Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain

Article abstract of DOI:10.1016/j.bmc.2016.06.007

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid–protein interactions in KRN7000/CD1d complexes.

Full text of DOI:10.1016/j.bmc.2016.06.007

Bioorganic & Medicinal Chemistry 24 (2016) 3687–3695
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and Th1-immunostimulatory activity of
a-galactosylceramide analogues bearing a halogen-containing or
selenium-containing acyl chain
a
b
a
Md. Imran Hossain a,b, Shinya Hanashima a, , Takuto Nomura , Sébastien Lethu , Hiroshi Tsuchikawa ,
⇑
Michio Murata a,b, , Hiroki Kusaka c, Shunsuke Kita c, Katsumi Maenaka c
⇑
a Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
b JST, ERATO, Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
c Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of CD1d ligand
the heavy atoms Br and Se in the acyl chain backbone of
synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells
to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crys-
tallographic probes to disclose precise atomic positions of alkyl carbons and lipid–protein interactions in
KRN7000/CD1d complexes.
a
-galactosylceramides (
a
-GalCers) were synthesized by incorporation of
-galactosyl-N-cerotoylphytosphingosine. The
Received 9 May 2016
Revised 3 June 2016
Accepted 3 June 2016
Available online 6 June 2016
a
Keywords:
Ó 2016 Elsevier Ltd. All rights reserved.
a-GalCer
Cytokines
CD1d
Natural product synthesis
Glycosylation
1. Introduction
disclosed that a simple alkyl chain of lipids sometimes plays a cru-
cial role in ligand–receptor interactions using a ultra-high resolu-
Marine invertebrates are a rich source of pharmacologically
active compounds. Agelasphins are a class of such natural prod-
ucts, isolated from the Okinawan marine sponge Agelas mauri-
tianus.1,2 These glycosphingolipids show potent in vivo antitumor
activity through stimulation of the host immune system. The struc-
tural optimizations of the agelasphins have led to the development
of a very potent immunostimulatory agent, KRN7000 (Fig. 1),3
which is a simple
receptor of KRN7000 has been identified as the antigen presenta-
tion protein CD1d.4,5 The
-GalCer–protein complex is further rec-
ognized by T-cell receptors (TCRs) expressed on invariant natural
tion protein–ligand complex (0.86–0.93 Å).8,9 However, with the
large CD1d/KRN7000/TCR complex, it would not be easy to prepare
high quality crystals necessary for high resolution density maps. To
address this issue, we designed a-GalCer probes, 3, 4, and 5, which
possess an electron-rich heavy atom (Br or Se) in the acyl chain
(Fig. 1). Br or Se is incorporated in 3, 4, and 5 by substituting a
C1 segment at C120, C180, or C260, respectively (Fig. 2). These posi-
tions are expected to act as milestones to determine the shape of
the acyl chain by X-ray crystallography, because the heavy atoms
are positioned at the beginning (C120) and middle (C180) of the
loop, and at the terminus (C260) of the chain.
a-galactosylceramide (a-GalCer). The innate
a
killer T cells (iNKT). The structure of this CD1d/
a
-GalCer/TCR tern-
The bromine substituent was employed because Br and a
methyl group have similar van der Waals radii (Br: 185 pm; CH3:
200 pm). The selenoether group was adopted in the place of the
methylene C120 or C180 because of the similar bond angles
(C–Se–C: 96.3°; C–C–C 112.6°) and van der Waals radii (Se:
190 pm; CH2: 200 pm), but it does have a slightly larger bond
length (C–Se: 194.5 pm; C–C: 154.0 pm). More importantly, the
distance between the two carbon atoms of a C–Se–C moiety is
289.8 pm, which is a little larger than the distance of 1,3-carbon
atoms in alkane by 34 pm, but should not significantly affect the
conformation of the whole acyl chain except for the selenoether
ary complex has been established by X-ray crystallography.6,7
However, the atomistic mechanism of ligand recognition by
CD1d remains elusive mainly due to ambiguity in the X-ray crystal
structures; the dispersed electron density of a bound
impeded the precise elucidation of the alkyl-chain conformation
in the deep binding pockets of CD1d (Fig. 2). We have recently
a-GalCer
⇑
Corresponding authors. Tel.: +81 6 6850 5789 (S.H.), +81 6 6850 5774 (M.M.).
E-mail addresses: hanashimas13@chem.sci.osaka-u.ac.jp (S. Hanashima), murata@
chem.sci.osaka-u.ac.jp (M. Murata).
http://dx.doi.org/10.1016/j.bmc.2016.06.007
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

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M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
O
are reported to inhibit the intrahepatic-stage infection of the
OH
HO
1'
R
KRN7000:R = CH3
1: R = F
2: R = Cl
rodent malaria parasites Plasmodium berghei,29 and IFN-
c inhibits
6''
HN
26'
O
20
hepatitis B virus replication.30 In addition, such CD1d-restricted
iNKT cell ligands are generally known as potent vaccine adjuvant.31
Therefore, the CD1d ligands that can fine-tune the balance of Th1/
Th2 cytokine secretion would be potential drug lead compounds.
The proportion of Th1/Th2 cytokine production is thought to
HO
1''
OH
HO
4C14H29
3: R = Br
O
3
OH
1
O
OH
HO
HO
depend on the stability of the CD1d/
Stronger stimulation for a longer period is required for the Th1
pathway (IFN- production) while a weaker and shorter stimula-
a
-GalCer/TCR complex.6,7,32
X
12'
Y
18'
C7H15
HN
O
5
OH
HO
c
O
C14H29
tion period results in simulation of the Th2 pathway (IL-4 produc-
tion).21 The X-ray crystal structure of the ternary complex has
revealed that the phytosphingosyl and acyl chains are incorporated
into the two deep hydrophobic binding pockets F0 and A0, respec-
tively, and the 3-OH of phytosphingosine is involved in the hydro-
gen bonding to Asp80 on CD1d5 and Arg95 of the TCR.6
OH
4: X = Se, Y = CH2
5: X = CH2, Y = Se
Figure 1. Structures of KRN7000 and its analogues 1–5 with a halogen or selenium
atom.
and its neighboring portions. Such heavy atoms at
a-GalCer probes
Furthermore, the a-galactoside moiety is directly presented to
are also expected to play a key role in phase determination in X-ray
crystallography by using the anomalous dispersions in single- or
multi-wavelength anomalous dispersion (SAD or MAD) methods.10
The selenoether moiety has been used previously as an analogue of
oxygen and sulfur atoms in protein ligands or in target proteins,
and selenoglycoside ligands including KRN7000 have been synthe-
sized11,12 to determine phasing of the protein crystals.13 On the
other hand, no X-ray crystallographic analysis has been reported
for the selenoether moiety used as a substitute for a methylene
segment.
