Synthesis and Evaluation of Liposomal Anti-GM3 Cancer Vaccine Candidates Covalently and Noncovalently Adjuvanted by αgalCer

Article abstract of DOI:10.1021/acs.jmedchem.0c01186

GM3, a typical tumor-associated carbohydrate antigen, is considered as an important target for cancer vaccine development, but its low immunogenicity limits its application. αGalCer, an iNKT cell agonist, has been employed as an adjuvant via a unique immune mode. Herein, we prepared and investigated two types of antitumor vaccine candidates: (a) self-adjuvanting vaccine GM3-αGalCer by conjugating GM3 with αGalCer and (b) noncovalent vaccine GM3-lipid/αGalCer, in which GM3 is linked with lipid anchor and coassembled with αGalCer. This demonstrated that βGalCer is an exceptionally optimized lipid anchor, which enables the noncovalent vaccine candidate GM3-βGalCer/αGalCer to evoke a comparable antibody level to GM3-αGalCer. However, the antibodies induced by GM3-αGalCer are better at recognition B16F10 cancer cells and more effectively activate the complement system. Our study highlights the importance of vaccine constructs utilizing covalent or noncovalent assembly between αGalCer with carbohydrate antigens and choosing an appropriate lipid anchor for use in noncovalent vaccine formulation.

Full text of DOI:10.1021/acs.jmedchem.0c01186

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Article
Synthesis and Evaluation of Liposomal Anti-GM3 Cancer Vaccine
Candidates Covalently and Noncovalently Adjuvanted by αGalCer
Xu-Guang Yin, Jie Lu, Jian Wang, Ru-Yan Zhang, Xi-Feng Wang, Chun-Miao Liao, Xiao-Peng Liu,
Zheng Liu, and Jun Guo*
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ABSTRACT: GM3, a typical tumor-associated carbohydrate
antigen, is considered as an important target for cancer vaccine
development, but its low immunogenicity limits its application.
αGalCer, an iNKT cell agonist, has been employed as an adjuvant
via a unique immune mode. Herein, we prepared and investigated
two types of antitumor vaccine candidates: (a) self-adjuvanting
vaccine GM3-αGalCer by conjugating GM3 with αGalCer and
(b) noncovalent vaccine GM3-lipid/αGalCer, in which GM3 is
linked with lipid anchor and coassembled with αGalCer. This demonstrated that βGalCer is an exceptionally optimized lipid anchor,
which enables the noncovalent vaccine candidate GM3-βGalCer/αGalCer to evoke a comparable antibody level to GM3-αGalCer.
However, the antibodies induced by GM3-αGalCer are better at recognition B16F10 cancer cells and more effectively activate the
complement system. Our study highlights the importance of vaccine constructs utilizing covalent or noncovalent assembly between
αGalCer with carbohydrate antigens and choosing an appropriate lipid anchor for use in noncovalent vaccine formulation.
used as a vaccine adjuvant in the treatment of cancer, viruses,
and bacteria.15,16 iNKT cells are a unique population of T cells
with immunomodulatory properties that link innate and
adaptive immune responses. Unlike conventional T cells that
see the peptides associated with MHC molecules, iNKT cells
recognize glycolipid epitopes present by CD1d, an MHC class I-
like molecule. The CD1d molecule is nonpolymorphic,
therefore, αGalCer can act as a “universal helper”.17 Upon
activation by glycolipids binding to CD1d, iNKT cells rapidly
release potent cytokines leading to downstream activation of a
broad spectrum of immune cells. Numerous strategies of
αGalCer-based cancer therapy have been developed.18−21
Clinical trials have demonstrated that iNKT cell agonists are
immunologically active in humans and have good safety
profiles.22 Recently, iNKT cell agonists have been identified as
potent immunological stimulants of a new class of carrier
molecules for the construction of fully synthetic self-adjuvant
conjugate vaccines.23−32 In these vaccines, Th epitope peptides
are not incorporated, and αGalCer plays a specific role: carrier
molecular and adjuvant. The iNKT cells provide cognate or
noncognate help to B-cells,33,34 representing a novel mode of
immunotherapy.
INTRODUCTION
■
Many cancer cells are expressed with aberrant levels and types of
carbohydrate structures on their surfaces, known as tumor-
associated carbohydrate antigens (TACAs). Due to the
structural conservatism and its important roles in tumorigenic
processes, TACAs have been recognized as promising targets for
the development of cancer vaccines.1−6 GM3, a sialylated
trisaccharide TACA, is highly expressed in a broad range of
human malignancies; furthermore, the presence of this molecule
in normal tissues is undetectable.7 A variety of GM3-targeting
cancer immunotherapies have been evaluated in the clinical
trials, showing evidence of a clinical effect.8,9 However, because
of its weak immunogenicity and T cell independence, the GM3
antigen alone is unable to initiate the adaptive immune system to
stimulate a robust immune response against tumor cells, thus the
common strategy in preparing the anti-GM3 vaccine is to
conjugate GM3 to immunogenic carriers, a modality known as
semisynthetic vaccines. Unfortunately, semisynthetic cancer
vaccines are suboptimal in some clinical trials, and the immune
responses to the TACAs vary significantly from person to
person. The possible reason might be the resulting anticarrier
antibody responses.10 To resolve this issue, fully synthetic
vaccines with well-defined molecular structures are pursued,
generally comprising an adjuvant, B cell epitopes, and Th cell
epitopes.11 These vaccines are able to eliminate inessential
epitopes and elicit more specific immune responses.
Received: August 12, 2020
Published: February 4, 2021
Adjuvants play an essential role in the improvement of vaccine
efficacy by providing appropriate “danger signals”.12−14
αGalCer, an iNKT cell glycolipid agonist, has been widely
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Scheme 1. (A) GM3 Covalently Conjugated with αGalCer: GM3-αGalCer and (B) Lipid-Modified GM3 Conjugates Non-
Covalently Co-Formulated with αGalCer: GM3-Pam/αGalCer, GM3-Pam2/αGalCer, GM3-Chol/αGalCer, and GM3-βGalCer/
αGalCer
In the present study, we extended the self-adjuvanting
approach to GM3, preparing GM3-αGalCer conjugate, of
which the antibody response and anticancer properties were
evaluated (Scheme 1A). Adjuvant and antigen codelivery to the
same antigen-presenting cells can break tolerance to self-antigen
and enhance antigen-specific response.35−37 Covalent con-
jugation is an important strategy for codelivery. In addition to
the self-adjuvanting (or covalent) strategy, an alternative
method could be to form a liposome in which a lapidated
antigen and lipophilic adjuvant are coassembled in a non-
covalent manner, simplifying the synthesis and avoiding the
modification of the adjuvant (Scheme 1B). A previous study
demonstrated that an amphiphilic vaccine comprising of an
antigen and adjuvant cargo linked to a lipophilic lipid tail is able
to increase the potency of subunit vaccines.38 Savage and co-
workers successfully developed the noncovalent vaccine against
Streptococcus pneumoniae serotype 14 polysaccharide, which
outperformed the clinically used semisynthetic vaccine in
potentiating the antibody response.39,40 The antigenic poly-
saccharide was modified by lipid-tail, potentially able to match
the amphiphilicity of the iNKT cell glycolipid agonist. However,
it is noteworthy that the effects of lipidation are manifold,41 and
the hydrophobicity bought about by lipidation is amendable to
the particular antigens, therefore, the lipid-anchor or hydro-
phobic foot appended needs to be optimized. For example, our
recent study showed that the dipalmitoyl-tailed glycopeptide
antigen MUC1 is more effective than the monopalmitoyl-tailed
and free MUC1 in potentiating immunological responses.42 In
contrast, BenMohamed and co-workers reported that the
antiviral T cell responses induced by the palmitoyl-tailed Th-
CTL chimeric epitopes are irrespective of the number of lipid
moieties.43 Therefore, whether the lipid anchor attached to
GM3 can trigger an optimal immune response remains to be
investigated.
In our ongoing study of anti-TACA vaccines,28,42,44 we
prepared a covalent vaccine candidate, GM3-αGalCer, that can
induce an effective antibody response. Furthermore, we also
hypothesized that an alternative vaccine modality, in which
GM3 antigen is modified with lipids of weak immunogenicity
and coformulated noncovalently with αGalCer in a liposome,
could elicit an antibody response as effective as that of GM3-
αGalCer. To evaluate the impact of lipidation and to identify an
optimal lipid anchor, we constructed four glycolipids: GM3-
Pam, GM3-Pam2, GM3-Chol, and GM3-βGalCer, which
appended with monopalmitoyl (Pam), dipalmitoyl (Pam2),
cholesterol, and βGalCer (the anomer of αGalCer), respectively
(Scheme 1B). The immune responses induced in mice by the
vaccine candidates were characterized by the anti-GM3
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Table 1. Synthesis of GM3 by [1 + 2] Glycosylation
a
b
c
1
Donor/acceptor = 1.2/1, −78 to −40 °C for 1 h. Isolated yield. Determined by H NMR spectroscopic analysis of the unpurified reaction
d
mixture. Ref 54.
antibody titers and the concentrations of cytokines (IFN-γ and
IL-4). The efficacies of the vaccine candidates were further
evaluated by the binding of the induced sera to B16F10 tumor
cells and the activation of complement-dependent cytotoxicity.
followed the previously reported procedures with slight
modifications (Scheme 2A).57−59 Sialodisaccharide 13 was
prepared by dibutyl phosphate sialoside 11 and 12 in a 83% yield
(α/β ratio 9/1), whose α-sialyl linkage was verified by NMR.
The chemical shifts of the H-3eq of sialic acid is 2.89 ppm (dd, J
= 12.1, 3.2 Hz) for α-glycoside and 2.58 ppm (dd, J = 12.1, 3.6
Hz) for β-glycoside. Additionally, the chemical shift of C-2 of
sialic acid is 97.3 ppm for α-glycoside and 99.4 ppm for β-
glycoside.59,60 Glycosylation of penta-O-acetylglucose (8) with
2-(2-azidoethoxy) ethan-1-ol and subsequent manipulation of
the protecting groups offered glycosyl acceptor 10.61 The NIS/
TfOH-promoted glycosylation between 13 and 10 provided the
fully protected form of GM3 antigen 14 with high β-selectivity.
The attempt to Staudinger reduction of azide 14 in the presence
of PMe3/wet MeOH led to an unidentified mixture probably
due to the high electrophilicity of the carbonyl group in
oxazolidinone. Therefore, our initial plan was to cleave the acetyl
and oxazolidinone protective group at the same condition.49,62
However, we found that treatment of 14 with a freshly prepared
NaOMe/MeOH solution (the concentration of NaOMe was
adjusted to be 2.0 mM) within 10 min at room temperature
provided methyl carbonate 15 in high yield, allowing acetyl
groups to be intact. The formation of methyl carbonate was
confirmed by HRMS and NMR (a clearly observed upfield-
shifted methyl group (δ = 1.86) of NHAc at the 5-position, and
appearance of the methyl group (δ = 3.68) at carbonate). This
result parallels Sugai and co-workers’ report on the selective
cleavage of oxazolidinone.63 Staudinger reduction of 15 and
amide-formation followed by global deprotection was un-
eventful, affording the desired lipid-anchored GM3 conjugates
GM3-αGalCer, GM3-βGalCer, GM3-Pam, and GM3-Pam2.
Because the alkene in cholesterol may be saturated under the
condition of catalytic hydrogenolysis, the above synthetic
sequence is not suitable for the preparation of GM3-Chol.
Therefore, an alternative route was carried out (Scheme 2B).
