Synthesis and Evaluation of Glycoconjugates Comprising N-Acyl-Modified Thomsen-Friedenreich Antigens as Anticancer Vaccines

Article abstract of DOI:10.1002/cmdc.201600094

Thomsen-Friedenreich (TF) antigen is an important tumor-associated carbohydrate antigen. Its low immunogenicity, however, limits its application in the development of anticancer vaccines. To solve this problem, several N-acyl-modified TF derivatives were synthesized and conjugated with carrier protein CRM197 (a mutated diphtheria toxoid cross-reactive material). The immunological results in BALB/c mice demonstrated that these modified TF antigen conjugates could stimulate the production of higher titers of IgG antibodies that cross-reacted with native TF antigen. These glycoconjugates showed strong lymphocyte proliferative response, suggesting that they can induce cellular immunity. Furthermore, the elicited antisera reacted strongly with TF-positive tumor cells (4T1). In particular, the N-monofluoroacetyl-modified TF conjugate 4-CRM197 showed the strongest complement-dependent cytotoxicity effect against 4T1 cells, implying the potential of this glycoconjugate as an anticancer vaccine. Recruiting the IgG army: Several N-modified TF antigen derivatives were synthesized and conjugated with the carrier protein CRM197. These glycoconjugates significantly increased the titers of antibodies than can recognize the native TF antigen and kill tumor cells, thus having the potential for use as carbohydrate-based anticancer vaccines.

Full text of DOI:10.1002/cmdc.201600094

DOI: 10.1002/cmdc.201600094
Full Papers
Synthesis and Evaluation of Glycoconjugates Comprising
N-Acyl-Modified Thomsen–Friedenreich Antigens as
Anticancer Vaccines
Shuang Sun+, Xiu-Jing Zheng+, Chang-Xin Huo, Chengcheng Song, Qin Li,* and Xin-
Shan Ye*[a]
Thomsen–Friedenreich (TF) antigen is an important tumor-as-
sociated carbohydrate antigen. Its low immunogenicity, how-
ever, limits its application in the development of anticancer
vaccines. To solve this problem, several N-acyl-modified TF de-
rivatives were synthesized and conjugated with carrier protein
CRM197 (a mutated diphtheria toxoid cross-reactive material).
The immunological results in BALB/c mice demonstrated that
these modified TF antigen conjugates could stimulate the pro-
duction of higher titers of IgG antibodies that cross-reacted
with native TF antigen. These glycoconjugates showed strong
lymphocyte proliferative response, suggesting that they can
induce cellular immunity. Furthermore, the elicited antisera re-
acted strongly with TF-positive tumor cells (4T1). In particular,
the N-monofluoroacetyl-modified TF conjugate 4-CRM197
showed the strongest complement-dependent cytotoxicity
effect against 4T1 cells, implying the potential of this glycocon-
jugate as an anticancer vaccine.
Introduction
Tumor-associated carbohydrate antigens (TACAs), aberrantly
expressed on the surface of cancer cells,[1] are correlated with
various stages of cancer development,[2] and are regarded as
important targets for the development of therapeutic anticanc-
er vaccines and cancer immunotherapies.[3] However, carbohy-
drate antigens are T-cell-independent and possess poor immu-
nogenicity; therefore, TACAs cannot induce T-cell-mediated im-
munity, which is critical for cancer immunotherapy.[4] Moreover,
because of the low level of TACA expression in normal tissues
and their high structural similarity to normal antigens, natural
TACAs were perceived as self-antigens and tolerated by the pa-
tient’s immune system.[5] One of the strategies to overcome
these problems is to improve the immune efficiency of TACAs
by conjugating them directly with suitable immunogenic carri-
ers such as proteins, peptides, and lipopeptides.[6,7] Another
strategy to enhance the immunogenicity of carbohydrate anti-
gens relies on the use of unnatural TACA derivatives, which
may be more immunogenic and generate antibodies cross-re-
active to natural TACAs. In 1986, Jennings’ research group re-
ported that modification of carbohydrate antigen structures
could improve the immunogenicity of vaccines.[8] Since then,
modification strategies such as the use of lactone or lactam
structures[9] and substitution of unnatural glycosidic linkages[10]
has been applied to many TACAs, proving the great potential
of this protocol.
Thomsen–Friedenreich (TF) antigen (b-d-Galp-(1!3)-a-d-Gal-
NAcp, 1) is a TACA that was initially reported as an oncofetal
blood-group-related disaccharide that is normally linked to
a serine or threonine residue of mucins. TF antigen is an im-
portant cancer-related marker expressed on breast and pros-
tate cancer cells.[11] The TF–KLH (keyhole limpet hemocyanin)
conjugate was evaluated as the vaccine candidate for breast,[12]
ovarian,[13] colorectal,[14] or prostate cancers.[15] As mentioned
above, to enhance the immunogenicity of TF antigen, many
formulations or modifications of TF antigen have been investi-
gated, including a clustered format,[16] a multimeric con-
struct,[17] a C-linked analogue,[18] and a fluorine-substituted ana-
logue.[19,20] However, the immunological results of all these
constructs are not satisfactory.
