Synthesis of RCAI-172 (C6 epimer of RCAI-147) and its biological activity

Article abstract of DOI:10.1016/j.bmc.2013.12.009

RCAI-147 is one of the hydroxylated analogues of KRN7000 which is known as a ligand for the activation of CD1d mediated invariant natural killer T cells (iNKT cells) and releases both T helper 1 (Th1) cytokines such as IFN-γ and T helper 2 (Th2) cytokines such as IL-4. KRN7000 has been anticipated as an antitumor drug or an adjuvant for viral infection such as influenza, because of its strong secretion of IFN-γ. In an interesting twist, it has been obvious in our previous paper that RCAI-147 induces much more Th2 cytokines (IL-4) than Th1 cytokines (IFN-γ) from iNKT cells compared to KRN7000, and shows fairly good result in the experimental autoimmune encephalomyelitis (EAE) test. Therefore, synthesis of RCAI-172 (C6-OH epimer of RCAI-147) was attempted to examine the biological activity. As a result, RCAI-172 was synthesized and its biological activity biased to Th2 response largely compared to that of KRN7000. However, this level decreased to approximately 61% compared to that of RCAI-147. And the clinical score of RCAI-172 for EAE suppression was disappointing. There exist seven chiral centers in the aglycon part of RCAI-172, and even though the change of configuration is just one position (C6-OH), the effect on both Th1/Th2 response and EAE test is fairly large.

Full text of DOI:10.1016/j.bmc.2013.12.009

Bioorganic & Medicinal Chemistry 22 (2014) 827–833
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of RCAI-172 (C6 epimer of RCAI-147) and its biological
activity
a
b
c
c
Masao Shiozaki a, , Takuya Tashiro , Hiroyuki Koshino , Tomokuni Shigeura , Hiroshi Watarai ,
⇑
Masaru Taniguchi c, Kenji Mori a
a Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
b Advanced Science Institute, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
c Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Yokohama Institute, Suehiro-cho 1-7-22, Tsurumi-ku,
Yokohama-shi, Kanagawa 230-0045, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
RCAI-147 is one of the hydroxylated analogues of KRN7000 which is known as a ligand for the activation
of CD1d mediated invariant natural killer T cells (iNKT cells) and releases both T helper 1 (Th1) cytokines
Received 15 October 2013
Revised 14 November 2013
Accepted 3 December 2013
Available online 11 December 2013
such as IFN-
drug or an adjuvant for viral infection such as influenza, because of its strong secretion of IFN-
interesting twist, it has been obvious in our previous paper that RCAI-147 induces much more Th2 cyto-
kines (IL-4) than Th1 cytokines (IFN- ) from iNKT cells compared to KRN7000, and shows fairly good
c
and T helper 2 (Th2) cytokines such as IL-4. KRN7000 has been anticipated as an antitumor
c
. In an
c
Keywords:
Glycolipids
Glycosylation
KRN7000
iNKT cells
Th1/Th2
result in the experimental autoimmune encephalomyelitis (EAE) test. Therefore, synthesis of RCAI-172
(C6-OH epimer of RCAI-147) was attempted to examine the biological activity. As a result, RCAI-172
was synthesized and its biological activity biased to Th2 response largely compared to that of
KRN7000. However, this level decreased to approximately 61% compared to that of RCAI-147. And the
clinical score of RCAI-172 for EAE suppression was disappointing. There exist seven chiral centers in
the aglycon part of RCAI-172, and even though the change of configuration is just one position (C6-
OH), the effect on both Th1/Th2 response and EAE test is fairly large.
EAE
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction1
effects of cytokines each other. Therefore, we need to develop more
effective antigens that bias the production of Th1 and Th2
Agelasphins are a series of the glycosphingolipids isolated from
the marine sponge (Agelas mauritianus), and they show potent ef-
fects on the immune system of mice.2 In 1995, KRN7000,3 one of
cytokines to one side or the other compared to KRN7000 which
induce NKT cells to produce both Th1- and Th2-type cytokines in
large quantities at the same time.
agelasphin related compounds having a structure of the
a
-galacto-
By the way, in our synthesis of the a-galactosylceramides, we
sylceramides, was developed by Kirin Brewery Co. in the process of
synthetic studies on agelasphins.
Invariant natural killer T (iNKT) cell is known as one of the lin-
eages of immunocytes.4 It produces immunoregulatory cytokines
both T helper 1 (Th1)-type [such as interferon-c (IFNc)] and Th2-
type [such as interleukin-4 (IL-4)] by the recognition of glycolipid
antigens such as KRN7000 presented by CD1d,5 which is an antigen
presenting protein for the T cell receptor (TCR) of iNKT cells. The
glycolipids stimulating the secretion of mainly Th1- or Th2-type
cytokines are expected to be promising anticancer drugs or immu-
nosuppressive agents and autoimmune disease treatment medi-
cine, respectively.6 It is thought that the balance of Th1 and Th2
cytokine production is important for therapeutic use. Because both
types of Th1 and Th2 may reduce or countervail the beneficial
found that two analogues [RCAI-147 and 160 in Fig. 1] of
KRN7000 induced highly biased Th2 cytokine secretion.7 As shown
in Figure 1, they have several hydroxy groups on the octadecyl
main chain and the C2 amide side chain, and showed good efficacy
in the experimental autoimmune encephalomyelitis (EAE) for a
mouse model of multiple sclerosis.8 In addition, we synthesized
both diastereomers of 4-deoxy-6-hydroxy analogues of KRN7000
(6R-isomer: RCAI-10, 6S-isomer: RCAI-45) in the early stage of
our structure-activity relationship (SAR) study on KRN7000, and
we found that the stereochemistry at C6-position highly influences
the potency of the immunostimulatory activity.9 Thereupon, we at-
tempted to synthesize C6-epimer (RCAI-172, 15) of RCAI-147.