After the removal of all the protecting groups, the GM3 antigen
was conjugated with 22, obtained from the reaction of p-
nitrophenyl chloroformate with cholesterol,64 to yield the
RESULTS AND DISCUSSION
■
The self-adjuvanting vaccine candidate GM3-αGalCer was
designed by following our previous research on an anti-STn
vaccine.28 As for the noncovalent candidates, Pam, Pam2, Chol,
and βGalCer were chosen as the lipid anchors. Notably, the anti-
MUC1 vaccine using a lysine-backbone-based dipalmitoyl lipid
anchor showed great potency in stimulating robust antibody
response.42 βGalCer, a weak iNKT cell agonist,45 was chosen
because of high structural similarity to αGalCer, which could
render GM3 similar pharmacokinetics to αGalCer. In addition,
GM3-βGalCer was used as a control in our study. In all lipid-
modified GM3 conjugates, the linkers bear no immunogenic
heterocycle in order to avoid immunological suppression.46
For the synthesis of GM3, highly regio- and stereoselective
glycosylation is desirable. For the widely used donor, 4O,5N-
acetyl oxazolidinone-protected p-toluenethiosialoside, the trans
orientation of the oxazolidinone group always leads to excellent
α selectivity of sialylation.47−49 The [1 + 2] synthetic route for
GM3 by using lactosyl as the acceptors has been reported due to
the short steps.50−55 On the basis of previous report, though the
lactosyl diol 2 bearing multiple benzyl ether of protective groups
as the acceptor only gave moderate yield of the trisaccharide
product, it was very difficult to isolate the anomers (α/β ratio 2/
1) by column chromatography.54 Instead of benzyl ether, these
acceptors with acetyl or benzoyl ester as the protective groups
were applied. Due to the disarming effect of acetyl or benzoyl
protective groups, both acceptors 3 and 4 afforded a mixture of
trisaccharide compounds (α:β ratio 1:2.5 or 1:1.6, respectively)
with a low yield (20−30%) (Table 1). This result was similar to a
previous report.56
The synthesis of lipid-modified GM3 conjugates is outlined in
Scheme 2. Instead of the [1 + 2] glycosylation reactions, an
alternative [2 + 1] synthetic sequence was carried out, which
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Scheme 2. Synthetic Routes to GM3-αGalCer, GM3-βGalCer, GM3-Pam, and GM3-Pam2 (A) and GM3-Chol (B)
targeted conjugate GM3-Chol. All the lipid-modified GM3
conjugates were purified by size-exclusion chromatography, and
their purities were confirmed by 1H and 13C NMR spectroscopy
(see the Supporting Information).
To prepare the coating antigen for ELISA (Scheme 3), the
terminal amino group derived from 21 was reacted with diethyl
squarate in EtOH/H2O at pH 8.0 resulting in squaric acid
monoamide 23,65 which was dissolved together with BSA in a
buffer solution at a pH of 9.5 to afford GM3-BSA conjugates (on
average 7.4 molecules of GM3 per molecule of BSA). The
loading of the GM3 antigen on BSA was determined by MALDI-
TOF mass spectrometry (see the Supporting Information).
To investigate the multivalency of the GM3 antigen, all the
GM3 conjugates were formulated into liposomes. In terms of
covalent modality, GM3-αGalCer was incorporated into
liposomes by hydration of a thin film of DSPC, cholesterol
(Chol), and GM3-αGalCer (molar ratios 5:4:1) in a HEPES
buffer (10 mm, pH 6.5) containing NaCl (145 mm), followed by
sonication for 15 min. The above formulation replacing GM3-
αGalCer with GM3-βGalCer was used as a control. For the
noncovalent modality, liposomes of mixtures of lipid-anchoring
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Scheme 3. Preparation of GM3-BSA Conjugate as Coating Antigen for ELISA
GM3 conjugates with αGalCer [molar ratios: DSPC/Chol/
αGalCer/GM3-Pam (or GM3-Pam2, GM3-Chol, GM3-
βGalCer) 5:4:1:1] in the HEPES buffer were prepared via the
same method as that of covalent modality. The contents of GM3
antigen (1.5 μg) and αGalCer (2 μg) were consistent in all
vaccine candidates. The physicochemical properties of the
vaccine candidate liposomes were evaluated (Figure S4). The
dynamic light scattering (DLS) confirmed that the liposomes are
sized with an approximate diameter range of 200−800 nm.
Furthermore, the surface charges represented by zeta potential
of liposomes were measured, all vaccine candidates had negative
zeta potentials.
Next, groups of five female BALB/c mice were immunized
intraperitoneally with freshly prepared liposomes at biweekly
intervals (days 1, 15, and 29), and sera were collected on days 14,
28, and 42. To determine whether the vaccine candidates can
promote cytokine production, the secretion of IL-4 and IFN-γ
after the first immunization was evaluated (Figure 1). All groups
produced a high level of IL-4 and IFN-γ compared to the control
GM3-βGalCer. This rapid production of cytokines indicated
that the iNKT cells were activated for all the vaccine candidates.
The cytokines induced between covalent and noncovalent
candidates were not significantly different, confirming that 6-
position conjugation exerts a marginal impact on the
immunological activity of αGalCer. Interestingly, although the
contents of αGalCer are consistent among the noncovalent
vaccine candidates, GM3-Chol/αGalCer is more potent than
GM3-βGalCer/αGalCer in promoting Th1-biasing cytokine
response, indicating that the physicochemical properties of
liposomes might affect the pharmacokinetics of αGalCer leading
to different Th1/Th2 profiles.66,67
We then measured the antibody titers of IgM and IgG in
serum by ELISA using GM3-BSA as the coating antigen (Figure
2). In terms of IgG, all noncovalent vaccine candidates but
GM3-βGalCer/αGalCer failed to generate any notable
amounts of anti-GM3 IgG responses in comparison to GM3-
βCalCer on days 14, 28, and 42. Interestingly, although the
dipalmitoyl lipid anchor exhibits high efficacy in cancer vaccines
targeting MUC1,42 the candidate GM3-Pam2/αGalCer did not
display significant potency. Consistent with our previous studies
on vaccines targeting STn28 and nicotine hapten,30 the self-
adjuvanting vaccine candidate GM3-αGalCer stimulated robust
IgG antibody response, leading to 6, 11, and 26-fold higher IgG
titers than GM3-βGalCer at days 14, 28, and 42, respectively.
GM3-βGalCer/αGalCer elicited an effective IgG response
similar to that of the covalent counterpart GM3-αGalCer. No
significant difference was observed between them, suggesting
Figure 1. (A) IL-4 and (B) IFN-γ production induced by the vaccine
candidates. The serum concentrations of cytokines were evaluated by
ELISA at the indicated time points after the first injection. The data are
indicated as the average value
SEM. Asterisks without brackets
indicate significant difference to the control GM3-βGalCer using one-
way ANOVA followed by Dunnett’s multiple comparison test. Asterisks
with brackets indicate significant difference using an unpaired two-
tailed Student’s t test. ns: not significant.
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Figure 2. (A) Anti-GM3 IgM and (B) IgG response after intraperitoneal immunization of female BALB/c mice at days 1, 15, and 29 with the vaccine
candidates. The data are indicated as the average SEM, and individual symbols represent individual mouse titers. Asterisks without brackets indicate
significant difference to control GM3-βGalCer (days 14, 28, and 42, respectively) using one-way ANOVA followed by Dunnett’s multiple comparison
test. Asterisks with brackets indicate significant difference using an unpaired two-tailed Student’s t test. ns: not significant.
that βCalCer is the optimal lipid anchor. Notably, a significant
increase was seen in the levels of IgG in mice receiving GM3-
αGalCer and GM3-βGalCer/αGalCer following the second
immunization, but the magnitude of the response only slightly
increased following the third immunization, which might
contribute to iNKT cell anergy.68−70 In constrast, with respect
to anti-GM3 IgM, the self-adjuvant vaccine candidate GM3-
αGalCer stimulates high levels of IgM production following the
first immunization, and the titers decrease gradually, indicating
an effective antibody isotype switching from IgM to IgG. In
contrast, GM3-βGalCer/αGalCer caused lower levels of IgM
production, observed at day 14, this level of production was
consistent until the end of the experiment. The pattern of IgG
subclasses was also analyzed (Figure 3). Except for these groups
immunized with GM3-αGalCer and GM3-βGalCer/αGalCer,
other noncovalent vaccine candidates induced no significant
levels of four IgG subtypes compared to GM3-βGalCer. No
significant difference was observed between GM3-αGalCer and
GM3-βGalCer/αGalCer for all IgG subtypes. Remarkably,
unlike our study with STn antigen, higher levels of IgG3 over
IgG1 were observed, which could be partly attributed to GM3
mediated suppression of T helper type 2-like NKT-cell
responses.71
The optimized lipid anchor in the noncovalent modality leads
to a potent vaccine candidate that induces comparable levels of
anti-GM3 IgG to covalent candidate GM3-αGalCer and also
demonstrated similar subtype profiles, which suggests that
noncovalent formulation is feasible as an alternative to self-
adjuvanting vaccines and is a potential approach for the facile
preparation of the vaccine. Importantly, although all non-
covalent vaccine candidates can provide appropriate danger
signals and stimulate the rapid production of comparable levels
of cytokines, the antibody response differs greatly, which could
be partly attributed to the different amphiphilicity and resultant
pharmacokinetics between lipid-modified GM3-lipid and
αGalCer. Remarkably, we found that GM3-Pam and GM3-
Chol are more soluble in MeOH than GM3-Pam2 and GM3-
βGalCer, while αGalCer is almost insoluble in MeOH.
Therefore, βGalCer might enable the amphiphilicity of lipid-
anchoring GM3 to be more analogous to that of αGalCer than
other lipid anchors, creating effective antigen/danger signal
coformulations.72
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Figure 4. FACS of the induced binding of sera to B16F10 cells by the
indicated vaccine candidates (negative control experiment was
performed with the sera before immunization (green) and PBS
(gray)). FITC-rabbit antimouse IgG was used for staining.
Figure 3. IgG subtype profiles at day 42. Data are represented as the
mean SEM in each group (n = 5), individual symbols represent
individual mouse titers. Asterisks without brackets indicate a significant
difference to control GM3-βGalCer using one-way ANOVA followed
by Dunnett’s multiple comparison test. Asterisks with brackets indicates
significant difference using unpaired two-tailed Student’s t test. ns: not
significant.
Figure 5. CDC for B16F10 and THP-1 cells that were activated by
antisera that were induced in mice through vaccine candidates. RC-
inactive: cell viability of inactivated rabbit complement, which was used
for the negative control experiment; RC, rabbit complement. Data are
Next, the ability of the antisera to recognize the GM3 antigen
presented on highly metastatic B16F10 melanoma cells was
evaluated by flow cytometry. As can be seen in Figure 4, antisera
obtained from immunization with GM3-αGalCer displayed
significant binding activity to GM3-expressing B16F10 cancer
cells, and the percentage of positive cells was 47.9% (Figure S7)
with enhanced mean fluorescent intensity (MFI: 45436).
Although the sera obtained from mice immunized with GM3-
βGalCer/αGalCer elicited IgG antibody titers equally as high as
GM3-αGalCer, a slightly impaired recognition of B16F10
cancer cells was observed (positive cells 34.1%, MFI: 29275).