the TCR at the surface of CD1d. Hydroxy groups of the galactose,
particularly 200-, 300-, and 400-OH, interact with Gly96, Ser30, and
Phe29 of the TCR.6
To improve the cytokine production activity and selectivity,
various derivatives of KRN7000 have been synthesized.33–37 Nota-
bly, modifications of the alkyl chains in the sphingosine and fatty
acyl moieties significantly influenced the balance of Th1/Th2 cyto-
kine production. Truncation of the acyl chain (C8) leads to higher
Th2 selectivity compared to full-length KRN7000.38 A shorter acyl
chain with a terminal hydroxy group or with the replacement of
the amide moiety with a triazole group also enhances Th2 selectiv-
ity.39,40 Similarly, a shorter alkyl chain of the phytosphingosine
chain elicits more Th2 responses.41 On the other hand, shortening
and/or introduction of an aromatic group in the fatty acid chain
(C8Ph) resulted in the highest Th1-biased cytokine production in
human NKT cell lines; MD simulations revealed that the interac-
tion between the phenyl group and the aromatic residues Tyr73
or Trp40 of CD1d could stabilize the ternary complex.42,43 Replace-
ment of the anomeric oxygen atom by a methylene group enhances
the selectivity toward Th1.36 Most of the synthetic variants that
show Th1/Th2 selectivity are modified at either the phy-
The CD1d/a-GalCer/TCR complex provides an ideal system to
investigate molecular mechanisms underlying Th1/Th2 immuno-
logical switching, in which the TCR regulates a wide range of
responses.14,15 Exogenous
a-GalCer antigens bound to CD1d are
presented to iNKT cells through TCRs at the cell surface, which
leads to the activation of iNKT cells.16 The activate cells induce
the production of cytokines such as interferon gamma (IFN-c),
interleukin (IL)-12, and granulocyte macrophage colony-stimulat-
ing factor (GM-CSF) of the Th1 immune pathway, and activation
of cellular immunity for tumor rejection.17–19 This ternary complex
also enhances the production of IL-4 and IL-13 that involve the Th2
pathway for humoral immunity.20,21 For this reason, a balance in
the production of Th1 and Th2 cytokines is a key point for control-
ling the immune responses in various diseases,22–24 including
cancer,18,25 diabete,26–28 and infections; CD1d-restricted iNKT cells
tosphigosine or acyl chain of
a-GalCer. As a consequence, these
structural alterations in the ceramide portion relevant to its bind-
ing affinity to CD1d may allow us to fine-tune the stability of the
ternary complex, which is thought to be a key factor for selective
cytokine production. To determine the exact atomic positions of
the acyl chain, we designed a-GalCer probes 3–5 (Fig. 1) preserving
most of the hydrophobic interactions in the ligand–protein com-
plex. Whether the Se or Br substitution influenced the binding to
CD1d was evaluated through a cytokine (IFN-c and IL-4) produc-
tion assay. In addition, we were also interested in the effect of
the electron negativity and possible halogen bond interactions at
the terminal halogens. We therefore prepared F and Cl substituted
derivatives 1 and 2 (Fig. 1). All of our synthetic
a-GalCer probes
successfully stimulated the production of Th1 and Th2 cytokines,
suggesting their potential use for co-crystallization with CD1d
to determine precise lipid–protein interactions by X-ray
crystallography.
2. Results
2.1. Chemical synthesis
The halogenated fatty acids were synthesized from reported
compounds 644 and 945 (Scheme 1). The hydroxy group of 6 was
converted to N-phenyl-tetrazolyl-sulfide (7), which was oxidized
to N-phenyl-tetrazolyl-sulfone (8). The benzyl hemiester 9 was
reduced to give 10, and the following oxidation yielded aldehyde
Figure 2. Electron density map of KRN7000 bound in human CD1d (PDB ID: 3HUJ).
Blue mesh shows the density of 1.0
r. The dispersed electron density prevents the
determination of atomic positions precisely. In this study, the heavy atoms Se and
Br are incorporated at C120 or C180, and at C260, respectively, to enhance the
electron density of the labeled positions.

M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
3689
(a)
Se
Se
n
Br
n
N
N
n
n = 4, 10
N
N
X
20: n = 4
(a)
OH
OTBS
21: n = 10
OTBS
6
7:
X = S
(b)
Br
OH
8
: X = SO2
n
O
O
22: n = 7
O
OBn
O
OBn
23: n = 13
(e)
OH
X
Y
(b)
OR
R
24: X = Se, Y = CH2
25: X = CH2, Y = Se
12 R = TBS
13: R = H
14: R = Ms
9: R = COOH
10: R = CH2OH
11: R = CHO
(f)
(g)
(c)
(d)
Scheme 2. Synthesis of selenocerotic acids 24 and 25. Reagents and conditions: (a)
Se, NaBH4, EtOH, 62%; (b) NaBH4, THF, H2O, 42% for 24 and 42% for 25.
O
OBn
(h)
To synthesize
a
-GalCer analogues, the key intermediate 2950
13
F
was prepared by glycosylation of donor 27 and acceptor 28
(Scheme 3). The protected galactose precursor (26) was obtained
by a reported method.51 Treatment of 26 with DAST and NBS
15
O
OH
yielded a mixture of
was separated for the use of the following reactions. The glycosyl
a,b-isomers (a/b = 4:1). The a-isomer 27
(i)
(i)
14
15
OMs
acceptor (28) was prepared from commercially available phy-
16
tosphingosine by following a reported method.52 Finally, the
a-
(j)
GalCer probes (1–5) were obtained by hydrogenation of compound
29, followed by amide coupling with suitable carboxylic acids (17,
18, 19, 24, and 25) in the presence of HBTU and base in moderate
yields.