Firstly we tried to develop an effective short-step route for 15
(C6 epimer of RCAI-147), because the synthetic steps of RCAI-147
were long and its total yield was poor. And then we examined
the biological activity of RCAI-172 in order to compare with that
of RCAI-147 (Fig. 1).
⇑
Corresponding author at present address: Saginomiya 3-31-2, Nakano-ku,
Tokyo 165-0032, Japan. Tel.: +81 333305616.
E-mail addresses: shiozaki@rcai.riken.jp, micheal1621@nifty.com (M. Shiozaki).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.009

828
M. Shiozaki et al. / Bioorg. Med. Chem. 22 (2014) 827–833
OH
HO
OH
HO
n
-C23H47
HO
n
-C25H51
O
O
OH
OH
OH
O
O
HO
HO
HN
HN
OH
HO
HO
O
O
6
n-C12H25
n-C14H29
OH OH
OH
RCAI-147
KRN7000
HO
HO
OH
HO
OH
HO
n
-C22H45
HO
O
n
-C23H47
O
OH
OH
O
OH
OH
OH
O
HO
HN
HO
HN
HO
HO
O
O
n
-C14H29
n-C12H25
6
OH
OH OH
RCAI-160
RCAI-172 (15)
Figure 1. Structures of KRN7000, RCAI-147, 160 and 172 (15).
OH
OH
OBn
OBn
O
c
OH
OBn
OBn
-C12H25
OR
d
f
b
RO
RO
O
6
n
-C12H25
n
n
-C12H25
OBn
OBn
OBn
OBn
OBn
OBn
1. R=TBDMS
2. R=H
5. R=H
6. R=TBDMS
3. R=H
4. R=Bn
a
e
N3
OBn
OBn
-C12H25
RO
g
n
OBn
OBn
7. R=TBDMS
8. R=H
Scheme 1. Reagents and conditions: (a) TBAF, THF, rt, 1 h, 98%; (b) 2,2-dimethoxypropane, p-TsOHꢀH2O, rt, 1 h, 91%; (c) BnBr, NaH, DMF, rt, 1 h, 65%; (d) aq 46% HF, CH2Cl2–
MeCN (1:1), rt, 30 min, 98%; (e) TBDMS-Cl, imidazole, catalytic DMAP, rt, 16 h, 99%; (f) Ph3P, DEAD (in toluene), LDPPA, THF, 0 °C, 30 min, quant; (g) TBAF, THF, rt, 50 min, 96%.
t
-Bu
Si
t
-Bu
t
-Bu
t
-Bu
Si
t
-Bu
O
O
Si
t
-Bu
O
O
O
O
O
O
O
n
-C23H47
O
O
O
a
c
BnO
CCl3
NH
BnO
BnO
NH OBn OBn
N3 OBn OBn
BnO
O
BnO
BnO
O
O
n
-C12H25
n
-C12H25
OBn OBn
OBn OBn
9
13
10. R=N3
b
11. R=NH2
HO
O
HO
OH
HO
OH
HO
n
-C23H47
n-C23H47
n
-C23H47
HOOC
O
O
O
O
OH
OH
NH OH OH
e
d
BnO
HO
NH OBn OBn
O
BnO
HO
O
O
6
n
-C12H25
n-C12H25
12
OBn OBn
OH OH
14
15 (RCAI-172)
Scheme 2. Reagents and conditions: (a) 8, MS 4 Å, CH2Cl2, rt, 30 min, then AgOTf, rt, 16 h, 91%; (b) Me3P, THF, rt, 2 h, then aq 1 M NaOH, rt, 2 h, 71%; (c) 12, EDAC, DMAP,
CH2Cl2–THF (1:1), rt, 3 h, 54%; (d) (1) aq HF, CH2Cl2–MeCN (1:1), rt, 3.5 h; (2) HFꢀpyridine (ꢁ70% HF, ꢁ30% pyridine), pyridine, THF, rt, 2 h, two steps 44%; (e) H2, Pd(OH)2/C, rt,
16 h, 82%.

M. Shiozaki et al. / Bioorg. Med. Chem. 22 (2014) 827–833
829
γ
Figure 2. The concentrations of IFN
c
and IL-4 upon administration of RCAI-172 using KRN7000 as a positive control.
Clinical score
5
DC + KRN7000
DC + RCAI-172
4
3
2
1
0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930
Days after immunization
Figure 3. Clinical score of RCAI-172 compared to KRN7000 for EAE suppression.

830
M. Shiozaki et al. / Bioorg. Med. Chem. 22 (2014) 827–833
to approximately 61% of RCAI-147, and RCAI-172 was tested for
2. Results and discussion
2.1. Synthesis
EAE8,12 as shown in Figure 3.
The cytokines secretion of RCAI-172 biased to more Th2 (IL-4)
than Th1 (IFNc
). This tendency was similar to that of RCAI-147,7
but the intencity of RCAI-172 was reduced to 61% of RCAI-147.
Therefore we anticipated that there might be a little difference be-
tween RCAI-147 and -172 in EAE test. But surprisingly the EAE
score of RCAI-172 was far worse than that of RCAI-147, namely al-
most the same as that of KRN7000. The amount of Th2 (IL-4) cyto-
kine secretion from iNKT cells mediated by RCAI-172 was reduced
to about 35.3% of KRN7000. Whereas that of RCAI-147 was 70% of
KRN7000. This difference in the amount of IL-4 should certainly af-
fect EAE score of RCAI-172 negatively. It is not known exactly why.
