Synthesis and biological activities of amino acids functionalized α-GalCer analogues

Article abstract of DOI:10.1016/j.bmc.2019.115141

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD

Full text of DOI:10.1016/j.bmc.2019.115141

Journal Pre-proofs
Synthesis and Biological Activities of Amino Acids Functionalized α-GalCer
Analogues
Weiwei Ma, Jingjing Bi, Chuanfang Zhao, Zhiguo Zhang, Tongxin Liu,
Guisheng Zhang
PII:
S0968-0896(19)31312-4
https://doi.org/10.1016/j.bmc.2019.115141
BMC 115141
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 July 2019
20 September 2019
23 September 2019
Please cite this article as: W. Ma, J. Bi, C. Zhao, Z. Zhang, T. Liu, G. Zhang, Synthesis and Biological Activities
of Amino Acids Functionalized α-GalCer Analogues, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.115141
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Synthesis and Biological Activities of Amino Acids
Functionalized α-GalCer Analogues
Weiwei Ma a, Jingjing Bi a,*, Chuanfang Zhao b, Zhiguo Zhang a, Tongxin Liu a,
Guisheng Zhang a, *
a
Key Laboratory of Green Chemical Media and Reactions, Ministry of Education,
Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine
Chemicals, Henan Key Laboratory of Organic Functional Molecule and Drug
Innovation, School of Chemistry and Chemical Engineering, Henan Normal University,
Xinxiang, Henan 453007, China.
b
School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing
100049, China.
* Corresponding author. E-mail address: bijingjing2008@126.com and zgs@htu.cn
Fax: (+86)-373-332-5250.
Abstract
Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating
the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines
upon the engagement of their T cell receptor with glycolipid antigens presented by
CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were
synthesized by incorporation of L-amino acid methyl esters in the C-6’ position of
glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate
iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated
that they were potent CD1d ligands and could stimulate murine spleen cells into a
higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-
GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3
showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with
α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as
efficient X-ray crystallographic probes to reveal the relationship between glycolipids

and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new
potent immunostimulating agents.
Keywords
α-GalCer, iNKT cells; Th1 response; amino acids functionalized α-GalCers.
1. Introduction
Regulation of iNKT-cells by glycolipids is a promising approach to treat cancer and
some autoimmune diseases.1,2 When the iNKT-cells are activated by glycolipid
antigens, they could rapidly secrete cytokines which could be classified as
proinflammatory T-helper 1 (Th1) type (e.g., IFN-γ, TNF α, IF-2) and
immunomodulatory T-helper 2 (Th2) type (e.g., IL-4, IL-10).3-6 However, these two
kinds of cytokines have opposing roles in vivo. The Th1 is involved in antitumor and
anti-infection effects, whereas the Th2 is suited to control the development of
autoimmune diseases. The negative cross-regulation of Th1 and Th2 cytokines and high
levels of both cytokines release are considered as a major limitation of glycolipids for
clinical applications.7-13 Consequently, a major challenge for glycolipid-based therapies
is the search for glycolipid antigens which can selectively control the release of Th1 or
Th2 cytokines.
Since the first synthetic α-galactosylceramide (α-GalCer, KRN7000, Figure 1) was
developed from systematic structure-activity relationship studies,14 various derivatives
of α-GalCer modifying on sugar part, ceramide moiety, galactose-ceramide linkage, or
the incorporation of unsaturation in the acyl chain, have been synthesized to improve
the cytokine production activity and selectivity.15-23 For example, α-GalCer analogues
with C-modified glycosidic linkages (α-C-GalCer) generated a higher Th1-biased
response and had been applied to the murine model for melanoma metastasis.24
Nevertheless, analogues containing truncated acyl and sphingosine chains (OCH),25
multiple cis-double bonds in the acyl chain (C20:2)26 or an amide group in the shorter
fatty-acylchain27 potently induced a Th2-biased cytokine response. Up to now, the

approximate immunological mechanism of α-GalCer is that by binding with the CD1d
protein, the binary complex is recognized by the T cell receptor (TCR) on iNKT-cells
to form the bioactive ternary complex. And then it stimulates iNKT cells to release Th1
and/or Th2 cytokines.28 The stability of the CD1d/glycolipid/TCR complex is an
important contributing factor to the proportion of Th1/Th2 cytokine production.29-32
The crystal structure of CD1d/α-GalCer/TCR complex33,34 shows that the 2, 3 and 4-
OH of the galactose unit form hydrogen bonds with TCR or CD1d while the 6-OH
shows no obvious interactions with TCR and CD1d, implying that chemical
modifications at this position are feasible. Savage’ group first reported that acetamide
group35 (PBS-57), different amide-linked fluorophores or biotin36 appended at the C6’
position of the sugar could also activate iNKT-cells and participate in CD1d and TCR
interactions, which could be used in studying glycosphingolipid transportation, but had
no obvious Th1/Th2-biased selectivity. Among the more recent reports, 37-44 the sugar
modified by C6’-substituted methyl or COOH showed minor cytokine secretion in vivo
in mice and resulted in no polarization.37 α-GalCer analogues modified by aryl amides,
carbamates and ureas at 6’-position α-GalCer analogues revealed a strongly biased Th1
response 3-12 times higher than α-GalCer39. However, acyl chain with length 12-13
(carbon atoms) at C6’ of α-GalCer or the 6’-amido group linked to an ethylene glycol
chain may have stronger ability to trigger Th2 immune response.42 Moreover, literature
search revealed that the presence of a triazole ring at position C6’43,44 sufficed to cause
superior antigenic activity than α-GalCer. C6’-triazole-substituted α-GalCer analogues
could induce both IL-4 and IFN-γ secretion in vivo, and elicited a small Th2 cytokine-
biased response. The 1,4-disubstituted triazole has a large dipole moment and is capable
of participating in hydrogen bond formation, dipole-dipole and -stacking interactions.
And a triazole moiety can act as H-bonding acceptor and form extra hydrogen bond
between compound and CD1d.44 These examples demonstrate that chemical
modifications at C6’ position are tolerated, but the relationship between the structural
changes of C6’-site substitution and cytokine release is not particularly clear.

Figure 1. The structure of α-GalCer and new designed analogues of α-GalCer.
Amino acids play significant roles in living organisms. Furthermore, in modern
medicinal chemistry, amino acids are indispensable components and become
increasingly prominent in new drugs discovery.45 For example, the oral bioavailability
of 5’-amino acid ester prodrugs of Decitabine (DAC) was greater than that of DAC.46
The phenylalanine-based organometallic complexes show anti-lung cancer and anti-
angiogenic activities by using L-amino acid transport system.47
The well-known crystal structure of the CD1d/α-GalCer/TCR complex indicated
that galactose part of α-GalCer was recognized by the T cell receptor (TCR) to form a
stable complex and the 6-OH group of galactose in the crystal structure of CD1d/α-
GalCer/TCR complex showed no obvious interactions with TCR.48 Structural analyses
of CD1d and TCR indicated that there were hydrophilic residues ASP-29, ASN-30,
ASP-51, GLN-52, LYS-68 and LYS-71 in the binding site of TCR from the crystal
structure of CD1d/α-GalCer/TCR complex. Therefore, the hydrophilic substitutes
introduced at C6’ of α-GalCer may form hydrogen bond interactions or salt-bridge
interaction with these residues, The introduced aryl groups may form π-cation, π-anion
interaction with these hydrophilic residues as well. Based on these analyses, several
glycolipids with different amino acid methyl esters were designed by introducing
hydrophilic groups at C6’ of α-GalCer with triazole as a linker (structures refer to Fig.
1), expecting to develop new glycolipid antigens and explore the influence of amino
acids on Th1/Th2 cytokines selectivity. Preliminary biological evaluation of these α-
GalCer derivatives containing different C6’-substituted triazole-moieties were also
carried out both in vivo and in vitro.