O
OH
X
2.2. Cytokine production assay
17: X = F
18: X = Cl
19: X = Br
To examine the immunostimulatory activity and Th1/Th2 selec-
tivity of the synthetic
a-GalCer analogues, cytokine productions
Scheme 1. Synthesis of halogenated fatty acids 17, 18, and 19. Reagents and
conditions: (a) 1-Phenyl-1H-tetrazole-5-thiol, Ph3P, DEAD, THF, 96%; (b) m-CPBA,
CH2Cl2, 84%; (c) BH3-THF, THF, 0 °C to rt, 82%; (d) PCC, sodium acetate, CH2Cl2, 80%;
(e) 8, LHMDS THF, ꢀ78 °C to rt, 80%; (f) TBAF, THF, 80%; (g) MsCl, pyridine, THF,
94%; (h) DAST, CH2Cl2, 0 °C to rt, 40%; (i) Pd(OH)2-C, Et3SiH, MeOH/CH2Cl2 = 3:5, 1 h
16: 80%, 17: 85% (j) KCl or KBr, (Bu4N)HSO4, H2O, toluene, 100 °C, 4 h, 18: 90%, 19:
93%.
were evaluated by using the primary culture of murine spleen cells
with KRN7000 as a positive control (Fig. 3).53 Five concentrations
(10–1000 nM) of analogues 1–5 were incubated with freshly pre-
pared mouse splenocytes and the amounts of cytokines produced
after incubation for 48 hours were determined by ELISA kits. All
of the compounds induced production of IFN-c at a significant level
(Fig. 3A). Fig. 3B shows the amounts of IL-4 produced in the pres-
ence of analogues 1–5. Although they stimulated IL-4 production,
their potency was grossly lower than KRN7000 in the concentra-
tions tested. When the stimulation of the produced cytokines are
compared at 100 nM with that of KRN7000 (Fig. 3C), all of the ana-
11. The Julia-Kocienski olefination reaction was applied to form
alkene 12 (E/Z = 3:1) from the precursors 8 and 11. The TBS protec-
tion of 12 was removed with TBAF to give 13. The fluoropenta-
cosanoic acid 17 was synthesized from the common intermediate
13 using the fluorinating agent diethylaminosulfur trifluoride
(DAST) and the subsequent hydrogenation procedures. Chloro or
bromo pentacosanoic acids 18 and 19 were synthesized by mesy-
lation of 13 using MsCl, and the yielded compound 14 was treated
with activated palladium hydroxide on carbon in the presence of
Et3SiH to yield the carboxylic acid 16. Finally, heating of compound
16 with excess amount of KCl or KBr in biphasic medium (toluene/
water) provided the desired chloro or bromo pentacosanoic acids
18 and 19, respectively.
logues showed higher selectivity toward IFN-
IL-4 = 2.9–5.0); in particular, analogue 1 showed the highest selec-
tivity for IFN- production (IFN- /IL-4 = 5.0). This potency is com-
selective ligand (IFN- /IL-4 = 5.80)
c production (IFN-c/
c
c
parable to the best IFN-
c
c
reported so far.54
3. Discussion
Our objective is to improve the accuracy of the 3D structure of
the
-GalCer chain bound to the A0 pocket of CD1d (Fig. 2). Toward
a
Selenocerotic acids 2446 and 25 were prepared in two steps
(Scheme 2): first dialkyl diselenides (20 and 21) were synthesized
from bromoalkanes and diselenide, the latter of which was pre-
pared in situ from metallic selenium and sodium borohydride.47
Bromoalkyl carboxylic acid (23) was synthesized according to the
similar synthetic scheme shown in Scheme 1 (see Supporting infor-
mation). The intermediates 20 and 21 thus obtained were next
reduced by sodium borohydride again and reacted with bro-
moalkyl carboxylic acids (22 and 23) to yield selenocerotic acids
24 and 25.48,49
this goal, we introduced a heavy atom into the acyl moiety of
KRN7000 for enhancing the electron diffraction in X-ray crystallog-
raphy. The high resolution crystal structure of the lipid binding
pocket would disclose the atomistic mechanism of lipid–protein
interactions and facilitate precise molecular designs for developing
a more specific CD1d ligand.
In the cytokine production assays, the overall IFN-c secretions
induced by the synthetic analogues 1–5 showed a roughly similar
rate to the standard KRN7000 (Fig. 3A). The result clearly indicates
that all of our synthetic analogues form the bioactive ternary

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M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
Ph
Ph
Ph
O
O
O
O
O
O
(b)
(a)
O
(c),(d)
O
BnO
O
BnO
N3 OBn
SPh
BnO
BnO
BnO
O
BnO
N3 OBn
F
C14H29
HO
OBn
C14H29
26
27
29
OBn
28
O
OH
HO
HN
X
Y
Z
O
HO
OH
OH
HO
O
1: X = CH2, Y = CH2, Z = F
2: X = CH2, Y = CH2, Z = Cl
3: X = CH2, Y = CH2, Z = Br
4: X = Se, Y = CH2, Z = CH3
5
: X = CH2, Y = Se, Z = CH3
Scheme 3. Reagents and conditions: (a) DAST, NBS, CH2Cl2, 40% (yield of
(d) 17, 18, 19, 24, or 25, HBTU, N-methylmorpholine, Et3N, (40–60%).
a-anomer); (b) AgOTf, SnCl2, MS 4Å, Et2O/THF = 5, 0 °C, 73%; (c) 20% Pd(OH)2-C, H2, MeOH/CH2Cl2;
complex with CD1d and TCR on NKT cells, and induce signaling
cascades for cytokine production. The perturbation induced by a
halogen atom at the C260 position or by a Se atom at C120 or C180
position is rather limited except for the reduced production of IL-
4 at higher doses (Fig. 3B), where the cytokine level in the presence
of the synthesized ligands was significantly lower than that of
KRN7000. The Th1/Th2 selectivity is often discussed with the
strength of the binding affinity of
CD1d/
-GalCer complex to TCR.24,32,36 Liang et al. reported that
the affinity between -GalCer and CD1d correlates well with IFN-
production by NKT cells, but less with IL-4 secretion.55 Thus, sim-
a-GalCer to CD1d, and of the
a
a
c
ilar IFN-c production rates of the synthesized ligands (1–5) and
KRN7000 imply their similar binding affinities to CD1d. According
to the previous X-ray structure of the CD1d and KRN7000 complex,
Koch et al. indicated that the binding of KRN7000 induces a slight
closure of the ligand binding groove that consists of two a
-helices.5
On the other hand, in the following complexation with TCR, the
KRN7000/CD1d structure is unaltered—a lock and key type of
interaction.6,32 Our synthetic analogues have all functional groups
required for the interaction between KRN7000/CD1d and TCR,
including 3-OH of sphingosine and 200-OH, 300-OH, and 400-OH of
galactose.36 Therefore, our synthetic analogues in complex with
CD1d would have similar conformation to that of KRN7000 in the
bound form. These results imply that small changes in the bond
lengths (C–X or C–Se vs C–C) or bond angle (C–Se–C vs C–C–C) in
the modified sites of KRN7000 do not significantly affect their
interaction mode. Thus, the X-ray co-crystallography of the syn-
thetic analogues (3, 4, and 5) in complex with CD1d is most likely
to mimic the ligand-binding mode of KRN7000 at the atomistic
level, and the electron-poor acyl chain atom is expected to be
clearly visualized by the electron-rich Br and Se atoms.