However, it is important to find the fact, namely, ‘the stereoiso-
mer at the only one position has a possibility to make an unex-
pected large influence on the biological activity’.
Therefore to examine more reliable and deep discussion con-
cerning the importance of the stereochemistry and functional
groups on the side chain by comparison of the other related syn-
thetic compounds, and also to examine more SAR studies, we need
to synthesize many other stereoisomers at C3, C4, C5 and/or C6
positions on the lipid chain of RCAI-147.
The tert-butyldimethylsilyl (TBDMS) group of the reported
compound 110 which was obtained by Grignard reaction of 2,3,4-
tri-O-benzyl-6-O-tert-butyldimethylsilyl-D
-galactose10 and dode-
cylmagnesium bromide, was deprotected with tetra-n-butylam-
monium fluoride (TBAF) to yield a triol 2 (98% yield). Both C1-
and C2-OH groups of compound 2 were protected as a 1,2-isopro-
pylidenedioxy group of compound 3 (91% yield) with 2,2-dime-
thoxypropane using a catalytic amount of p-toluenesulfonic acid
monohydrate (TsOHꢀH2O) in order to distinguish between two sec-
ondary alcohols, C2-OH and C6-OH. The remaining C6-OH of 3 was
benzylated with benzyl bromide using sodium hydride as a base to
give 4 (65% yield). The 1,2-isopropylidenedioxy group of 4 was
deprotected with aqueous 46% hydrofluoric acid (HF) to afford diol
5 (98% yield), which was treated with TBDMS-Cl and imidazole as a
base to give a silyl ether 6 (99% yield). Treatment of 6 with diethyl
azodicarboxylate (DEAD in toluene), triphenylphosphine (Ph3P),
and then diphenylphosphoryl azide [DPPA, (PhO)2P(O)N3] in tetra-
hydrofuran (THF) at 0 °C for 30 min afforded azide 7 (quant)
accompanying the inversion of configuration from R to S at C2-
OH position.7 Deprotection of TBDMS group from 7 with TBAF in
THF afforded primary alcohol 8 as a glycosyl acceptor (Scheme 1).
Glycosylation of 8 with imidate 9, obtained easily from 2,3-di-
In addition, the other mouse model tests for human autoim-
mune diseases such as type I diabetes, systemic lupus erythemato-
sus (SLE) and/or rheumatoid arthritis may possibly find out the
effectiveness of RCAI-172.
O-benzyl-4,6-O-(di-tert-butyl)silylene-
D
-galactopyranose7
and
2.3. Discussion
CCl3CN using Cs2CO3 as a base, was performed to afford
a
-anomer
10 (91% yield) by use of silver trifluoromethanesulfonate (AgOTf).
The azide group of 10 was reduced to amine 11 (71% yield) by
treatment with trimethylphosphine (Me3P) and successively aque-
ous NaOH. Reaction of 11 with (2R,3R)-(isopropylidenedioxy)hexa-
cosanoic acid 127 in the presence of N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide hydrochloride (EDAC) as a dehydrating agent
yielded amide 13 (54% yield). Treatment of 13 with aqueous HF in
CH2Cl2–CH3CN, and then HFꢀpyridine (ꢁ70% HF, ꢁ30% pyridine)
yielded a tetraol 14. The six benzyl groups of 14 were deprotected
by using 20% palladium hydroxide on carbon [Pd(OH)2/C] under
hydrogen in THF to afford 15 (RCAI-172) (Scheme 2).
Although it has not been clarified yet how the extra hydroxy
groups of RCAI-172 (15) interact with the residues of CD1d with-
in a 15/CD1d complex, it was obvious that the stereochemistry
of C6 hydroxy group influenced the immunostimulatory activity.
When the relationship between the configuration of C4 hydroxy
group and bioactivity of KRN7000 was investigated, its 4R-iso-
mer induced larger amounts of cytokines than that of C4 epimer
(4S-isomer).13 According to the X-ray analysis, the C4 hydroxy
group of KRN7000 makes no hydrogen-bonding interaction with
any residues in human TCR/KRN7000/CD1d complex.14 However,
it is essential for the glycolipid to stimulate human iNKT cells.15
It is thought that the inversion of the stereochemistry may
change the binding conformation of glycolipid/CD1d complex,
and also may cause to change the cytokine Th1/Th2 profiles se-
creted by iNKT cells, even if the hydroxyl group does not in-
volved in the hydrogen-bonding network.
2.2. Biological activity
The cytokine-producing (IFN
of mice in vivo of RCAI-172 was investigated using KRN7000 as a
positive control as shown in the Figure 2 (IFN and IL-4), and also
c and IL-4) activity from iNKT cells
c
The iNKT cells secrete both Th1 and Th2 cytokines (such as IFN
and IL-4, respectively) by stimulation of the -galactosylcera-
mides. In our previous report (cf. Ref. 7), for better EAE suppression
effect, it is important to decrease the secretion amount of IFN less
c
RCAI-172 was tested for the suppression activity of the symptoms
associated with experimental autoimmune encephalomyelitis
(EAE)8 as shown in Figure 3.
a
c
These glycolipid antigens were administered (2
phosphate buffered saline (PBS) solutions (10 g/mL, 200
caudal vein. Serum concentrations of IFN and IL-4 were measured
l
g/mouse) as
than 5–7% of KRN7000, and also to hold the secretion amount of IL-
4 more than 60–70% of KRN7000 as shown in RCAI-147 and -160.
l
lL) into
c
In other words, not only the less secretion of IFN
c but also the
at the indicated time points. Data are means SD from three
mice.11
more secretion of IL-4 gave the better clinical score of EAE
suppression.
