Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

Article abstract of DOI:10.1002/cbic.201500499

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.

Full text of DOI:10.1002/cbic.201500499

DOI: 10.1002/cbic.201500499
Full Papers
Chemical Synthesis of GM2 Glycans, Bioconjugation with
Bacteriophage Qb, and the Induction of Anticancer
Antibodies
Zhaojun Yin,[a] Steven Dulaney,[a] Craig S. McKay,[b] Claire Baniel,[a] Katarzyna Kaczanowska,[b]
Sherif Ramadan,[a, c] M. G. Finn,[b] and Xuefei Huang*[a]
This manuscript is dedicated to Prof. Koji Nakanishi for his 90th birthday.
The development of carbohydrate-based antitumor vaccines is
an attractive approach towards tumor prevention and treat-
ment. Herein, we focused on the ganglioside GM2 tumor-asso-
ciated carbohydrate antigen (TACA), which is overexpressed in
a wide range of tumor cells. GM2 was synthesized chemically
and conjugated with a virus-like particle derived from bacterio-
phage Qb. Although the copper-catalyzed azide–alkyne cyclo-
addition reaction efficiently introduced 237 copies of GM2 per
Qb, this construct failed to induce significant amounts of anti-
GM2 antibodies compared to the Qb control. In contrast, GM2
immobilized on Qb through a thiourea linker elicited high
titers of IgG antibodies that recognized GM2-positive tumor
cells and effectively induced cell lysis through complement-
mediated cytotoxicity. Thus, bacteriophage Qb is a suitable
platform to boost antibody responses towards GM2, a repre-
sentative member of an important class of TACA: the ganglio-
side.
Introduction
Aberrant glycosylation is a hallmark of many human can-
cers.[1–4] Tumor-associated carbohydrate antigens (TACAs) are
attractive targets for antitumor vaccines, due to their high
levels of expression in tumor cells.[5–8] However, the develop-
ment of an effective carbohydrate-based antitumor vaccine is
extremely challenging. In nature, TACAs are often expressed as
a heterogeneous mixture. As a result, it is difficult to obtain
sufficient quantities of TACAs in conjugatable forms through
isolation. In addition, there are concerns of highly active trace
contaminants present in isolated samples. Thus, synthesis be-
comes critical to produce these complex molecules.[9–10]
for a long time. To induce high-affinity IgG antibodies, a typical
approach is to conjugate TACAs with carriers containing helper
T (Th) cell epitopes, which include immunogenic proteins,[10–11]
peptides,[6,12–13] multiple antigenic glycopeptides,[14–15] nanopar-
ticles,[16–18] polymers,[18–20] and polysaccharides.[21] Recently, we
have demonstrated that self-assembled virus-like particles
(VLPs) could be used to deliver a TACA, the Tn antigen, to the
immune system and generate powerful antibody respons-
es.[22–25] The induced antibodies bound strongly with Tn-ex-
pressing tumor cells, resulting in tumor cell death and protec-
tion of immunized mice from tumor development.[22]
In addition to the challenge of accessing TACAs, the immu-
nological obstacle is that TACAs are Tcell-independent B cell
antigens.[5–8] When administered alone, they generally produce
low titers of low-affinity IgM antibodies, which do not persist
Building on the success of the VLP–Tn studies, we became
interested in testing whether the VLP platform could potently
induce antibody responses against another important family of
TACAs, that is, the gangliosides,[3] as represented by GM2. GM2
[a] Dr. Z. Yin,+ S. Dulaney,+ C. Baniel, S. Ramadan, Prof. Dr. X. Huang
Department of Chemistry, Michigan State University
578 S. Shaw Lane, Room 426, East Lansing, MI 48824-1322 (USA)
E-mail: xuefei@chemistry.msu.edu
[b] Dr. C. S. McKay, Dr. K. Kaczanowska, Prof. Dr. M. G. Finn
School of Chemistry and Biochemistry, Georgia Institute of Technology
901 Atlantic Drive, Atlanta, GA 30332-0400 (USA)
[c] S. Ramadan
Current address:
Chemistry Department, Faculty of Science, Benha University
Benha, Qaliobiya (Egypt)
[+] These authors contributed equally to this work.
contains
a sialic-acid-terminated branched tetrasaccharide
linked to a ceramide chain. GM2 is expressed on the surfaces
of a wide range of human cancers, which include cancer cells
of neuroectodermal origin (melanoma, sarcoma, and neuro-
blastoma), as well as epithelial cancers, such as breast and
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500499: synthetic procedures and charac-
terization data of GM2 and Qb–GM2 conjugates; NMR and MS spectra for
key compounds.