2. Results and Discussion
2.1. Computational modeling and activity predication
To predict the binding ability of newly designed α-GalCer analogues with CD1d and
TCR, a docking study was performed with the Surflex-Dock program in SYBYL-X
software (Tripos Inc., St. Louis, MO, USA). As shown in Table S1, the TotalScore of
the newly designed α-GalCer analogues 1-4 was higher than α-GalCer, which means
these newly designed glycolipids had stronger CD1d, TCR affinities than α-GalCer.
Detailed structure analyses indicated that the binding interaction of newly designed
glycolipids was very similar to α-GalCer (Fig. 2). For Gly-α-GalCer (1), there was
hydrophilic interaction between MET-69 and the newly introduced groups. For Ser-α-
GalCer (2), the newly introduced hydroxyl formed a hydrogen bond with GLN-52 of
TCR and enhanced their TCR affinity. For Lys-α-GalCer (3), it was hoped that the
ammonium would form hydrogen bonds with polar residues in TCR. Unexpectedly it
formed a hydrogen bond with the carbonyl of LYS-65 of CD1d and the methyl formed
π…H-C interaction with aryl residue HIS-68 of CD1d, which strengthened their CD1d
binding interaction. Similar to Lys-α-GalCer, there was π…H-C interaction between
the newly introduced group in Phe-α-GalCer (4) and HIS-68 and MET-69 of CD1d.

Ser-α-GalCer (2)
Gly-α-GalCer (1)
Lys-α-GalCer (3)
Phe-α-GalCer (4)
Figure 2. Structure analyses of ternary complexes of newly designed glycolipids (shown in cyan) within
the mCD1d (shown in green) and TCR (shown in orange). Hydrogen bond interactions between ligand
and residues around it were labeled in red dot line and the carbon skeleton of the ligands was shown in
blue.
2.2. Chemistry
We synthesized the desired glycolipid analogues starting from the synthesis of
propiolic acid amides 7 in Scheme 1. Propiolic acid 5 condensed with different L-
amino-acid methyl esters 6 in the presence of DCC and DIPEA49 to afford propiolic
acid amides 7a-d with good yields.
O
R
O
R
(a)
+
H2N
N
OH
COOMe
COOMe
H
6a
6b
6c
6d
, R = H
5
7a
7b
7c
7d
, R = H
, R = CH2OH
, R = (CH2)4NHCBz
, R = CH2Ph
, R = CH2OH
, R = (CH2)4NHCBz
, R = CH2Ph
Scheme 1. Reagents and conditions: (a) DCC, DIPEA, CH2Cl2, 0 oC-r.t.; 82% for 7a, 79% for 7b, 75%
for 7c, 80% for 7d. DCC, dicyclohexylcarbodiimide; DIPEA, N,N-diisopropylethylamine.

Synthesis of glycosyl donor 12 starting from the known 1,2,3,4,6-penta-O-acetyl-D-
galactose 8 was shown in Scheme 2. The galactose pentaacetate and p-thiocresol were
catalyzed by boron trifluoride diethyl etherate to afford thio-β-D-galactopyranoside 9
in a good yield. Subsequent deacetylation under Zemplén conditions yielded 10
quantitatively. Treatment of thiogalactoside 10 with p-toluenesulfonyl chloride50 and
then benzyl bromide yielded compound 11 in two steps with a total yield of 69%. Then
51
the compound 11 reacted with sodium azide in DMF
to afford the donor 12 in
excellent yield.
OH
HO
OAc
O
OAc
O
AcO
AcO
AcO
AcO
(a)
(b)
O
STol
HO
OAc
STol
OH
OAc
OAc
8
10
9
N3
OTs
O
BnO
BnO
(e)
(c, d)
O
STol
STol
BnO
BnO
OBn
OBn
12
11
.
o
Scheme 2. Reagents and conditions: (a) p-Thiocresol, BF3 OEt2, CH2Cl2, 0 C-r.t., 69%; (b) NaOMe,
MeOH, r.t., quantitative; (c) TsCl, Py., r.t.; (d) NaH, BnBr, DMF, r.t., 69% for 2 steps; (e) NaN3, DMF,
o
65 C, 90%. TsCl, 4-toluene sulfonyl chloride; Py., pyridine; BnBr, benzyl bromide; DMF, N,N-
dimethylformamide.
52
With glycosyl donor 12 and our sphingosine acceptor 13
in hand, amino acids
functionalized α-GalCer analogues could be prepared in Scheme 3. Firstly, the coupling
of C6’ azido galactose donor 12 with acceptor 13 was carried out successfully in the
presence of NIS/TfOH to provide compound 14 with 65% yield with α-configuration
as a major product. 53 Then, the reaction of azide 14 and propiolic acid amides 7 in the
presence of CuSO4 and sodium ascorbate provided the desired C6’-triazole-substituted
α-GalCer analogues 15a-15d in excellent yields. Final debenzylation by catalytic
hydrogenolysis and debenzoylation with NaOMe in MeOH afforded the desired
analogues 1-4 in approximate 88-93% yields.

H
N
R
O
(CH2)24CH3
NH OBz
(b)
N3
O
O
BzO
BzO
N3
O
(a)
BnO
BnO
(CH2)24CH3
OBz
O
COOMe
+
HN
STol
HO
7a
7b
7c
7d
, R = H
, R = CH2OH
, R = (CH2)4NHCBz
, R = CH2Ph
BzO
C
14H29
O
OBn
C14H29
OBz
OBz
14
12
13
R
R
O
O
COOMe
COOMe
N
N
H
N
H
N
N
N
(c, d)
N
N
O
O
HN
BnO
BnO
(CH2)24CH3
HO
HO
(CH2)24CH3
OH
O
O
HN
OBz
C
BnO
HO
O
O
14H29
C14H29
OBz
OH
15a
15b
15c
15d
, R = H
, R = CH2OH
, R = (CH2)4NHCBz
, R = CH2Ph
1
2
3
4
, R = H
, R = CH2OH
, R = (CH2)4NH2 Lys--GalCer
, R = CH2Ph Phe--GalCer
Gly--GalCer
Ser--GalCer
Scheme 3. Reagents and conditions: (a) NIS, TfOH, CH2Cl2, 0oC-r.t., 65%; (b) 7, CuSO4, sodium
ascorbate, THF:H2O = 1:1, 65 oC, 88% for 15a, 83% for 15b, 85%; for 15c, 86% for 15d; (c) Pd(OH)2,
H2, r.t., (d) NaOMe, MeOH, r.t., 91% for 1, 93% for 2, 92% for 3, 91% for 4 (over 2 steps). NIS, N-
iodosuccinimide; TfOH, trifluoromethanesulfonic acid.
2.3. Biological evaluation
2.3.1. Cytotoxicity and cell reproductive activity assay
The α-GalCer/CD1d binary complex formed by α-GalCer and CD1d protein interacts
with T cell receptor (TCR) of iNKT cells to activate immune responses.28 This activity
produces a large number of Th1 or Th2 cytokines release. Before the cytokine
production assays, the MTT assay was carried out to evaluate the cytotoxicity and cell
reproductive activity of these amino acids functionalized α-GalCer analogues on
murine splenocytes.54 Cell viability was examined after splenocytes treated with these
novel glycolipid analogues for 48 h at concentrations of 1, 0.1, and 0.01 µM,
respectively. As the results illustrate in Figure 3, all of the four glycolipid analogues
were found no cytotoxicity to the splenocytes at experimental concentration. The
splenocyte cells proliferation increased in a dose-dependent manner, and at the
concentration of 1 µM, these compounds all revealed acceptable cell proliferative
activity.