Compound 15 (RCAI-172) has two successive (5S,6R)-5,6-dihy-
droxy groups next to C4-hydroxy group on the phytosphingosine
main-chain and also (2R,3R)-2,3-dihydroxyhexacosanamide on
It is difficult to estimate better clinical score of EAE test from
these few facts about the secretion amount of the cytokines IFN
and IL-4. However we feel that there may be an upper limit of IFN
and a lower limit of IL-4, for example, such as ‘the amount of IFN
c
c
c
the amide side chain. As shown in Figure 2, the amount of IFN
c
secretion mediated by RCAI-172 decreased to approximately 4.1%
of KRN7000 (cf, approximately 5% of KRN7000 in the case of
RCAI-147),7 and the IL-4 production amount was down to approx-
imately 35.3% of KRN7000 (cf, remaining approximately 70% of
KRN7000 in the case of RCAI-147).7 The cytokine secretion of
RCAI-172 biased to Th2 response (almost 8.6 times; cf. approxi-
mately 14 times in the case of RCAI-147)7 as compared to that of
KRN7000. Therefore, the Th2 response of RCAI-172 was reduced
has to be less than 5% of KRN7000, and the amount of IL-4 has to be
at least more than 70% of KRN7000’.
In fact, a compound (RCAI-151)7 increased IL-4 secretion
amount to 138% of KRN7000. But the secretion amount of IFN
c
was ꢁ41% of KRN7000, and RCAI-151 biased to Th2 response
(about 3.4 times compared to KRN7000). However this compound
showed only weak suppression effect against EAE symptom. It may

M. Shiozaki et al. / Bioorg. Med. Chem. 22 (2014) 827–833
831
be thought to be causally related to exceeding the upper limit of
IFN
(1H, m), 4.52–4.94 (6H, m), 7.10–7.44 (15H, m). ESI-MS: m/z
683.4 [M+Na]+. HR ESI-MS: calcd for C42H60O6Na: 683.4288; ob-
served: 683.4289.
c.
Anyway, we may need to investigate the immunostimulatory
activities of the other diastereomers of RCAI-147.
4.1.3. (2R,3S,4R,5S,6R)-1,2-(Isopropylidenedioxy)-3,4,5,6-
(tetrabenzyloxy)octadecane (4)
3. Conclusion
To a solution of 3 (3.55 g, 5.37 mmol) in dry DMF (35 mL) were
added NaH [60% oil dispersion, 656 mg (NaH: 393 mg, 16.4 mmol)]
and BnBr (1.84 g, 10.7 mmol) at 0 °C, and the mixture was stirred
for 1 h at room temperature. The reaction mixture was diluted
with EtOAc, which was washed with water and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column. Elu-
tion with hexane–EtOAc (19:1, then 9:1) gave 4 (2.61 g, 65%) as an
RCAI-172, C6 epimer of RCAI-147, was synthesized from a
known compound (1)10 in 11 steps. It is obvious that the cytokine
secretion ability of RCAI-172 biases to the Th2 response as shown
in Figure 2. However the IL-4 production was reduced to ꢁ35% of
KRN7000. This value was almost half compared to ꢁ70% in the case
of RCAI-147.7 In addition, RCAI-172 did not show the effectiveness
for the EAE suppression.8,12 There exist seven chiral centers in the
aglycon part of RCAI-172. Surprisingly, it is obvious that the change
of configuration in just one position (C6-OH) has a fairly large ef-
fect both on the Th1/Th2 response and for the EAE suppression.
oil. IR m
max(KBr) 2924, 2853, 1516, 1496, 1455, 1067 cmꢂ1. 1H NMR
(500 MHz, C6D6) d 0.92 (3H, t, J 6.8 Hz), 1.31 (19H, bs), 1.38 (3H, s),
1.48 (3H, s), 1.52 (1H, m), 1.70 (1H, m), 1.86 (1H, m), 3.81 (1H, m),
3.90–4.01 (5H, m), 4.49 (1H, m), 4.53, 4.58 (2H, AB-q, J 11.6 Hz),
4.68, 4.86 (2H, AB-q, J 11.8 Hz), 4.73 (2H, s), 4.74, 4.80 (2H, AB-q,
J 11.5 Hz), 7.09–7.42 (20H, m). ESI-MS: m/z 773.5 [M+Na]+. HR
ESI-MS: calcd for C49H66O6Na: 773.4757; observed: 773.4745.
4. Experimental
4.1. General
4.1.4. (2R,3S,4S,5S,6R)-3,4,5,6-(Tetrabenzyloxy)octadecane-1,2-
diol (5)
IR spectra were measured with a Jasco FT/IR-460 plus spec-
trometer. 1H NMR and 13C NMR spectra were recorded with a Var-
ian VNMRS-500 spectrometer using tetramethylsilane (TMS) as an
internal standard. Mass spectra and high resolution MS were re-
corded with Jeol JMS-SX102A or Bruker BioAPEX II 70e FT-ICR mass
spectrometers. Optical rotation value was measured with a Jasco P-
1010 polarimeter. Column chromatography was carried out using
silica gel 60 N (70–230 mesh ASTM, using ca 10–50 times weight
of substrate if there is no indication) under a slightly elevated pres-
sure for elution. Preparative TLC was carried out on a plate (Merck,
Silica gel 60 F254, 0.5 mm thick).
