3.40
(m, 17H), 3.25 (s, 3H), 3.13 (s, 1H), 2.37 (t, J = 6.8, 2H), 1.88
(dt, J =14.8, 6.4, 2H), 1.66 (dt, J = 14.8, 7.2, 2H), 1.26 (d, J = 6.4
Hz, 3H), 1.15 (d, J = 6.0 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 155.89, 139.26, 138.57, 138.48,
132.99, 128.53, 128.34, 128.30, 127.73, 127.61, 127.51,
127.40, 127.28, 127.23, 117.61, 116.30, 99.68, 99.51, 94.91,
88.28, 80.84, 80.53, 80.30, 80.04, 79.92, 79.56, 79.32, 79.06,
77.98, 75.07, 74.82, 71.55, 69.05, 68.90, 66.46, 61.81, 59.21,
59.07, 58.08, 53.53, 44.68, 31.74, 26.06, 18.81, 18.34, 18.03,
16.64. HRMS m/z calcd for C55H69ClO13Na [M+Na]+: 995.4319,
found: 995.4324.
4.1.2 Synthesis of compound 8. A mixture of compound donor 2
(347 mg, 0.94 mmol), acceptor 7 (500 mg, 0.63 mmol), and 4 Å
molecular sieve (500 mg) in CH2Cl2 (8 mL) cooled down to -25
°C and N-iodosuccinimide (NIS, 418 mg, 1.86 mmol) and triflic
acid (22 µL, 0.25 mmol) were added. After stirring for 15 min at
-25 °C, the reaction mixture was warmed to room temperature
and stirred for another 10 min when TLC indicated the
completion of the reaction. The reaction mixture was quenched
by Et3N, filtered, and concentrated under reduce pressure.
Chromatography (petroleum ether/ethyl acetate 3:1) afforded the
title compound 8.
4.1.4 Synthesis of compound 11. To a solution of compound 10
(435 mg, 0.45 mmol) in dry DMF (5 mL) were added NaN3 (286
mg, 4.4 mmol) and TBAI (163 mg, 0.44 mmol). After stirring
overnight at 60 °C, the reaction mixture was diluted with EtOAc
(15 mL), washed with brine, dried over MgSO4, filtered and
concentrated under reduce pressure. Chromatography (petroleum
ether/ethyl acetate 1:4) afforded the title compound 11.
4.1.2.1
p iodophenyl
(3,4 di O acetyl-
4.1.4.1
(6-(4‐ hydroxyphenyl)-1-azido-5-hexyne)-yl
2 O methyl α L fucopyranosyl)‐ (1→3)‐ (2,4‐ di‐ O‐ benz
yl‐ α‐ L‐ rhamnopyranosyl)‐ (1→3)‐ (4‐ O‐ benzyl‐ 2‐ O‐ me
thyl)‐ α‐ L‐ rhamnopyranoside (8). Obtained from compound
donor 2 and acceptor 7 following the procedure described above.