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prostate cancers.[7,26,27] The wide expression of GM2 on multi-
ple types of cancers renders it an intriguing target for develop-
ing a potentially universal anticancer vaccine. In addition, clini-
cal studies have shown that elevated levels of anti-GM2 IgM
antibodies are strongly associated with prolonged survival of
melanoma patients.[28–29] Both passive administration of anti-
GM2 monoclonal antibodies[30] and active immunity gained
through vaccination[28,31] could lead to favorable prognosis,
such as tumor regression or longer disease-free intervals.
These clinical outcomes have inspired the drive towards GM2-
based anticancer vaccines.[28,32–34]
Our synthesis commenced with lactoside 5,[40] which was de-
rived from d-lactose and subsequently transformed to diol 2
through protecting group manipulations (Scheme 2A). Sialyla-
tion of acceptor 2 was performed with thiosialoside donor 3.
Initial coupling of 2 and 3 was mediated by using N-iodosucci-
nimide (NIS) and triflic acid as the promoter, which gave de-
The generation of antibodies is a highly complex process.
Many structural features of the construct can significantly
impact the results of antibody responses. Livingston and co-
workers showed that anti-GM2 antibody titers were highly de-
pendent upon the carrier moiety of the vaccine construct.[35]
The Lo-Man group demonstrated that GM2 coupled with a Th
epitope through a copper-catalyzed azide–alkyne cycloaddition
(CuAAC) reaction gave good titers of anti-GM2 antibodies.[34]
Yet, when the same Th cell peptide was conjugated with two
GM2 molecules, despite the higher valency, it failed to elicit
detectable levels of IgM or IgG antibodies in mice, even after
repeated immunizations. Thus, the structure of a vaccine con-
struct needs to be carefully designed and evaluated. Herein,
we report our results using synthetic GM2 antigens arrayed
over the surface of the VLP bacteriophage Qb capsid for the
induction of antitumor antibodies.
Results and Discussion
Prior anti-GM2 vaccine studies have primarily utilized GM2
glycan extracted from mammalian tissues[28,32] or enzymatically
synthesized.[33–34,36] Chemical synthesis can bestow flexibility in
functionalizing the antigen for immunological investigations.
Although GM2 glycans have been chemically synthesized pre-
viously,[37–39] with the need for stereoselective sialylation and
formation of branched glycans, its preparation in a conjugata-
ble form is not a trivial task. Our synthetic target was the GM2
tetrasaccharide 1, bearing a reducing end free amine, which
was prepared by regioselective sialylation of the lactosyl diol
acceptor 2 by sialyl donor 3, followed by glycosylation of the
4’-OH by galactosamine (GalN) donor 4 (Scheme 1).
Scheme 2. Synthesis of GM2 tetrasaccharide 1. a) NaN3, DMF; b) NaOMe,
MeOH; c) acetone, p-TsOH, 2,2-dimethoxypropane; d) NaH, BnBr, DMF;
e) TFA, CH2Cl2, (44% for five steps); f) sialyl donor 3, p-TolSCl, AgOTf, À408C,
MeCN (65%); g) GalN donor 4, p-TolSCl, AgOTf, À788C, CH2Cl2, Et2O (63%);
h) NaOH, THF; i) Ac2O, TEA, MeOH; j) PMe3, NaOH; k) Pd(OH)2, H2 (54% for
four steps).
Scheme 1. Retrosynthetic analysis of GM2 tetrasaccharide 1.
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sired a-sialoside 6 in 42% yield, along with 8% of the b-
anomer. The stereochemistry of the newly formed glycosyl
linkage of 6 was assigned based on the 3-bond coupling con-
stant between C1 and H3ax of sialic acid (3JC1,H3ax =8 Hz), as well
as that between H7 and H8 of sialic acid (3JH7,H8 =7.9 Hz).[41–42]