The fluorinated modifications may lead to high immunoge-
nicity due to the absence of fluorine in most organisms, given
the similar atomic radius and lipophilicity of the fluorine rela-
tive to hydrogen.[19,21] Recently, our group revealed that the
suitable modifications of Tn, STn, and GM3 antigens, including
fluorine-containing acyl group modifications, could significant-
ly improve immunogenicity; the resulting mouse antisera were
able to recognize tumor cells that express the native anti-
gens.[21] To explore the enhancement of TF-antigen-based vac-
cine immunogenicity without the need for a glyco-engineering
technique, several N-acyl-modified (propionyl, fluoroacetyl, di-
fluoroacetyl, and trifluoroacetyl) TF saccharide derivatives were
designed and synthesized (Figure 1) in this study, and the
immune efficacy of their corresponding CRM197 (a mutated
diphtheria toxoid cross-reactive material) conjugates were eval-
uated. An allyl group was introduced as the linker. Ozonolysis
[a] S. Sun,+ X.-J. Zheng,+ C.-X. Huo, C. Song, Prof. Q. Li, Prof. X.-S. Ye
State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences, Peking University,
Xue Yuan Road No. 38, Beijing 100191 (China)
E-mail: xinshan@bjmu.edu.cn
liqin@bjmu.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600094.
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Scheme 2. Synthesis of glycoconjugates. Reagents and conditions: a) O3,
MeOH, À728C; b) PBS, pH 7.4, NaBH3CN, CRM197 or BSA, RT, 24 h.
Figure 1. Natural TF antigen 1 and modified TF derivatives 2–6.
presence of sodium cyanoborohydride. The loading level of
these carbohydrate antigens, as determined by MALDI-TOF
mass spectrometry, was on average 4–11 carbohydrate mole-
cules per CRM197/BSA molecule (Table S1, Supporting Informa-
tion).
of the double bond in the allyl group provides an aldehyde
functionality that facilitates the conjugation of TF saccharides
to carrier proteins via reductive amination.
Recently, the Wong research group found that C34 (Fig-
ure S1), a chemically synthesized glycolipid, could serve as
a promising adjuvant for the Globo H vaccine, eliciting specific
antibodies.[22] Therefore, the immune efficacy of unmodified
and modified TF-CRM197 conjugates was evaluated by using
C34 as the adjuvant. Thus, groups of six female BALB/c mice
were vaccinated intramuscularly four times at both hind legs
at biweekly intervals with TF-CRM197 glycoconjugates and
C34. To test antibodies recognizing the native antigen, the an-
tibody titers of the pooled sera of each group or the serum an-
tibody of each mouse were determined by ELISA, with plates
coated with the glycoconjugate 2-BSA. To test antibodies
against the modified carbohydrate antigens, plates coated by
the corresponding modified TF-BSA conjugates were used in-
stead. As shown in Figure 2a and Table S2, for IgG titers of in-
dividual mouse against the native antigen 2-BSA after the 4th
vaccination, the median antibody level of 3-CRM197, 4-
CRM197, 5-CRM197, and 6-CRM197 exhibited a clear enhance-
ment relative to that of 2-CRM197 against 2-BSA. The IgG titers
of pooled sera were also analyzed (Figure 2b and Table S3),
showing similar results. All antibody titers elicited by the modi-
fied TF-CRM197 conjugates against the corresponding modi-
fied TF-BSA were higher than that elicited by 2-CRM197
against 2-BSA. The antibody titers elicited by 3-CRM197, 4-
CRM197, and 6-CRM197 against their corresponding modified
Results and Discussion
The synthesis of TF saccharide 2 and its derivatives 3–6 is
shown in Scheme 1. The stereoselective glycosyl coupling reac-
tion of galactosyl bromide 7[10d] and benzylidene-protected ac-
ceptor 8[10d,21a] promoted by silver(I) trifluoromethanesulfonate
provided the b-linked disaccharide 9 smoothly. The subsequent
ZemplØn deacetylation and removal of 4,6-O-benzylidene
acetal afforded the unmodified TF antigen 2 in 93% isolated
yield. After cleavage of the N-acetyl group in compound 2
with 2n aqueous sodium hydroxide, the obtained free amino
functionality was acylated by the corresponding fluorinated
carboxylic methyl esters or propionic anhydride, yielding the
desired TF saccharide analogues 3–6.