In contrast to the IFN-c production, all of the analogues induced
IL-4 secretion in smaller amounts as compared with KRN7000
under the middle and higher ligand concentrations (Fig. 3B). In
other words, the significant suppression of the Th2 cytokine secre-
tion makes the synthetic ligands biased toward IFN-c production
(Fig. 3C). The structural basis of the selectivity in the cytokine pro-
duction has been speculated so far. As shown in Fig. 4, structural
superposition of the Th1-selective ligand C8PhF55 (PDB code:
3GMO)56 and non-selective KRN7000 (PDB code: 3HE6)7 bound
in a murine CD1d showed a slight difference. It is believed that
the position of the sugar moiety, as the main recognition site by
Figure 3. Cytokines produced due to the designed and synthesized
a-GalCer
analogues 1–5 by murine splenocytes. The amount of cytokine production, (pg/mL)
of IFN- (A) and IL-4 (B) are summarized. (C) The relative rate of cytokine
production of -GalCers analogues against KRN7000, at 100 nM concentrations are
summarized. IFN- /IL-4; 1; 5.0, 2; 3.8, 3; 4.4, 4; 3.3, 5; 2.9.
c
a
c

M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
5. Experimental
3691
5.1. General
The commercially available reagents were used without further
purification. The 1H and 13C NMR spectra were measured on the
ECA-500 and ECS-400 spectrometer (JEOL, Tokyo) with CDCl3 as a
solvent unless otherwise indicated. Chemical shifts are given in
ppm (d) and coupling constants (J) are in Hz. The following abbre-
viations were used to designate the multiplicities: s = singlet,
d = doublet, t = triplet, q = quartet, quint = quintuplet, m = multi-
plet, br = broad. High resolution mass spectra were obtained on
LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, CA).
5.2. Synthetic procedures
5.2.1. 5-(12-tert-Butyldimethylsilyloxydodecylthio)-1-phenyl-
1H-tetrazole (7)
To a solution of 6 (2.04 g, 6.45 mmol) in THF (30 mL) added
PPh3 (2.03 g, 7.74 mmol), diethyl azodicarboxylate (1.35 mL;
2.2 M solution in toluene, 7.74 mmol), and 1-phenyl-1H-tetra-
zole-5-thiol (1.38 g, 7.74 mmol) at 0 °C was stirred for 2 h at room
temperature under argon atmosphere. Then, the mixture was
diluted with Et2O and the organic phase was washed with water
and brine, dried over MgSO4, filtered and concentrated. The crude
residue was purified by silica gel column chromatography (4%
EtOAc in hexane) to afford 7 (2.94 g, 6.19 mmol, 96%) as a sticky
mass. 1H NMR (500 MHz, CDCl3) d 7.62–7.48 (m, 5H), 3.58 (t,
J = 6.6 Hz, 2H), 3.42–3.34 (m, 2H), 1.80 (dt, J = 15.0, 7.5 Hz, 2H),
1.54–1.46 (m, 2H), 1.46–1.37 (m, 2H), 1.35–1.19 (m, 14H), 0.88
(s, 9H), 0.03 (s, 6H); 13C NMR (126 MHz, CDCl3) d 154.55, 133.8,
130.1, 129.8, 123.9, 63.4, 33.5, 32.9, 29.7, 29.6, 29.5, 29.2, 29.1,
28.7, 26.1, 25.9, 18.4, ꢀ5.1; HRMS calculated for C25H44N4OSSiNa:
499.3005, found 499.3002 [M+Na]+.
Figure 4. (A) Chemical structure of C8PhF, a Th1-selective ligand. (B) Superposed
structures of murine CD1d complexed with Th1-selective ligand C8PhF (pink ribbon
and magenta wire) and that with KRN7000 (sky-blue ribbon and blue wire); PDB
codes are 3GMO and 3HE6. C260 of KRN7000 is shown with a dark green ball and
C120/C180 are shown with gray balls, which corresponded to halogen and selenium
positions of the synthetic analogues, respectively.
TCR, influences the CD1d–TCR interaction, and consequently mod-
ulates the stability of the CD1d/a
-GalCer/TCR complex.6,32 In the
CD1d–C8PhF complex, the modified acyl chain induces a small,
but significant shift of the sugar moiety from the position found
with KRN7000 (Fig. 4B). The cytokine assay results of a series of
C8PhF-related compounds uniformly showed a reduction of IL-4
5.2.2. 5-(12-tert-Butyldimethylsilyloxydodecylsulfonyl)-1-
phenyl-1H-tetrazole (8)
secretion, but a diverse response of IFN-
c
secretion.55 For ana-
m-Chloroperoxybenzoic acid (75%, 7.15 g, 31.05 mmol) was
added to the solution of 7 (2.59 g, 6.21 mmol) in CH2Cl2 (70 mL)
at 0 °C under argon atmosphere. After the reaction mixture was
stirred overnight at room temperature, 10% aq Na2S2O3 (15 mL)
was added and stirred for additional 30 min. Sat. NaHCO3
(20 mL) was added before extraction with CH2Cl2. The organic
phase was washed sequentially with 10% aq Na2S2O3, sat NaHCO3,
and brine. The organic phase was dried over Na2SO4, filtered, and
filtrate was evaporated and the residue was purified by silica gel
column chromatography (3% EtOAc in hexane) to afford 8 (2.66 g,
5.24 mmol, 84%) as a white solid. 1H NMR (500 MHz, CDCl3) d
7.72–7.66 (m, 2H), 7.66–7.55 (m, 3H), 3.75–3.68 (m, 2H), 3.59 (t,
J = 6.6 Hz, 2H), 1.99–1.90 (m, 2H), 1.49 (s, 4H), 1.38–1.21 (m,
14H), 0.89 (s, 9H), 0.04 (s, 6H); 13C NMR (126 MHz, CDCl3) d
153.6, 133.1, 131.5, 129.8, 125.1, 63.4, 56.1, 32.1, 29.7, 29.6, 29.5,
29.3, 29.0, 28.3, 26.1, 25.9, 22.1, 18.5, ꢀ5.1; HRMS calculated for
logues 1–5 that showed similar cytokine production profiles to
that of C8PhF, interaction of the acyl chain with the A0 pocket
might be slightly weakened by the halogen or Se atom, and the
resultant structural change could induce a slight shift in the posi-
tion of the sugar head group upon the CD1d/ligand/TCR complex-
ation, which possibly led to attenuation of the Th2 pathway. It is
worth noting that a simple heavy atom substitution in the acyl
chain of a-GalCers significantly influences the Th1/Th2 selectivity.
Knowledge on structural basis in selective activation of iNKT cells
potentially help develop drugs that modulate immune pathways
including those necessary for cancer18,25 and malaria29 treatments.