Column chromatography gave title compound as yellow syrup
(532 mg, 81%). 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 8.8 Hz,
2H), 7.30 – 7.19 (m, 16 H), 6.78 (d, J = 9.2 Hz, 2H), 5.42 (d, J =
1.4 Hz, 1H), 5.25 – 5.18 (m, 4H), 5.08 (d, J = 11.6 Hz, 1H), 4.97
(d, J = 2.4Hz, 1H), 4.76 (d, J = 11.6 Hz, 1H), 4.58 – 4.53 (m, 3H),
4.19 – 4.13 (m, 2H), 4.03 (dd, J = 9.6, 3.2 Hz, 1H), 3.92 – 3.85
(2,3,4‐ tri‐ O‐ methyl‐ α‐ L‐ fucopyranosyl)‐ (1→3)‐ (2,4‐ di
‐ O‐ benzyl‐ α‐ L‐ rhamnopyranosyl)‐ (1→3)‐ (4‐ O‐ benzyl‐
2‐ O‐ methyl)‐ α‐ L‐ rhamnopyranoside (11). Obtained from
compound 10 following the procedure described above. Column
chromatography gave title compound as yellow syrup (357 mg,
1
82%). H NMR (400 MHz, CDCl3) δ 7.33 – 7.17 (m, 19H), 6.92
(t, J = 8.8 Hz, 2H), 5.50 – 5.39 (m, 1H), 5.26 – 5.09 (m, 3H),
4.80 (d, J = 11.6 Hz, 1H), 4.66 – 4.47 (m, 3H), 4.19 (dd, J = 12.2,
8.2 Hz, 2H), 4.07 – 4.00 (m, 1H), 3.89 (dq, J = 12.4, 6.0 Hz, 1H),
3.76 (s, 1H), 3.72 – 3.42 (m, 16H), 3.32 – 3.22 (m, 5H), 3.15 (s,
1H), 2.39 (t, J = 6.8 Hz, 2H), 1.71 (dt, J =15.2, 6.0, 2H), 1.61 (dt,
J = 14.8, 6.4, 2H), 1.26 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.4 Hz,
3H), 0.90 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
155.91, 139.27, 138.59, 138.48, 133.00, 128.54, 128.35,
128.31, 127.74, 127.62, 127.52, 127.41, 127.27, 127.24,
117.67, 116.30, 99.69, 99.52, 94.90, 88.25, 80.88, 80.52,
80.31, 80.05, 79.91, 79.58, 79.31, 79.08, 77.98, 75.08, 74.84,
71.55, 69.06, 68.90, 66.46, 61.82, 59.21, 59.08, 58.09, 53.54,
51.16, 28.15, 25.97, 19.10, 18.35, 18.04, 16.65. HRMS m/z
(m, 1H), 3.74– 3.57 (m, 5H), 3.50 – 3.44 (m, 5H), 3.18 (s, 3H),
2.05 (s, 3H), 1.95 (s, 3H), 1.28 (d, J = 6.4 Hz, 3H), 1.15 (d, J =
6.4 Hz, 3H), 0.68 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 170.63, 170.15, 156.29, 139.24, 138.49, 138.29,
137.89, 128.66, 128.60, 128.30, 127.41, 127.24, 118.71,
99.64, 99.35, 94.80, 84.92, 80.20, 79.94, 79.89, 79.73, 78.15,
75.78, 75.18, 74.85, 71.57, 71.34, 70.28, 69.06, 68.98, 64.57,
59.33, 58.96, 21.10, 20.83, 18.31, 18.06, 16.05. The 1H and 13
NMR spectra were consistent with those reported [25].
C
calcd for C55H69N3O13Na [M
+
Na]+: 1002.4728, found:
4.1.3 Synthesis of compound 10. A stock solution of catalyst was
prepared by mixing PPh3 (9.9 mg, 38 µmol), CuI (14.4 mg, 76
µmol) and PdCl2(PPh3)2 (26.4 mg, 38 µmol) in freshly distilled
Et3N (18 mL) and stirring this solution at 40 °C for 15 min. Then,
12.2 mL of this stock solution was added to a solution of 6-
chloro-1-hexyne 5 (0.6 mL, 5.1 mmol) and trisaccharide 9 (500
mg, 0.51 mmol) in freshly distilled Et3N (6.0 mL). The reaction
was then stirred at 40 °C for 2 h. The reaction was diluted with
1002.4723.
4.1.5 Synthesis of compound 1. Palladium hydroxide on carbon
(10%, wetted with ca. 50% water) was added to a solution of
compound 11 (150 mg, 0.15 mmol) in EtOAc (0.8 mL) and
EtOH (0.2 mL) under nitrogen. The mixture was stirred at room
temperature under atmospheric pressure H2 (0.4 MPa) for 24 h.
Then, the catalyst was filtered through Celite and the solvent was
removed under reduced pressure. Crude was purified by column
chromatography (CH2Cl2/MeOH 1:1) to give compound 1.