CRM197, which was approved by the USFDA as vaccine car-
rier protein, was selected as the carrier protein because of its
well-proven clinical validation. Bovine serum albumin (BSA)
conjugates were also prepared as capture reagents for ELISAs
carried out in the immunological studies. The glycoconjugates
were prepared by a two-step procedure (Scheme 2). Thus, sac-
charides 2–6 were ozonized to generate the aldehyde inter-
mediates. Without any purification, the obtained aldehydes
were coupled with carrier proteins (CRM197 or BSA) via reduc-
tive amination in aqueous phosphate buffer (pH 7.4) in the
Scheme 1. Synthesis of TF saccharide and its analogues. Reagents and conditions: a) 4 Š MS, toluene/nitromethane (1:1), AgOTf, À158C to RT, 4 h, 70%;
b) 30% NaOMe/MeOH, MeOH, RT, 30 min; c) 60% AcOH, 608C, 30 min, 93% (two steps); d) 2n NaOH; e) methyl ester of the corresponding carboxylic acid,
MeOH, Et3N, reflux; 42% for 4, 95% for 5, 90% for 6; for the preparation of 3: NaHCO3, propionic anhydride, MeOH, 08C, 98%.
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respectively (e.g., for splenocytes from mice immunized with
4-CRM197, 4-CRM197 would be taken as the recall antigen) at
a final glycoconjugate concentration of 2 mgmLÀ1. As shown in
Figure 3, the optical densities of lymphocyte proliferation of all
Figure 3. The lymphocyte proliferative response after immunization. Pooled
splenocytes from each group were employed to detect the proliferation abil-
ity. Triplicate wells were arranged. Each bar represents each group’s stimula-
tion index (SI) by taking different glycoconjugate as recall antigen. SI was
calculated based on the following formula: SI=mean absorption of cells cul-
tured with different modified TF-CRM197 antigen divided by the mean ab-
sorption of cells cultured with 2-CRM197. a: Corresponding recall antigen ac-
cording to each group’s immunogen; e.g., for splenocytes from mice immu-
nized with 4-CRM197, 4-CRM197 would be taken as recall antigen.
Figure 2. a) Serum antibody titers against 2-BSA of individual mouse immu-
nized with 2-CRM197, 3-CRM197, 4-CRM197, 5-CRM197 and 6-CRM197 after
the 4th vaccination. Each data point represents the average titer of two de-
tections of an individual mouse, and each series of black lines represents
the median with interquartile range of a group of six mice. b) Pooled sera
IgG titers against 2-BSA (hollow bar) and against modified TF–BSA (dotted
bar) after the 4th vaccination with different CRM197 conjugates. The titers
were separately detected twice, and three parallel micropores were ar-
ranged. Each column represents the meanÆSD of two detections.
groups immunized with the modified TF-CRM197 were higher
than that of the group immunized with 2-CRM197. In particu-
lar, the mice immunized with 4-CRM197 showed the strongest
lymphocyte proliferative response. The data suggest that these
modified TF-CRM197 conjugates could induce cellular immuni-
ty, which should benefit tumor therapy.
To determine whether the mouse sera after vaccination can
recognize the native TF antigen presented on cancer cells, flow
cytometry assays were used to analyze the binding affinity of
the immune serum antibodies for breast mammary carcinoma
cells 4T1 expressing the native TF antigen.[27] The results dem-
onstrated that all the antisera elicited by 2-CRM197 or modi-
fied TF-CRM197 reacted strongly with the TF-positive tumor
cells (Figure 4). The mean fluorescence intensity (MFI) values of
antisera elicited by 3-CRM197, 4-CRM197, and 6-CRM197 were
107, 116, and 136, respectively, while the MFI value of antisera
elicited by 2-CRM197 was 86 (Table S4), indicating that anti-
bodies elicited by modified TF-CRM197 could bind 4T1 cells
stronger than that elicited by 2-CRM197. These data confirmed
the specificity of the antibody response to tumor cells.
TF-BSA after the fourth vaccination were almost fourfold
higher than that induced by 2-CRM197 against 2-BSA, which
indicated that the immunogenicity was increased by the modi-
fication strategy. The modified TF antigen conjugates pro-
duced high anti-modified-TF antibody titers with reasonable
cross-recognition efficiency, resulting in an increase in anti-TF
IgG antibodies. In this work, there were no detectable IgM an-
tibodies in the sera diluted at 1:100. From the viewpoint of
tumor therapy, IgG responses are more desirable because of
their properties such as immunological memory, affinity matu-
ration, and antibody-dependent cell-mediated cytotoxici-
ty.[5,23–25]
The lymphocyte proliferative response was used to evaluate
the antigen-specific cellular immune response.[26] The magni-
tude of the proliferative response was expressed as the stimu-
lation index (SI). Pooled splenocytes from each group mice
were stimulated with 2-CRM197 and the modified TF-CRM197
To investigate whether the modified TF-CRM197-provoked
immune response or antibodies are useful for cancer immuno-
therapy, we assessed the capacity of antisera derived from
mice vaccinated with TF or modified TF-CRM197 to mediate
the killing of tumor cells, by analyzing complement-dependent
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Figure 4. Serological IgG analysis results on 4T1 cells after the fourth immu-
nization by flow cytometry.