Figure 3. Effect of analogues 1-4 on splenocyte toxicity and proliferation. Splenocytes were treated
with analogues 1-4 for 48 h, and proliferation of splenocytes was estimated with the MTT method.
2.3.2. Cytokine production assay
To determine the immunostimulatory activity of the new synthetic analogues 1-4, the
in vitro assays were first manipulated using the primary culture of murine spleen cells.
55 Three concentrations (0.01-1 µM) of analogues were incubated with freshly prepared
mouse splenocytes and the level of cytokine productions was evaluated by ELISA kits
after incubation for 48 hours. α-GalCer was used as a positive control in this assay. As
shown in Figure 4, cytokine estimation revealed that all of the four analogues exhibited
iNKT-cell stimulating activity by concentration-dependent increases and maximized at
1 µM, just like α-GalCer. 54 It showed the efficacy to release IFN-γ and IF-4 that was
comparable to the standard α-GalCer. As can be seen from Figure 4A, Gly-α-GalCer
(1) presented a similar ability to induce the release of IFN-γ comparing to α-GalCer.
Compounds 2, 3 and 4, with serine, lysine and phenylalanine methyl ester substitution
respectively, were found to produce more significant IFN- cytokine levels at all three
concentrations. In comparison with the IFN- production, the levels of IL-4 cytokine
secretion induced by 1, 3 and 4 were inferior to those observed for α-GalCer (Figure
4B). However, Ser-α-GalCer (2) showed a similar tendency and ability to induce the
release of IL-4 in mouse splenocytes. Lys-α-GalCer (3) induced only a half of the
amount of IL-4 secretion induced by α-GalCer. Further analysis revealed that Lys-α-
GalCer (3) exhibited a notable increase in IFN- and greatly decrease in IL-4 at all three

concentrations compared to that of α-GalCer. On the other hand, the significant
compress of the Th2 cytokine secretion made these novel synthetic glycolipid
analogues biased toward Th1 cytokine production. The relative rates of cytokine
secretion levels at 0.01 μM concentration were shown in Figure 4C. The IFN-γ/IL-4
ratios of α-GalCer analogues 1-4 were 2.1, 1.7, 4.9 and 3.3, respectively. These results
clearly demonstrated that all of the synthetic analogues had significant effects on
forming the bioactive ternary complex with CD1d and TCR on iNKT cells, and induce
signaling cascades for cytokine production in vitro.
0.01 M
0.1 M
1 M
0.01 M
0.1 M
1 M
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
IFN-γ
IL-4
(A)
(B)
2.5
2.0
1.5
1.0
0.5
0.0
(C)
200
150
100
50
0
α-GalCer
1
2
3
4
α-GalCer
1
2
3
4
α-GalCer
1
2
3
4
Compound
Compound
Compound
Figure 4. Cytokine release from C57BL/6 murine splenocytes stimulated with compound 1-4. IFN-γ and
IL-4 concentrations were measured after 48 h treatment. The secretion amount (pg/mL) of IFN-γ (A) and
IL-4 (B) are summed up. (C) The relative proportion of cytokine production of α-GalCer and its
analogues at 0.01 μM concentration is summed up.
In addition, to further examine the immunostimulatory activity and Th1/Th2
selectivity of the synthetic α-GalCer analogues 1-4 in vivo, the serum levels of IFN-γ
and IL-4 were measured by ELISA kits at several time points in C57BL/6 mice
following intraperitoneal injection. Similar to previous reports,25,52 after α-GalCer was
injected, the secretion amount of IL-4 peaked at 3 h and IFN-γ production had a delayed
but elevated release in C57BL/6 mice from 6 to 24 h. As shown in Figure 5A, all the
four glycolipids could induce Th1 and Th2 cytokine secretion in vivo effectively.
Compound 1, 2 and 4 induced kinetic release of cytokines in agreement with α-GalCer
with a short period of IL-4 peaked at 3 h, followed by high IFN-γ production after a
long period (6-24 h) of time. It is interesting to note that compound 3 stimulated the
release of IL-4 peaked at 6 h. The injection of nonpolar amino acid substituted α-GalCer

analogues 1 and 4 produced levels of the two kinds of cytokine that were similar to
those elicited by α-GalCer, which demonstrated that compound 1 and 4 had equally
powerful abilities to stimulate iNKT cells. Serine substituted analogue Ser-α-GalCer
(2) induced noticeably increased production of both IFN-γ and IL-4. Interestingly, Lys-
α-GalCer (3) showed greatly reduced production of IL-4 while the production of IFN-
γ still remained relatively high (3886 pg/mL), compared with α-GalCer (2925 pg/mL).
In Figure 5B, it can be seen more clearly that compound 1 and compound 4 appeared
to induce comparable IFN-γ levels as α-GalCer at 24 hours after injection. Compound
2 (4755 pg/mL) and compound 3 revealed more effective IFN-γ release concerning α-
GalCer. The levels of Th2 cytokine IL-4 induced by compound 2 was apparently higher
than α-GalCer. While compound 3 revealed IL-4 secretion at a low level during the
mice testing in vivo. The ratio of IFN-γ/IL-4 at the maximum value was used to estimate
Th1/Th2 selectivity of the synthetic α-GalCer analogues 1-4 (Figure 5C). It's worth
noting that compound 3 contained a higher ratio of IFN-γ/IL-4 (5.6/1) compared to α-
GalCer (2.5/1), while compound 2 contained a low ratio of IFN-γ/IL-4 (1.8/1).
Meanwhile, the area under curve (AUC) ratio of IFN-γ/IL-4 cytokine release for α-
GalCer is 13.2/1, which is consistent with the previous report. Lys-α-GalCer (3)
showed high AUC ratio (23.6/1), suggesting compound 3 resulted in remarkable Th1-
biased response (see Supporting Information Figure S33). Thus, the addition of an
amino acid methyl esters moiety at the 6’-position of the galactose did indeed affect the
cytokine secretions and alkaline amino acid lysine led to a marked functional Th1
polarization in vivo.

(A)
5000
4000
3000
2000
1000
0
5000
4000
3000
2000
1000
0
5000
4000
3000
2000
1000
0
IFN-γ
IL-4
-GalCer
Control
1: Gly-α-GalCer
IFN-γ
IL-4
IFN-γ
IL-4
0
6
12
18
24
30
36
42
48
54
0
6
12
18
24
30
36
42
48
54
0
6
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54
Time (h)
Time (h)
Time (h)
5000
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1000
0
5000
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1000
0
5000
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1000
0
2: Ser-α-GalCer
4: Phe-α-GalCer
3: Lys-α-GalCer
IFN-γ
IL-4
IFN-γ
IL-4
IFN-γ
IL-4
0
6
12
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30
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42
48
54
0
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Time (h)
Time (h)
Time (h)
(B)
(C)
6
5
4
3
2
6000
3000
2500
2000
1500
1000
500
1
2
3
4
1
2
3
4
5000
4000
3000
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1000
0
α-GalCer
α-GalCer
0
α-GalCer
1
2
3
4
0
6
12
18
24
30
36
42
48
54
0
6
12
18
24
30
36
42
48
54
Time (h)
Time (h)
Figure 5. Biological assay of α-GalCer and its analogues in vivo. (A, B) IFN-γ and IL-4 secretion in vivo
after intraperitoneal injection of α-GalCer or its analogues 1-4 (100 μg/kg) in C57BL/6 mice. Each group
includes 3 mice and their serum samples were tested at the indicated time individually. (C) The peak
production ratio of IFN-/IL-4 for α-GalCer and analogues 1-4.
3. Conclusions
In summary, a series of L-amino-acid methyl ester-substituted C6’-triazolyl α-
GalCer analogues have been designed by computational modeling and successfully
synthesized using a copper-catalyzed azide-alkyne cycloaddition and glycosylation
coupling reactions with acceptable total yields. Cytotoxicity and cell reproductive
activity assay displayed that all of the four glycolipid analogues had no cytotoxicity to
the splenocytes at experimental concentrations. What is surprising is that they have
acceptable splenocyte cell proliferative activities. The immunostimulatory activities of
α-GalCer and the new synthetic analogues 1-4 were investigated both in vitro and in

