ARTICLE IN PRESS
JID: CCLET
[m5G;June 3, 2021;3:28]
H. Lin, H. Hong, L. Feng et al.
Chinese Chemical Letters xxx (xxxx) xxx
as a substrate in our experiment. Therefore, Man-NHDNP (7) was
enzymatically transformed into 5ʹ-N-DNP-substituted sialic acid 8
in the presence of sodium pyruvate in excellent yield (91%).
The synthesis of the DNP-modified GM3 is provided in
Scheme 2. An efficient chemoenzymatic “1+2” approach was ap-
plied to assemble the trisaccharide because the glycosyltrans-
ferases required for this biochemical transformation could be eas-
ily obtained either by recombinant expression in Escherichia coli
(E. coli) or commercially. Accordingly, the lactose acceptor 10 was
prepared with an azidoethyl group as a linker at the reducing
end following a reported procedure [31]. The GM3-NHDNP ana-
log 11a was assembled by two-step, one-pot enzymatic transfor-
mation. First, the sialic acid derivatives 8 were converted to the
CMP-Neu5R derivatives 9a by Neisseria meningitidis CMP-sialic acid
synthetase (NmCSS) according to a previously published proce-
dure [32]. Then, the lactose acceptor 10 and the recombinant en-
zyme Pasteurella multocida α-(2→3)-sialyltransferase (PmST1) were
added into the reaction mixture, which was incubated for 12 h at
37 °C. After quenching the reaction and a brief purification using a
Bio-P2 column, the azido group on the reducing end of the 5ʹ-N-
DNP-substituted GM3 analog 11a was reduced to the correspond-
ing aminoethyl group, affording GM3-NHDNP 12a in 77% yield. Fol-
lowing a similar procedure, the natural GM3-NHAc 12b was pre-
pared, as well. All of the intermediates and final compounds were
characterized by 1H and 13C NMR and HRMS.
Fig. 1. Design and structure of the GM3-NHDNP glycoconjugate vaccine construct.
Scheme 1. a) 1-Fluoro-2,4-dinitrobenzene, acetone, 2 mol/L Na2CO3, r.t., 1 h, 83%;
b) D-Mannosamine, EDCI, Et3N, MeOH, r.t., 18 h, 30%; c) Sialic acid aldolase, MgCl2
(20 mmol/L), Tris-HCl buffer (100 mmol/L, pH 7.5), sodium pyruvate, DTT, 37 °C,
24 h, 91%.
lustrated the potential collaborative role of endogenous antibodies
in vaccine development.
The GM3 analogs were conjugated to Keyhole limpet hemo-
cyanin (KLH) or human serum albumin (HSA) via the bifunctional
glutaryl ester method, which is an established conjugation method
that was not expected to affect the immunological properties of
the conjugates [33]. Briefly, the GM3 analogs 12a or 12b were re-
acted with a large excess (15.0 equiv.) of disuccinimidyl glutarate
(DSG) in a solution of DMF and PBS buffer (4:1) to afford the cor-
responding monoesters 13a or 13b, respectively. The monoesters
were then mixed with KLH or HSA in phosphate buffer saline (PBS)
to generate conjugates 1–4, which were purified by centrifugal fil-
ter devices (10 kDa for HSA conjugates and 30 kDa for KLH conju-
gates). The antigen loading percentages of the resulting glycoconju-
gates 1–4 were determined by the Svennerholm method (described
in the Supporting information), which were calculated to be 3%,
6%, 6.1% and 4.9%, respectively.
Ganglioside GM3 is a common TACA that is highly expressed
by several types of malignancies, such as lung, brain and breast
cancers, as well as melanomas [27]. The expression of GM3 in-
fluences the development and proliferation of cancer cells and is
positively correlated with tumor malignancy. GM3 is one of the
50 most investigated cancer antigens for the development of can-
cer immunotherapies and has been widely targeted in vaccine de-
velopment [28,29]. Based on previous work related to non-natural
TACA-based cancer vaccines and cell-surface glycoengineering, we
designed and synthesized a new 5ʹ-N-DNP-modified GM3 antigen
(GM3-NHDNP) for use in cancer immunotherapy (Fig. 1). The in-
corporation of the DNP hapten into the structure of GM3 had mul-
tiple purposes. First, the artificial GM3-NHDNP antigen could elicit
strong immune responses and overcome immune tolerance be-
cause of its unnatural structure. Second, the targeted delivery of
the GM3-NHDNP antigen to APCs could be achieved in an ADE
manner to further enhance the antigen-presenting process in the
presence of endogenous anti-DNP antibodies. Finally, the surface of
cancer cells could be glycoengineered to express the GM3-NHDNP
antigen using established metabolic approaches. Thus, it was ex-
pected that the endogenous anti-DNP antibodies, together with the
elicited endogenous anti-GM3-NHDNP antibodies, would synergis-
tically promote tumor binding and phagocytosis after effective vac-
cinations. In this context, we communicate the chemoenzymatic
synthesis of 5ʹ-N-DNP-modified GM3 and the evaluation of the im-
mune activity of the GM3-NHDNP glycoconjugate in the presence
of anti-DNP antibodies as a potential carbohydrate-based cancer
vaccine.
To evaluate the immunological activities of our glycoconjugate
vaccine candidates, we created three groups of mice. Group 1 (con-
trol group), which consisted of five mice, were immunized with
natural GM3-NHAc-KLH (3). The mice in the second group were
immunized with GM3-NHDNP-KLH (1) only, while the mice in the
third group were first pre-immunized with DNP-ovalbumin (OVA)
to establish a high level of anti-DNP antibodies (Fig. S2 in Sup-
porting information) and then immunized with conjugate 1. All
of these conjugates were premixed with Freund’s complete adju-
vant to activate the vaccine. Mouse blood samples were collected
on days 0, 21 and 28, and the specific antibodies were determined
by enzyme-linked immunosorbent assays (ELISA). The ELISA results
from the antisera of the three groups on Day 28 are summarized in
Fig. 2. As expected, the mice in group 1 produced the lowest num-
ber of GM3-specific antibodies (approx. 9784, Fig. 2A), while the
mice in group 2 generated a significantly higher number of anti-
bodies specifically against GM3-NHDNP (approx. 164502, Fig. 2B).
Therefore, the specific antibody production increased nearly 17-
fold compared to the mice in group 1. This result revealed that the
artificial DNP-modified GM3 demonstrated a significantly improved
immunogenicity compared to GM3, which was in consistent with
a previously published report [34]. For the mice in group 3 that
were pre-immunized with the DNP-OVA conjugate, the highest
production of GM3-NHDNP-specific antibodies was observed (ap-
prox. 424294, Fig. 2C), which was approximately 2.6-fold higher
than the mice in group 2. This result indicated that the endoge-
The synthesis of the DNP-modified GM3 analog required a
key intermediate: 5ʹ-N-DNP-substituted sialic acid containing an
aminocaproic acid linker (8). The intermediate was prepared
by
a chemoenzymatic method, as shown in Scheme 1. First,
aminocaproic acid was reacted with 1-fluoro-2,4-dinitrobenzene in
the presence of sodium bicarbonate to afford intermediate 6 in 83%
yield, after which 6 was coupled to D-mannosamine using 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide (EDCI) as the condensa-
tion reagent in the presence of triethylamine, affording the DNP-
modified mannosamine derivative (Man-NHDNP) 7, albeit in only
30% yield. Bacterial sialic acid aldolase typically has a broad sub-
strate specificity [30], so it was able to recognize Man-NHDNP (7)
2