cytotoxicity (CDC). Cytotoxicity is shown with percent cell lysis
determined by lactate dehydrogenase (LDH) assay. For the
CDC experiment, the rabbit complement was used as effector
cells, and 4T1 cells were incubated with pooled sera from each
group of mice after the fourth vaccination and the rabbit com-
plement.[28] As depicted in Figure 5a, percent cell lysis of
pooled sera from 4-CRM197 was found to be the highest
(74%) among the five groups of mice, whereas the percent
cell lysis of sera from 2-CRM197 was only 57%. To further
verify the ability of lysing cells, three serum concentrations
were employed to conduct the CDC assay. As shown in Fig-
ure 5b, the cell lysis capacity of sera elicited by 4-CRM197 was
higher than that of sera elicited by 2-CRM197 at three serum
concentrations. IgG2b was detected in the sera elicited by 4-
CRM197 at 1:100 dilution (Figure S2), which might be the
reason for its high CDC activity.
Figure 5. CDC assays against 4T1 cells. Data are the meanÆSEM of three in-
dependent experiments. In these studies, cytotoxicity is shown with percent
cell lysis determined by lactate dehydrogenase (LDH) assay. Triplicate wells
were arranged in each assay. a) Cell lysis of pooled sera (1:10 dilution) after
the fourth immunization of different CRM197 glycoconjugates. b) Cell lysis
of pooled sera of mice immunized with 2-CRM197 or 4-CRM197 at various
dilutions, as indicated.
Conclusions
Several N-modified TF antigens and their conjugates with carri-
er protein CRM197 were designed and synthesized. The vacci-
nation results in mice showed that suitable structural modifica-
tions to the native TF antigen can enhance its immunogenicity.
Compared with the unmodified TF conjugate (2-CRM197), the
modified TF-CRM197 conjugates induced higher titers of anti-
bodies against the modified and unmodified antigens. The an-
tisera could react strongly with the TF-antigen-positive tumor
cells 4T1. Among the synthetic glycoconjugates, 4-CRM197
showed the strongest lymphocyte proliferative response and
the highest CDC effect after immunization. These results indi-
cate that 4-CRM197 may hold great promise as a candidate for
an anticancer vaccine. We expect that the antigen modification
strategy could find wide application in the development of
carbohydrate-based vaccines and other types of vaccines.
staining with ceric ammonium molybdate for visualization. Column
chromatography was performed with 200–300 mesh silica gel or
C18 (Merck). Optical rotations were measured on a Rudolph Re-
search Analytical Autopol IV automatic polarimeter. 1H NMR and
13C NMR spectra were recorded on Bruker AVANCE III-400 or III-600
spectrometers at ambient temperature. Chemical shifts (ppm) were
referenced to the residual proton signal of the solvent. High-reso-
lution mass spectrometry (HRMS) was performed on a Thermo Sci-
entific LTQ Orbitrap Discovery instrument. Matrix-assisted laser de-
sorption/ionization time of flight (MALDI-TOF) MS was performed
on an AB SCIEX 5800 spectrometer.