vivo. Initial in vitro experiments showed that all of the synthetic analogues exhibited
effective iNKT-cell stimulation and Lys-α-GalCer (3) revealed a notably higher IFN-
γ/IL-4 ratio (4.9) at all three concentrations compared to that of α-GalCer. In vivo,
compound 1 and 4 exhibited stimulating abilities similar to α-GalCer and compound 2
was more potent than α-GalCer in producing both IFN-γ and IL-4. Compound 3
demonstrated excellent enlargement of IFN-γ secretion and reduction of IL-4 secretion,
proving that compound 3 is a fantastic immunostimulant for Th1-biased response,
consistent with previous in vitro experiments. These biological results suggest that
modification at the C6’ position with different L-amino-acid methyl esters can have a
significant effect on cytokine bias, which may pave the way for developing new potent
immunostimulating agents. Future work will involve producing more amino acid or
short-chain polypeptides modified glycolipids at C-6’ for understanding of the cytokine
profile release bias and the relationship between glycolipids and CD1d proteins.
4. Experimental Section
4.1. Chemistry and synthesis
All chemicals were purchased from commercial sources and used without further
treatment unless otherwise stated. All moisture sensitive reactions were carried out in a
flame dried flask under a nitrogen atmosphere. Anhydrous solvents were purified
according to standard procedures before use. Flash chromatography was carried out on
SiO2 (silica gel 200-300 mesh). All reactions were monitored by TLC with GF254 silica
gel coated plates and/or by charring following immersion in a 10% ethanolic solution
of sulfuric acid. 1H NMR and 13C NMR spectra were recorded on Bruker Avance/600
(1H: 600 MHz, 13C: 150 MHz at 25 ºC) or Bruker Avance/400 (1H: 400 MHz, 13C: 100
1
MHz at 25 ºC) and TMS as an internal standard. Chemical shifts of H NMR spectra
were given in ppm relative to a TMS internal standard to the residual solvent peak
(abbreviation in spectra: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad).
All high-resolution mass spectra (HRMS) were measured on a mass spectrometer by

using electrospray ionization (ESI-oa-TOF), and the purity of all samples used for
HRMS (>95%) was confirmed by 1H NMR and 13C NMR spectroscopic analysis.
4.1.1. General procedure for the synthesis of compounds 7a-7d
To a solution of propiolic acid 5 (100.0 mg, 1.4 mmol) in dry CH2Cl2 (5 mL) was
added molecular sieve (4 Å) under an inert atmosphere at 0 °C. The reaction mixture
was stirred 30 minutes at 0 °C. In another flask, the corresponding methyl ester
hydrochloride 6a-6d (1.6 mmol) and DCC (309.2 mg, 1.5 mmol) were dissolved in
CH2Cl2 (2 mL). Then DIPEA (193.9 mg, 1.5 mmol) was added. This solution was added
slowly to the propiolic acid-containing mixture. The reaction was stirred overnight
while the ice bath was allowed to warm to room temperature. After the completion of
the reaction, the mixture was filtered, washed with CH2Cl2 and the solvent was
evaporated. The residue was purified by a short flash silica gel column chromatography
(petroleum ether /EtOAc = 3/2) to give 7a-7d.
Methyl propioloylglycinate 7a. This product was obtained as a slight yellow liquid
in 82% yield. 1H NMR (600 MHz, CDCl3) δ 6.60 (s, 1H), 4.08 (d, J = 4.8 Hz, 2H), 3.77
13
(s, 3H), 2.86 (s, 1H). C NMR (150 MHz, CDCl3) δ 169.44, 152.07, 76.67, 74.31,
52.63, 41.34. HRMS (ESI), m/z calcd. for C6H8NO3 ([M+H]+) 142.0499, found:
142.0497.
Methyl propioloyl-L-serinate 7b. This product was obtained as a slight yellow liquid
1
in 79% yield. H NMR (600 MHz, CDCl3) δ 6.99 (d, J = 6.0 Hz, 1H), 4.72-4.67 (m,
1H), 4.03 (ddd, J = 11.3, 6.0, 3.5 Hz, 1H), 3.94 (ddd, J = 11.3, 5.8, 3.4 Hz, 1H), 3.80
(s, 3H), 2.91 (s, 1H), 2.69 (t, J = 6.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 170.08,
152.11, 76.60, 74.68, 62.64, 54.71, 53.01. HRMS (ESI), m/z calcd. for C7H10NO4
([M+H]+) 172.0604, found: 172.0608.
Methyl N6-((benzyloxy)carbonyl)-N2-propioloyl-L-lysinate 7c. This product was

1
obtained as a slight yellow liquid in 75% yield. H NMR (600 MHz, CDCl3) δ 7.41-
7.28 (m, 5H), 6.60 (d, J = 7.2 Hz, 1H), 5.10 (s, 2H), 4.79 (s, 1H), 4.61 (dd, J = 12.6, 7.6
Hz, 1H), 3.76 (s, 3H), 3.21-3.17 (m, 2H), 2.82 (s, 1H), 1.98-1.83 (m, 1H), 1.83-1.68
13
(m, 1H), 1.52 (m, 2H), 1.46-1.34 (m, 1H). C NMR (100 MHz, CDCl3) δ 171.85,
156.55, 151.87, 136.31, 128.28, 127.86, 127.81, 74.20, 66.35, 52.38, 52.16, 40.03,
30.97, 29.10, 22.02. HRMS (ESI), m/z calcd. for C18H22N2NaO5 ([M+Na]+) 369.1421,
found: 369.1426.
Methyl propioloyl-L-phenylalaninate 7d. This product was obtained as a slight
yellow liquid in 80% yield. 1H NMR (600 MHz, CDCl3) δ 7.29 (t, J = 7.2 Hz, 2H), 7.25
(d, J = 7.2 Hz, 1H), 7.11 (d, J = 7.2 Hz, 2H), 6.68 (d, J = 7.8 Hz, 1H), 4.90-4.86 (m,
1H), 3.71 (s, 1H), 3.16 (dd, J = 14.1, 5.7 Hz, 1H), 3.09 (dd, J = 14.0, 6.0 Hz, 1H), 2.83
(s, 1H). 13C NMR (100 MHz, CDCl3) δ 170.96, 151.36, 135.22, 129.18, 128.62, 127.25,
74.02, 53.41, 52.48, 37.50. HRMS (ESI), m/z calcd. for C13H14NO3 ([M+H]+) 232.0968,
found: 232.0971.
4.1.2. p-Tolyl 2,3,4,6-tetra-O-acetyl-α-D-glactopyranoside 9. 50
To a solution of 1,2,3,4,6-penta-O-acetyl-D-galactose 8 (10.46 g, 26.8 mmol) in dry
CH2Cl2 (20 mL) at 0 ºC were added p-thiocresol (3.99 g, 32.2 mmol) and BF3·Et2O (6.8
mL, 53.6 mmol). The reaction mixture was allowed to warm to r.t. and stirred overnight.
After the completion of the reaction, the reaction mixture was quenched with saturated
aqueous sodium bicarbonate sequentially. The organic layer was washed sequentially
with water and brine, then was dried with anhydrous sodium sulfate and concentrated
in vacuo. The residue was purified by flash column chromatography (petroleum ether
/EtOAc = 4/1) to afford product 9 (8.43 g, 69%) as a white powder. Mp 98-103 ºC. 1H
NMR (600 MHz, CDCl3) δ 7.41 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 5.40 (s,
1H), 5.21 (t, J = 9.6 Hz, 1H), 5.03 (d, J = 9.6 Hz, 1H), 4.64 (d, J = 9.6 Hz, 1H), 4.24-
4.15 (m, 1H), 4.14-4.05 (m, 1H), 3.91 (t, J = 6.2 Hz, 1H), 2.34 (s, 3H), 2.11 (s, 3H),
2.10 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H).HRMS (ESI), m/z calcd. for C21H27O9S ([M+H]+)
455.1370, found: 455.1372.