Regioselectivity was confirmed by the correlation between C2
of sialic acid with H3’ of the lactose unit in the HMBC NMR
spectrum. In order to improve the sialylation yield, various re-
action conditions were examined. Whereas changing the sol-
vent, reaction time, or temperature did not lead to significant
enhancements, the combination of p-TolSCl/AgOTf[43–44] as the
promoter system improved the yield of 6 to 65%. Recently,
modified sialyl donors with groups such as 4-O,5-N-oxazolidi-
none and 5-N-trifluoroacetyl have been shown to give high
yields and stereoselectivities in sialylation reactions.[45–49] Donor
3 has the advantage that no additional synthetic steps were
needed to adjust the protecting groups on C5 of sialic acid,
while achieving good yield and stereoselectivity. With trisac-
charide 6 in hand, glycosylation by the GalN donor 4 was car-
ried out by using the p-TolSCl/AgOTf promoter system, produc-
ing the protected GM2 7 in 63% yield, with the new glycosidic
3
bond being exclusively b (1JH1,C1 of GalN =161.4 Hz,[50] JH1,H2 of GalN
=
8.8 Hz).
Compound 7 was deprotected in four steps, starting from
the hydrolysis of O-acetyl groups concomitant with Troc re-
moval (Scheme 2B). The newly freed amino group on GalN
was selectively acetylated with acetic anhydride in methanol.
Finally, Staudinger reduction of the azido group and global
debenzylation with Pearlman’s catalyst provided the fully de-
protected GM2 tetrasaccharide 1 in 54% yield over the four
deprotection steps.
With the GM2 glycan in hand, we prepared a GM2 conjugate
vaccine with the VLP bacteriophage Qb as the carrier, as we
have previously shown that Qb is superior to several other VLP
platforms in boosting anti-Tn immunity.[23] Our initial approach
for bioconjugation utilized the CuAAC reaction, due to its high
reaction rate, mild reaction conditions, and bioorthogonal
nature.[51–52] GM2 1 was treated with activated ester 8 to attach
an azide moiety to the reducing end for bioconjugation (GM2
9, 77% yield; Scheme 3A). Subsequently, 9 was coupled with
Scheme 3. Synthesis of GM2–Qb conjugates. a) NaHCO3, H2O; b) CuSO4, sodium ascorbate, THPTA, PBS buffer (CuAAC conditions); c) thiophosgene, NaHCO3,
CHCl3/H2O; d) Na2B4O7 buffer (pH 8.5).
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the alkyne-functionalized Qb 10 under CuAAC conditions,
which introduced approximately 237 copies of GM2 antigen to
each Qb capsid (Scheme 3B). The remaining free alkyne
groups on Qb were capped with 3-azidopropan-1-ol 12 to
afford Qb–GM2 13.
Next, the ability of Qb–GM2 13 to generate anti-GM2 anti-
bodies was evaluated. C57BL/6 mice were immunized subcuta-
neously with three biweekly injections of Qb–GM2 13, and
sera from these mice were collected one week after the final
boost injection. The control group of mice received the uncon-
jugated Qb only. For enzyme-linked immunosorbent assay
(ELISA) analysis of serum antibodies, a bovine serum albumin
(BSA) conjugate of GM2 (BSA–GM2 14) was prepared through
To better understand the Qb–GM2 13 vaccine, the epitope
profiles of antibodies generated were screened by ELISA. BSA
conjugates to structural components of GM2—N-acetyl galac-
tosamine (GalNAc),[24] lactose, GM3, and BSA-triazole[22]—were
synthesized and immobilized on ELISA plates. Although there
was some IgG binding to BSA–GalNAc, BSA–GM3, and BSA–
GM2, the binding to BSA–triazole was significantly stronger
(Figure 1). This suggests that the triazole linker is the dominant
epitope among the components analyzed.
To avoid antibody responses to the triazole linker, alternative
strategies were explored. Previously, we showed that reducing
the number of triazoles on the Qb by removing the triazole
used to cap the unreacted alkynes did not lead to enhanced
anti-glycan responses.[22] Therefore, we utilized another biocon-
jugation approach to ligate GM2 to Qb. Treatment of GM2
1 with thiophosgene converted the amine group to isothiocya-
nate[54] in 85% yield (Scheme 3C). The resulting GM2 15 was
incubated with wild-type Qb particle 16 at pH 8.5 to afford the
Qb–GM2 conjugate 17. This reaction proceeded smoothly, in-
troducing an average of 220 copies of GM2 per Qb particle
(Scheme 3C).