Allyl
(2,3,4,6-tetra-O-benzoyl-b-d-galactopyranosyl)-(1!3)-2-
acetamido-4,6-O-benzylidene-2-deoxy-a-d-galactopyranoside
(9): A solution of bromide 7[10d] (1.0 g, 1.52 mmol) and compound
8[10d] (0.5 g, 1.43 mmol) in a dry mixed solvent of nitromethane/tol-
uene (25 mL, 1:1, v/v) in the presence of 4 Š molecular sieves
(2.5 g) under argon atmosphere was stirred for 30 min. Then the
temperature of the solvent was adjusted to À158C, and AgOTf
(0.5 g, 1.95 mmol) in a solution of nitromethane/toluene was
added dropwise. The mixture was stirred for 4 h. Then bromide 7
(1.0 g, 1.52 mmol) and AgOTf (0.5 g, 1.95 mmol) were added again,
Experimental Section
Chemistry
General: Solvents were dried according to standard methods. Ana-
lytical thin-layer chromatography (TLC) was carried out on
0.25 mm silica gel 60 F254 plates (Merck), using UV light and/or
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and the resulting mixture was stirred until TLC analysis indicated
that the reaction was complete. Et3N (0.5 mL) was added. After the
mixture was filtered through a Celite pad and concentrated, the
residue was purified by column chromatography on silica gel with
petroleum ether/acetone (2:1, v/v) to give product 9 as a colorless
concentrated again. The residue was purified by C18 reversed-
phase column chromatography (MeOH/H2O, 1:4, v/v) to give 4 as
a white solid (25.3 mg, 42% over two steps): ½aŠ25 = +97.68 (c=
D
1
1.17, H2O); H NMR (400 MHz, D2O): d=6.00–5.76 (m, 1H), 5.31 (dd,
J=17.3, 1.4 Hz, 1H), 5.21 (d, J=10.4 Hz, 1H), 4.98–4.79 (m, 3H),
4.47–4.37 (m, 2H), 4.21 (d, J=3.1 Hz, 1H), 4.20–4.14 (m, 1H), 4.09
(dd, J=11.1, 3.1 Hz, 1H), 4.04–3.96 (m, 2H), 3.86 (t, J=5.2 Hz, 1H),
3.79–3.64 (m, 4H), 3.63–3.60 (m, 1H), 3.59–3.53 (m, 1H), 3.46 ppm
(dd, J=9.9, 7.8 Hz, 1H); 13C NMR (100 MHz, D2O): d=171.12 (d, J=
18.7 Hz), 133.68, 118.08, 104.67, 96.32, 79.82 (d, J=180.7 Hz), 77.10,
75.04, 72.62, 70.77, 70.65, 68.73, 68.65, 68.50, 61.20, 61.03,
48.39 ppm; HRMS (ESI) calcd for C17H28NO11FNa [M+Na]+:
464.1544, found: 464.1542.
1
oil (0.9 g, 70%): H NMR (400 MHz, CDCl3): d=8.10–7.28 (m, 25H),
5.99 (d, J=2.6 Hz, 1H), 5.94–5.74 (m, 2H), 5.60 (dd, J=10.3, 3.3 Hz,
1H), 5.43 (d, J=7.9 Hz, 1H), 5.39 (s, 1H), 5.25 (dd, J=17.2, 1.5 Hz,
1H), 5.18 (t, J=8.2 Hz, 2H), 5.12 (d, J=3.4 Hz, 1H), 4.70 (dd, J=
11.3, 6.8 Hz, 1H), 4.62 (ddd, J=11.4, 7.8, 3.3 Hz, 1H), 4.50–4.36 (m,
3H), 4.17–4.10 (m, 3H), 3.98 (dd, J=12.9, 6.0 Hz, 1H), 3.76 (dd, J=
12.5, 1.3 Hz, 1H), 3.53 (s, 1H), 1.41 ppm (s, 3H). The spectroscopic
data are in agreement with those reported in the literature.[29]
Allyl O-(b-d-galactopyranosyl)-(1!3)-2-acetamido-2-deoxy-a-d-
galactopyranoside (2): To a solution of disaccharide 9 (574 mg,
0.62 mmol) in MeOH (38 mL) was added six drops of 33% NaOMe/
MeOH. The resulting mixture was stirred at room temperature for
30 min. Then 1n HCl aqueous solution was added dropwise to
adjust the pH to 7. The solvent was removed under reduced pres-
sure. The residue was dissolved in 60% aqueous AcOH, and the
mixture was held at reflux at 608C for 30 min. The solvent was
then evaporated under reduced pressure. The residue was purified
by C18 reversed-phase column chromatography (H2O as eluent) to
afford disaccharide 2 as a white solid (244 mg, 93%): 1H NMR
(400 MHz, D2O): d=6.07–5.92 (m, 1H), 5.28 (d, J=17.3 Hz, 1H),
5.19 (d, J=10.5 Hz, 1H), 4.87 (d, J=3.8 Hz, 1H), 4.39 (d, J=7.7 Hz,
1H), 4.27 (dd, J=11.1, 3.7 Hz, 1H), 4.21–4.10 (m, 2H), 4.02–3.93 (m,
3H), 3.84 (d, J=3.3 Hz, 1H), 3.71–3.63 (m, 4H), 3.61–3.55 (m, 1H),
3.55–3.52 (m, 1H), 3.45 (dd, J=9.8, 7.8 Hz, 1H), 1.95 ppm (s, 3H).