EtOAc (50 mL), washed with aq. HCl (2 M, 2×30 mL) and brine
(30 mL), and dried (Na2SO4). The solvent was removed under
reduced pressure. Chromatography (petroleum ether/ethyl acetate
1:2) afforded the title compound 10.
4.1.5.1
(6-(4‐ hydroxyphenyl)-1-hexylamine)-yl
(2,3,4‐ tri‐ O‐ methyl‐ α‐ L‐ fucopyranosyl)‐ (1→3)‐ α‐ L-
rhamnopyranosyl‐ (1→3)‐ (2‐ O‐ methyl)‐ α‐ L‐ rhamnopyran
oside (1). Obtained from compound 11 following the procedure
described above. Column chromatography gave title compound
as white solid (82 mg, 78%).
4.1.3.1
(6-(4‐ hydroxyphenyl)-1-chloro-5-hexyne)-yl
(2,3,4‐ tri‐ O‐ methyl‐ α‐ L‐ fucopyranosyl)‐ (1→3)‐ (2,4‐ di
‐ O‐ benzyl‐ α‐ L‐ rhamnopyranosyl)‐ (1→3)‐ (4‐ O‐ benzyl-
2‐ O‐ methyl)‐ α‐ L‐ rhamnopyranoside (10). Obtained from

6
1H NMR (400 MHz, D2O) δ 7.32 –7.23 (m, 2H), 7.14 –7.07
4.2. Biological evaluation
ACCEPTED MANUSCRIPT
(d, m, 2H), 5.72 (s, 1H), 5.42 (d, J = 3.6 Hz, 1H), 5.10 (s, 1H),
4.21 – 4.06 (m, 3H), 3.96 – 3.79 (m, 5H), 3.74 (m, 1H), 3.69 –
3.47 (m, 15H), 2.98 (t, J = 7.4 Hz, 2H), 2.62 (t, J = 7.4 Hz,
2H), 1.71 – 1.55 (m, 4H), 1.46 –1.33 (m, 7H), 1.30 (d, J = 6.4
Hz, 3H), 1.24 (d, J = 6.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 154.68, 136.36, 129.46, 116.42, 102.21, 100.58,
95.31, 82.40, 80.79, 80.18, 79.20, 79.04, 78.86, 77.35, 73.57,
72.26, 71.66, 71.22, 69.32, 69.10, 67.53, 61.98, 60.14, 59.00,
57.75, 40.16, 34.99, 31.36, 28.66, 27.69, 26.55, 18.01, 17.95,
16.80. HRMS m/z calcd for C34H57NO13Na [M + Na]+: 710.3728,
found: 710.3722.
Female 3-week-old (NIH) mice free of common viral
pathogens were acquired from Beijing Vital River Laboratory
Animal Technology Co., Ltd. and were bred at 22 ± 0.5 °C on a
12/12 h light-dark cycle from 7 a.m. to 7 p.m.. All procedures
and protocols were approved by the Institutional Animal Care
and Use Committee. Vaccine doses were formulated to contain
0.5 µg, 2.0 µg, 5.0 µg of PG-tb1 in 100 µL of formulations. Mice
were immunized two or three times at 2 week intervals with
vaccine formulation (100 µL) administered by subcutaneous
injections. Enzyme Linked Immunosorbent Assay (ELISA)-
ELISAs were conducted on serum samples which were collected
after the 2nd and 3rd injections. We coated ELISA plates with
PG-BSA (5 µg/mL) in carbonate buffer (pH 9.6). After washing
with PBS containing 0.1% Tween (PBST), wells were filled with
100 µL/well of serial dilutions of sera (starting from 10−3). BSA
(1%) in PBST was used for dilutions to prevent non-specific
binding. Plates were sealed and incubated for 1 h at room
temperature. After washing with PBST, a reporter antibody (anti-
mouse IgG, HRP conjugate) in 1% BSA PBST, at a dilution of
1/10000 was applied and plates were incubated for 1 h at room
temperature. Plates were washed again with PBST and color
developed with a HRP substrate system for 15 min. The reaction
was stopped with 2 M sulphuric acid and absorbance was
measured at 450 nm in an ELISA plate reader.