To a solution of 4 (2.61 g, 3.48 mmol) in CH2Cl2–MeCN (1:1,
140 mL) was added aq 46% HF (2.2 mL) at room temperature. The
mixture was stirred for 15 min at room temperature, diluted with
CH2Cl2, which was washed with satd aq NaHCO3 and brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo
to give 5 (2.41 g, 98%) as an oil. IR
mmax(KBr) 3537, 2926, 2854,
1515, 1456 cmꢂ1 1H NMR (500 MHz, CDCl3) d 0.88 (3H, t, J
.
7.0 Hz), 1.14–1.32 (19H, m), 1.48–1.68 (3H, m), 3.43 (1H, dd, J
5.0, 11.3 Hz), 3.48 (1H, m), 3.62 (1H, dd, J 6.4, 11.3 Hz), 3.68–3.71
(2H, m), 3.91 (1H, m), 4.09 (1H, dd, J 3.2, 7.4 Hz), 4.39, 4.50 (2H,
AB-q, J 11.6 Hz), 4.43, 4.52 (2H, AB-q, J 11.4 Hz), 4.65, 4.78 (2H,
AB-q, J 11.6 Hz), 4.77, 4.80 (2H, AB-q, J 10.8 Hz), 7.23–7.34 (20H,
m). ESI-MS: m/z 733.4 [M+Na]+. HR ESI-MS: calcd for C46H62O6Na:
733.4444; observed: 733.4439.
4.1.1. (2R,3S,4R,5S,6R)-3,4,5-(Tribenzyloxy)octadecane-1,2,6-
triol (2)
To a solution of 1 (4.44 g, 6.04 mmol) in THF (80 mL) was added
TBAF (1.0 M in THF, 22 mL). After stirring for 1 h at room temper-
ature, the reaction mixture was diluted with EtOAc, washed with
water and brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a residue, which was chromato-
graphed on a silica gel column. Elution with hexane–EtOAc (3:1,
4.1.5. (2R,3S,4S,5S,6R)-1-tert-Butyldimethylsilyloxy-3,4,5,6-
(tetrabenzyloxy)octadecan-2-ol (6)
To a solution of 5 (2.41 g, 3.39 mmol) in dry CH2Cl2 (140 mL)
was added TBDMS-Cl (1.02 g, 6.77 mmol), imidazole (690 mg,
10.1 mmol) and a catalytic amount of DMAP (7 mg). The mixture
was stirred for 16 h at room temperature. The reaction mixture
was diluted with CH2Cl2, which was washed with water and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a residue, which was chromatographed on a silica gel
column. Elution with hexane–EtOAc (19:1, then 3:2) gave 6
then 1:1) gave 2 (3.67 g, 98%). IR
mmax(KBr) 3465, 2925, 2853,
1457, 1065 cmꢂ1 1H NMR (500 MHz, CDCl3) d 0.88 (3H, t, J
.
7.0 Hz), 1.26 (19H, bs), 1.35–1.62 (3H, m), 3.54 (1H, dd, J 2.0,
7.4 Hz), 3.58 (1H, dd, J 4.9, 11.2 Hz), 3.64 (1H, m), 3.71 (1H, dd, J
5.7, 11.3 Hz), 3.76 (1H, t, J 3.2 Hz), 3.92 (1H, m), 4.10 (1H, dd, J
2.7, 7.4 Hz), 4.55, 4.73 (2H, AB-q, J 11.6 Hz), 4.58, 4.83 (2H, AB-q,
J 11.0 Hz), 4.72, 4.78 (2H, AB-q, J 11.0 Hz), 7.27–7.36 (15H, m).
ESI-MS: m/z 643.4 [M+Na]+. HR ESI-MS: calcd for C39H56O6Na:
643.3975; observed: 643.3952.
(2.77 g, 99%). IR
mmax(KBr) 3538, 2925, 2854, 1518, 1508,
1456 cmꢂ1 1H NMR (500 MHz, CDCl3) d 0.03 (3H, s), 0.04 (3H, s),
.
0.88 (9H, s, and 3H, t, J 7.0 Hz), 1.10–1.32 (20H, m), 1.55–1.63
(2H, m), 3.49 (1H, dd, J 6.4, 10.8 Hz), 3.52 (1H, br, OH), 3.65–3.69
(3H, m), 3.84 (1H, dd, J 1.5, 4.2 Hz, C3-H), 3.92 (1H, t, J 6.6 Hz, chan-
ged to dt, J 1.5, 6.6 Hz, on addition of D2O, C2-H), 4.14 (1H, dd, J 4.2,
6.4 Hz, C4-H), 4.41-4.83 (8H, m, benzylic protons), 7.22-7.33 (20H,
m). ESI-MS: m/z 847.5 [M+Na]+. HR ESI-MS: calcd for C52H76O6-
SiNa: 847.5309; observed: 847.5299.
4.1.2. (2R,3S,4R,5S,6R)-1,2-(Isopropylidenedioxy)-3,4,5-
(tribenzyloxy)octadecan-6-ol (3)
A solution of 2 (3.67 g, 5.15 mmol) in 2,2-dimethoxypropane
(80 mL) containing p-toluenesulfonic acid monohydrate (70 mg)
was stirred for 1.5 h at room temperature, and diluted with EtOAc,
which was washed with satd aq NaHCO3 and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column. Elu-
tion with hexane–EtOAc (9:1, then 6:1) gave 3 (3.55 g, 91%). IR
4.1.6. (2S,3S,4S,5S,6R)-2-Azido-1-tert-butyldimethylsilyloxy-
3,4,5,6-(tetrabenzyloxy)octadecane (7)
m
max(KBr) 3628, 2926, 2855, 1457, 1067 cmꢂ1 1H NMR (500 MHz,
.