This result indicated that the covalent linker between
carbohydrate antigen and adjuvant was important for cancer
cell recognition, consistent with a previous study by Boons et
al.73
represented as the mean
SEM in each group (n = 4). Asterisks
without brackets indicate significant differences to RC using one-way
ANOVA followed by Dunnett’s multiple comparison test. Asterisks
with brackets indicate significant differences using an unpaired two-
tailed Student’s t test. ns: not significant.
the complement system and stimulate significant cytotoxicity to
B16F10 cells (cell lysis rate 60%), which was much higher than
preimmune serum (cell lysis rate 41%). Under the same
conditions, no significant cytotoxicity was observed for the
antisera from GM3-βGalCer/αGalCer. Meanwhile, acute
monocytic leukemia (THP-1) was used as the control due to
the fact that it lacked the GM3 antigen on the cell surface.74,75
There was no statistically significant cytotoxicity observed in
THP-1 cells of each group. This result demonstrated that the
antisera of GM3-αGalCer exhibited a moderate cytokine toxicity
to B16F10 cells, but no cytotoxicity to other cells which lacked
GM3 antigen.
Finally, to evaluate the ability of activate the complement
system, the complement-dependent cytotoxicity (CDC) assay
was assessed and the percent of lysed cell was determined by
applying a tetrazolium bromide (MTT) assay. As shown in
Figure 5, antisera from GM3-αGalCer could effectively activate
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10:1, v/v) to provide the unprotected glucosyl derivative as
intermediate (575 mg, 38% for two steps). To a stirred solution of
the unprotected glucosyl derivative (440 mg, 1.5 mmol) in anhydrous
CH3CN (2 mL) was added benzaldehyde dimethyl acetal (2 mL, 1.32
mmol) and a catalytic amount of CSA (3.5 mg, 0.015 mmol) at rt. After
stirring at rt for 2 h, the reaction mixture was quenched by Et3N (4.2 μL,
0.03 mmol) and diluted with DCM (10 mL). After being washed with
saturated NaHCO3 (10 mL), the organic layer was dried (MgSO4),
concentrated, and purified by silica chromatography (DCM/MeOH,
30:1, v/v) to give 9 (520 mg, 90.6%) as a white solid; 1H NMR (600
MHz, CDCl3): δ 7.57−7.43 (m, 2H), 7.37 (d, J = 6.4 Hz, 3H), 5.53 (s,
1H), 4.45 (d, J = 7.8 Hz, 1H), 4.33 (dd, J = 10.6, 5.0 Hz, 1H), 4.04 (dt, J
= 11.5, 3.7 Hz, 1H), 3.79 (dt, J = 26.6, 9.9 Hz, 3H), 3.74−3.62 (m, 3H),
3.54 (dt, J = 15.9, 8.8 Hz, 2H), 3.46 (dd, J = 9.8, 4.9 Hz, 1H), 3.39 (t, J =
5.0 Hz, 2H). 13C NMR (150 MHz, CDCl3): δ 136.87, 129.23, 128.29,
126.25, 103.47, 101.83, 80.37, 74.41, 72.91, 70.15, 69.94, 69.14, 68.56,
66.35, 50.51. ESI-MS calcd. for C17H23N3O7Na+ [M + Na]+ 404.14,
found 404.19.
2-(2-Azido-ethoxy)-ethyl 2,3,6-Tri-O-benzyl-β-D-glucopyranoside
(10). Benzylidene acetal 9 (520 mg, 1.36 mmol) was dissolved in DMF
(5 mL), and NaH (60% in mineral oil, 163.4 mg, 4.08 mmol) was added
at 0 °C. After 5−10 min, benzyl bromide (648 μL, 5.4 mmol) was added
dropwise at 0 °C and the reaction mixture was allowed to stir for 0.5 h at
rt. After completion, the reaction was quenched with MeOH (5 mL).
The solvent was evaporated under reduced pressure, and the residue
was purified by silica chromatography (PE/EA, 2:1, v/v) to give the
fully protected glucosyl derivative 9′ (564 mg, 80.2%) as a white solid;
1H NMR (400 MHz, CDCl3): δ 7.48 (d, J = 6.4 Hz, 2H), 7.40−7.31 (m,
9H), 7.28 (dd, J = 13.1, 5.5 Hz, 4H), 7.20 (s, 1H), 5.55 (s, 1H), 4.92
(dd, J = 11.2, 8.5 Hz, 2H), 4.79 (dd, J = 11.2, 8.6 Hz, 2H), 4.56 (d, J =
7.7 Hz, 1H), 4.34 (dd, J = 10.5, 5.0 Hz, 1H), 4.02 (dt, J = 9.8, 4.3 Hz,
1H), 3.84−3.71 (m, 3H), 3.69 (d, J = 7.2 Hz, 3H), 3.61 (t, J = 5.2 Hz,
2H), 3.48 (t, J = 8.1 Hz, 1H), 3.40 (td, J = 9.6, 5.0 Hz, 1H), 3.28 (q, J =
4.1 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 138.37, 137.19, 128.82,
128.20, 128.17, 128.12, 127.90, 127.55, 127.50, 125.88, 104.13, 100.96,
81.87, 81.27, 80.66, 75.03, 74.97, 70.29, 69.86, 69.32, 68.62, 65.87,
50.50. ESI-MS calcd. for C31H35N3O7Na+ [M + Na]+ 584.24, found
584.38. Then, compound 9′ (50 mg, 0.1 mmol) was dissolved in dry
DCM (5 mL), followed by addition of Et3SiH (189 μL, 1.2 mmol). The
mixture was stirred at rt for 10 min and then cooled to −5 °C, and BF3·
OEt2 (25 μL, 0.2 mmol) was added and stirred at −5 °C for another 6 h.
The reaction was quenched by Et3N (50 μL) and washed with saturated
NaHCO3. The organic layer was dried over MgSO4 and concentrated.
The residue was purified by silica chromatography (PE/EA, 2:1, v/v) to
give 10 as a colorless oil (42 mg, 79.7%). 1H NMR (400 MHz, CDCl3):
δ 7.46−7.24 (m, 15H), 4.95 (dd, J = 17.3, 11.2 Hz, 2H), 4.72 (d, J =
11.2 Hz, 2H), 4.63−4.52 (m, 2H), 4.47 (d, J = 6.7 Hz, 1H), 4.04 (dt, J =
10.9, 4.2 Hz, 1H), 3.83−3.72 (m, 2H), 3.69 (dd, J = 10.3, 5.3 Hz, 2H),
3.63−3.57 (m, 2H), 3.45 (q, J = 4.8, 4.3 Hz, 3H), 3.29 (dd, J = 6.3, 4.1
Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 138.47, 138.43, 137.79,
128.48, 128.35, 128.30, 127.99, 127.92, 127.79, 127.66, 127.61, 103.77,
83.81, 81.48, 75.20, 74.47, 73.94, 73.56, 71.25, 70.37, 70.08, 69.85,
69.05, 50.57. ESI-MS calcd. for C31H37N3O7Na+ [M + Na]+ 586.25,
found 586.21.
p-Tolyl (Methyl 5-Acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbon-
yl-3,5-dideoxy-D-glycero-α,β-D-galacto-non-2-ulopyranosylonate)-
(2 → 3)-(2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-galactopyra-
noside (13). The donor 13 was prepared as previously described.59 13
was purified by silica flash chromatography with toluene and EA (6/1 to
3/1) as the eluent to give 13 as a white solid (α-glycoside 347 mg, 75%;
β-glycoside 37 mg, 8%). 13α: 1H NMR (600 MHz, CDCl3): δ 8.20−
8.11 (m, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.48 (dd, J = 7.9, 5.4 Hz, 4H),
7.44−7.30 (m, 6H), 7.09−6.99 (m, 2H), 5.53 (d, J = 5.6 Hz, 2H), 5.34
(s, 2H), 4.92 (d, J = 9.7 Hz, 1H), 4.58 (dd, J = 9.6, 3.5 Hz, 1H), 4.51−
4.42 (m, 2H), 4.36 (d, J = 12.3 Hz, 1H), 4.12 (dd, J = 14.5, 8.3 Hz, 2H),
4.08 (d, J = 3.5 Hz, 1H), 3.99 (dd, J = 12.2, 6.3 Hz, 1H), 3.70 (d, J = 21.8
Hz, 2H), 3.53−3.47 (m, 1H), 3.44 (s, 3H), 2.89 (dd, J = 12.1, 3.2 Hz,
1H), 2.43 (s, 3H), 2.34 (d, J = 13.5 Hz, 4H), 2.19 (s, 3H), 2.08−2.00
(m, 5H). 13C NMR (150 MHz, CD2Cl2): δ 172.01, 171.08, 170.76,
170.20, 168.91, 165.35, 153.79, 138.57, 138.26, 133.92, 133.60, 130.66,
CONCLUSION
■
Given the unique immunology of iNKT cells and the valuable
target, GM3, for diverse cancer immunotherapy, we evaluated
two vaccine modalities to deliver antigen GM3 and adjuvant
αGalCer to draining lymph nodes. The covalent vaccine
candidate GM3-αGalCer is capable of promoting a high level
of anti-GM3 IgG response, which exhibits strong binding
activity to cancer cells and efficient activation of the complement
system, underlining the importance of covalent linkers for the
improvement of cancer vaccines. However, the limitation of
covalent vaccine is that the ratio of antigen and adjuvant is fixed,
and in some cases, the adjuvant’s activity may be significantly
influenced by the modification with linker. To facilitate the
preparation of vaccines, an alternative modality was also
explored. Although the induced antibodies have a slightly
reduced recognition of B16F10 cancer cells, the noncovalent
vaccine candidate GM3-βGalCer/αGalCer with the optimized
lipid anchor could potentiate a comparable magnitude of IgG
titer to GM3-αGalCer, suggesting that lipid anchors play an
important role in altering antigen pharmacokinetics, and that
lipidation of carbohydrate antigens has great potential in the
preparation of simple and effective iNKT cell-based synthetic
carbohydrate vaccines.
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Information. All reactions were
carried out under a dry Ar atmosphere using oven-dried glassware and
magnetic stirring. The solvents were dried before use as follows: THF
and Et2O were heated at reflux over sodium benzophenone ketyl;
toluene was heated at reflux over sodium; dichloromethane was dried
over CaH2. Anhydrous N,N-diisopropylethylamine and triethylamine
were used directly as purchased. Commercially available reagents were
used without further purification unless otherwise noted. Aluminum
TLC sheets (silica gel 60 F254) of 0.2 mm thickness were used to
monitor the reactions. The spots were visualized with short wavelength
UV light or by charring after spraying with a solution prepared from one
of the following solutions: phosphomolybdic acid (5.0 g) in 95% EtOH
(100 mL); p-anisaldehyde solution (2.5 mL of p-anisaldehyde, 2 mL of
AcOH, and 3.5 mL of conc. H2SO4 in 100 mL of 95% EtOH); or
ninhydrin solution (0.3 g ninhydrin in 100 mL of n-butanol; add 3 mL
AcOH). Flash chromatography was carried out with silica gel 60 (230−
400 ASTM mesh). NMR spectra were obtained on a 400-MHz or 600-
MHz spectrometer. Proton chemical shifts are reported in ppm from an
internal standard of residual chloroform (7.26 ppm) or methanol (3.31
ppm), and carbon chemical shifts are reported using an internal
standard of residual chloroform (77.0 ppm) or methanol (49.1 ppm).
Proton chemical data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, and br = broad), coupling constant, and integration.