4.1.6 Synthesis of compound 12. Amine 1 (50 mg, 0.073 mmol)
was dissolved in a 1:1 solution of ethanol/water (2 mL), and 3,4-
diethoxy-3-cyclobutene-1, 2-dione (55 µL, 0.37 mmol,
5
equivalent) was added. A saturated solution of sodium carbonate
was then added in 2 µL aliquots, allowing the reaction to stir for
5 min between additions. The addition was continued until TLC
analysis indicated the free amine had been completely converted
into a faster moving product. The reaction mixture was then
concentrated and purified by
a hydrophobic interaction
chromatography Sephadex LH-20 column using distilled water as
eluent. The product was lyophilized to afford the PG-tb1 squarate
monomethyl ester 12.
4.1.6.1
dioxocyclobut-1-en))-yl
((6-(4 hydroxyphenyl)-1-aminohexyl)-(2-ethoxy-3,4-
Acknowledgments
(2,3,4 tri O methyl α‐ L‐ fucopyranosyl)‐ (1→3)‐ α‐ L-
rhamnopyranosyl‐ (1→3)‐ (2‐ O‐ methyl))‐ α‐ L‐ rhamnopyra
noside (12). Obtained from compound 1 following the procedure
described above. The product was purified by a hydrophobic
interaction chromatography Sephadex LH-20 column and was
lyophilized to gave title compound 12 as white solid (54 mg,
92%). 1H NMR (600 MHz, D2O) δ 6.87 (d, J =16.0 Hz, 4H), 5.43
(s, 1H), 5.35 (s, 1H), 5.00 (s, 1H), 4.54 (d, J = 20.6 Hz, 2H), 4.10
(d, J = 14.0 Hz, 2H), 3.93 (s, 1H), 3.80 (d, J = 20.4 Hz, 3H), 3.68
(s, 2H), 3.63 – 3.33 (m, 18H), 3.22 (s, 1H), 2.32 (s, 2H), 1.55 –
0.91 (m, 22H).13C NMR (100 MHz, D2O) δ 188.96, 188.52,
182.89, 182.66, 176.91, 176.50, 173.12, 172.81, 154.11, 136.36,
129.20, 116.63, 102.45, 97.86, 94.82, 79.92, 78.48, 78.10, 76.81,
71.46, 71.22, 70.37, 69.96, 69.41, 66.87, 61.45, 58.52, 57.13,
56.78, 44.30, 34.62, 31.08, 30.08, 28.35, 25.92, 17.24, 15.68,
15.47, 15.39. HRMS m/z calcd for C40H60NO16 [M-H]-: 810.3912,
found: 810.3906.
This work was financially supported by the Natural Science
Foundation of China (21502139, 21402140, 31501544),
International Science & Technology Cooperation Program of
China (2013DFA31160), the Foundation (No. 2016IM102) of
Key Laboratory of Industrial Fermentation Microbiology of
Ministry of Education and Tianjin Key Lab of Industrial
Microbiology (Tianjin University of Science & Technology),
Laboratory Innovation Foundation of Undergraduate (No.
1604A306) (Tianjin University of Science & Technology),
Science and Technology Commissioner Foundation of Tianjin
(15JCTPJC56200), National Science and Technology Major
Project (2015ZX09J15105-002-003). The authors are thankful to
the Research Centre of Modern Analytical Technology, Tianjin
University of Science and Technology for NMR measurements
and MALDI-TOF analysis.
References
4.1.7 General procedure for the synthesis and characterization of
glycoconjugates
1. Ling, R.; Yoshida, M.; Mariano, P. S. J. Org. Chem. 1996, 61,
4439–4449. Global tuberculosis control: WHO report 2016, World
CRM197 (30 mg, 0.52 µmol) was dissolved in borate buffer
(Na2B4O7 0.07 M, KHCO3 0.035 M, pH 9.0). When all protein
had dissolved, compound 12 (8 mg, 9 µmol) was added and the
solution was slowly stirred at room temperature for 72 h.