4. Conclusion
We established the synthesis of a novel class of
bearing halogen-containing and selenium-containing fatty acid
derivatives. The analogues showed efficient stimulation for IFN-
a-GalCer probes
C
25H44N4O3SSiNa: 531.2903, found 531.2903 [M+Na]+.
c
5.2.3. 13-Benzyloxy-13-oxotridecanoic acid (9)
secretion from murine spleen cells, which was comparable to
KRN7000. The data suggests that synthetic analogues could be uti-
lized to examine the atomistic interactions between KRN7000 and
CD1d. The heavy atoms will help analyze the lipid–protein interac-
tions by enhancing the poor electron density of the lipid tail. They
will also be a good reporter in determining atomic positions pre-
cisely. Further study on X-ray crystallography is currently under-
way to elucidate the mechanism of atomistic interactions and
To a solution of tridecanedioic acid (5.0 g, 20.5 mmol) in THF
(30 mL) at 0 °C added benzylbromide (2.44 mL, 20.5 mmol) and
diazabicyclo[5.4.0]undec-7-ene (3.05 mL, 20.5 mmol) was stirred
at room temperature for overnight. Then, water was added, and
the reaction mixture was extracted with EtOAc, dried over MgSO4,
filtered, and concentrated. The crude residue was purified by silica
gel column chromatography (16% EtOAc in hexane) to afford 9
(3.10 g, 9.42 mmol, 46%) as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.41–7.28 (m, 5H), 5.11 (s, 2H), 2.34 (td, J = 7.5, 2.9 Hz,
the conformations of the
form.
a-GalCer ligands in the CD1d complex

3692
M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
4H), 1.62 (dt, J = 15.0, 7.3 Hz, 4H), 1.39–1.19 (m, 14H); 13C NMR
(101 MHz, CDCl3) d 179.8, 173.8, 136.2, 128.6, 128.2, 66.2, 34.5,
J = 14.7, 7.3 Hz, 2H), 1.55 (dt, J = 13.3, 6.7 Hz, 2H), 1.43–1.20 (m,
32H);13C NMR (126 MHz, CDCl3) d 173.7, 136.2, 130.4, 129.9,
128.6, 128.2, 66.1, 63.2, 34.5, 29.9, 29.8, 29.7, 29.6, 29.6, 29.4,
29.6, 29.26 (d), 27.3, 25.9, 25.1; HRMS calculated for C32H54O3Na:
509.4073, found 509.4071 [M+Na]+.
34.1, 29.39, 25.1, 24.7; HRMS calculated for
C20H30O4Na:
357.2144, found 357.2141 [M+Na]+.
5.2.4. Benzyl 13-hydroxytridecanoate (10)
A solution of 9 (2.69 g, 8.05 mmol) in THF (20 mL) was cooled at
0 °C and borane-tetrahydrofuran complex (1 M solution in THF,
16 mL) was added over 20 min with maintaining the cooling tem-
perature. After stirring at room temperature for 2 h, the reaction
mixture was quenched with water, extracted with EtOAc, dried
over MgSO4, filtered, and concentrated. The crude residue was
purified by silica gel column chromatography (16% EtOAc in hex-
ane) to afford 10 (2.12 g, 6.61 mmol, 82%) as a white solid. 1H
NMR (500 MHz, CDCl3) d 7.40–7.28 (m, 5H), 5.11 (s, 2H), 3.63 (t,
J = 6.6 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.69–1.59 (m, 2H), 1.59–
1.51 (m, 2H), 1.39–1.20 (m, 16H); 13C NMR (126 MHz, CDCl3) d
173.8, 136.2, 66.2, 63.2, 34.5, 34.5, 32.9, 32.9, 29.6, 29.5, 29.3,
29.2; 25.8, 25.0 HRMS calculated for C20H32O3Na: 343.2351, found
343.2345 [M+Na]+.
5.2.8. Benzyl 25-(methylsulfonyloxy)pentacos-13-enoate (14)
To a solution of 13 (510 mg, 1.1 mmol) in pyridine (6.5 mL)
and THF (10 mL) added methanesulfonyl chloride (297 lL,
3.83 mmol) at 0 °C was stirred overnight at room temperature.
Then, solvent was evaporated and the residue was purified by sil-
ica gel column chromatography (10% EtOAc in hexane and 50%
EtOAc in hexane) to afford 14 (588 mg, 1.04 mmol, 94%) as a
sticky mass. 1H NMR (500 MHz, CDCl3) d 7.40–7.28 (m, 5H),
5.41–5.32 (m, 2H), 5.11 (s, 2H), 4.21 (t, J = 6.6 Hz, 2H), 2.99 (s,
3H), 2.34 (t, J = 7.6 Hz, 2H), 2.03–1.92 (m, 4H), 1.79–1.69 (m,
2H), 1.68–1.57 (m, 2H), 1.43–1.14 (m, 32H); 13C NMR
(126 MHz, CDCl3) d 173.5, 140.5, 130.3, 128.5, 128.1, 70.1, 66.0,
37.3, 34.3, 32.6, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.3,
29.2 29.1, 29.1, 29.0, 25.4, 24.9; HRMS calculated for
C
33H56O5SNa: 587.3848, found 587.3743 [M+Na]+.
5.2.5. Benzyl 13-oxotridecanoate (11)
To a solution of 10 (2.65 g, 8.28 mmol) in dichloromethane
(50 mL) at 0 °C added pyridinium chlorochromate (3.41 g,
15.81 mmol), and sodium acetate (339 mg, 4.14 mmol) was stirred
overnight at room temperature. Then, the precipitate was sepa-
rated by filtration and washed with EtOAc. The filtrate was concen-
trated and purified by silica gel column chromatography (8% EtOAc
in hexane) to afford 11 (2.12 g, 6.67 mmol, 80%) as an oil. 1H NMR
(500 MHz, CDCl3) d 9.75 (s, 1H), 7.42–7.28 (m, 5H), 5.11 (s, 2H),
2.41 (t, J = 7.4 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.62 (dd, J = 14.6,
7.3 Hz, 4H), 1.36–1.19 (m, 14H); 13C NMR (126 MHz, CDCl3) d
202.8, 173.6, 136.0, 128.4, 128.1, 66.0, 43.8, 34.3, 29.4, 29.3, 29.3,
29.1, 29.1, 29.0, 24.9, 22.0.; HRMS calculated for C20H30O3Na:
341.2195, found 341.2185 [M+Na]+.