Chemical shifts were referenced on residual solvent peaks: CDCl3 (δ
= 7.26 ppm for 1H NMR and 77.00 ppm for 13C NMR), CD2Cl2 (δ =
1
5.32 ppm for H NMR and 53.84 ppm for 13C NMR), CD3OD (δ =
3.31 ppm for 1H NMR and 49.00 ppm for 13C NMR), D2O (δ = 4.79
ppm for 1H NMR). Matrix-assisted laser desorption/ionization time-of-
flight (MALDI-TOF) MS was performed on an AB SCIEX 5800
spectrometer (Shimadzu AXIMA Assurance).
2-(2-Azido-ethoxy)-ethyl 4,6-O-Benzylidene β-D-Glucopyranoside
(9). To a solution of penta-O-acetylglucose 8 (2 g, 5.12 mmol) and 2-(2-
azidoethoxy) ethan-1-ol (0.88 g, 6.6 mmol) in anhydrous DCM (10
mL) was added BF3·Et2O (0.82 mL, 6.66 mmol). The reaction mixture
was stirred at rt for 24 h and diluted with saturated aqueous NaHCO3
(10 mL). The organic layer was separated, washed with aqueous
NaHCO3 (10 mL), dried over Na2SO4, and concentrated. The crude
product was dissolved in 0.5 M NaOMe (5 mL) and stirred at rt for 0.5
h. After completion, the reaction was neutralized to pH 6−8 with 1 M
HCl. After the solvent was evaporated under reduced pressure, the
residue was purified by flash column chromatography (DCM/MeOH,
1958
https://dx.doi.org/10.1021/acs.jmedchem.0c01186
J. Med. Chem. 2021, 64, 1951−1965

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
130.41, 129.87, 129.46, 128.84, 128.55, 126.81, 101.47, 97.35, 86.13,
75.29, 73.90, 71.75, 69.83, 69.54, 68.46, 68.32, 63.95, 59.02, 36.91,
24.82, 21.55, 21.30, 20.97, 20.65. 13β: 1H NMR (600 MHz, CDCl3): δ
8.08−7.95 (m, 2H), 7.64−7.56 (m, 1H), 7.53−7.43 (m, 4H), 7.43−
7.31 (m, 5H), 7.16 (t, J = 7.3 Hz, 1H), 7.07 (d, J = 7.9 Hz, 2H), 5.84 (t, J
= 1.9 Hz, 1H), 5.64 (s, 1H), 5.59−5.51 (m, 1H), 5.32 (t, J = 9.7 Hz,
1H), 5.09 (dd, J = 12.1, 2.6 Hz, 1H), 4.81 (d, J = 10.0 Hz, 1H), 4.49 (dd,
J = 12.4, 3.8 Hz, 3H), 4.41−4.35 (m, 1H), 4.33−4.27 (m, 1H), 4.19−
4.10 (m, 1H), 3.90 (dd, J = 12.1, 9.3 Hz, 1H), 3.72 (s, 1H), 3.63 (s, 1H),
3.50 (dd, J = 11.3, 9.5 Hz, 1H), 3.19 (s, 3H), 2.58 (dd, J = 12.1, 3.6 Hz,
1H), 2.35 (d, J = 2.6 Hz, 4H), 2.32 (s, 3H), 2.14 (s, 3H), 2.08 (s, 4H),
2.03 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 171.39, 171.07, 170.93,
169.96, 169.31, 165.70, 164.79, 153.35, 138.42, 137.69, 134.56, 133.02,
130.34, 129.71, 129.46, 129.14, 128.36, 128.09, 126.67, 126.43, 100.95,
99.39, 84.77, 75.89, 75.55, 75.10, 74.17, 74.09, 71.88, 69.67, 69.05,
68.25, 63.13, 58.96, 52.62, 36.64, 29.63, 24.32, 21.27, 21.02, 20.86,
20.60. ESI-MS calcd. for C46H49NO18SNa+ [M + Na]+ 958.25, found
958.27.
2-(2-Azido-ethoxy)-ethyl (Methyl 5-Acetamido-7,8,9-tri-O-acetyl-
5-N,4-O-carbonyl-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulo-
pyranosylonate)-(2 → 3)-(2-O-benzoyl-4,6-O-benzylidene-β-D-gal-
actopyranosyl)-(1 → 4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside
(14). Glycosyl donor 13 (80 mg, 0.086 mmol) and acceptor 10 (57
mg, 0.10 mmol) were dissolved in anhydrous DCM (3 mL) containing
activated 4 Å powdered molecular sieves (100 mg) under argon
atmosphere and then cooled to −20 °C, stirred 1 h, and followed by
addition of NIS (38 mg, 0.172 mmol) and TfOH (2.5 μL, 0.025 mmol).
The reaction mixture was stirred at −20 °C for 20 min and gradually
raised to 0 °C for a period of 2 h, then quenched with triethylamine (20
μL) and warmed to rt. The mixture was diluted with DCM (5 mL) and
filtered through a pad of Celite. The filtrate was washed with 20%
aqueous Na2S2O3 solution, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The resulting residue was
purified by flash column chromatography (6:1, DCM/EA, v/v) using
silica gel to give 14 (72 mg, 60%) as a light-yellow solid. 1H NMR (600
MHz, CDCl3): δ 8.14 (d, J = 7.6 Hz, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.52
(dd, J = 6.7, 3.0 Hz, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.37−7.27 (m, 8H),
7.27−7.14 (m, 10H), 5.56 (dt, J = 13.5, 9.6 Hz, 2H), 5.48 (dd, J = 10.1,
7.9 Hz, 1H), 5.34 (s, 1H), 5.09 (d, J = 11.3 Hz, 1H), 5.02 (d, J = 8.0 Hz,
1H), 4.89 (dd, J = 15.1, 11.2 Hz, 2H), 4.67 (d, J = 11.2 Hz, 1H), 4.50−
4.39 (m, 3H), 4.35 (d, J = 7.9 Hz, 1H), 4.29 (d, J = 11.9 Hz, 1H), 4.20
(d, J = 12.0 Hz, 1H), 4.06 (d, J = 12.2 Hz, 1H), 3.99 (dd, J = 12.4, 7.1
Hz, 1H), 3.95 (dd, J = 11.6, 4.1 Hz, 1H), 3.88 (dd, J = 11.1, 6.7 Hz, 2H),
3.77−3.72 (m, 1H), 3.72−3.62 (m, 3H), 3.60 (d, J = 10.7 Hz, 1H), 3.56
(t, J = 5.2 Hz, 1H), 3.52 (d, J = 10.2 Hz, 1H), 3.49 (s, 3H), 3.45 (dd, J =
10.9, 5.9 Hz, 1H), 3.40 (t, J = 8.5 Hz, 1H), 3.36 (d, J = 5.7 Hz, 2H), 3.23
(q, J = 5.2 Hz, 2H), 2.91 (dd, J = 12.1, 3.3 Hz, 1H), 2.44 (s, 3H), 2.19 (s,
3H), 2.00 (s, 3H), 1.78 (d, J = 12.9 Hz, 1H), 1.74 (s, 3H). 13C NMR
(150 MHz, CDCl3): δ 171.94, 170.82, 170.20, 168.40, 164.98, 153.40,
139.08, 138.39, 137.75, 133.27, 129.94, 128.85, 128.54, 128.18, 128.02,
127.92, 127.43, 127.38, 126.92, 126.44, 103.49, 101.06, 100.74, 97.04,
83.11, 81.73, 77.77, 77.21, 77.00, 76.79, 75.18, 75.01, 74.73, 74.45,
74.30, 73.02, 72.92, 72.81, 71.41, 71.15, 70.32, 69.82, 69.15, 69.00,
68.62, 67.83, 66.15, 63.67, 58.81, 52.91, 50.56, 36.81, 24.64, 21.36,
20.61. ESI-MS calcd. for C70H78N4O25Na+ [M + Na]+ 1397.49, found
1397.75.
(d, J = 11.2 Hz, 2H), 4.43 (dd, J = 10.9, 4.5 Hz, 2H), 4.37−4.32 (m,
1H), 4.31−4.23 (m, 2H), 4.11 (d, J = 12.5 Hz, 1H), 4.01 (ddd, J = 15.0,
7.6, 3.5 Hz, 3H), 3.97−3.85 (m, 3H), 3.81−3.71 (m, 1H), 3.66 (d, J =
18.6 Hz, 5H), 3.60−3.49 (m, 6H), 3.47 (s, 1H), 3.44−3.36 (m, 1H),
3.30 (d, J = 8.0 Hz, 2H), 3.23 (q, J = 3.5, 2.3 Hz, 2H), 2.68 (dd, J = 12.7,
4.5 Hz, 1H), 2.20 (s, 3H), 2.02 (s, 3H), 1.86 (d, J = 4.6 Hz, 6H), 1.63 (d,
J = 12.4 Hz, 1H). 13C NMR (150 MHz, CDCl3): δ 170.69, 170.27,
170.05, 168.33, 164.88, 155.24, 139.07, 138.60, 138.34, 137.82, 133.10,
129.86, 128.78, 128.48, 128.25, 128.17, 128.02, 127.97, 127.86, 127.67,
127.63, 127.39, 126.92, 126.44, 103.48, 100.82, 100.79, 96.78, 83.05,
81.68, 75.29, 74.50, 74.26, 73.36, 73.02, 72.47, 72.34, 71.65, 70.96,
70.31, 69.82, 68.99, 68.67, 67.27, 67.04, 66.21, 62.52, 55.10, 52.78,
50.56, 49.41, 38.20, 29.53, 23.23, 21.35, 20.75, 20.65. ESI-MS calcd. for
C71H82N4O26Na+ [M + Na]+ 1429.51, found 1429.39.