Ph3P (2.01 g, 7.66 mmol) and DEAD (2.2 M in toluene, 3.4 mL,
7.48 mmol) were added to a solution of 6 (1.40 g, 1.70 mmol) in
dry THF (34 mL) under argon at room temperature. DPPA (2.11 g,
7.66 mmol) was added to this solution at 0 °C. After stirring for
30 min at 0 °C, the mixture was concentrated in vacuo to give a
C6D6) d 0.92 (3H, t, J 7.0 Hz), 1.31 (19H, bs), 1.36 (3H, s), 1.47
(3H, s), 1.52 (2H, m), 1.65 (1H, m), 3.69 (1H, dd, J 2.6, 5.8 Hz),
3.80–3.86 (3H, m), 3.93 (1H, m), 4.05 (1H, dd, J 6.3, 8.3 Hz), 4.52

832
M. Shiozaki et al. / Bioorg. Med. Chem. 22 (2014) 827–833
mixture, which was chromatographed on a silica gel (85 g) column.
temperature, diluted with water, and extracted with CHCl3, which
was washed with H2O and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give a mixture, which
was chromatographed on a silica gel column. Elution with hex-
ane–EtOAc (4:1, then 1:1) afforded 11 (1.33 g, 71%) as a gum. IR
Elution with hexane–EtOAc (19:1) gave 7 (1.44 g, quantitatively) as
an oil. IR
m .
max(KBr) 2926, 2855, 2098, 1455 cmꢂ1 1H NMR
(500 MHz, CDCl3) d 0.04 (6H, s), 0.88 (3H, s), 0.88 (3H, t, J 7.1 Hz),
0.90 (9H, s), 1.19–1.30 (20H, m), 1.55–1.68 (2H, m), 3.53 (1H, m),
3.69 (1H, dd, J 3.2, 6.4 Hz), 3.76 (1H, m), 3.84 (1H, m), 4.01–4.05
(2H, m), 4.39–4.82 (8H, m), 7.17–7.32 (20H, m). ESI-MS: m/z
872.5 [M+Na]+. HR ESI-MS: calcd for C52H75N3O5SiNa: 872.5374;
observed: 872.5380.
m
max(KBr) 3031, 2926, 2856, 1496, 1455, 1362 cmꢂ1 1H NMR
.
(500 MHz, CDCl3) d 0.88 (3H, t, J 6.8 Hz) 1.00 (9H, s), 1.05 (9H, s),
1.17–1.30 (20H, m), 1.55–1.63 (2H, m), 3.25 (1H, m), 3.36 (1H,
dd, J 8.3, 9.5 Hz), 3.54 (1H, s), 3.55 (1H, m), 3.66 (1H, dd, J 3.3,
6.0 Hz), 3.75 (1H, dd, J 4.7, 5.6 Hz), 3.81 (1H, dd, J 2.8, 9.9 Hz),
3.96–4.01 (4H, m), 4.08 (1H, dd, J 1.9, 12.5 Hz), 4.40–4.83 (10H,
m), 7.19–7.42 (30H, m). ESI-MS: m/z 1192.7 [M+H]+. HR ESI-MS:
calcd for C74H102NO10Si: 1192.7273; observed: 1192.7250.
4.1.7. (2S,3S,4S,5S,6R)-2-Azido-3,4,5,6-
(tetrabenzyloxy)octadecan-1-ol (8)
To a solution of 7 (1.44 g, 1.70 mmol) in THF (30 mL) was added
TBAF (1.0 M in THF, 5.1 mL). After stirring for 50 min at room tem-
perature, the reaction mixture was concentrated in vacuo, and di-
luted with EtOAc, which was washed with water and brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo
to give a residue, which was chromatographed on a silica gel col-
umn. Elution with hexane–EtOAc (9:1, then 4:1) gave 8 (1.20 g,
4.1.10. (2S,3S,4S,5S,6R,20R,30R)-2-[20,30-
(Isopropylidenedioxy)hexacosanoylamino]-3,4,5,6-
(tetrabenzyloxy)octadecyl 2,3-di-O-benzyl-4,6-O-(di-tert-
butyl)silylene-a-D-galactopyranoside (13)
To a solution of amine 11 (729 mg, 0.628 mmol) and (2R,3R)-
(isopropylidenedioxy)hexacosanoic acid (12, 883 mg, 1.88 mmol)
in dry CH2Cl2–THF (1:1, 70 mL) were added DMAP (921 mg,
7.54 mmol) and EDAC (1.45 g, 7.54 mmol). The mixture was stirred
for 3 h at room temperature. The reaction mixture was diluted
with CH2Cl2, which was washed with water and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a mixture, which was chromatographed on a silica gel column. Elu-
tion with hexane–EtOAc (19:1, then 7:1) gave 13 (547 mg, 54%) as
96%). IR m .
max(KBr) 3526 (br), 2925, 2853, 2097, 1455, 1065 cmꢂ1
1H NMR (500 MHz, CDCl3) d 0.89 (3H, t, J 7.0 Hz), 1.09–1.30 (20H,
m), 1.53 (1H, m), 1.65 (1H, m), 2.77 (1H, t, J 5.6 Hz, OH), 3.46
(1H, m), 3.67–3.75 (3H, m), 3.83 (1H, t, J 3.8 Hz), 3.95 (1H, m),
4.06 (1H, dd, J 3.3, 7.7 Hz), 4.47 (2H, s), 4.48, 4.54 (2H, AB-q, J
11.4 Hz), 4.61, 4.78 (2H, AB-q, J 11.5 Hz), 4.77 (2H, s), 7.22–7.33
(20H, m). ESI-MS: m/z 758.5 [M+Na]+. HR ESI-MS: calcd for C46H61-
N3O5Na: 758.4509; observed: 758.4492.