With Qb–GM2 17 in hand, mice were immunized. In contrast
to Qb–GM2 13, ELISA analysis of post-immune sera showed
good anti-GM2 IgG and IgM antibody responses, with IgG as
the main antibody type (Figure 2A). The subclasses of IgG anti-
bodies were also determined. The levels of IgG2 antibodies
(IgG2b and IgG2) were much higher than those of IgG1 and
IgG3, suggesting a more Th1-weighted immune response (Fig-
ure 2B).[55–56] This is likely due to the ability of Qb to encapsu-
late single-stranded Escherichia coli RNA in the interior, which
are potent agonists of Toll-like receptors 7 and 8 for immune
potentiation favoring a Th1 response.[57] The antibodies elicited
by Qb–GM2 17 were able to bind with multiple types of GM2-
positive tumor cells, as determined by flow cytometry (Fig-
reductive amination with glutaraldehyde,[53] with an average of
11 GM2 glycans coupled to BSA. ELISA analysis showed no sig-
nificant binding to BSA–GM2 14 by any post-immune sera,
compared to the control sera from mice immunized with Qb
only. To test serum binding with GM2 expressed in its native
environment, that is, on the tumor cell surface, flow cytometry
analysis of all sera were performed. None of the sera was able
to bind with GM2-positive human lymphoma Jurkat cells, even
at a relatively high concentration (1:10 dilution). These results
demonstrated that Qb–GM2 13 was unable to elicit high titers
of anti-GM2 antibodies in vivo.
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the sialic acid motif contains the major recognition sites of
GM2. This observation is consistent with a literature report in
which the removal of sialic acid from GM2 abrogated the bind-
ing by anti-GM2 polyclonal antibodies.[34]
To assess the therapeutic potential of anti-GM2 antibodies,
we evaluated the complement-dependent cytotoxicity against
tumor cells. The classical pathway of complement activation is
triggered by multivalent binding between the C1 complex and
the Fc region of antibodies.[58] Compared to other IgG subclass-
es, the IgG2 antibodies in mice have the strongest abilities to
initiate the complement cascade.[59] As shown in Figure 2E, the
antibodies induced by Qb–GM2 17 were able to efficiently kill
GM2-positive Jurkat cells by the complement mechanism.
The CuAAC reaction and the triazole linker have been com-
monly used in carbohydrate-based vaccines.[23–24,60–65] In our
recent studies of Qb–Tn conjugates, we observed that the tri-
azole-linked Qb–Tn failed to induce antibodies capable of rec-
ognizing Tn expressed on tumor cell TA3HA, which was attrib-
uted to the possible hindrance of Tn-specific B cell binding to
the vaccine construct by anti-triazole antibodies.[22] The inabili-
ty of the triazole-containing Qb–GM2 13 to generate anti-GM2
antibodies was consistent with the Qb–Tn results, suggesting
that the detrimental effect of triazole on anti-TACA immunity
was not restricted to a small antigen such as Tn, which con-
tains only a monosaccharide N-acetyl galactosamine linked
with serine or threonine. Although the exact reasons for the
suppressive effect of triazole on anti-GM2 antibody responses
Figure 1. ELISA analysis of the epitope profiles of post-immune sera from
mice immunized with triazole linked Qb–GM2 conjugate 13 and thiourea-
linked Qb–GM2 17, respectively. For 13, the anti-triazole antibody level was
significantly higher for than other types of antibodies, such as anti-GM2 or
anti-GM3 antibodies (p<0.0001). Qb-GM2 17 induced significantly higher
anti-GM2 antibodies (p=0.002) but much lower levels of anti-triazole anti-
bodies (p<0.0001) than did 13. Sera from each group were analyzed at
1600-fold dilution. The average of optical density value and SEM were
shown. Statistics were performed by Student’s t-test.
ure 2C and D), whereas sera from the control mice receiving
Qb or the pre-immunized mice did not show any tumor cell
recognition.
The epitope profiles of antibodies induced by Qb–GM2 17
were analyzed by ELISA (Figure 1). The antibodies exhibited
strongest binding to BSA–GM3, but the recognition of BSA–
GalNAc and BSA–lactose was much weaker. This suggests that
Figure 2. Immunological evaluation of Qb–GM2 conjugate vaccine 17. A) IgM and IgG titers of anti-GM2 antibodies tested by ELISA. Sera from mice immu-
nized with wild-type Qb particle were tested as a control. B) The levels of anti-GM2 IgG subclasses as determined by ELISA. Sera were tested at 1:1000 dilu-
tion. C) Binding of GM2-expressing Jurkat cells and D) MCF-7 cells with representative mouse sera diluted at 1:20. Gray filled: pre-immune sera and sera from
mice immunized with Qb only; solid line: day 35 sera from a mouse immunized with Qb–GM2 17. E) Complement-dependent toxicity against Jurkat cells as
~
&
measured by LDH assay. Sera from two mice immunized with Qb-GM2 17 are shown (mouse 1: , mouse 2: ). Pre-immune serum was utilized as a control
*
(
). Sera from mice immunized with Qb gave similar results as the pre-immune sera.