5.2.9. Benzyl 25-fluoropentacos-13-enoate (15)
To a solution of 13 (100 mg, 0.22 mmol) in CH2Cl2 (10 mL)
added diethylaminosulfur trifluoride (DAST; 48 lL, 0.39 mmol) at
0 °C was stirred for 5 h at room temperature. Then, solvent was
evaporated and the residue was purified by silica gel column chro-
matography (4% EtOAc in hexane) to afford 15 (76 mg, 0.16 mmol,
71%) as a white solid. 1H NMR (500 MHz, CDCl3) d 7.40–7.28 (m,
5H), 5.38 (t, J = 3.7 Hz, trace), 5.34 (t, J = 4.5 Hz, trace), 5.11 (s,
2H), 4.43 (dt, J = 47.4, 6.2 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 2.04–
1.92 (m, 4H), 1.74–1.59 (m, 4H), 1.45–1.20 (m, 32H); 13C NMR
(126 MHz, CDCl3) d 173.7, 136.2, 130.4 129.96, 128.6, 128.2, 84.3
(JC–F = 164.1 Hz), 66.1, 34.5, 32.7, 30.6, 30.5, 29.9, 29.8, 29.7, 29.6,
29.6, 29.4, 29.3, 27.3, 25.3, 25.2, 25.1; HRMS calculated for
C
32H53FO2Na: 511.4030, found 511.4026 [M+Na]+.
5.2.6. Benzyl 25-(tert-butyldimethylsilyloxy)pentacos-13-
enoate (12)
5.2.10. 25-((Methylsulfonyl)oxy)pentacosanoic acid (16)
To a solution of 14 (240 mg, 0.53 mmol) in CH2Cl2/MeOH (5:3 v/
v, 10 mL) added Pd(OH)2 (20 wt%, 240 mg) and triethylsilane
(0.84 mL, 5.3 mmol) was stirred at room temperature for 30 min.
Then, the catalyst was removed by filtration through Celite pad,
and filtrate was evaporated and the residue was purified by silica
gel column chromatography (10% MeOH in CH2Cl2) to afford 16
(155 mg, 0.43 mmol, 80%) as a white solid. 1H NMR (500 MHz,
CDCl3) d 4.21 (t, J = 6.6 Hz, 2H), 2.98 (s, 3H), 2.34 (t, J = 7.5 Hz,
2H), 1.74 (dt, J = 14.6, 6.7 Hz, 2H), 1.62 (dd, J = 14.9, 7.3 Hz, 2H),
1.45–1.36 (m, 2H), 1.31 (d, J = 54.7 Hz, 40H); 13C NMR (126 MHz,
CDCl3) d 178.5, 70.1, 37.3, 33.8, 29.7, 29.5, 29.4, 29.3, 29.2, 29.1,
29.0, 25.4, 24.7; HRMS calculated for C26H52O5SNa: 499.3535,
found 499.3539 [M+Na]+.
A solution of 8 (1.79 g, 3.53 mmol) and 11 (1.36 g 4.27 mmol) in
THF was cooled at ꢀ78 °C and LiHMDS (1 M in THF, 3.53 mL) was
added over 15 min under argon atmosphere. After stirring for
3 h, the reaction mixture was warmed up and stirred overnight
at room temperature. The reaction mixture was quenched with
NH4Cl solution and extracted with Et2O. After removal of the sol-
vent, the residue was purified by silica gel column chromatography
(2% EtOAc in hexane) to afford 12 (1.7 g, 2.83 mmol, 80%) as an oil.
1H NMR (500 MHz, CDCl3) d 7.40–7.27 (m, 5H), 5.38 (t, J = 3.7 Hz,
trace), 5.35 (t, J = 5 Hz, trace), 5.11 (s, 2H), 3.60 (t, J = 6.7 Hz, 2H),
2.35 (t, J = 7.5 Hz, 2H), 1.96 (dt, J = 7.3, 3.6 Hz, 4H), 1.70–1.59 (m,
2H), 1.55–1.47 (m, 2H), 1.37–1.20 (m, 32H), 0.92–0.87 (m, 9H),
0.06–0.03 (m, 6H); 13C NMR (126 MHz, CDCl3) d 173.7, 136.2,
130.4, 129.9, 128.6, 128.2, 66.1, 63.4, 34.4, 33.0, 32.7, 29.9, 29.7,
29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 29.3, 29.2, 27.3, 26.1, 25.9, 25.0,
18.5, -5.1; HRMS calculated for C38H68O3SiNa: 623.4938, found
623.4939 [M+Na]+.
5.2.11. 25-Fluoropentacosanoic acid (17)
To a solution of 15 (76 mg, 0.16 mmol) in CH2Cl2/MeOH
(5:3 v/v; 5 mL) added Pd(OH)2 (20 wt%, 76 mg) and triethylsilane
(299 lL, 1.6 mmol) was stirred for 30 min at room temperature.
5.2.7. Benzyl 25-hydroxypentacos-13-enoate (13)
To a solution of 12 (358 mg, 0.596 mmol) in THF added TBAF
(1 M in THF, 894 lL) at 0 °C was stirred overnight at room temper-
ature. Then, solvent was evaporated and the residue was purified
by silica gel column chromatography (16% EtOAc in hexane) to
afford 13 (231 mg, 0.48 mmol, 80%) as a sticky mass. 1H NMR
(500 MHz, CDCl3) d 7.39–7.29 (m, 5H), 5.38 (t, J = 3.7 Hz, trace),
5.34 (t, J = 4.5 Hz, trace), 5.11 (s, 2H), 3.63 (t, J = 6.6 Hz, 2H), 2.35
(t, J = 7.5 Hz, 2H), 1.97 (ddd, J = 12.4, 10.0, 3.4 Hz, 4H), 1.62 (dd,
Then, the catalyst was removed by filtration through Celite pad,
and filtrate was concentrated and the residue was purified by silica
gel column chromatography (10% MeOH in CH2Cl2) to afford 17
(56 mg, 0.14 mmol, 85%) as a white solid. 1H NMR (300 MHz,
CDCl3) d 4.43 (dt, J = 47.4, 6.2 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H),
1.77–1.54 (m, 4H), 1.49–1.17 (m, 40H); 13C NMR (126 MHz, CDCl3)
d 175.3, 82.0 (JC–F = 163.8 Hz) 34.2, 32.9, 29.6, 29.3, 29.2, 28.9, 28.3,
24.8; HRMS calculated for C25H49FO2Na: 423.3717, found 423.3716
[M+Na]+.