an oil. IR m .
max(KBr) 2924, 2853, 1677, 1466, 1455 cmꢂ1 1H NMR
4.1.8. (2S,3S,4S,5S,6R)-2-Azido-3,4,5,6-(tetrabenzyloxy)octadecyl
2,3-di-O-benzyl-4,6-O-(di-tert-butyl)silylene-a-D-
galactopyranoside (10)
(500 MHz, CDCl3) d 0.879 (3H, t, J 7.0 Hz), 0.883 (3H, t, J 7.0 Hz),
0.96 (9H, s), 1.02 (9H, s), 1.13–1.31 (65H, m, containing 3H singlet
at 1.30 ppm), 1.38 (3H, s), 1.39–1.70 (4H, m), 3.50 (1H, s), 3.66 (1H,
m), 3.70 (1H, m), 3.74 (1H, dd, J 3.0, 10.1 Hz), 3. 83 (1H, dd, J 2.4,
3.9 Hz), 3.90 (1H, dd, J 9.1, 11.5 Hz), 3.94 (1H, dd, J 4.0, 10.3 Hz),
3.99 (2H, bs), 4.09 (1H, dd, J 4.0, 6.5 Hz), 4.21 (1H, m), 4.31 (1H,
m), 4.35–4.81 (16H, m), 7.00 (1H, d, J 8.3 Hz, NH), 7.18–7.41
(30H, m). ESI-MS: m/z 1665.1 [M+Na]+. HR ESI-MS: calcd for C103-
H155NO13SiNa: 1665.1165; observed: 1665.1166.
(1) A solution of 2,3-di-O-benzyl-4,6-O-(di-tert-butyl)silylene-
D-galactopyranose (1.47 g, 2.94 mmol) and CCl3CN (3.0 mL,
29.7 mmol) in CH2Cl2 (30 mL) containing Cs2CO3 (1.11 g,
3.41 mmol) as a base was stirred for 16 h at room temperature,
and the reaction mixture was diluted with CH2Cl2, which was
washed with H2O, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give crude trichloroacetimidoyl
2,3-di-O-benzyl-4,6-O-(di-tert-butyl)silylene-
D-galactopyranoside
4.1.11. (2S,3S,4S,5S,6R,20R,30R)-2-[20,30-
(9), which was employed for the next reaction without further
purification.
(Dihydroxy)hexacosanoylamino]-3,4,5,6-
(tetrabenzyloxy)octadecyl 2,3-di-O-benzyl-a-D-
(2) To a solution of azido-alcohol 8 (1.27 g, 1.73 mmol) and the
above obtained imidate (9) in dry CH2Cl2 (25 mL) was added MS
4 Å (dry powder, 3.0 g). After stirring for 30 min at room tempera-
ture, AgOTf (266 mg, 1.04 mmol) was added to this mixture. The
mixture was stirred for 16 h at room temperature, and filtered.
The filter cake was washed with CH2Cl2. The combined filtrate
was washed with satd aq NaHCO3, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give a mixture which was
chromatographed on a silica gel column. Elution with hexane–
galactopyranoside (14)
(1) To a solution of 13 (540 mg, 0.329 mmol) in CH2Cl2–CH3CN
(1:1, 200 mL) were added water (4 mL) and aq 46% HF (16 mL).
After stirring for 3.5 h at room temperature, the reaction mixture
was diluted with CHCl3 (200 mL), and the whole was washed with
satd aq NaHCO3, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a mixture.
(2) The above obtained mixture was dissolved in dry THF
(100 mL). Dry pyridine (1.80 g) and HF-pyridine complex (ꢁ70%
HF, ꢁ30% pyridine, 1.64 g) were added to this THF solution. After
stirring for 2 h at room temperature, the reaction mixture was di-
luted with CHCl3, which was washed with satd aq NaHCO3, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo
to give a mixture, which was chromatographed on a silica gel col-
umn. Elution with hexane–EtOAc (2:3, then 1:5) gave 14 (213 mg,
EtOAc (19:1, then 9:1) gave 10 (1.92 g, 91%) as an oil. IR
m
max(KBr)
2928, 2857, 2099, 1671, 1496, 1472, 1455 cmꢂ1
.
1H NMR
(500 MHz, CDCl3) d 0.88 (3H, t, J 6.9 Hz), 0.98 (9H, s), 1.04 (9H,
s), 1.10–1.32 (20H, m), 1.50–1.65 (2H, m), 3.49–3.53 (2H, m),
3.67 (1H, dd, J 6.9, 9.8 Hz), 3.73 (1H, dd, J 3.9, 6.3 Hz), 3.78–3.83
(2H, m), 3.94–4.07 (6H, m), 4.39–4.55 (5H, m), 4.61–4.82 (9H,
m), 7.15–7.41 (30H, m). ESI-MS: m/z 1240.7 [M+Na]+. HR ESI-MS:
calcd for C74H99N3O10SiNa: 1240.6997; observed: 1240.6995.
two steps 44%) as a gum. IR
m
max(KBr) 3393 (br), 3063 (w), 3031
(w), 2924, 2852, 1648, 1523, 1497, 1466, 1455 cmꢂ1
.