(2S,3S,4R)-3,4-Dibenzyloxy-2-hexacosanoylamino-octadecane-
(2,3-di-O-benzyl-6-deoxy-6-N-(2-(2-(methyl 5-Acetamido-7,8,9-tri-
O-acetyl-4-O-methoxycarbonyl-3,5-dideoxy-D-glycero-α-D-galac-
to-non-2-ulopyranosylonate)-(2 → 3)-(2-O-benzoyl-4,6-O-benzyli-
dene-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzyl-β-D-gluco-
pyranosyloxy)-ethoxy)-ethylamino)-oxohexanamido-α-D-galacto-
pyranoside (16). To a solution of 15 (20 mg, 0.014 mmol) in 5 mL of
wet MeOH was added PMe3 (140 μL, 0.14 mmol) at rt, and the
resulting solution was stirred for 3 h. Then, the solvent was
concentrated under reduced pressure. The residue was then subjected
to high vacuum at 30 °C for 24 h to remove the Me3PO byproduct. The
residue was dissolved in DCM (2 mL) and followed by addition of 16′
(α-GalCer-selenoester, 10 mg, 0.0067 mmol) and DIPEA (2.4 μL,
0.014 mmol). The reaction mixture was set to stir for 0.5 h at rt, then,
the mixture was evaporated and purified by column chromatography
(DCM/MeOH 20:1, v/v) to give 16 (18 mg, 98%) as a white solid. 1H
NMR (400 MHz, CDCl3): δ 8.09 (d, J = 7.8 Hz, 2H), 7.61 (t, J = 7.5 Hz,
1H), 7.57−7.45 (m, 3H), 7.44−7.31 (m, 32H), 7.28 (dd, J = 4.8, 2.2
Hz, 5H), 7.24−7.16 (m, 3H), 6.44 (s, 1H), 6.01 (d, J = 9.4 Hz, 2H),
5.58 (s, 1H), 5.48 (t, J = 9.0 Hz, 1H), 5.40 (s, 1H), 5.34−5.27 (m, 1H),
5.23 (d, J = 9.4 Hz, 1H), 5.17 (d, J = 11.1 Hz, 1H), 4.97 (d, J = 8.0 Hz,
1H), 4.90 (d, J = 11.5 Hz, 1H), 4.85 (d, J = 3.6 Hz, 1H), 4.81 (d, J = 7.6
Hz, 1H), 4.78−4.72 (m, 2H), 4.68 (d, J = 13.1 Hz, 1H), 4.62 (d, J = 12.3
Hz, 1H), 4.56−4.51 (m, 1H), 4.47 (dd, J = 12.8, 5.3 Hz, 1H), 4.38 (d, J
= 7.7 Hz, 1H), 4.32 (t, J = 11.7 Hz, 1H), 4.16 (d, J = 12.2 Hz, 1H),
4.10−4.01 (m, 2H), 4.01−3.87 (m, 3H), 3.82 (t, J = 9.9 Hz, 1H), 3.73
(s, 3H), 3.71−3.61 (m, 2H), 3.58 (d, J = 12.0 Hz, 3H), 3.52−3.40 (m,
2H), 3.16 (s, 1H), 2.73 (dd, J = 12.5, 3.6 Hz, 1H), 2.25 (s, 3H), 2.13 (s,
2H), 2.05 (d, J = 8.4 Hz, 4H), 1.99−1.93 (m, 1H), 1.90 (d, J = 7.9 Hz,
4H), 1.79 (s, 6H), 1.70−1.61 (m, 2H), 1.57 (d, J = 5.9 Hz, 3H), 1.28 (d,
J = 8.6 Hz, 63H), 0.92 (t, J = 6.7 Hz, 6H). 13C NMR (100 MHz,
CDCl3): δ 173.69, 173.07, 172.69, 170.65, 170.24, 170.06, 168.37,
164.90, 155.27, 139.07, 138.49, 137.89, 133.12, 132.04, 130.04, 129.88,
128.79, 128.52, 128.42, 128.36, 128.30, 128.25, 128.05, 127.98, 127.89,
127.83, 127.78, 127.70, 127.64, 126.98, 126.45, 103.55, 100.83, 99.38,
96.86, 83.16, 81.79, 79.90, 75.80, 75.34, 74.64, 74.36, 73.65, 73.40,
73.07, 72.47, 72.37, 72.10, 71.70, 71.04, 70.12, 69.71, 68.84, 68.72,
68.59, 68.42, 67.31, 67.14, 66.28, 62.57, 55.08, 52.77, 50.45, 49.57,
39.64, 39.08, 38.27, 36.71, 35.91, 35.72, 31.91, 29.72, 29.69, 29.45,
29.35, 25.72, 24.96, 24.75, 23.23, 22.67, 21.34, 20.74, 20.65, 14.10.
(2S,3S,4R)-3,4-Dibenzyloxy-2-hexacosanoylamino-octadecane-
(2,3-di-O-benzyl-6-deoxy-6-N-(2-(2-(methyl 5-Acetamido-7,8,9-tri-
O-acetyl-4-O-methoxycarbonyl-3,5-dideoxy-D-glycero-α-D-galac-
to-non-2-ulopyranosylonate)-(2 → 3)-(2-O-benzoyl-4,6-O-benzyli-
dene-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzyl-β-D-gluco-
pyranosyloxy)-ethoxy)-ethylamino)-oxohexanamido-β-D-galacto-
pyranoside (17). The preparation of 17 is similar to that of 16. White
solid, yield (45 mg, 95%). 1H NMR (600 MHz, CDCl3): δ 8.07 (d, J =
7.7 Hz, 2H), 7.58 (t, J = 7.6 Hz, 1H), 7.48 (dd, J = 19.0, 11.5 Hz, 4H),
7.32 (ddq, J = 33.6, 24.3, 7.3 Hz, 40H), 7.18 (d, J = 5.0 Hz, 3H), 6.78 (d,
J = 7.4 Hz, 1H), 6.07 (d, J = 5.8 Hz, 1H), 6.00 (d, J = 8.8 Hz, 1H), 5.55
(d, J = 7.7 Hz, 1H), 5.46 (t, J = 9.1 Hz, 1H), 5.38 (s, 1H), 5.32 (d, J = 9.7
Hz, 1H), 5.28 (d, J = 9.6 Hz, 1H), 5.15 (d, J = 11.2 Hz, 1H), 4.94 (d, J =
8.0 Hz, 1H), 4.86 (dd, J = 18.2, 11.1 Hz, 2H), 4.80 (s, 2H), 4.77 (s, 1H),
4.73−4.62 (m, 5H), 4.59 (d, J = 11.5 Hz, 1H), 4.51 (d, J = 11.5 Hz, 1H),
4.49−4.22 (m, 7H), 4.13 (d, J = 12.2 Hz, 1H), 4.03 (dt, J = 17.4, 9.4 Hz,
4H), 3.94 (t, J = 8.9 Hz, 3H), 3.88 (d, J = 3.4 Hz, 1H), 3.79 (d, J = 7.5
2-(2-Azido-ethoxy)-ethyl (Methyl 5-Acetamido-7,8,9-tri-O-acetyl-
4-O-methoxycarbonyl-3,5- dideoxy-D-glycero-α-D-galacto-non-2-
ulopyranosylonate)-(2 → 3)-(2-O-benzoyl-4,6-O-benzylidene-β-D-
galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside
(15). To a solution of 14 (20 mg, 0.015 mmol) in anhydrous MeOH (3
mL) was added freshly prepared solution of 1 M NaOMe (6 μL), and
the mixture was stirred at rt for 5−10 min, then neutralized with
Amberlite 15 (H+) resin. The resin was filtered off and washed with
DCM/MeOH (1/1, v/v), the filtrate was concentrated to provide a
residue 15 that was used directly in the next step without further
operation. 1H NMR (400 MHz, CDCl3): δ 8.06 (d, J = 7.7 Hz, 2H),
7.56 (t, J = 7.4 Hz, 1H), 7.52−7.40 (m, 5H), 7.30 (ddt, J = 24.2, 13.0,
7.4 Hz, 14H), 7.16 (t, J = 3.3 Hz, 3H), 5.59−5.50 (m, 1H), 5.44 (dd, J =
10.1, 7.9 Hz, 1H), 5.35 (s, 1H), 5.27−5.18 (m, 2H), 5.11 (d, J = 11.2
Hz, 1H), 4.94 (d, J = 8.0 Hz, 1H), 4.86 (dd, J = 11.2, 7.3 Hz, 2H), 4.68
1959
https://dx.doi.org/10.1021/acs.jmedchem.0c01186
J. Med. Chem. 2021, 64, 1951−1965

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 2H), 3.71 (d, J = 7.8 Hz, 4H), 3.63 (d, J = 8.5 Hz, 1H), 3.57 (d, J =
15.2 Hz, 5H), 3.50−3.36 (m, 3H), 3.36−3.13 (m, 6H), 2.70 (dd, J =
12.7, 4.5 Hz, 1H), 2.22 (s, 3H), 2.03 (s, 4H), 1.87 (d, J = 10.3 Hz, 6H),
1.74−1.58 (m, 5H), 1.56−1.41 (m, 9H), 1.26 (d, J = 19.3 Hz, 84H),
0.90 (t, J = 6.9 Hz, 7H). 13C NMR (150 MHz, CDCl3): δ 173.90,
172.92, 172.83, 170.73, 170.35, 170.28, 170.13, 168.37, 164.95, 155.28,
139.04, 138.50, 138.25, 137.86, 133.20, 129.91, 128.84, 128.39, 128.03,
127.86, 127.73, 127.05, 126.47, 104.19, 103.55, 100.82, 96.88, 83.19,
81.77, 80.75, 80.46, 78.83, 78.11, 77.28, 75.40, 75.18, 74.72, 74.31,
73.45, 73.11, 73.00, 72.89, 72.55, 72.35, 72.20, 71.90, 71.72, 71.03,
70.17, 69.70, 69.01, 68.87, 68.72, 67.30, 67.13, 66.77, 66.30, 65.95,
62.62, 55.14, 54.57, 52.84, 50.13, 49.44, 42.73, 39.84, 39.11, 38.26,
36.61, 35.66, 31.95, 29.39, 29.32, 26.01, 25.65, 24.96, 24.67, 23.27,
22.72, 21.40, 20.80, 20.71, 18.66, 17.36, 14.16.
29.64, 29.62, 29.55, 29.50, 29.37, 29.32, 29.29, 28.86, 25.78, 25.63,
23.18, 22.64, 22.29, 21.31, 20.71, 20.63, 14.08. ESI-MS calcd. for
C109H156N4O29Na+ [M + Na]+ 2008.08, found 2008.09.
2-(2-N-tert-Butyloxycarbonylamido-ethoxy)-ethyl-(methyl 5-
Acetamido-7,8,9-tri-O-acetyl-4-O-methoxycarbonyl-3,5-dideoxy-D-
glycero-α-D-galacto-non-2-ulopyranosylonate)-(2 → 3)-(2-O-ben-
zoyl-4,6-O-benzylidene-β-D-galactopyranosyl)-(1 → 4)- 2,3,6-tri-O-
benzyl-β-D-glucopyranoside (20). To the solution of azide 15 (50 mg,
0.036 mmol) in 2 mL DCM/MeOH/H2O (1/1/0.01, v/v/v) was
added PMe3 (360 μL, 0.36 mmol) under the argon atmosphere. The
reaction mixture was stirred at rt for 2 h, and then the solvent was
removed under reduced pressure. Then the residue was dissolved in 2
mL of DCM, followed by addition of Boc2O (15.8 mg, 0.072 mmol)
and DIPEA (12 μL, 0.072 mmol). The reaction mixture was stirred at rt
for 2 h. The solvents were evaporated under reduced pressure, and the
resulting residue was purified by flash column chromatography (DCM/
MeOH, 40:1, v/v) using silica gel to give 20 (48 mg, 89%) a colorless
solid. 1H NMR (600 MHz, CDCl3): δ 5.53 (s, 1H), 5.44 (t, J = 9.1 Hz,
1H), 5.35 (s, 1H), 5.28−5.23 (m, 1H), 5.17 (d, J = 9.9 Hz, 1H), 5.11 (d,
J = 11.1 Hz, 1H), 4.93 (d, J = 7.9 Hz, 2H), 4.86 (dd, J = 11.2, 3.7 Hz,
2H), 4.68 (d, J = 11.0 Hz, 1H), 4.46−4.38 (m, 2H), 4.38−4.29 (m,
2H), 4.26 (d, J = 12.0 Hz, 1H), 4.10 (d, J = 12.1 Hz, 1H), 4.05−3.98 (m,
1H), 3.97 (d, J = 3.7 Hz, 1H), 3.90 (dd, J = 18.6, 10.2 Hz, 3H), 3.75 (q, J
= 10.0 Hz, 1H), 3.69 (s, 2H), 3.68−3.55 (m, 1H), 3.53 (s, 2H), 3.52−
3.35 (m, 2H), 3.18 (d, J = 20.3 Hz, 2H), 2.69 (dd, J = 12.8, 4.6 Hz, 1H),
2.20 (s, 3H), 2.02 (s, 3H), 1.85 (d, J = 11.2 Hz, 6H), 1.65 (s, 4H). 13C
NMR (150 MHz, CDCl3): δ 170.28, 169.86, 169.65, 167.95, 164.47,
154.86, 138.71, 138.09, 137.46, 132.72, 129.49, 128.39, 128.11, 127.87,
127.81, 127.64, 127.59, 127.25, 126.54, 126.06, 103.14, 100.47, 100.40,
96.38, 82.72, 81.29, 74.92, 74.18, 73.91, 72.96, 72.64, 72.08, 71.97,
71.25, 70.59, 69.66, 68.48, 68.34, 68.30, 66.85, 66.66, 65.84, 62.14,
54.72, 52.39, 49.08, 39.84, 37.86, 27.99, 22.87, 20.98, 20.28. ESI-MS
calcd. for C76H92N2O28Na+ [M + Na]+ 1503.57, found 1503.39.