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Sera were tested by flow cytometry on GM2-bearing Jurkat (kindly
provided by Profs. Barbara Kaplan and Norbert Kaminski, Michigan
State University) and MCF-7 (kindly provided by Prof. Olivera J.
Finn, University of Pittsburgh) tumor cell lines. Cells were incubat-
ed with 1:20 diluted mice sera on ice for 30 min and then labeled
with goat anti-mouse IgG conjugated with FITC (BioLegend,
405305) for 30 min. Acquisition of cells was performed with LSR II
(BD), and data were analyzed with FlowJo software (Tree Star, Inc.).
need further investigation, these results indicate that caution
should be taken in applying CuAAC chemistry in future glycan-
based vaccine design.
Compared to GM2 vaccine candidates reported to
date,[28,32–34] the Qb–GM2 17 elicited similar total titers of anti-
GM2 IgG antibodies and binding to GM2-positive tumor cells.
Conjugates such as KLH–GM2 produced more IgG1 and IgG3
in human patients.[35] Qb–GM2 17 elicited higher titers of IgG2,
which can be potentially advantageous for future clinical appli-
cations, as mouse IgG2s have been recognized as the most ef-
ficient IgG subclass to induce effector functions against tumor
cells.[66]
Complement-dependent cytotoxicity: Mice sera were diluted
with DMEM medium (10% FBS, without phenol red), mixed with
105 Jurkat cells and incubated on ice for 45 min. The 96-well plate
was then centrifuged, and the supernatant was discarded. A final
concentration of 10% baby rabbit complement (Cedarlane,
CL3441-S) in DMEM medium was added and incubated at 378C for
4 h. After centrifugation, 50 mL of the supernatant was transferred
to a new 96-well plate, mixed with 50 mL of a lactose dehydrogen-
ase substrate (CytoTox 96 nonradioactive cytotoxicity kit, G1780,
Promega) and incubated at room temperature for 15 min, followed
by addition of 50 mL stopping buffer. The plate was then read at
490 nm. The percentage of specific cell lysis was calculated as
follows: [(AÀC)/(BÀC)]”100, where A represents absorbance ob-
tained from mouse sera, B represents maximal lysis obtained by
treating Jurkat cells with lysis buffer from kit, and C represents
spontaneous lysis by treating Jurkat cells with complement only.
Conclusion
In conclusion, we have established an efficient chemical syn-
thesis of GM2 glycans. The synthetic approach can bestow flex-
ibilities to prepare GM2 derivatives such as GM2 lactones[67–68]
in the future to further enhance the immunogenicity of the
antigen. In order to develop a GM2-based vaccine, our first-
generation approach utilized the CuAAC reaction, linking 237
copies of GM2 onto a VLP carrier protein-bacteriophage Qb.
However, no significant anti-GM2 antibodies were generated
compared to the control. To overcome this obstacle, isothio-
cyanate chemistry was employed to introduce the GM2 glycan
onto Qb. The resulting Qb–GM2 conjugate, 17, was able to
induce high titers of anti-GM2 antibodies, in particular IgG2 an-
tibodies. The antibodies produced were capable of binding
GM2-expressing tumor cells and exhibited complement-depen-
dent cytotoxicity, lysing the tumor cells. Therefore, these re-
sults demonstrate that bacteriophage Qb can be an effective
vaccine platform for a GM2-based vaccine. Studies are ongoing
to optimize the GM2 antigen structure, as well as the vaccine
construct, to further enhance vaccine efficacy.
Acknowledgement
We are grateful to the National Cancer Institute (R01A149451–
01A1) for financial support of our work. The authors declare no
competing financial interests.
Keywords: antibodies
synthesis · vaccines
·
carbohydrates
·
immunology
·
[1] A. Cazet, S. Julien, M. Bobowski, J. Burchell, P. Delannoy, Breast Cancer
Res. 2010, 12, 204.
[2] S. Hakomori, Adv. Exp. Med. Biol. 2001, 491, 369–402.
[3] S. Hakomori, Y. Zhang, Chem. Biol. 1997, 4, 97–104.
[4] A. Vasconcelos-dos-Santos, I. A. Oliveira, M. C. Lucena, N. R. Mantuano,
S. A. Whelan, W. B. Dias, A. R. Todeschini, Front. Oncol. 2015, 5, 138.
[5] Z. Guo, Q. Wang, Curr. Opin. Chem. Biol. 2009, 13, 608–617.