M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
3693
5.2.12. 25-Chloropentacosanoic acid (18)
became colorless. A solution of 17-bromoheptadecanoic acid (23)
(150 mg, 0.42 mmol) and triethylamine (0.069 mL, 0.48 mmol) in
water (0.5 mL) was added dropwise and the mixture was stirred
overnight. The mixture was then quenched with 4 M HCl and
extracted with Et2O (ꢁ3). Combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated. The crude
residue was purified by chromatography on silica gel (gradient
hexane/EtOAc 10:0 to 5:5) to afford 17-(octylselenyl)heptade-
canoic acid 25 (80 mg, 0.173 mmol, 42%) as a white solid. 1H
NMR (400 MHz, CDCl3) d 2.54 (t, J = 7.5 Hz, 4H), 2.34 (t, J = 7.5 Hz,
2H), 1.63 (m, 7.3 Hz, 6H), 1.45–1.18 (m, 34H), 0.87 (t, J = 6.8 Hz,
3H); 13C NMR (101 MHz, CDCl3) d 179.0, 33.9, 31.9, 30.8, 30.1,
29.7, 29.3, 24.8, 24.1, 22.7, 14.2; HRMS calculated for
To a solution of 17 (60 mg, 0.109 mmol) in toluene/H2O = 2:3
(1.5 mL) added KCl (203.1 mg, 2.72 mmol) and tetrabutylammo-
nium hydrogensulfate (12.95 mg, 0.038 mmol) was heated at
100 °C for 4 h. Then, the reaction mixture was cooled to room tem-
perature, solvent was removed and the residue was purified by sil-
ica gel column chromatography (2% MeOH in CH2Cl2) to afford 18
(40 mg, 0.098 mmol, 90%) as a white solid. 1H NMR (400 MHz,
CDCl3) d 3.52 (t, J = 6.8 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.83–1.70
(m, 2H), 1.62 (dd, J = 14.8, 7.3 Hz, 2H), 1.41 (dd, J = 10.1, 5.1 Hz,
2H), 1.37–1.17 (m, 38H); 13C NMR (101 MHz, CDCl3) d 178.4,
45.2, 33.7, 32.6, 29.8 29.7, 29.6, 29.5, 29.4, 29.2, 29.0, 28.9, 26.8,
24.6; HRMS calculated for C25H49ClO2Na: 439.3421, found 439.
3420 [M+Na]+.
C25H50O2SeNa: 485.2976, found [M+Na] 485.2975.
5.2.13. 25-Bromopentacosanoic acid (19)
5.2.17. (2-Azido-3,4-di-O-benzyl-1-O-(2,3-di-O-benxyl-4,6-O-
To a solution of 17 (50 mg, 0.105 mmol) in toluene/H2O = 2:3
(1.5 mL) added KBr (119 mg, 2.62 mmol) and tetrabutylammo-
nium hydrogensulfate (12.5 mg, 0.037 mmol) was heated at
100 °C for 4 h. Then, the mixture was cooled to room temperature,
solvent was removed, and the residue was purified by silica gel col-
umn chromatography (2% MeOH in CH2Cl2) to afford 19 (45 mg,
0.098 mmol, 93%) as a white solid. 1H NMR (500 MHz, CDCl3) d
3.40 (t, J = 6.9 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.85 (dt, J = 8.9,
6.9 Hz, 2H), 1.63 (dt, J = 15.0, 7.5 Hz, 2H), 1.41 (dd, J = 9.2, 4.3 Hz,
2H), 1.35–1.21 (m, 38H); 13C NMR (126 MHz, CDCl3) d 180.5,
34.0, 33.7, 32.8, 32.3, 31.5, 29.7, 29.6, 29.5, 29.2, 29.0, 28.7, 24.7;
HRMS calculated for C25H49BrO2Na: 483.2916, found 483.2914
[M+Na]+.
benzyllidene-
triol (29)
a-D-galactopyranosyl)-D-ribo-octadecane-1,3,4-
A mixture of 28 (1.74 g, 3.32 mmol) and 27 (1.63 g, 3.62 mmol)
was co-evaporated with toluene and dissolved in Et2O/THF = 5:1
(72 mL) dried with MS 4Å under argon atmosphere. AgOTf
(2.58 g, 10.05 mmol) and SnCl2 (1.2 g, 6.36 mmol) were added to
the reaction mixture at 0 °C. After stirring for 2 h at 0 °C, the TLC
analysis (6% hexane in EtOAc) of the reaction mixture shows full
consumption of compound 27. The reaction was quenched with
sat. NaHCO3 and extracted with EtOAc, and the organic phase
was washed with brine, dried over MgSO4, filtered, and concen-
trated under reduced pressure. The crude residue was purified by
silica gel column chromatography (7% EtOAc in hexene) to afford
29 (1.53 g, 1.72 mmol, 73%) as a white solid. 1H NMR (500 MHz,
CDCl3) d 7.51 (dd, J = 7.7, 1.8 Hz, 2H), 7.42–7.19 (m, 23H), 5.45 (s,
1H), 4.96 (d, J = 3.4 Hz, 1H), 4.83 (m, 2H), 4.73 (d, J = 12.3 Hz, 1H),
4.70–4.62 (m, 2H), 4.58 (dd, J = 13.7, 9.6 Hz, 2H), 4.49 (d,
J = 11.5 Hz, 1H), 4.17–4.14 (m, 1H), 4.11–3.97 (m, 3H), 3.92–3.85
(m, 1H), 3.80 (s, 1H), 3.66 (m, 5H), 1.75–1.60 (m, 1H), 1.54 (s,
2H), 1.48–1.35 (m, 1H), 1.35–1.08 (m, 23H), 0.88 (t, J = 6.9 Hz, 3H).
5.2.14. Dioctyldiselenide (20)
Elemental selenium (147.5 mg, 1.87 mmol) was suspended in
ethanol (6 mL) and NaBH4 was slowly added (297.4 mg,
7.86 mmol) under argon atmosphere while H2 gas was evolved vig-
orously. The mixture was stirred until complete dissolution of sele-
nium. Second portion of the elemental selenium (147.5 mg,
1.87 mmol) was then added and the mixture was stirred for
20 min at room temperature, and followed by heating at 65 °C
for 5 min with continuous argon bubbling. 1-Bromooctane
(719 mg, 3.74 mmol) was then slowly added under vigorous stir-
ring and the mixture was stirred for 1 h at 65 °C. Then, the reaction
mixture was filtered and the residue was washed with hexane. The
filtrate was concentrated and the crude residue was purified by
chromatography on silica gel (hexane) to afford 20 (450 mg,
1.17 mmol, 62%) as a sticky mass. 1H NMR (400 MHz, CDCl3) 1H
NMR (400 MHz, CDCl3) d 3.13–3.04 (m, 1H), 2.95–2.85 (m, 3H),
1.80 (dt, J = 14.9, 7.3 Hz, 1H), 1.77–1.67 (m, 3H), 1.32 (m, 20H),
0.88 (t, J = 6.9 Hz, 6H); 13C NMR (101 MHz, CDCl3) d 31.8, 31.0,
30.5, 30.3, 29.5, 29.2, 29.1, 22.6, 14.1; HRMS calculated for
5.2.18. General procedure for amide coupling reaction
2 equiv of HBTU, Et3N, and N-methylmorpholine were added to
the solution of deprotected galactosyl sphingosine 29 and one of
the long chain fatty acid (17, 18, 19, 24 and 25) in THF. The reaction
mixture was stirred overnight at room temperature. Upon full con-
sumption of starting materials confirmed by TLC analysis, the
crude residue was concentrated and purified by chromatography
on silica gel (MeOH/CH2Cl2 = 10:1) to afford the corresponding
amides.