The spectroscopic data are in agreement with those reported in
the literature.[26]
Allyl
O-(b-d-galactopyranosyl)-(1!3)-2-difluoroacetamido-2-
solution of (26.3 mg,
deoxy-a-d-galactopyranoside (5):
A
2
0.062 mmol) in 2n aqueous NaOH (1.5 mL) was stirred and held at
reflux for 6 h. Then the mixture was neutralized with 1m aqueous
HCl (to pH 7). After concentration under reduced pressure, the resi-
due was dissolved in MeOH (2 mL). Then Et3N (0.3 mL) and methyl
difluoroacetate (0.16 mL) were added successively under argon at-
mosphere. The mixture was stirred at reflux overnight, and then
concentrated in vacuum. The residue was dissolved in H2O and
concentrated again. The residue was purified by C18 reversed-
phase column chromatography (MeOH/H2O, 1:4, v/v) to give 5 as
a white solid (28.5 mg, 95% over two steps): ½aŠ25 = +91.38 (c=
D
1.35, H2O); 1H NMR (400 MHz, D2O): d=6.06 (t, J=53.5 Hz, 1H),
5.92–5.82 (m, 1H), 5.26 (dd, J=17.3, 1.6 Hz, 1H), 5.16 (dd, J=10.4,
1.4 Hz, 1H), 4.91 (d, J=3.8 Hz, 1H), 4.38 (d, J=7.8 Hz, 1H), 4.34
(dd, J=11.1, 3.7 Hz, 1H), 4.18 (d, J=2.6 Hz, 1H), 4.16–4.10 (m, 1H),
4.06 (dd, J=11.1, 3.1 Hz, 1H), 4.00–3.90 (m, 2H), 3.81 (d, J=3.1 Hz,
1H), 3.74–3.60 (m, 4H), 3.56 (dd, J=7.8, 4.2 Hz, 1H), 3.51 (dd, J=
9.9, 3.4 Hz, 1H), 3.41 ppm (dd, J=9.9, 7.8 Hz, 1H); 13C NMR
(100 MHz, D2O): d=165.24 (t, J=25.6 Hz), 133.58, 118.07, 110.64,
108.17 (t, J=247.5 Hz), 95.97, 76.93, 75.00, 72.54, 70.72, 70.61,
68.63, 68.58, 68.47, 61.15, 60.98, 48.88 ppm; HRMS (ESI) calcd for
C17H27NO11F2Na [M+Na]+: 482.1444, found: 482.1442.
Allyl O-(b-d-galactopyranosyl)-(1!3)-2-propionylamido-2-deoxy-
a-d-galactopyranoside (3): A solution of 2 (45 mg, 0.11 mmol) in
2n NaOH aqueous solution (3 mL) was stirred and held at reflux
for 6 h. The mixture was then neutralized with 1n aqueous HCl (to
pH 7). After concentration under reduced pressure, the residue was
dissolved in MeOH (7 mL). Then NaHCO3 (22 mg, 0.25 mmol) was
added and propionic anhydride (56 mL, 0.43 mmol) was added
under argon atmosphere at 08C. After stirring for 1 h, propionic an-
hydride (56 mL, 0.43 mmol) was added again. Then the mixture was
stirred for another 3 h and concentrated under reduced pressure.
The residue was purified by C18 reversed-phase column chromatog-
raphy (H2O as eluent) to give 3 as a white solid (47.3 mg, 98%
Allyl
O-(b-d-galactopyranosyl)-(1!3)-2-trifluoroacetamido-2-
solution of (23.2 mg,
deoxy-a-d-galactopyranoside (6):
A
2
0.055 mmol) in 2n aqueous NaOH (1.3 mL) was stirred and held at
reflux at 1008C for 6 h. Then the mixture was neutralized with 1m
aqueous HCl (to pH 7). After concentration under reduced pres-
sure, the residue was dissolved in MeOH (1.8 mL). Then Et3N
(0.14 mL) and methyl trifluoroacetate (0.14 mL) were added succes-
sively under argon atmosphere. The mixture was stirred at reflux
overnight, and then concentrated in vacuum. The residue was dis-
solved in H2O and concentrated again. The residue was purified by
C18 reversed-phase column chromatography (MeOH/H2O, 1:4, v/v)
over two steps): ½aŠ25 = +98.28 (c=2.25, H2O); 1H NMR (400 MHz,
D
D2O): d=5.95–5.83 (m, 1H), 5.28 (dd, J=17.3, 1.3 Hz, 1H), 5.18 (d,
J=10.5 Hz, 1H), 4.86 (d, J=3.7 Hz, 1H), 4.40 (d, J=7.7 Hz, 1H),
4.27 (dd, J=11.1, 3.7 Hz, 1H), 4.20–4.06 (m, 2H), 4.03–3.88 (m, 3H),
3.83 (d, J=3.3 Hz, 1H), 3.75–3.62 (m, 4H), 3.62–3.57 (m, 1H), 3.54
(dd, J=10.0, 3.4 Hz, 1H), 3.45 (dd, J=9.8, 7.8 Hz, 1H), 2.28–2.16 (m,
2H), 1.11–0.95 ppm (m, 3H); 13C NMR (100 MHz, D2O): d=178.48,
133.66, 117.93, 104.61, 96.39, 77.00, 74.97, 72.50, 70.67, 70.66,
68.76, 68.59, 68.43, 61.19, 60.99, 48.52, 29.12, 9.45 ppm; HRMS (ESI)
calcd for C18H31NO11Na [M+Na]+: 460.1789, found: 460.1794.