(2S,3S,4R)-3,4-Dihydroxy-2-hexacosanoylamino-octadecane-
(2,3-di-O-benzyl-6-deoxy-6-N-(2-(2-((5-acetamido-3,5-dideoxy-D-
glycero-α-D-galacto-non-2-ulopyranosylonic acid)-(2 → 3)-(β-D-
galactopyranosyl)-(1 → 4)-β-D-glucopyranosyloxy)-ethoxy)-ethyl-
amino)-oxohexanamido-α-D-galactopyranoside (GM3-αGalCer).
Glycolipid conjugate 16 (16 mg, 0.0067 mmol) was dissolved in
DCM/MeOH (1/1, v/v) and carefully degassed. Pearlman’s catalyst
(17 mg, 20% Pd on carbon, normally contain 50% water) was added.
The suspension was stirred under H2 atmosphere for 16 h. After
restoring Ar atmosphere, the reaction was filtered through a pad of
Celite which was prewashed with DCM/MeOH (1:1, v/v). The residue
obtained was dissolved in NaOMe (4 mL, 1:1, v/v) and stirred at 45 °C
for 3−4 h. A freshly prepared solution of NaOMe [prepared by addition
of 11.5 mg (0.5 mmol) sodium metal to 1 mL of MeOH, and cooled to
rt], it was neutralized with Amberlite 15 (H+) resin. The resin was
filtered off and washed with DCM/MeOH (1:1, v/v). The filtrate and
washings were combined and concentrated in vacuo to provide the
residue, to which was added 0.4 mL of THF/H2O (1:1, v/v) followed
by 0.1 mL of 2.0 M aqueous solution of NaOH at rt. After the solution
was stirred at rt overnight, it was neutralized with 1.0 M aqueous
solution of HCl to pH 7. Then, the reaction mixture was concentrated
in vacuo, then purified on a LH-20 column using DCM/MeOH (1:1, v/
v) as the eluent to give GM3-αGalCer after lyophilization as a white
solid (7 mg, 62% over 3 steps). 1H NMR (400 MHz, CDCl3/CD3OD
1/1): δ 4.90 (t, J = 4.2 Hz, 1H), 4.45 (d, J = 7.9 Hz, 1H), 4.38 (d, J = 7.6
Hz, 1H), 4.19 (s, 1H), 4.01 (q, J = 6.5, 6.1 Hz, 3H), 3.95−3.46 (m,
21H), 3.42 (d, J = 4.9 Hz, 2H), 2.79 (d, J = 12.7 Hz, 1H), 2.06 (d, J = 7.2
Hz, 5H), 1.94 (d, J = 12.4 Hz, 1H), 1.65 (s, 14H), 1.27 (s, 99H), 0.96 (t,
J = 7.5 Hz, 2H), 0.89 (t, J = 6.7 Hz, 10H). 13C NMR (100 MHz,
CDCl3/CD3OD 1/1): δ 103.79, 102.91, 99.75, 79.95, 77.71, 76.36,
75.22, 74.93, 74.83, 74.15, 74.01, 73.32, 72.83, 72.09, 71.79, 70.85,
69.88, 69.50, 69.31, 69.21, 68.69, 68.56, 68.15, 66.86, 64.29, 63.95,
63.81, 63.18, 63.10, 61.17, 60.72, 52.70, 52.35, 52.05, 50.49, 39.75,
39.63, 39.07, 36.17, 35.33, 34.29, 31.80, 31.79, 31.11, 29.70, 29.58,
29.52, 29.49, 29.46, 29.35, 29.24, 29.20, 29.07, 25.87, 25.79, 25.05,
22.50, 21.91, 18.51. HR ESI-MS calcd. for C83H154N4O30Na+ [M +
Na]+ 1710.0540, found 1710.0514.
2-(2-N-Hexadecanamide-ethoxy)-ethyl-(methyl 5-Acetamido-
7,8,9-tri-O-acetyl-4-O-methoxycarbonyl-3,5-dideoxy-D-glycero-α-
D-galacto-non-2-ulopyranosylonate)-(2 → 3)-(2-O-benzoyl-4,6-O-
benzylidene-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzyl-β-D-
glucopyranoside (18). To the DCM/1,4-dioxane (2 mL, 3/1, v/v)
solution of terminal amine prepared by Staudinger reduction of azide
15 (30 mg, 0.021 mmol) was added palmitic acid 18′ (6.7 mg, 0.026
mmol), EDC (6.2 mg, 0.032 mmol), and HOBt (4.4 mg, 0.032 mmol)
at rt. The reaction mixture was raised to 40 °C and stirred at 40 °C for
2−3 h, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (DCM/MeOH 20:1, v/v)
using silica gel to give 18 as a white solid. (32 mg, 89% over 2 steps). 1H
NMR (400 MHz, CDCl3): δ 8.23−7.88 (m, 2H), 7.57 (t, J = 7.4 Hz,
1H), 7.47 (dt, J = 15.3, 6.7 Hz, 4H), 7.40−7.21 (m, 18H), 7.20−7.12
(m, 3H), 5.83 (s, 1H), 5.54 (t, J = 6.7 Hz, 1H), 5.44 (dd, J = 10.1, 8.0
Hz, 1H), 5.35 (s, 1H), 5.25 (dd, J = 9.7, 2.2 Hz, 1H), 5.15 (dd, J = 20.1,
10.4 Hz, 2H), 4.93 (d, J = 8.0 Hz, 1H), 4.85 (dd, J = 14.6, 11.0 Hz, 2H),
4.68 (d, J = 11.1 Hz, 1H), 4.43 (dd, J = 10.8, 3.7 Hz, 2H), 4.37−4.32 (m,
1H), 4.31−4.21 (m, 2H), 4.11 (d, J = 12.3 Hz, 1H), 4.01 (ddd, J = 12.8,
8.3, 3.3 Hz, 3H), 3.96−3.87 (m, 3H), 3.81−3.73 (m, 1H), 3.73−3.23
(m, 15H), 2.69 (dd, J = 12.7, 4.6 Hz, 1H), 2.20 (s, 3H), 2.02 (s, 3H),
1.98 (td, J = 7.4, 2.1 Hz, 2H), 1.87 (s, 3H), 1.85 (s, 3H). 13C NMR (150
MHz, CDCl3): δ 173.21, 170.69, 170.27, 170.07, 168.34, 164.87,
155.26, 139.02, 138.40, 138.27, 137.83, 133.13, 129.89, 128.79, 128.51,
128.29, 128.26, 128.04, 127.98, 127.67, 127.63, 126.98, 126.45, 103.54,
100.84, 100.80, 96.79, 83.13, 81.76, 77.35, 75.38, 74.70, 74.29, 73.34,
73.05, 72.47, 72.35, 71.66, 70.97, 70.14, 69.86, 68.81, 68.68, 67.23,
67.06, 66.25, 62.55, 55.12, 52.79, 49.45, 38.97, 38.26, 36.55, 31.91,
29.67, 29.50, 29.35, 25.63, 23.25, 22.69, 21.37, 20.77, 20.67, 14.13. ESI-
MS calcd. for C87H114N2O27Na+ [M + Na]+ 1641.75, found 1641.77.
2-(2-N-(N-α, ε-Bis-hexadecanamide)-L-lysyl)-ethoxy)-ethyl-
(methyl 5-Acetamido-7,8,9-tri-O-acetyl-4-O-methoxycarbonyl-3,5-
dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonate)-(2 → 3)-
(2-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranosyl)-(1 → 4)-
2,3,6-tri-O-benzyl-β-D-glucopyranoside (19). The preparation of 19
1
is similar to that of 18. Yield (48 mg, 84%). H NMR (400 MHz,
CDCl3): δ 8.20−7.94 (m, 2H), 7.61−7.53 (m, 1H), 7.52−7.41 (m,
4H), 7.40−7.27 (m, 9H), 7.26 (d, J = 4.7 Hz, 6H), 7.19−7.12 (m, 3H),
6.51 (t, J = 5.6 Hz, 1H), 6.36 (d, J = 7.8 Hz, 1H), 5.76 (t, J = 5.8 Hz,
1H), 5.53 (ddd, J = 9.1, 6.1, 2.7 Hz, 1H), 5.44 (dd, J = 10.1, 7.9 Hz, 1H),
5.36 (s, 1H), 5.32 (d, J = 9.5 Hz, 1H), 5.26 (dd, J = 9.5, 2.3 Hz, 1H),
5.12 (d, J = 11.3 Hz, 1H), 4.93 (d, J = 8.0 Hz, 1H), 4.84 (dd, J = 11.2, 4.2
Hz, 2H), 4.68 (d, J = 11.1 Hz, 1H), 4.43 (dd, J = 10.7, 3.5 Hz, 2H),
4.38−4.23 (m, 4H), 4.16−4.08 (m, 1H), 4.07−3.97 (m, 3H), 3.96−
3.85 (m, 3H), 3.74 (dd, J = 16.3, 10.4 Hz, 1H), 3.69 (s, 3H), 3.68−3.62
(m, 1H), 3.62−3.55 (m, 2H), 3.53 (s, 3H), 3.49−3.36 (m, 2H), 3.31
(dd, J = 9.4, 4.2 Hz, 3H), 3.22−3.10 (m, 2H), 2.68 (dd, J = 12.7, 4.6 Hz,
1H), 2.19 (s, 3H), 2.16 (dd, J = 8.5, 6.9 Hz, 2H), 2.10 (dd, J = 8.6, 6.8
Hz, 3H), 2.02 (s, 3H), 1.86 (d, J = 2.8 Hz, 6H), 1.63−1.53 (m, 4H),
1.25 (d, J = 3.6 Hz, 48H), 0.88 (t, J = 6.8 Hz, 6H). 13C NMR (100 MHz,
CDCl3): δ 173.48, 173.37, 171.82, 170.66, 170.29, 170.23, 170.05,
168.36, 164.88, 155.24, 139.09, 138.53, 138.26, 137.85, 133.10, 129.99,
129.86, 128.76, 128.49, 128.28, 128.24, 128.20, 128.12, 128.02, 127.96,
127.89, 127.66, 127.49, 126.93, 126.42, 103.43, 100.79, 96.86, 83.06,
81.75, 75.26, 74.59, 74.26, 73.43, 73.04, 72.51, 72.33, 71.71, 71.00,
70.06, 69.49, 68.95, 68.70, 67.38, 67.12, 66.24, 62.55, 55.06, 52.74,
52.57, 49.44, 39.18, 38.63, 38.20, 36.74, 36.51, 32.11, 31.88, 29.66,
1960
https://dx.doi.org/10.1021/acs.jmedchem.0c01186
J. Med. Chem. 2021, 64, 1951−1965

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(2S,3S,4R)-3,4-Dihydroxy-2-hexacosanoylamino-octadecane-
(2,3-di-O-benzyl-6-deoxy-6-N-(2-(2-((5-acetamido-3,5-dideoxy-D-
glycero-α-D-galacto-non-2-ulopyranosylonic acid)-(2 → 3)-(β-D-
galactopyranosyl)-(1 → 4)-β-D-glucopyranosyloxy)-ethoxy)-ethyl-
amino)-oxohexanamido-β-D-galactopyranoside (GM3-βGalCer).