Conjugation of BSA (20 mg, 0.33 µmol) and compound 12 (5 mg,
6 µmol) followed the same procedure above. After that, to
remove the low molecular mass material, the reaction mixture
was further purified by ultrafiltration using centrifugal filter
devices (30K Amicon Ultra, Millipore) against deionized water
(10 mL) where the final volume after concentration was no
greater than 1 mL (centrifugation at 4 ℃, 3000 × g, 7 times, ~ 30
min each time). The final retentate was lyophilized to afford
glycoconjugate as white solid (31.4 mg PG-CRM197 and 23 mg
PG-BSA). The characterization of the glycoprotein was carried
out by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). Hapten to protein ratio was determined by
MALDI-TOF-MS in positive linear ion mode using sinapinic
acid (SA) as matrix.
Health
Organization,
link:
http://www.who.int/tb/publications/global_
report/en/index.html, 2016.
2. C. Aagaard, T. Hoang, J. Dietrich, P. J. Cardona, A. Izzo, G.
Dolganov, G. K. Schoolnik, J. P. Cassidy, R. Billeskov, P.
Andersen, Nat. Med. 17 (2011) 189–194.
3. Brosch, S. V. Gordon, T. Garnier, K. Eiglmeier, W. Frigui, P.
Valenti, S. D. Santos, S. Duthoy, C. Lacroix, C. Garcia-Pelayo, J.
K. Inwald, P. Golby, J. N. Garcia, R. G. Hewinson, M. A. Behr,
M. A. Quail, C. Churcher, B. G. Barrell, J. Parkhill, S. T. Cole,
Proc. Natl. Acad. Sci. USA. 104 (2007) 5596–5601.
4. D. Sinsimer, G. Huet, C. Manca, L. Tsenova, M. S. Kool, N.
Kurepina, B. Kana, B. Mathema, S. A. E. Marras, B. N.
Kreiswirth, C. Guilhot, G. Kaplan. Infect. Immun. 76 (2008)
3027–3036.
5. M. B. Reed, P. Domenech, C. Manca, H. Su, A. K. Barczak, B. N.
Kreiswirth, G. Kaplan, C. E. Barry III. Nature. 431 (2004) 84–87.
6. Lyle, F. R. U.S. Patent 5 973 257, 1985; Chem. Abstr. 1985, 65,
2870. K.J. De, P. E. de Haas, A. H. de Ru, P. A. van Veelen, S. D.
Van. Mol. Cell. Proteomics. 13 (2014) 2632–2645.
7. C. Garzelli, N. Lari, L. Rindi. Tuberculosis. 97 (2016) 147–153.

8. J. Bordóna, M.W. Plankey, M. Young, R. M. Greenblatt, M.
Nissum, P. Costantino, R. Rappuoli, G. Spraggon. Proc. Natl.
Acad. Sci. USA. 109 (2012) 5229–5234.
22. R. Adamo, M. Tontini, G. Brogioni, M. R. Romano, G. Costantini,
E. Danieli, D. Proietti, F. Berti, P. Costantino. J. Carbohydr.
Chem. 30 (2011) 249–280.
ACCEPTED MANUSCRIPT
C.Villacres, A. L. French, J. Zhang, G. Brock, S. Appana, B.
Herold, H. Durkin, J. E. Golub, R. Fernandez-Botran, Cytokine.
56 (2011) 325–331.
9. K.W. Jo, Y. Hong, Y.J. Jung, B. Yoo, C.K. Lee, Y. G. Kim, S. K.
Yang, J. S. Byeon, K. J. Kim, B. D. Ye, S. D. Lee, W. S. Kim, D.
S. Kim,T. S. Shim, Resp. Med. 107 (2013) 1797–1802.
10. L. Tsenova, E. Ellison, R. Harbacheuski, A.L.Moreira, N.
Kurepina, M. B. Reed, B. Mathema, C.E. Barry III, and G.