[6] T. Buskas, P. Thompson, G.-J. Boons, Chem. Commun. 2009, 5335–5349.
[7] Z. Yin, X. Huang, J. Carbohydr. Chem. 2012, 31, 143–186; and references
therein.
[8] C.-C. Liu, X.-S. Ye, Glycoconjugate J. 2012, 29, 259–271.
[9] D. P. Galonic, D. Y. Gin, Nature 2007, 446, 1000–1007.
[10] S. J. Danishefsky, J. R. Allen, Angew. Chem. Int. Ed. 2000, 39, 836–863;
Angew. Chem. 2000, 112, 882–912.
[11] P. O. Livingston, G. Ragupathi, Human Vaccines 2006, 2, 137–143; and
references therein.
[12] B. L. Wilkinson, S. Day, L. R. Malins, V. Apostolopoulos, R. J. Payne,
Angew. Chem. Int. Ed. 2011, 50, 1635–1639; Angew. Chem. 2011, 123,
1673–1677.
[13] N. Gaidzik, U. Westerlind, H. Kunz, Chem. Soc. Rev. 2013, 42, 4421–4442;
and references therein.
[14] R. Lo-Man, S. Vichier-Guerre, S. Bay, E. Deriaud, D. Cantacuzene, C. Le-
clerc, J. Immunol. 2001, 166, 2849–2854.
[15] R. Lo-Man, S. Vichier-Guerre, R. Perraut, E. Deriaud, V. Huteau, L. BenMo-
hamed, O. M. Diop, P. O. Livingston, S. Bay, C. Leclerc, Cancer Res. 2004,
64, 4987–4994.
Experimental Section
Immunization of mice: Pathogen-free C57BL/6 female mice age
6–10 weeks were obtained from Charles River and maintained in
the University Laboratory Animal Resources facility of Michigan
State University. All animal care procedures and experimental pro-
tocols were approved by the Institutional Animal Care and Use
Committee (IACUC) of Michigan State University. Groups of five
C57BL/6 mice were injected subcutaneously under the scruff on
day 0 with 0.1 mL of various Qb constructs as emulsions in com-
plete Freund’s adjuvant (Sigma–Aldrich, F5881), and boosters were
given subcutaneously under the scruff on days 14 and 28 with vari-
ous Qb constructs (0.1 mL) as emulsions in incomplete Freund’s ad-
juvant (Sigma–Aldrich, F5506). All GM2 vaccine constructs adminis-
tered had the same amounts of GM2 antigen (4 mg). Serum sam-
ples were collected on days 0 (before immunization), 7, and 35.
The final bleeding was done by cardiac bleed. Statistical analysis of
immune responses of various groups were performed by Student’s
t-test.
Antibody detection by ELISA and flow cytometry: Sera were
tested as described previously for anti-Tn and anti-triazole antibod-
ies by ELISA. The titer was determined by regression analysis with
the log10 dilution plotted against optical density.
[16] R. P. Brin¼s, A. Sundgren, P. Sahoo, S. Morey, K. Rittenhouse-Olson, G. E.
Wilding, W. Deng, J. J. Barchi, Bioconjugate Chem. 2012, 23, 1513–1523.
[17] S. Sungsuwan, Z. Yin, X. Huang, ACS Appl. Mater. Interfaces 2015, 7,
17535–17544.
ChemBioChem 2016, 17, 174 – 180
www.chembiochem.org
179
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[18] A. L. Parry, N. A. Clemson, J. Ellis, S. S. R. Bernhard, B. G. Davis, N. R. Ca-
meron, J. Am. Chem. Soc. 2013, 135, 9362–9365.
[43] X. Huang, L. Huang, H. Wang, X.-S. Ye, Angew. Chem. Int. Ed. 2004, 43,
5221–5224; Angew. Chem. 2004, 116, 5333–5336.
[19] Q. Qin, Z. Yin, P. Bentley, X. Huang, MedChemComm 2014, 5, 1126–
1129.
[20] L. Nuhn, S. Hartmann, B. Palitzsch, B. Gerlitzki, E. Schmitt, R. Zentel, H.
Kunz, Angew. Chem. Int. Ed. 2013, 52, 10652–10656; Angew. Chem.
2013, 125, 10846–10850.
[44] B. Sun, B. Srinivasan, X. Huang, Chem. Eur. J. 2008, 14, 7072–7081.