The preparation of GM3-βGalCer is similar to that of GM3-αGalCer.
3)-(β-D-galactopyranosyl)-(1 → 4)-β-D-glucopyranoside (GM3-
Chol). Trisaccharide 21 (12 mg, 0.014 mmol) was added 35% aqueous
trifluoroacetic acid solution (500 μL, TFA/H2O, 35/65, v/v) and kept
10−15 min at rt, then equal volume toluene was added, concentrated
via rotary evaporator. The residue was further dried at rt for 5 h under
high vacuum. Then, the residue was dissolved in DCM/DMF (1/1, 2
mL, v/v), followed by cholesteryl nitrophenyl carbonate 2264 (24 mg,
0.044 mmol) and DIPEA (3.8 μL, 0.022 mmol). The mixture was kept
stirring until TLC analysis (CHCl3/MeOH/0.2% CaCl2 50:50:1)
showed the complete consumption of amine. The residue was
coevaporated with toluene three times to remove the DMF and washed
with Et2O to remove the redundant cholesterol. The desired product
GM3-Chol was obtained from further purification by size exclusion
column chromatography (LH-20, eluted by DCM/MeOH 1:1), white
solid (11.5 mg, 70% over 2 steps). 1H NMR (600 MHz, CDCl3
/CD3OD 1/1): δ 5.33 (s, 1H), 4.34 (d, J = 45.7 Hz, 3H), 3.97 (s, 2H),
3.90−3.33 (m, 22H), 3.14 (d, J = 7.8 Hz, 1H), 2.76 (d, J = 29.6 Hz, 1H),
2.29 (s, 2H), 1.99 (d, J = 9.4 Hz, 5H), 1.83 (s, 3H), 1.66−0.73 (m,
30H), 0.66 (s, 3H). 13C NMR (100 MHz, CD3OD): δ 123.14, 104.84,
104.04, 77.41, 76.75, 76.42, 76.23, 76.05, 75.43, 75.09, 72.12, 70.93,
70.68, 70.59, 70.41, 69.56, 69.11, 64.64, 64.29, 62.46, 62.44, 57.86,
57.29, 55.58, 53.70, 53.40, 51.36, 49.43, 49.21, 49.00, 48.79, 48.57,
43.51, 40.85, 40.41, 39.41, 38.00, 37.09, 36.83, 32.95, 32.74, 28.99,
28.86, 25.03, 24.65, 22.89, 22.65, 22.42, 22.30, 21.88, 19.51, 18.95,
18.08, 14.11, 12.88, 12.03. HR ESI-MS calcd. for C55H92N2O22Na+ [M
+ Na]+ 1155.6034, found 1155.6053.
2-(2-N-(2-Ethoxy-3,4-dioxocyclobut-1-en-1-yl)-aminoethoxy)-
ethyl-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulo-
pyranosylonic acid)-(2 → 3)-(β-D-galactopyranosyl)-(1 → 4)-β-D-
glucopyranoside (23). The crude amine prepared by deprotection of
Boc group in trisaccharide 21 (20 mg, 2.43 μmol) was dissolved in
EtOH/H2O (v/v = 1/1, 1 mL). A solution of aqueous NaHCO3
(saturated) was added until reaching pH = 8.0. Then the mixture was
treated with 3,4-diethoxy-3-cyclobutene-1,2-dione (12.4 mg, 7.32
μmol). After stirring for 1.5 h at rt, the mixture was neutralized by
AcOH. The solvent was evaporated under reduced pressure, and the
residue was triturated with EA to removed nonpolar impurities. The
crude product was dissolved in minimal MeOH, and the pure product
was precipitated after acetone was gradually added. The precipitate was
dried to provide 23 (18 mg, 87%) as a colorless solid. 1H NMR (600
MHz, CD3OD): δ 4.74 (dd, J = 11.0, 6.7 Hz, 2H), 4.43 (d, J = 8.0 Hz,
1H), 4.35 (d, J = 7.8 Hz, 1H), 4.10−4.02 (m, 1H), 4.01−3.45 (m,
21H), 3.42 (s, 1H), 3.26 (s, 1H), 2.86 (d, J = 12.4 Hz, 1H), 2.01 (s, 3H),
1.72 (d, J = 11.4 Hz, 1H), 1.46 (t, J = 7.1 Hz, 3H). ESI-MS calcd. for
C33H51N2O23− [M − H]− 843.30, found 842.73.
GM3-BSA. To the solution of squaric acid monoamide 23 (8 mg,
0.0095 mmol) in 5 mL of 70 mM Na2B4O7 and 35 mM KHCO3
aqueous solution was added BSA (21 mg, 0.31 μmol) at rt. After being
incubated at rt for 48 h, the mixture was centrifuged using the Microcon
(Millipore) under 6000g for 15 min. And the resulting aqueous solution
was lyophilized to give GM3-BSA conjugate. The average loading of
GM3 on BSA was 7.4, the value was estimated by MALDI-TOF-MS.
Immunological Test. Materials and Reagents. DSPC was
purchased from TCI. Cholesterol was purchased from Energy
Chemical. Peroxidase-conjugated AffiniPure goat antimouse kappa,
IgA, IgE, IgG1, IgG2a, IgG2b, and IgG3 antibodies were purchased
from Southern Biotechnology, and peroxidase-conjugated AffiniPure
goat antimouse kappa antibody IgG and IgM were purchased from
Jackson ImmunoResearch. Mouse IL-4 and IFN-γ ELISA kits were
obtained from BD Pharmingen. B16F10 cells was purchased from
China Infrastructure of Cell Line.
Liposomal Formulation of Vaccine Candidates. Liposomal
formulations of vaccine candidates were prepared by following a
previously reported protocol.27,29,41 As for covalent vaccine candidate
GM3-αGalCer and control GM3-βGalCer, the liposomes consist of
DSPC, cholesterol, and GM3-αGalCer (or GM3-βGalCer) in a molar
ratio of 5:4:1. In terms of noncovalent vaccine candidates, the
liposomes consist of DSPC, cholesterol, αGalCer, and GM3-Pam
(GM3-Pam2, GM3-Chol, or GM3-βGalCer) in a molar ratio of
5:4:1:1. After the mixture of corresponding ingredients was dissolved in
1
White solid, yield (21 mg, 78% over 3 steps). H NMR (600 MHz,
CDCl3/CD3OD 1/1): δ 4.41 (d, J = 7.8 Hz, 1H), 4.34 (d, J = 7.5 Hz,
2H), 4.18 (d, J = 6.8 Hz, 3H), 4.08 (s, 1H), 3.97 (s, 3H), 3.90−3.80 (m,
3H), 3.74 (d, J = 10.1 Hz, 4H), 3.69−3.45 (m, 21H), 3.39 (s, 5H), 2.75
(d, J = 12.8 Hz, 1H), 2.24 (d, J = 27.6 Hz, 11H), 2.02 (d, J = 3.6 Hz,
4H), 1.89 (d, J = 12.0 Hz, 1H), 1.61 (s, 10H), 1.49 (s, 1H), 1.23 (s,
121H), 0.84 (t, J = 6.8 Hz, 13H). 13C NMR (150 MHz, CDCl3/
CD3OD 1/1, v/v): δ 176.06, 175.49, 174.97, 171.53, 104.44, 104.40,
103.53, 99.05, 80.42, 77.13, 76.02, 75.68, 75.47, 74.77, 74.53, 73.98,
73.84, 73.73, 72.67, 71.92, 71.81, 70.59, 70.15, 69.94, 69.81, 69.55,
69.28, 68.77, 67.78, 64.35, 61.96, 61.30, 53.13, 50.99, 40.35, 39.74,
36.95, 36.11, 32.54, 32.10, 30.33, 30.26, 29.98, 29.95, 26.61, 25.78,
23.25, 22.67, 14.36. HR ESI-MS calcd. for C83H154N4O30Na+[M + Na]+
1710.0540, found 1710.0516.
2-(2-N-Hexadecanamide-ethoxy)-ethyl-(5-acetamido-3,5-di-
deoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonic acid)-(2 →
3)-(β-D-galactopyranosyl)-(1 → 4)-β-D-glucopyranoside (GM3-
Pam). The preparation of GM3-Pam is similar to that of GM3-
αGalCer. White solid, yield (12 mg, 67% over 3 steps). 1H NMR (600
MHz, CD3OD): δ 4.62 (s, 2H), 4.42 (d, J = 7.8 Hz, 1H), 4.34 (d, J = 7.9
Hz, 1H), 4.04 (d, J = 9.7 Hz, 1H), 3.98 (dd, J = 10.0, 5.6 Hz, 1H), 3.94−
3.80 (m, 6H), 3.79−3.66 (m, 9H), 3.56 (ddt, J = 18.5, 13.0, 7.5 Hz,
10H), 3.48 (d, J = 8.9 Hz, 1H), 3.41 (d, J = 8.9 Hz, 2H), 3.35 (q, J = 5.4
Hz, 2H), 3.26 (t, J = 8.7 Hz, 2H), 2.84 (d, J = 12.8 Hz, 1H), 2.19 (t, J =
7.5 Hz, 2H), 2.00 (s, 3H), 1.72 (s, 1H), 1.58 (s, 2H), 1.28 (s, 26H), 0.89
(t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CD3OD): δ 104.80, 104.02,
80.69, 77.35, 76.76, 76.24, 76.01, 74.65, 74.44, 72.67, 70.90, 70.55,
70.33, 69.81, 69.53, 69.07, 68.71, 64.28, 62.44, 61.70, 53.68, 41.81,
39.96, 36.79, 32.77, 30.48, 30.35, 30.17, 30.02, 26.74, 23.43, 22.31,
14.14. HR ESI-MS calcd. for C43H78N2O21Na+ [M + Na]+ 981.4989,
found 981.4968.
2-(2-N-(N-α, ε-Bis-hexadecanamide)-L-lysyl)-ethoxy)-ethyl-(5-
acetamido-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosy-
lonic acid)-(2 → 3)-(β-D-galactopyranosyl)-(1 → 4)-β-D-glucopyr-
anoside (GM3-Pam2). The preparation of GM3-Pam2 is similar to that
1
of GM3-αGalCer. White solid, yield (8 mg, 49% over 3 steps). H
NMR (400 MHz, CDCl3/CD3OD 1/1, v/v): δ 4.44−4.24 (m, 5H),
4.04−3.49 (m, 25H), 3.45 (d, J = 8.9 Hz, 1H), 3.41−3.34 (m, 2H), 3.13
(t, J = 6.9 Hz, 3H), 2.77 (d, J = 12.9 Hz, 1H), 2.35 (t, J = 7.4 Hz, 1H),
2.22 (q, J = 9.7, 7.7 Hz, 3H), 2.13 (t, J = 7.6 Hz, 2H), 2.05−1.95 (m,
5H), 1.89−1.73 (m, 4H), 1.58 (p, J = 7.5 Hz, 8H), 1.48 (t, J = 7.3 Hz,
2H), 1.25 (d, J = 14.1 Hz, 76H), 0.95−0.76 (m, 14H). 13C NMR (100
MHz, CDCl3/CD3OD): δ 104.32, 103.29, 80.41, 78.20, 76.67, 76.05,
75.47, 75.17, 74.04, 73.77, 71.95, 70.36, 69.81, 68.98, 63.91, 61.98,
61.24, 41.21, 39.47, 39.24, 36.68, 36.43, 32.25, 32.08, 30.00, 29.98,
29.96, 29.94, 29.89, 29.87, 29.81, 29.73, 29.67, 29.57, 29.15, 27.44,
26.34, 26.17, 23.28, 22.96, 14.04. HR ESI-MS calcd. for
C65H120N4O23Na+ [M + Na]+ 1347.8236, found 1347.8279.