to give 6 as a white solid (23.6 mg, 90% over two steps): ½aŠ25 = +
D
80.28 (c=1.18, H2O); 1H NMR (600 MHz, D2O): d=5.97–5.88 (m,
1H), 5.31 (dd, J=17.3, 1.5 Hz, 1H), 5.24–5.18 (m, 1H), 4.99 (d, J=
3.8 Hz, 1H), 4.43 (d, J=7.8 Hz, 1H), 4.40 (dd, J=11.1, 3.7 Hz, 1H),
4.25 (d, J=2.9 Hz, 1H), 4.19 (dd, J=12.8, 5.4 Hz, 1H), 4.14 (dd, J=
11.1, 3.1 Hz, 1H), 4.05–3.98 (m, 2H), 3.87 (d, J=3.3 Hz, 1H), 3.78–
3.67 (m, 4H), 3.62 (dd, J=7.8, 4.3 Hz, 1H), 3.57 (dd, J=9.9, 3.4 Hz,
1H), 3.47 ppm (dd, J=9.9, 7.9 Hz, 1H); 13C NMR (150 MHz, D2O):
d=159.97 (q, J=37.8 Hz), 134.29, 118.87, 116.53 (q, J=286.0 Hz),
105.43, 96.52, 77.46, 75.74, 73.26, 71.46, 71.30, 69.40, 69.33, 69.24,
61.88, 61.74, 50.20 ppm; HRMS (ESI) calcd for C17H30N2O11F3 [M+
NH4]+: 495.1796, found: 495.1807.
Allyl
O-(b-d-galactopyranosyl)-(1!3)-2-fluoroacetamido-2-
solution of (58.6 mg,
deoxy-a-d-galactopyranoside (4):
A
2
0.138 mmol) in 2n aqueous NaOH (4 mL) was stirred and held at
reflux for 6 h. Then the mixture was neutralized with 1m aqueous
HCl (to pH 7). After concentration under reduced pressure, the resi-
due was dissolved in MeOH (2 mL). Then Et3N (0.8 mL) and methyl
fluoroacetate (0.22 mL) were added successively under argon at-
mosphere. The mixture was stirred at reflux overnight, and then
concentrated in vacuum. The residue was dissolved in H2O and
General procedure for the preparation of glycoconjugates with
CRM197 and BSA: Ozone was generated and bubbled into a solu-
tion of compound 2–6 (2.0 mg) in dry MeOH (1.0 mL) at À728C
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until a blue color appeared and remained for 15 min. Then nitro-
gen was then bubbled into the solution until the blue color disap-
peared. Dimethyl sulfide (two drops) was added at À728C, and the
mixture was stirred for 10 min. The mixture was allowed to return
to room temperature and condensed. The resulting crude alde-
hydes were used directly for the next step without further purifica-
tion.
blood cell lysis buffer on ice. Pooled splenocytes from each group
were seeded into a 96-well flat-bottom micro-titer plate at 5.0”106
cellsmLÀ1 in 100 mL RPMI-1640 culture medium. Glycoconjugates
(TF and modified TF conjugated with CRM197) as stimuli were
added into plate at a final concentration of 2 mgmLÀ1 and cultured
at 378C for 48 h. Triplicate wells were arranged. CCK-8 reagent
(10 mL, Dojindo Laboratories, Japan) was added to each well, and
the plates were further incubated for another 3 h at 378C. The op-
tical density was measured by using a microplate reader at
l 450 nm (reference wavelength: 600 nm). The magnitude of prolif-
erative response was expressed as the stimulation index (SI): the
mean absorbance of cells cultured with different modified TF-
CRM197 antigen divided by the mean absorbance of cells cultured
with 2-CRM197.
A solution of the above aldehyde (4.0 mg) prepared from ozoniza-
tion, CRM197 (2.0 mg) and NaCNBH3 (4.0 mg) in PBS (0.5 mL, 0.1m,
pH 7.4) was gently shaken in the dark for 24 h at room tempera-
ture, and then dialyzed against PBS at 48C (molecular weight
cutoff: 14000 Da, six changes of PBS). Coating antigen TF or modi-
fied TF disaccharides conjugated with BSA at 1 mg:1 mg in PBS.
After dialysis, glycoconjugates were evaluated by SDS-PAGE to fur-
ther determine their purity (Figure S3). Before immunizing mice,
the glycoconjugates were filtered through a 0.22 mm microfiltration
membrane to keep these immunogens sterile.