[45] P. K. Kancharla, C. Navuluri, D. Crich, Angew. Chem. Int. Ed. 2012, 51,
11105–11109; Angew. Chem. 2012, 124, 11267–11271.
[46] X.-T. Zhang, Z.-Y. Gu, G.-W. Xing, Carbohydr. Res. 2014, 388, 1–7; and ref-
erences therein.
[21] R. A. De Silva, Q. Wang, T. Chidley, D. K. Appulage, P. R. Andreana, J. Am.
Chem. Soc. 2009, 131, 9622–9623.
[47] H. Tanaka, Y. Nishiura, T. Takahashi, J. Am. Chem. Soc. 2006, 128, 7124–
7125.
[22] Z. Yin, W. S. Wright, C. McKay, C. Baniel, K. Kaczanowska, P. Bentley, J. C.
Gildersleeve, M. G. Finn, L. BenMohamed, X. Huang, ACS Chem. Biol.
2015, 10, 2364–2372.
[23] Z. Yin, M. Comellas-Aragones, S. Chowdhury, P. Bentley, K. Kaczanowska,
L. BenMohamed, J. C. Gildersleeve, M. G. Finn, X. Huang, ACS Chem. Biol.
2013, 8, 1253–1262.
[48] C. De Meo, M. Farris, N. Ginder, B. Gulley, U. Priyadarshani, M. Woods,
Eur. J. Org. Chem. 2008, 3673–3677.
[49] C.-C. Lin, K.-T. Huang, C.-C. Lin, Org. Lett. 2005, 7, 4169–4172.
[50] K. Bock, C. Pedersen, J. Chem. Soc. Perkin Trans. 2 1974, 293–297.
[51] M. G. Finn, V. V. Fokin, Chem. Soc. Rev. 2010, 39, 1231–1232 and referen-
ces therein.
[24] Z. Yin, H. G. Nguyen, S. Chowdhury, P. Bentley, M. A. Bruckman, A. Mier-
mont, J. C. Gildersleeve, Q. Wang, X. Huang, Bioconjugate Chem. 2012,
23, 1694–1703.
[25] A. Miermont, H. Barnhill, E. Strable, X. Lu, K. A. Wall, Q. Wang, M. G.
Finn, X. Huang, Chem. Eur. J. 2008, 14, 4939–4947.
[26] S. Zhang, C. Cordon-Cardo, H. S. Zhang, V. E. Reuter, S. Adluri, W. B.
Hamilton, K. O. Lloyd, P. O. Livingston, Int. J. Cancer 1997, 73, 42–49.
[27] G. Ritter, P. O. Livingston, Semin. Cancer Biol. 1991, 2, 401–409.
[28] P. O. Livingston, G. Y. C. Wong, S. Adluri, Y. Tao, M. Padavan, R. Parente,
C. Hanlon, M. J. Calves, F. Helling, G. Ritter, H. F. Oettgen, L. J. Old, J.
Clin. Oncol. 1994, 12, 1036–1044.
[52] V. Hong, S. I. Presolski, C. Ma, M. G. Finn, Angew. Chem. Int. Ed. 2009, 48,
9879–9883; Angew. Chem. 2009, 121, 10063–10067.
[53] I. Migneault, C. Dartiguenave, M. J. Bertrand, K. C. Waldron, Biotech-
niques 2004, 37, 790–802.
[54] D. F. Smith, D. A. Zopf, V. Ginsburg, Methods Enzymol. 1978, 50, 169–
171.
[55] D. J. Lefeber, B. Benaissa-Trouw, J. F. G. Vliegenthart, J. P. Kamerling,
W. T. M. Jansen, K. Kraaijeveld, H. Snippe, Infect. Immun. 2003, 71, 6915–
6920.
[56] T. Germann, M. Bongartz, H. Dlugonska, H. Hess, E. Schmitt, L. Kolbe, E.
Kçlsch, F. J. Podlaski, M. K. Gately, E. Rüde, Eur. J. Immunol. 1995, 25,
823–829.
[57] M. P. Schçn, M. Schçn, Oncogene 2008, 27, 190–199.
[58] V. N. Schumaker, P. Zavodszky, P. H. Poon, Annu. Rev. Immunol. 1987, 5,
21–42.
[59] J. L. Jansen, A. P. Gerard, J. Kamp, W. P. Tamboer, P. G. Wijdeveld, J. Im-
munol. 1975, 115, 387–391.