2-(2-N-tert-Butyloxycarbonylamido-ethoxy)-ethyl-(5-acetamido-
3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonic acid)-
(2 → 3)-(β-D-galactopyranosyl)-(1 → 4)-β-D-glucopyranoside (21).
The preparation of 21 from 20 is similar to that of GM3-αGalCer.
1
White solid, yield (14 mg, 84%, over 3 steps). H NMR (600 MHz,
CD3OD): δ 4.43 (d, J = 7.8 Hz, 1H), 4.35 (t, J = 7.2 Hz, 1H), 4.09. −
3.96 (m, 3H), 3.89 (dt, J = 7.8, 4.6 Hz, 4H), 3.85−3.80 (m, 3H), 3.79−
3.71 (m, 7H), 3.65 (tt, J = 18.6, 10.0 Hz, 9H), 3.60−3.45 (m, 10H),
3.41 (d, J = 9.3 Hz, 2H), 3.29−3.17 (m, 4H), 2.77 (dd, J = 12.9, 4.3 Hz,
1H), 2.00 (s, 3H), 1.88 (t, J = 11.8 Hz, 1H), 1.43 (s, 9H).13C NMR
(150 MHz, CD3OD): δ 175.26, 172.51, 105.02, 104.26, 100.02, 80.72,
77.74, 76.79, 76.44, 76.22, 75.30, 74.69, 72.83, 72.62, 71.16, 71.03,
70.68, 70.02, 69.81, 69.42, 68.74, 64.71, 62.42, 61.85, 53.69, 41.18,
28.76, 22.67. ESI-MS calcd. for C32H55N2O22− [M − H]− 819.33, found
819.80.
2-(2-N-Cholesterylcarbamate-ethoxy)-ethyl-(5-acetamido-3,5-
dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosylonic acid)-(2 →
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Article
a mixture of DCM/MeOH (1:1, v/v, 2 mL), the solvents were removed
under reduced pressure through rotary evaporation, which generated a
thin lipid film on the flask wall. This film was hydrated and subjected to
freeze/thaw cycles to produce multilamellar vesicles; the film was
hydrated with HEPES buffer (20 mM, pH 7.5) containing NaCl (150
mM), and the mixture was sonicated 15 min at rt before use.
Immunization of Mice. Female BALB/c mice of 6−8 weeks age used
for immunological studies were purchased from Laboratory Animal
Centre of Huazhong Agriculture University. All mice were used
according to the animal ethics guidelines (Regulations of Hubei
Province on the Administration of Experimental Animals and Measures
of Hubei Province on Administration of Laboratory Animal Licenses).
Groups of five female BALB/c mice (age 6−8 weeks) were bred in the
Laboratory Animal Centre of Huazhong Agriculture University. Mice
were immunized by intraperitoneal injection on days 1, 15, and 29. The
mice were bled on day 0 before initial immunization and on days 14, 28,
and 42 after boost immunizations. In addition, sera collected at 2 and 24
h after the first injection were analyzed for the secretion of cytokines
IFN-γ and IL-4. Mouse blood samples were clotted to obtain antisera
that were stored at −80 °C before use.
In vivo Cytokine Assay. The cytokine levels in sera were evaluated
using ELISA kits (IFN-γ and IL-4, BD Pharmingen) according to the
manufacturer’s protocol. Briefly, 96-well plates were coated with
capture antibodies dissolved in the coating buffer per well and
incubated overnight at 4 °C. The wells were then blocked with BSA for
1 h at rt. After blocking, 100 μL/well of standard, sera, or control were
added and incubated for 2 h at 28 °C. After washing, the working
detector (detection antibody and Sav-HRP reagent) was added to each
well. The plates were incubated for 1 h at 28 °C. Then, the plates were
washed, and the tetramethyl benzidine (TMB) substrate solution was
added. The reactions were stopped after 30 min at 28 °C with a
stopping solution. The absorbance was measured at 450 nm by the plate
reader.
with medium without FBS, then incubated with these antisera (diluted
1:25, 50 μL per well) from vaccinated mice for 1 h. After washing with
PBS solution, the rabbit complement (at a rabbit sera dilution of 1:50)
in 1% BSA/PBS was added. The rabbit complement inactivated by
treatment at 65 °C for 30 min was used as the control. After incubation
for 2 h, 0.5% MTT solution in PBS was added (20 μL/well) and
incubated for 2 h. After removing the medium, DMSO was added (150
μL/well) and the absorption was analyzed at the wavelength of 490 nm.
The survival rate of cells was measured with the following formula. All
assays were conducted in four independent experiments. Cytotoxicity
(%) = [1-(experimental OD/control OD)] × 100.
Statistical Analysis. Data reported in the figures were analyzed, and
charts were generated using Prism 6 (GraphPad Software). All values
and error bars are mean SEM. Significance between two groups was
determined by unpaired two-tailed Student’s t test. Significance of
multiple groups in comparison to the control group was evaluated using
one-way ANOVA followed by a Dunnett’s multiple comparison test.
Asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤
0.001) indicate significant differences.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01186.
■
sı
MALDI-TOF mass spectrometry determination of the
average level of GM3 incorporated on BSA protein,
ELISA evaluation of anti-GM3 antibody levels of IgA and
IgE on Day 42, and 1H and 13C NMR spectra of the new
compounds (PDF)
Molecular formula strings data (CSV)
ELISA Procedure. A 96-well microtiter plate (Costar type 3590,
Corning Inc.) was first coated with GM3-BSA (2 μg/plate), which had
been dissolved in a 0.1 M bicarbonate buffer (pH = 9.6). The plate was
then incubated at 4 °C overnight. The plate was then washed three
times with PBST (0.05% Tween-20 in PBS), followed by the addition of
3% (w/v) BSA in PBS to each well and incubation at 37 °C for 1 h. After
the plate was washed again with PBST, serially diluted sera were added
to microwells (100 μL/well) and incubated for 1 h at 37 °C and washed
(three times). A 1:5000 diluted solution of horseradish peroxidase
(HRP)-conjugated goat antimouse IgG (γ-chain specific) or IgM (μ-
chain specific), IgG1, IgG2a, IgG2b, IgG3, IgA, or IgE in 0.1% BSA/
PBS (100 μL per well) was added to each well, respectively. The plate
was incubated for 1 h at 37 °C. After a final wash (three times),
substrate solutions that were freshly prepared with a 9.5 mL critic buffer
at pH 5.0, 0.5 mL (1.6 mg/mL) TMB, and 32 μL 3% (w/v) urea
hydrogen peroxide were added to the wells (100 μL per well). Color
was allowed to develop in the dark for 8 min and then a solution of 2 M
H2SO4 was added to quench the reaction. The optical density was
measured at 450 nm. The antibody titer was defined as the highest
dilution showing an absorbance of 0.1, after subtracting the
background. Preimmunization sera have been used as the negative
control.
FACS Assay Protocol. B16F10 cells were cultured in RPMI-1640
containing 10% FBS and 1% antibiotic. After, B16F10 cells were
harvested and washed with FACS buffer (PBS containing 5% FBS). The
cells were incubated with normal or anti-GM3 serum induced by the
vaccine candidates (1:10 dilution in FACS buffer, contained PBS (1×),
10% FBS, and 0.1% sodium azide) for 1 h at 4 °C. Then cells were
washed three times with 1% BSA/PBS and incubated with FITC-linked
goat antimouse kappa antibody (Alexa Fluor 488-conjugated Goat
Anti-Mouse IgG (H+L), Jackson ImmunoResearch, diluted 1:50) for
30 min at 4 °C. After washing three times with 1% BSA in PBS, the cells
were added to the FACS buffer and analyzed by flow cytometry (BD
Calibur).
AUTHOR INFORMATION
Corresponding Author
■
Jun Guo − Key Laboratory of Pesticide & Chemical Biology,
Ministry of Education, Hubei International Scientific and
Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China; orcid.org/0000-0002-2097-5054;
Email: jguo@mail.ccnu.edu.cn
Authors
Xu-Guang Yin − Key Laboratory of Pesticide & Chemical
Biology, Ministry of Education, Hubei International Scientific
and Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
Jie Lu − Key Laboratory of Pesticide & Chemical Biology,
Ministry of Education, Hubei International Scientific and
Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
Jian Wang − Key Laboratory of Pesticide & Chemical Biology,
Ministry of Education, Hubei International Scientific and
Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
Complement Dependent Cytotoxicity Assay (CDC). B16F10 cells
or THP-1 cells76 (1 × 104 cell/well) were seeded in the wells of the 96-
well plate. After incubation at 37 °C overnight, the plate was washed
1962
https://dx.doi.org/10.1021/acs.jmedchem.0c01186
J. Med. Chem. 2021, 64, 1951−1965

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Ru-Yan Zhang − Key Laboratory of Pesticide & Chemical
Biology, Ministry of Education, Hubei International Scientific
and Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
kin-4; iNKT, invariant natural killer T; MHC, major
histocompatibility complex; MTT, 3-(4,5-dimethyl-2-thiazol-
yl)-2,5-diphenyl-2-H-tetrazolium bromide; NIS, N-iodosuccini-
mide; PE, petroleum ether; PBS, phosphate-buffered saline;
SEM, standard error of measurement; TACAs, tumor-associated
carbohydrate antigens; TfOH, trifluoromethanesulfonic acid;
Th, T helper; TLR, toll-like receptor
Xi-Feng Wang − Key Laboratory of Pesticide & Chemical
Biology, Ministry of Education, Hubei International Scientific
and Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
Chun-Miao Liao − Key Laboratory of Pesticide & Chemical
Biology, Ministry of Education, Hubei International Scientific
and Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
Xiao-Peng Liu − Key Laboratory of Pesticide & Chemical
Biology, Ministry of Education, Hubei International Scientific
and Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China
Zheng Liu − Key Laboratory of Pesticide & Chemical Biology,
Ministry of Education, Hubei International Scientific and
Technological Cooperation Base of Pesticide and Green
Synthesis, International Joint Research Center for Intelligent
Biosensing Technology and Health, College of Chemistry,
Central China Normal University, Wuhan, Hubei 430079, P.
R. China; orcid.org/0000-0002-3696-339X
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01186
Funding
This work was supported by the National Key Research and
Development Program of China (2017YFA0505200), the
National Natural Science Foundation of China (21977034
and 21772056), Wuhan Bureau of Science and Technology
(2018060401011323, 2020020601012217), the self-deter-
mined research funds of CCNU from the colleges’ basic
research and operation of MOE (CCNU20TS016 and
CCNU20TS014), and Program of Introducing Talents of
Discipline to Universities of China (111 program, B17019).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
αGalCer, α-galactosylceramide; APCs, antigen-presenting cells;
BSA, bovine serum albumin; CDC, complement-dependent
cytotoxicity; CTL, cytotoxic T lymphocyte; DCs, dendritic cells;
DCM, dichloromethane; DIPEA, diisopropylethylamine;
DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; EA, ethyl
acetate; ELISA, enzyme-linked immunosorbent assay; EDC, 1-
ethyl-3-(3-(dimethylamino)propyl)-carbodiimide; FACS, fluo-
rescence activated cell sorter; FBS, fetal serum albumin; HEPES,
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; HOBt, 1-
hydroxy-1H-benzotriazole; IFN-γ, interferon-γ; IL-4, interleu-
1963
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