Flow cytometry assays: Flow cytometry was used to analyze the
binding of the immune serum to 4T1 cancer cells that express the
native TF antigen. Single-cell suspensions were obtained by digest-
ing cells with 1% EDTA; 5”105 4T1 cells per tube were washed
with 3% fetal calf serum (FCS) in PBS and incubated with 25 mL
1:20 diluted test sera (sera from pre-immunization and 14 days
after the fourth vaccination) for 30 min on ice. After two washes
with 3% FCS in PBS, 25 mL of 1:30-diluted secondary antibody goat
anti-mouse IgG (g-chain specific) Alexa-488 (Jackson) was added,
and the mixture was incubated for 30 min on ice. After washing
twice, the percentage of positive cells and mean fluorescence in-
tensity (MFI) of stained cells were analyzed using a FACScan
(Becton Dickinson).
The epitope ratios of the glycoconjugates were determined by
MALDI-TOF MS analysis, and protein content was determined by
BCA assay. The carbohydrate loading of each glycoconjugate was
calculated according to Equation (1):
M
r ðglycoconjugateÞÀM
loading ½%Š ¼
r ðproteinÞ Â 100
ð1Þ
Mr ðglycoconjugateÞ
Complement-dependent cytotoxicity (CDC) assays: CDC was as-
sayed on 4T1 cells expressing TF antigen with a non-radioactive cy-
totoxicity assay kit (Promega). 4T1 cells were seeded into 96-well
round-bottom microtiter plates (Corning) at 4”105 cellsmLÀ1 in
25 mL RPMI-1640 culture medium. Diluted pooled immunological
sera from each group were added and incubated at 378C for 1 h
(the final concentration of sera was 1:10). After washing cells twice,
rabbit complement (1:20) was added to the cells and incubated at
378C for another 4 h. The content of lactate dehydrogenase (LDH)
of supernatants was tested according to specifications of the man-
ufacturer. The assays were performed in triplicate. The percentage
of cell lysis was calculated by Equation (2):
Immunological studies
Immunization of mice: Six- to eight-week-old female BALB/c mice
(Number: SCXKjing2012-0001, SPF/VAF) were obtained from the
Department of Laboratory Animal Science, Peking University
Health Science Center. Groups of six mice were immunized by in-
tramuscular injection in the hind legs four times at two-week inter-
vals with different preparations mixed with 2 mg of 2-CRM197 or
modified TF-CRM197 glycoconjugate and 2 mg of C34. Mice were
bled 14 days after the fourth immunization. Serum was isolated
from blood. After the complement was inactivated at 378C for
30 min the serum was stored at À808C. All of the experiments
were approved by the Institutional Animal Care and Use Commit-
tee of Peking University.
ðexptl: releaseÞÀðspontaneous releaseÞ
ð2Þ
cell lysis ½%Š ¼
 100
ðmax: releaseÞÀðspontaneous releaseÞ
Serological assays: Serum titers were analyzed using enzyme-
linked immunosorbent assay (ELISA). 2-BSA or modified TF-BSA
were coated at ELISA plates and incubated at 48C overnight. After
washing three times with PBST (0.05% Tween-20 in PBS), 3% BSA
in PBS was added to block each well at 378C for 1 h. After washing
three times, serial twofold diluted sera were added to the wells
and incubated at 378C for 1 h. The plates were washed and incu-
bated with a 1:5000 dilution of horseradish peroxidase conjugated
goat anti-mouse IgG (g-chain specific; Southern Biotechnology As-
sociates, Inc., Birmingham, AL, USA) at 378C for 1 h. The prepara-
tions were washed and developed with o-phenylenediamine (OPD)
substrate in the dark for 15 min, terminated with 2m H2SO4, and
then optical density (OD) was read at l 490 nm. The antibody titer
was defined as the highest dilution showing an absorbance of 0.1,
after subtracting background. Every concentration was carried out
in triplicate wells. The assays were conducted separately twice.
Acknowledgements
The materials CRM197 and C34 were generously provided by Dr.
Chung-Yi Wu (Genomics Research Center, Academia Sinica,
Taiwan). This work was supported financially by grants from the
Ministry of Science and Technology of China (2012CB822100 and
2013CB910700), and the National Natural Science Foundation of
China (21232002 and 81473084).
Keywords: anticancer
agents
·
carbohydrates
·
glycoconjugates · TF antigens · vaccines
Lymphocyte proliferation assays: Spleen-derived lymphocytes of
six mice from each group were used to detect the proliferation
ability. Briefly, spleens were removed, minced and passed through
a fine steel mesh to obtain a homogeneous cell suspension under
aseptic conditions. Erythrocytes were lysed with Tris·NH4Cl red
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Received: February 14, 2016
Revised: March 17, 2016
Published online on April 14, 2016
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