4.1.3. p-Tolyl α-D-glactopyranoside 10. 50
To a solution of compound 9 (7.99 g, 17.6 mmol) in MeOH (80 mL) was added 1M
solution of sodium methoxide in MeOH until pH 9-10 was reached. The reaction
mixture was stirred at r.t. (the whole process was closely monitored by TLC). After the
completion of the reaction, amberlite IR-120 (H+) was added to neutralize the mixture.
The exchange resin was then filtered off and the solvent was removed in vacuo gave
1
the crude residue 10 (5.04 g, quant) as a white solid. Mp 140-143 ºC. H NMR (600
MHz, MeOD) δ 7.47 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 4.52 (d, J = 9.6 Hz,
1H), 3.90 (d, J = 3.2 Hz, 1H), 3.78-3.70 (m, 2H), 3.59 (t, J = 9.4 Hz, 1H), 3.55 (t, J =
6.0 Hz, 1H), 3.50 (dd, J = 9.2, 3.3 Hz, 1H), 2.32 (s, 3H). HRMS (ESI), m/z calcd. for
C13H19O5S ([M+H]+) 287.0948, found: 287.0952.
4.1.4. p-Tolyl 2,3,4-tri-O-benzyl-6-O-tosyl-α-D-glactopyranoside 11.
To a solution of compound 10 (2.86 g, 10 mmol) in anhydrous pyridine (10 mL) was
added TsCl (3.81 g, 20 mmol) at 0 °C.[50] The mixture was allowed to reach room
temperature and was stirred overnight. After the completion of the reaction, the solvent
was removed in vacuo to give a crude residue as an oil. To a solution of the crude
product from the previous step in DMF (10 mL) was added sodium hydride (2.40 g,
60%, 60 mmol) at 0 oC. After 5 min, BnBr (6.0 mL, 50 mmol) was added. The reaction
mixture was allowed to warm to r.t. and stirred for 6 h, then the mixture was quenched
by addition of MeOH (40 mL). The reaction mixture was then concentrated in vacuo
(co-evaporation with toluene) and the residue was dissolved in CH2Cl2. The resulting
solution was washed with brine, dried with anhydrous sodium sulfate and concentrated
in vacuo. The residue was purified by flash column chromatography (petroleum ether
/EtOAc = 10/1) to afford product 11 (5.12 g, 69%) as a white solid. Mp 105-109 ºC. 1H

NMR (600 MHz, CDCl3) δ 7.74 (d, J = 8.4 Hz, 2H), 7.37 (t, J = 7.4 Hz, 4H), 7.34-7.27
(m, 13H), 7.20 (dd, J = 7.0, 2.2 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 4.94 (d, J = 11.2 Hz,
1H), 4.78-4.68 (m, 4H), 4.50 (dd, J = 21.0, 10.4 Hz, 2H), 4.12 (dd, J = 9.8, 6.1 Hz, 1H),
4.06 (dd, J = 9.8, 6.7 Hz, 1H), 3.90 (d, J = 2.4 Hz, 1H), 3.83 (t, J = 9.4 Hz, 1H), 3.63
(t, J = 6.4 Hz, 1H), 3.57 (dd, J = 9.2, 2.6 Hz, 1H), 2.40 (s, 3H), 2.30 (s, 3H). 13C NMR
(150 MHz, CDCl3) δ 145.15, 138.30, 138.07, 137.51, 132.47, 132.29, 130.20, 129.82,
129.67, 128.57, 128.43, 128.35, 128.91, 128.08, 127.89, 127.86, 127.66, 88.04, 83.84,
77.01, 75.68, 75.47, 74.42, 73.04, 68.00, 21.72, 21.20. HRMS (ESI), m/z calcd. for
C41H42NaO7S2 ([M+Na]+) 733.2264, found: 733.2265.
4.1.5. p-Tolyl 2,3,4-tri-O-benzyl-6-azide-α-D-glactopyranoside 12.
To a solution of compound 11 (2.99 g, 4.22 mmol) in dry DMF (5 mL) was added
sodium azide (1.37 g, 5.5 mmol) at 65 oC overnight. 51 Then the solvent was removed
in vacuo and the residue was dissolved in EtOAc. The resulting solution was washed
with brine, dried with anhydrous sodium sulfate and concentrated in vacuo. The residue
was purified by flash column chromatography (petroleum ether /EtOAc = 10/1) to
afford product 12 (2.23 g, 90%) as a white solid. Mp 113-118 ºC. 1H NMR (600 MHz,
CDCl3) δ 7.52-7.00 (m, 19H), 5.03 (d, J = 11.4 Hz, 1H), 4.86 (d, J = 10.2 Hz, 1H),
4.82-4.72 (m, 3H), 4.62-4.57 (m, 2H), 3.89 (t, J = 9.4 Hz, 1H), 3.80 (s, 1H), 3.61 (dd,
J = 12.8, 7.4 Hz, 1H), 3.45 (t, J = 6.2 Hz, 1H), 3.17 (dd, J = 12.3, 5.7 Hz, 1H), 2.32 (s,
3H). 13C NMR (150 MHz, CDCl3)  138.41, 138.22, 137.73, 132.78, 130.01, 129.75,
128.64, 128.50, 128.47, 128.45, 128.22, 127.96, 127.92, 127.89, 127.76, 88.61, 84.28,
77.43, 75.83, 74.47, 73.77, 73.28, 51.41, 21.28. HRMS (ESI), m/z calcd. for
C34H35N3NaO4S ([M+Na]+) 604.2240, found: 604.2240.
4.1.6. (2S,3S,4R)-2-(N-hexacosanoylamino)-3,4-di-O-benzoyl-1-O-(2,3,4-tri-O-be
nzyl-6-azide-α-D-galactopyranosyl)-1,3,4-octadecanetriol 14.

To a solution of donor 12 (145.3 mg, 0.25 mmol) and acceptor 13 (180.9 mg, 0.2
mmol) in dry CH2Cl2 (4 mL) was added freshly dried 4Å molecular sieves (20 mg)
under nitrogen, then the mixture was stirred at 0 °C for 1 h. After addition of NIS (54
mg, 0.24 mmol) and TfOH (10.2 mL, 0.02 mmol) at 0 °C, the reaction mixture was
continuously stirred at 0 °C until the donor was completely consumed on TLC analysis.
The mixture was filtered, washed with CH2Cl2, and the combined filtrate and washings
were washed with saturated Na2CO3 solution, saturated Na2S2O3 solution and brine,
dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column
chromatography (petroleum ether /EtOAc = 10/1) to afford product 14 (176.9 mg, 65%)
as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 8.03 (d, J = 7.8 Hz, 2H), 7.94 (d, J =
7.8 Hz, 2H), 7.59 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.45 (t, J = 7.2 Hz, 2H),
7.41-7.23 (m, 13H), 7.20-7.15 (m, 3H), 6.98 (d, J = 9.0 Hz, 1H), 5.68 (d, J = 8.8 Hz,
1H), 5.36 (s, 1H), 4.95 (d, J = 11.4 Hz, 1H), 4.78 (d, J = 11.4 Hz, 2H), 4.72-4.52 (m,
5H), 3.98 (dd, J = 10.2, 3.0 Hz, 1H), 3.87-3.76 (m, 4H), 3.67 (d, J = 10.2 Hz, 1H), 3.51
(dd, J = 12.0, 8.4 Hz, 1H), 3.01 (dd, J = 12.3, 4.5 Hz, 1H), 2.20 (t, J = 7.2 Hz, 2H),
2.09-2.06 (m, 2H), 1.91-1.88 (m, 2H), 1.33-1.16 (m, 68H), 0.88 (t, J = 6.4 Hz, 6H). 13C
NMR (150 MHz, CDCl3) δ 173.06, 166.29, 165.37, 138.53, 138.00, 133.30, 132.92,
130.07, 129.79, 129.74, 128.53, 128.45, 128.38, 128.36, 128.30, 128.11, 127.91,
127.71, 127.61, 99.19, 78.36, 76.45, 74.90, 74.60, 73.92, 73.48, 73.28, 72.13, 70.21,
68.70, 51.29, 48.47, 36.76, 31.91, 29.72, 29.68, 29.64, 29.60, 29.56, 29.53, 29.46, 29.37,
29.34, 28.06, 25.77, 25.65, 22.67, 14.10. HRMS (ESI), m/z calcd. for C85H125N4O10
([M+H]+) 1361.9390, found:1361.9383.
4.1.7. General procedure for the synthesis of compounds 15a-15d
To a mixture of compound 14 (204.1 mg, 0.15 mmol) and the corresponding
compounds 7a-7d (0.20 mmol) in H2O-THF (1:1, 5 mL) were added sodium ascorbate
(30 mg, 0.15 mmol) and CuSO4·5H2O (19 mg, 0.075 mmol). The reaction mixture was
stirred vigorously in dark at 65 oC for 4 h. After removal of THF under reduced pressure,
water (20 mL) was added, and the product was extracted with EtOAc. The combined