Kaplan. J. Infect. Dis. 192 (2005) 98–106.
11. D. Aguilar, M. Hanekom, D. Mata, N. C. Gey van Pittius, P. D.
van Helden, R. M. Warren, R. Hernandez-Pando. Tuberculosis. 90
(2010) 319–325.
12. I. Tomotada, G. Louis, A. Kentaro, N. Noriko, C. Luz, C. Jorge, S.
Patricia, W. Takayuki, A. T. Carmen, S. Marie-Anne, A. J. M.
David, and H. G. Robert. PLOS One. 7 (2012) e49651.
13. Z. H. Huang, L. Shi, J. W. Ma, Z. Y. Sun, H. Cai, Y. X. Chen, Y.
F. Zhao, Y. M. Li, J. Am. Chem. Soc. 134 (2012) 8730–8733.
14. I. Tavernaro, S. Hartmann, L. Sommer, H. Hausmann, C. Rohner,
M. Ruehl, A. Hoffmann-Roedere, S. Schlecht, Org. Biomol.
Chem. 13 (2014) 81–97.
23. G.H. Veeneman, S.H. Van Leeuwen，H. Zuurmond，J.H. Van
Boom. J. Carbohydr. Chem. 9 (1990) 783-796.
24. L.F.Tietze, C. Schröter, S. Gabius, U. Brinck, A. Goerlach-Graw,
H.J. Gabius. Bioconjugate Chem. 2 (1991) 148–153.
25. S. Barroso, R. Castelli, M. P. Baggelaar, D. Geerdink, B. Horst,
E. Casas-Arce, H. S. Overkleeft, G. A. van der Marel, J. D. C.
Code, A. J. Minnaard. Angew. Chem. Int. Ed. 51 (2012) 11774–
11777.
26. S.C. Ranade, A. V. Demchenko. J. Carbohydr. Chem. 32 (2013)
1-43.
27. P. Kamath, P. Diedrich, O. Hindsgaul, Glycoconjugate J. 13
(1996) 315–319.
28. L. Tietze, M. Arlt, M. Beller, K. H. Glusenkamp, E. Jahde, M. F.
Rawjewsky. Chem Ber. 124 (1991) 1215–1221.
29. M. Nitz, D. R. Bundle, J. Org. Chem. 66 (2001) 8411–8423.
15. P. Farkas, J. Korcová, J. Kronek, S. Bystrický, Eur. J. Med. Chem.
45 (2010) 795–799.
16. N. Simonney, P. Chavanet, C. Perronne, M. Leportier, F. Revol, J.
L. Herrmann, P. H. Lagrange. Tuberculosis. 87 (2007) 109 –122.
17. A. Weintraub. Carbohyd. Res. 338 (2003) 2539–2547.
18. D. Gao, J. Zeng, X. Wang, Y. Liu, W. Li, W. Li, Y. Hu, N. Gao,
Y. Diao, Z. Wang, W. Jiang, J. Chen, G. Jin. Eur. J. Med. Chem.
120 (2016) 111–120.
Appendix A. Supplementary Data
Supplementary data related to this article can be found at
19. S. Crotti, H. Zhai, J. Zhou, M. Allan, D. Proietti, W. Pansegrau, Q.
Y. Hu, F. Berti, R. Adamo. Chem. Bio. Chem. 15 (2014) 836–843.
20. G. Giannini, R. Rappuoli, G. Ratti. Nucleic Acids Res. 12 (1984),
4063–4969.
Click here to remove instruction text...
21. E. Malito, B. Bursulaya, C. Chen, P. Lo Surdo, M. Picchianti, E.
Balducci, M. Biancucci, A. Brock, F. Berti, M. J. Bottomley, M.

ACCEPTED MANUSCRIPT
An original neoglycoconjugate of PG-CRM197 has been synthesized.
The application of squaric acid chemistry afforded conjugates in good yield.
The conjugate without any adjuvant induced high antigen-specific IgG levels in mice.