5.2.19. 1-O-
2S,3S,4R-phytosphingosine (1)
(15 mg, 17.42
mol 53%), white solid; 1H NMR (500 MHz, CDCl3,
a-D-Galactopyranosyl-2-N-(25-fluoropentacosanoyl)-
C
16H34Se2Na: 509.0991, found 509.0990 [M+Na]+.
l
CD3OD) d 4.88 (d, J = 3.8 Hz, 1H), 4.45 (t, J = 6.2 Hz, 1H), 4.35 (t,
J = 6.2 Hz, 1H), 4.23–4.14 (m, 1H), 3.88 (ddd, J = 15.2, 6.9, 2.7 Hz,
2H), 3.84–3.77 (m, 2H), 3.77–3.65 (m, 4H), 3.62–3.50 (m, 2H),
2.21 (t, J = 7.6 Hz, 2H), 1.79–1.53 (m, 6H), 1.48–1.18 (m, 67H),
0.88 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3, CD3OD) d
180.6, 99.8, 83.4 (JC–F = 163.5 Hz) 75.1, 74.2, 73.8, 71.2, 70.2, 69.7,
68.9, 67.1, 61.5, 37.7, 36.0, 31.7, 30.3, 30.2, 29.6, 29.5, 29.4, 29.3,
29.2, 29.1, 29.0, 25.7, 22.4, 13.2, 8.0; HRMS calculated for
5.2.15. 11-(Tetradecylselenyl)undecanoic acid (24)
To synthesize compound 24, 1-bromotetradecane was trans-
formed into diselenide 21 by following the procedure as of com-
pound 20. Without further purification, the ditetradecyldiselenide
21 was used for the reaction with carboxylic acid 22 in a manner
similar to that described for compound 25 to give 24 (42%, white
solid). 1H NMR (400 MHz, CDCl3) d 2.54 (t, J = 7.5 Hz, 4H), 2.34 (t,
J = 7.5 Hz, 2H), 1.63 (m, 7.3 Hz, 6H), 1.45–1.18 (m, 34H), 0.87 (t,
J = 6.8 Hz, 3H), 13C NMR (101 MHz, CDCl3) d 178.9, 33.8, 31.8, 30.7,
30.0, 29.6, 29.5, 29.4 29.2, 29.1, 29.0, 24.7, 24.0, 22.6, 14.1.
C
49H96FNO9Na: 884.7069, found 884.7065 [M+Na]+.
5.2.20. 1-O-
2S,3S,4R-phytosphingosine (2)
(15 mg, 17.09
mol, 53%), white solid; 1H NMR (400 MHz,
a-D-Galactopyranosyl-2-N-(25-chloropentacosanoyl)-
5.2.16. 17-(Octylselenyl)heptadecanoic acid (25)
l
A solution of NaBH4 (22.7 mg, 0.6 mmol) in THF/water 1:1
(0.072 mL) was added to a solution of 20 (45 mg, 0.12 mmol) in
THF (0.5 mL), and the mixture was stirred until the solution
CDCl3) d 4.85 (d, J = 3.8 Hz, 1H), 4.21–4.12 (m, 1H), 3.90–3.64 (m,
2H), 3.63–3.57, 3.52 (t, J = 4.0 Hz, 2H), 2.20 (t, J = 7.7 Hz, 2H),
1.78–1.68 (m, 2H), 1.68–1.52 (m, 4H), 1.47–1.37 (m, 3H),

3694
M. I. Hossain et al. / Bioorg. Med. Chem. 24 (2016) 3687–3695
1.37–1.05 (m, 54H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz,
CDCl3) d 162.8, 56.2, 47.0, 46.2, 43.8, 43.7, 43.6, 43.4, 43.3, 43.1,
41.5, 39.8, 31.3, 20.58, 17.87, 8.80; HRMS calculated for
Acknowledgements
We thank Mr. Y. Shibata for his help in organic synthesis, Drs. Y.
Todokoro, N. Inazumi, and A. Ito for maintenance and measure-
ments for NMR and MS, and Mr. D. Takahashi and his colleague
(KAC Inc., Kyoto, Japan) for bioassay. This work was supported in
part by Japan Science and Technology Agency (JST) ERATO, Lipid
Active Structure Project and by JSPS KAKENHI Grant Numbers
26560448, 25242073 and 15K01801.
C
49H96ClNO9Na: 900.6774, found 900.6771 [M+Na]+.
5.2.21. 1-O-
2S,3S,4R-phytosphingosine (3)
(15 mg, 16.24
mol, 43%), white solid; 1H NMR (400 MHz,
a-D-Galactopyranosyl-2-N-(25-bromopentacosanoyl)-
l
CDCl3, CD3OD) d 4.91 (d, J = 3.8 Hz, 1H), 4.20 (d, J = 4.6 Hz, 1H),
4.03–3.86 (m, 2H), 3.84–3.71 (m, 6H), 3.55 (d, J = 4.0 Hz, 2H),
3.50–3.37 (m, 2H), 3.39–3.27 (m, 2H), 2.21 (t, J = 7.7 Hz, 2H),
1.99–1.76 (m, 2H), 1.73–1.50 (m, 4H), 1.43 (d, J = 7.5 Hz, 3H),
1.28 (d, J = 11.3 Hz, 59H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR
(101 MHz, CDCl3, CD3OD) d 174.1, 99.4, 74.4, 71.6, 70.4, 69.9,
69.4, 68.6, 67.0, 61.5, 50.1, 36.1, 33.5, 32.5, 32.2, 31.5, 29.3, 29.1,
29.0, 28.9, 28.3, 27.7, 25.4, 22.2, 13.5; HRMS calculated for
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.06.007.
References and notes
C
49H96BrNO9Na: 946.2090, found 946.2099 [M+Na]+.
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CD3OD) d 4.85 (d, J = 3.7 Hz, 1H), 4.17 (dt, J = 6.3, 4.5 Hz, 1H),,
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