organic layers were dried over anhydrous Na2SO4 and evaporated in vacuo. The crude
product was purified by a flash silica gel column chromatography (petroleum ether
/EtOAc = 1:4) to give 15a-15d as a slight yellow oil.
(2S,3S,4R)-2-(N-hexacosanoylamino)-3,4-di-O-benzoyl-1-O-(2,3,4-tri-O-benzyl-6-
(4-((N-methyl glycinate)carbonyl)-1H-1,2,3-triazolyl)-α-D-galactopyranosyl)-1,3,
4- octadecanetriol 15a.
This product was obtained as a slight yellow liquid in 88% yield. 1H NMR (600 MHz,
CDCl3) δ 8.05 (s, 1H), 8.03 (d, J = 7.2 Hz, 2H), 7.91 (d, J = 7.2 Hz, 2H), 7.65-7.55 (m,
2H), 7.51 (t, J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 2H), 7.37-7.34 (m, 6H), 7.32-7.29 (m,
4H), 7.25 (d, J = 6.6 Hz, 2H), 7.19-7.16 (m, 3H), 6.67 (d, J = 9.0 Hz, 1H), 5.62 (d, J =
9.0 Hz, 1H), 5.30 (s, 1H), 4.96 (d, J = 11.0 Hz, 1H), 4.75 (d, J = 12.0 Hz, 2H), 4.65 (t,
J = 11.4 Hz, 2H), 4.59-4.53 (m, 3H), 4.35 (d, J = 5.9 Hz, 2H), 4.20 (t, J = 4.8 Hz, 2H),
4.15 (t, J = 12 Hz, 1H), 3.98 (d, J = 9.6 Hz, 1H), 3.81(d, J = 10.0 Hz, 1H), 3.77 (s, 3H),
3.68 (s, 1H), 3.56 (d, J = 10.8 Hz, 1H), 3.34 (d, J = 10.8 Hz, 1H), 2.20 (t, J = 7.2 Hz,
2H), 1.88-1.85 (m, 2H), 1.65-1.62 (m, 2H), 1.25-1.19 (m, 68H), 0.87 (t, J = 6.4 Hz,
6H). 13C NMR (150 MHz, CDCl3) δ 173.03, 169.85, 166.53, 165.50, 160.19, 142.53,
138.43, 138.01, 137.90, 133.42, 133.06, 129.91, 129.75, 129.69, 128.67, 128.54,
128.45, 128.37, 128.34, 128.20, 128.12, 127.77, 127.71, 127.66, 126.86, 121.59, 99.13,
78.32, 77.29, 77.08, 76.87, 76.16, 74.70, 74.45, 74.02, 73.64, 73.36, 72.41, 69.83, 68.61,
52.38, 50.77, 48.42, 40.89, 36.74, 31.95, 29.77, 29.73, 29.68, 29.65, 29.61, 29.58, 29.52,
29.39, 28.18, 25.74, 25.64, 22.72, 14.15. HRMS (ESI), m/z calcd. for C91H132N5O13
([M+H]+) 1502.9816, found: 1502.9832.
(2S,3S,4R)-2-(N-hexacosanoylamino)-3,4-di-O-benzoyl-1-O-(2,3,4-tri-O-benzyl-6-
(4-((N-methyl L-serinate)carbonyl)-1H-1,2,3-triazolyl)-1,3,4-octadecanetriol 15b.
This product was obtained as a slight yellow liquid in 83% yield. 1H NMR (600 MHz,
CDCl3) δ 8.03 (d, J = 7.8 Hz, 2H), 8.02 (s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.93 (d, J =

7.8 Hz, 2H), 7.58 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 7.2 Hz, 2H),
7.40-7.36 (m, 6H), 7.35-7.30 (m, 5H), 7.28-7.25 (m, 1H), 7.18-7.14 (m, 3H), 7.10 (t, J
= 7.2 Hz, 2H), 6.70 (d, J = 9.0 Hz, 1H), 5.60 (d, J = 9.6 Hz, 1H), 5.22 (d, J = 9.6 Hz,
1H), 4.96 (d, J = 11.4 Hz, 1H), 4.91-4.82 (m, 1H), 4.77 (d, J = 11.6 Hz, 1H), 4.66 (d, J
= 11.6 Hz, 1H), 4.61 (d, J = 11.6 Hz, 2H), 4.57 (d, J = 12.0 Hz, 2H), 4.46 (d, J = 11.8
Hz, 1H), 4.40 (t, J = 9.2 Hz, 1H), 4.29 (dd, J = 13.8, 9.2 Hz, 1H), 4.20 (d, J = 13.6 Hz,
1H), 4.06 (s, 2H), 3.92 (dd, J = 9.8, 2.9 Hz, 1H), 3.82 (s, 3H),3.79-3.77 (m, 1H), 3.40
(d, J = 10.2 Hz, 1H), 2.98 (d, J = 9.6 Hz, 1H), 2.31-2.28 (m, 1H), 2.16-2.13 (m, 1H),
1.81-1.76 (m, 2H), 1.63 (s, 2H), 1.39-1.07 (m, 68H), 0.88 (t, J = 5.0 Hz, 6H). 13C NMR
(150 MHz, CDCl3) δ 173.44, 170.53, 166.65, 165.22, 159.62, 142.55, 138.39, 137.87,
137.65, 133.34, 133.06, 129.83, 129.71, 129.58, 128.67, 128.57, 128.55, 128.42,
128.32, 128.19, 128.03, 127.65, 126.48, 98.11, 78.18, 77.21, 77.00, 76.79, 75.58, 74.46,
74.39, 74.04, 73.62, 72.92, 71.78, 70.52, 67.80, 62.91, 54.66, 52.59, 51.74, 47.75, 36.62,
31.88, 29.70, 29.65, 29.61, 29.58, 29.52, 29.48, 29.41, 29.32, 29.27, 28.03, 25.60, 25.55,
22.64, 14.07. HRMS (ESI), m/z calcd. for C92H133N5NaO14 ([M+Na]+) 1554.9741,
found:1554.9778.
(2S,3S,4R)-2-(N-hexacosanoylamino)-3,4-di-O-benzoyl-1-O-(2,3,4-tri-O-benzyl-6-
(4-((N-methyl N6-((benzyloxy)carbonyl)-L-lysinate)carbonyl)-1H-1,2,3-triazolyl)-
α-D-galactopyranosyl)-1,3,4-octadecanetriol 15c.
This product was obtained as a slight yellow liquid in 85% yield. 1H NMR (400 MHz,
CDCl3) δ 8.03 (d, J = 7.6 Hz, 2H), 7.99 (s, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.66-7.42 (m,
5H), 7.37-7.28 (m, 18H), 7.26-7.24 (m, 2H), 7.22-7.13 (m, 3H), 6.91 (d, J = 9.6 Hz,
1H), 5.64 (d, J = 9.2 Hz, 1H), 5.38-5.17 (m, 1H), 5.08-5.05 (m, 3H), 4.97 (d, J = 11.2
Hz, 1H), 4.84-4.43 (m, 8H), 4.42-4.24 (m, 2H), 4.13 (t, J = 6.2 Hz, 1H), 3.98 (dd, J =
10.0, 3.4 Hz, 1H), 3.83 (d, J = 9.6 Hz, 1H), 3.76 (br s, 3H), 3.69 (s, 1H), 3.52 (d, J =
9.2 Hz, 1H), 3.31 (dd, J = 11.0, 2.6 Hz, 1H), 3.18 (dd, J = 12.7, 6.4 Hz, 2H), 2.20-2.15
(m, 2H), 2.11-2.05 (m, 2H), 1.62-1.60 (m, 2H), 1.55-1.53 (m, 2H), 1.43-1.40 (m, 2H),
1.41-1.33 (m, 2H), 1.33-1.13 (m, 68H), 0.87 (t, J = 6.8 Hz, 6H). 13C NMR (100 MHz,