[60] E. Kaltgrad, S. Sen Gupta, S. Punna, C. Y. Huang, A. Chang, C. H. Wong,
M. G. Finn, O. Blixt, ChemBioChem 2007, 8, 1455–1462.
[61] R. D. Astronomo, E. Kaltgrad, A. Udit, S.-K. Wang, K. J. Doores, C.-Y.
Huang, R. Pantophlet, J. C. Paulson, C. H. Wong, M. G. Finn, D. R. Burton,
Chem. Biol. 2010, 17, 357–370.
[29] P. C. Jones, L. L. Sze, P. Y. Liu, D. L. Morton, R. F. Irie, J. Natl. Cancer Inst.
1981, 66, 249–254.
[30] R. Irie, T. Matsuki, D. Morton, Lancet 1989, 333, 786–787.
[31] P. O. Livingston, E. J. J. Natoli, M. J. Calves, E. Stockert, H. F. O. Oettgen,
L. J. Old, Proc. Natl. Acad. Sci. USA 1987, 84, 2911–2915.
[32] P. B. Chapman, D. M. Morrissey, K. S. Panageas, W. B. Hamilton, C. Zhan,
A. N. Destro, L. Williams, R. J. Israel, P. O. Livingston, Clin. Cancer Res.
2000, 6, 874–879.
[33] J. R. Rich, W. W. Wakarchuk, D. R. Bundle, Chem. Eur. J. 2006, 12, 845–
858.
[34] S. Bay, S. Fort, L. Birikaki, C. Ganneau, E. Samain, Y. M. Coic, F. Bon-
homme, E. Deriaud, C. Leclerc, R. Lo-Man, ChemMedChem 2009, 4, 582–
587.
[62] T. Lipinski, T. Luu, P. I. Kitov, A. Szpacenko, D. R. Bundle, Glycoconjugate
J. 2011, 28, 149–164.
[35] F. Helling, S. Zhang, A. Shang, S. Adluri, M. Calves, R. Koganty, B. M. Lon-
genecker, T. J. Yao, H. F. Oettgen, P. O. Livingston, Cancer Res. 1995, 55,
2783–2788.
[36] S. Jacques, J. R. Rich, C.-C. Ling, D. R. Bundle, Org. Biomol. Chem. 2006,
4, 142–154.
[37] J. C. Castro-Palomino, G. Ritter, S. R. Fortunato, S. Reinhardt, L. J. Old,
R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1997, 36, 1998–2001; Angew.
Chem. 1997, 109, 2081–2085.
[38] Y. S. Cho, Q. Wan, S. J. Danishefsky, Bioorg. Med. Chem. 2005, 13, 5259–
5266.
[63] Q. Wang, Z. Zhou, S. Tang, Z. Guo, ACS Chem. Biol. 2012, 7, 235–240.
[64] Q. Y. Hu, M. Allan, R. Adamo, D. Quinn, H. L. Zhai, G. X. Wu, K. Clark, J.
Zhou, S. Ortiz, B. Wang, E. Danieli, S. Crotti, M. Tontini, G. Brogioni, F.
Berti, Chem. Sci. 2013, 4, 3827–3832.
[65] H. Cai, Z. Y. Sun, M. S. Chen, Y. F. Zhao, H. Kunz, Y. M. Li, Angew. Chem.
Int. Ed. 2014, 53, 1699–1703; Angew. Chem. 2014, 126, 1725–1729.
[66] F. Nimmerjahn, J. V. Ravetch, Science 2005, 310, 1510–1512.
[67] G. Ragupathi, M. Meyers, S. Adluri, L. Howard, C. Musselli, P. O. Living-
ston, Int. J. Cancer 2000, 85, 659–666.
[68] G. Ritter, E. Boosfeld, R. Adluri, M. Calves, H. F. Oettgen, L. J. Old, P. Liv-
ingston, Int. J. Cancer 1991, 48, 379–385.
[39] M. Sugimoto, M. Numata, K. Koike, Y. Nakahara, T. Ogawa, Carbohydr.
Res. 1986, 156, C1–5.
[40] B. Sun, B. Yang, X. Huang, Sci. China Chem. 2012, 55, 31–35.
[41] H. Paulsen, H. Tietz, Angew. Chem. Int. Ed. Engl. 1982, 21, 927–928;
Angew. Chem. 1982, 94, 934–935.
Manuscript received: September 29, 2015
Accepted article published: November 5, 2015
Final article published: December 4, 2015
[42] G.-J. Boons, A. V. Demchenko, Chem. Rev. 2000, 100, 4539–4566.
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