CDCl3) δ 172.96, 172.19, 166.46, 165.40, 159.70, 156.40, 142.54, 138.35, 137.94,
137.81, 136.69, 133.34, 132.97, 129.87, 129.82, 129.66, 128.58, 128.45, 128.40,
128.36, 128.29, 128.25, 128.08, 128.04, 127.95, 127.92, 127.68, 127.61, 127.55,
126.75, 99.16, 78.21, 77.20, 76.03, 74.64, 74.37, 73.91, 73.55, 73.29, 72.27, 69.78,
68.67, 66.43, 52.32, 51.69, 50.61, 48.38, 40.62, 36.65, 32.07, 31.86, 29.70, 29.68, 29.64,
29.60, 29.57, 29.52, 29.51, 29.43, 29.30, 28.11, 25.65, 25.59, 22.63, 22.57, 14.06.
HRMS (ESI), m/z calcd. for C103H147N6O15 ([M+H]+) 1708.0919, found: 1708.0933.
(2S,3S,4R)-2-(N-hexacosanoylamino)-3,4-di-O-benzoyl-1-O-(2,3,4-tri-O-benzyl-6-
(4-((N-methyl L-phenylalaninate)carbonyl)-1H-1,2,3-triazolyl)-α-D-galactopyran
osyl) -1,3,4-octadecanetriol 15d.
This product was obtained as a slight yellow liquid in 86% yield. 1H NMR (600 MHz,
CDCl3) δ 8.03 (d, J = 7.6 Hz, 2H), 7.99 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.59 (t, J =
7.2 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.48 (m, 3H), 7.34-7.32 (m, 8H), 7.31-7.27 (m,
8H), 7.23-7.14 (m, 6H), 7.03 (d, J = 9.4 Hz, 1H), 5.69 (d, J = 9.6 Hz, 1H), 5.30 (s, 1H),
5.02 (dd, J = 13.4, 6.6 Hz, 1H), 4.97 (d, J = 11.4 Hz, 1H), 4.77 (d, J = 10.2 Hz, 2H),
4.71 (d, J = 12.0 Hz, 1H), 4.66 (d, J = 11.6 Hz, 1H), 4.61 (d, J = 11.6 Hz, 1H), 4.53 (d,
J = 11.0 Hz, 2H), 4.35-4.30 (m, 2H), 4.16 (t, J = 6.0 Hz, 1H), 4.00 (dd, J = 9.8, 2.4 Hz,
1H), 3.84 (d, J = 10.0 Hz, 1H), 3.69 (d, J = 12.0 Hz, 4H), 3.54 (d, J = 9.6 Hz, 1H), 3.36
(d, J = 9.6 Hz, 1H), 3.21 (d, J = 6.0 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H), 1.91-1.89 (m,
2H), 1.71-1.59 (m, 2H), 1.25-1.19 (m, 68H), 0.88 (t, J = 6.4 Hz, 6H). 13C NMR (150
MHz, CDCl3) δ 172.92, 171.43, 166.51, 165.49, 159.50, 142.44, 138.30, 137.76, 135.82,
133.36, 132.99, 129.83, 129.81, 129.67, 129.55, 129.18, 128.60, 128.58, 128.54,
128.45, 128.36, 128.26, 128.17, 128.04, 127.71, 127.65, 127.63, 127.08, 126.71, 98.88,
78.10, 76.15, 74.63, 74.44, 74.05, 73.56, 73.32, 72.00, 69.61, 68.01, 53.11, 52.20, 50.61,
48.28, 38.26, 36.66, 31.86, 29.69, 29.64, 29.59, 29.57, 29.51, 29.44, 29.34, 29.30, 27.86,
25.71, 25.60, 22.63, 14.06. HRMS (ESI), m/z calcd. for C98H138N5O13 ([M+H]+)
1593.0286, found: 1594.0273.

4.1.8. General procedure for the synthesis of compounds 1-4
To a solution of the corresponding compounds 15a-15d (0.13 mmol) in EtOAc-
MeOH (1:1, 5 mL) was added 10% palladium(II) hydroxide (100 mg) under hydrogen
atmosphere at ambient temperature for 8 h. After filtration, the solvent was evaporated
to give the crude residue as a colorless oil. To a solution of the crude residue from the
previous step in anhydrous methanol (2 mL) was added a 1M solution of sodium
methoxide in MeOH until pH 9-10 was reached. The reaction mixture is stirred at r.t.
for 4 h. After completion, the reaction was neutralized with amberlite IR-120 (H+),
dissolved with CH2Cl2, and filtered. The filtrate was concentrated under reduced
pressure to afford 1-4.
(2S,3S,4R)-2-(N-hexacosanoylamino)-1-O-(6-(4-((N-methyl glycinate)carbonyl)-
1H-1,2,3-triazolyl)-α-D-galactopyranosyl)-1,3,4-octadecanetriol 1.
This product was obtained as a white foam solid in 91% yield. Mp 112-115 oC. 1H
NMR (600 MHz, MeOD/CDCl3 = 5/1) δ 8.34 (s, 1H), 4.83 (br s, 2H), 4.64 (d, J = 6.0
Hz, 2H), 4.23 (t, J = 6.0 Hz, 1H), 4.15 (d, J = 6.0 Hz, 1H), 4.11-4.06 (m, 1H), 3.86 (s,
1H), 3.80 (d, J = 9.2 Hz, 1H), 3.76 (br s, 4H), 3.58-3.44 (m, 2H), 3.40 (s, 2H), 2.30-
2.11 (m, 2H), 1.67-1.46 (m, 4H), 1.30-1.25 (m, 68H), 0.87 (t, J = 6.4 Hz, 6H). 13C NMR
(150 MHz, MeOD/CDCl3 = 5/1) δ 175.41, 171.32, 162.51, 143.02, 128.13, 100.48,
75.08, 72.72, 70.78, 70.53, 70.40, 69.54, 67.89, 52.71, 52.14, 51.00, 41.52, 37.13, 32.76,
32.74, 30.59, 30.53, 30.51, 30.44, 30.39, 30.28, 30.18, 30.15, 26.76, 26.69, 23.43, 14.38.
HRMS (ESI), m/z calcd. for C56H105N5NaO11 ([M+Na]+) 1046.7703, found:1046.7712.
(2S,3S,4R)-2-(N-hexacosanoylamino)-1-O-(6-(4-((N-methyl L-serinate)carbonyl)-
1H-1,2,3-triazolyl)-α-D-galactopyranosyl)-1,3,4-octadecanetriol 2.
This product was obtained as a white foam solid in 93% yield. Mp 121-125 oC. 1H
NMR (600 MHz, MeOD/CDCl3 = 5/1) δ 8.35 (d, J = 12.0 Hz, 1H), 4.84-4.21 (m, 3H),

4.65 (s, 2H), 4.36-4.12 (m, 1H), 4.14-4.01 (m, 2H), 4.01-3.91 (m, 1H), 3.88 (d, J = 12.6
Hz, 1H), 3.81-3.75 (m, 4H), 3.59-3.36 (m, 4H), 2.32-2.09 (m, 2H), 1.73-1.47 (m, 4H),
1.28-1.15 (m, 68H), 0.87 (s, 6H). 13C NMR (150 MHz, MeOD/CDCl3 = 5/1) δ 175.43,
171.76, 161.86, 142.97, 128.17, 100.41, 75.10, 72.74, 70.79, 70.63, 70.57, 70.43, 69.54,
67.94, 62.71, 55.66, 53.01, 52.31, 51.04, 49.43, 37.12, 32.76, 32.74, 30.60, 30.53, 30.52,
30.46, 30.44, 30.30, 30.18, 30.14, 26.78, 26.71, 23.44, 23.43. HRMS (ESI), m/z calcd.
for C57H107N5NaO12 ([M+Na]+)1076.7808, found: 1076.7828.
(2S,3S,4R)-2-(N-hexacosanoylamino)-1-O-(6-(4-((N-methyl L-lysinate)carbonyl)-
1H-1,2,3-triazolyl)-1H-1,2,3-triazolyl)-α-D-galactopyranosyl)-1,3,4-octadecanetri
ol 3.
This product was obtained as a white foam solid in 92% yield. Mp 128-130 oC. 1H
NMR (400 MHz, MeOD) δ 3.83 (s, 1H), 4.83 (d, J = 2.6 Hz, 1H), 4.70-4.64 (m, 3H),
4.24 (t, J = 6.1 Hz, 1H), 4.06 (d, J = 4.8 Hz, 1H), 3.90 (s, 1H), 3.80-3.74 (m, 5H), 3.58-
3.46 (m, 3H), 3.40 (dd, J = 10.1, 3.2 Hz, 1H), 2.96 (t, J = 7.0 Hz, 2H), 2.37-2.15 (m,
2H), 2.10-1.96 (m, 1H), 1.98-1.85 (m, 1H), 1.78-1.72 (m, 2H), 1.66-1.45 (m, 6H), 1.40-
1.29 (m, 68H), 0.89 (t, J = 6.4 Hz, 6H). 13C NMR (100 MHz, MeOD/CDCl3 = 1/1) δ
174.37, 172.28, 167.45, 160.90, 141.83, 127.35, 99.37, 73.47, 71.97, 69.64, 69.50,
69.39, 68.45, 67.11, 52.20, 51.74, 51.35, 49.78, 39.20, 36.25, 31.80, 31.79, 31.68, 31.03,
29.68, 29.59, 29.51, 29.39, 29.34, 29.31, 29.23, 29.20, 29.11, 26.97, 26.67, 25.83, 22.50,
22.46, 13.60. HRMS (ESI), m/z calcd. for C60H115N6O11 ([M+H]+) 1095.8618,
found:1095.8626.
(2S,3S,4R)-2-(N-hexacosanoylamino)-1-O-(6-(4-((N-methyl L-phenylalaninate)ca
rbonyl)-1H-1,2,3-triazolyl)-α-D-galactopyranosyl)-1,3,4-octadecanetriol 4.
This product was obtained as a white foam solid in 91% yield. Mp 125-129 oC. 1H
NMR (600 MHz, MeOD/CDCl3 = 5/1) δ 8.29 (s, 1H), 7.26 (t, J = 6.9 Hz, 2H), 7.21 (d,
J = 6.6 Hz, 3H), 4.90 (t, J = 6.0 Hz, 2H), 4.83 (d, J = 2.8 Hz, 1H), 4.62 (d, J = 6.0 Hz,

2H), 4.23 (t, J = 6.0 Hz, 1H), 4.11 (d, J = 4.2 Hz, 1H), 3.84 (s, 1H), 3.80 (dd, J = 9.8,
2.6 Hz, 1H), 3.75 (dd, J = 10.2, 2.6 Hz, 1H), 3.71 (s, 3H), 3.53-3.47 (m, 2H), 3.42 (d, J
= 3.0 Hz, 1H), 3.25 (dd, J = 13.4, 5.6 Hz, 1H), 3.17 (dd, J = 13.6, 7.6 Hz, 1H), 2.30-
2.13 (m, 2H), 1.59-1.50 (m, 4H), 1.34-1.25 (m, 68H), 0.87 (t, J = 6.6 Hz, 6H). 13C NMR
(150 MHz, MeOD/CDCl3 = 5/1) δ 175.35, 172.76, 161.63, 142.80, 137.22, 129.98,
129.34, 128.10, 127.80, 100.45, 74.91, 72.74, 70.71, 70.43, 70.26, 69.50, 67.85, 54.60,
52.81, 52.09, 50.97, 38.41, 37.15, 32.73, 32.72, 30.60, 30.49, 30.42, 30.26, 30.19, 30.15,
30.12, 27.86, 26.75, 26.67, 23.41, 14.38, 14.37. HRMS (ESI), m/z calcd. for
C63H112N5O11 ([M+H]+) 1114.8353, found: 1114.8380.
4.2. Splenocyte proliferation assay
Murine spleen cells were collected from female C57BL/6 mice. The mice were
sacrificed and the spleens were immediately collected. Then they were made into a
single-cell suspension by passing through a 100 µM cell strainer. Red Blood Cell (RBC)
was lyzed with RBC Lysis buffer (0.5 M ammonium chloride, 10 mM potassium
bicarbonate, and 0.1 mM disodium ethylene diamine tetraacetic acid, pH 7.2) for 5 min
at 4 °C. The cells were washed with PBS three times by centrifugation (1000 rpm, 10
oC, 5 min), and the resulting pellet was resuspended. Splenocytes were seeded into 96-
well plates (1×105/well) and incubated with indicated doses of synthetic compounds in
RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin solution.
After incubation for 48 h at 37 oC, 20 µL of MTT (5 mg/mL in PBS) solution was added
into each well and incubated for another 4 h. Following, the plates were centrifuged
and the supernatant was removed. To each well, 100 µL of a DMSO solution was added
to dissolve the formazan crystals and the plate was shaken for 10 min to fully dissolve
formazan and homogenize. The optical density of the dissolved formazan crystals was
measured at a wavelength of 570 nm. Splenocyte cells cultured with culture media were
selected as controls, and their viabilities were defined as 100%. And the relative cell
viability (%) was expressed as a percentage relative to the untreated control cells.

4.3. In vitro cytokine production assay
The in vitro production of IFN-γ and IL-4 were examined using murine spleen cells.
Splenocytes were seeded into 96-well plates (1×105/well) and incubated with indicated
doses of α-GalCer and synthetic compounds in RPMI 1640 supplemented with 10%
o
FBS and 1% penicillin-streptomycin solution. After incubation for 48 h at 37 C,
cytokine concentrations were determined using ELISA (eBioscience) according to the
general procedures.
4.4. In vivo cytokine production assay
All mouse experimental procedures were performed in accordance with the
Regulations for the Administration of Affairs Concerning Experimental Animals
approved by the State Council of the People’s Republic of China. Female C57BL/6 (B6)
mice weighing 25-28 g, 7 to 8 weeks old, were housed in an animal facility, given free
access to water and food under specific pathogen-free conditions. Stock solutions of
glycolipids in DMSO at 1 mg/mL were prepared and kept at -20 oC. For research, the
stock solution was further diluted with PBS. B6 mice were injected intraperitoneally
with 100μg/kg of the glycolipids, and serum samples were obtained at indicated times
after injection. The serum concentrations of IFN-γ and IL-4 were assessed by ELISA
kit (eBioscience). Every group contains 3 mice and their serum of indicated times was
detected individually.
4.5. Computational methods 57
The X-ray crystal structure of the CD1d and TCR (PDB code: 3HE6) was obtained
from the Protein Data Bank (PDB, www.rcsb.org). The initial structure of α-GalCer
was extracted from The X-ray crystal structure of 3HE6, while the initial structures of
its analogues 1, 2, 3 and 4 were built by modifying the structure of α-GalCer with Sybyl
package. Structures of protein and ligands were minimized with Powell method and

Tripos force field provided in Sybyl (Termination for protein minimization: Gradient
was set to 1 kcal/(mol*Å) or Max Iterations was set to 1000; Termination for ligands
minimization: Gradient was set to 0.05 kcal/(mol*Å) or Max Iterations was set to 100).
The Gasteiger-Hückel charge was added for both protein and ligands. Protomol was
built with default parameters (threshold was set to 0.5 Å, and bloat was set to 10.0 Å),
and the Surflex-docking was performed with default input options except for the post-
docking minimization flag. The bindings of α-GalCer analogues with CD1d and TCR
were estimated using a variety of scoring functions within the Total Score. Pictures of
complexes in the article were drawn with PyMol software (https://pymol.org/2/).
Acknowledgments
We thanks the NNSFC (21702051, U1604285 and 21877206), the Key Scientific and
Technological Project of Henan Province (17A350006), the Postdoctoral Science
Foundation of Henan Province (001802033), the Henan Normal University Research
Foundation for the Doctoral Program (qd15108), the Henan Normal University Science
Foundation for Young Scholars (2016QK10). We thank Prof. Min Fang from Institute
of Microbiology, Chinese Academy of Sciences for in vivo cytokine production assay.
Supplementary data
Supplementary data associated with this article can be found, in the online version,
at http://dx.doi.org/....
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