1H NMR
(500 MHz, CDCl3) d 0.879 (3H, t, J 7.0 Hz), 0.882 (3H, t, J 7.0 Hz),
1.01–1.30 (62H, m), 1.30–1.70 (4H, m), 2.11 (1H, dd, J 4.6, 8.0 Hz,
OH), 2.49 (1H, s, OH), 3.42 (1H, dd, J 2.8, 3.9 Hz, on addition of
D2O), 3.44 (1H, d, J 5.6 Hz, OH), 3.47 (1H, dd, J 3.9, 11.7 Hz, on addi-
tion of D2O), 3.53–3.66 (6H, m), 3.70 (1H, dd, J 3.1, 9.8 Hz), 3.77
(1H, dd, J 3.7, 9.8 Hz), 3.80 (1H, t, J 3.1 Hz), 3.85 (1H, d-like, J
5.8 Hz, OH), 3.91 (1H, d, J 2.5 Hz), 4.01 (1H, dd, J 3.4, 5.1 Hz), 4.22
(1H, dd, J 3.9, 10.2 Hz), 4.33 (1H, d, J 11.8 Hz), 4.48–4.78 (13H,
4.1.9. (2S,3S,4S,5S,6R)-2-Amino-3,4,5,6-
(tetrabenzyloxy)octadecyl 2,3-di-O-benzyl-4,6-O-(di-tert-
butyl)silylene-a-D-galactopyranoside (11)
To a solution of 10 (1.92 g, 1.58 mmol) in dry THF (30 mL) was
added Me3P (1.0 M solution in THF, 8.3 mL). After stirring for 2 h at
room temperature, aq NaOH (1.0 M solution in H2O, 30 mL) was
added to this solution. The mixture was stirred for 2 h at room

M. Shiozaki et al. / Bioorg. Med. Chem. 22 (2014) 827–833
833
m), 6.81 (1H, d, J 8.8 Hz, NH), 7.20–7.40 (30H, m). ESI-MS: m/z
1484.9 [M+Na]+. HR ESI-MS: calcd for C92H135NO13Na:
1484.9831; observed: 1484.9845.
jected on day 1 and day 3. Mice were observed daily until day 30
for clinical score as 0–5 graduations with 0.5 for intermediate
scores. 0: No clinical signs, 1: flaccid tail, 2: hind limb weakness
or abnormal gait, 3: complete hind limb paralysis, 4: complete hind
limb paralysis + forelimb weakness or paralysis, 5: moribund or
deceased. In order to analyze the effect of glycolipid on EAE, each
glycolipd-pulsed GM-CSF induced DC (GM-DC) (5 ꢄ 105 cells/
mouse) were intravenously injected on day 5 and day 3. GM-DC
were induced by bone marrow by culturing with GM-CSF (10 ng/
mL) for 5 days, and then enriched by CD11c MACS beads (Miltenyi
Biotec). Each glycolipid (100 ng/mL) was pulsed by culturing GM-
DC (5 ꢄ 106/mL) for 24 h.
4.1.12. (2S,3S,4S,5S,6R,20R,30R)-2-[20,30-
(Dihydroxy)hexacosanoylamino]-3,4,5,6-
(tetrahydroxy)octadecyl a-D-galactopyranoside (15) (RCAI-172)
A solution of 14 (70 mg, 0.048 mmol) in THF (30 mL) containing
Pd(OH)2/C (20 wt.%, Degussa type, wet, ꢁ50%, 140 mg) was stirred
for 20 h under hydrogen atmosphere (balloon) at room tempera-
ture, and the reaction mixture was concentrated in vacuo to give
a crude mixture, which was dissolved in pyridine. The pyridine
solution was filtered, and the filter cake was washed with pyridine.
The combined filtrate was concentrated in vacuo to give a crude
product 15 to which dry Et2O (10 mL) was added. The mixture
was stirred for 16 h at room temperature to yield a powder, which
was washed with Et2O for several times to give 15 (36 mg, 82%) as
Acknowledgments
We thank Drs. Takemichi Nakamura and Yayoi Hongo, The
Institute of Physical and Chemical Research (RIKEN), for measure-
ments of high-resolution mass spectra.
a powder. ½a 2D5
ꢃ
+53.1 (c 1.0, pyridine). IR
mmax(KBr) 3315 (br), 2920,
2850, 1638, 1562, 1468 cmꢂ1
.
1H NMR (500 MHz, pyridine-d5): d
0.87 (6H, t, J 7.0 Hz), 1.20–1.39 (58H, m), 1.50–1.62 (2H, m),
1.72–1.84 (2H, m), 1.95–2.02 (4H, m), 4.32–4.38 (3H, m), 4.39–
4.43 (2H, m), 4.45–4.50 (3H, m), 4.52–4.55 (2H, m), 4.58 (1H, d, J
2.9 Hz), 4.62 (1H, d, J 9.3 Hz), 4.66 (1H, dd, J 3.9, 9.7 Hz), 4.71
(1H, d, J 4.9 Hz), 4.75 (1H, dd, J 6.1, 10.8 Hz), 4.86 (1H, dd, J 3.1,
9.0 Hz), 5.38 (1H, m), 5.64 (1H, d, J 3.9 Hz), 5.90 (1H, s, low and
broad), 8.70 (1H, d, J 9.0 Hz, NH). 13C NMR (125 MHz, pyridine-
d5): d 14.31, 22.97, 26.49, 26.52, 29.64, 29.95, 30.02, 30.08, 30.12,
30.22, 30.25, 30.31, 32.15, 32.66, 34.58, 50.66, 62.65, 67.91,
70.30, 70.99, 71.74, 72.91, 73.15, 73.19, 73.50, 74.12, 74.40,
76.07, 101.22, 173.96. ESI-MS: m/z 944.7 [M+Na]+. HR ESI-MS:
calcd for C50H99NO13Na: 944.7014; observed: 944.7031. Both com-
pounds, RCAI-172 and -1477 are stable at room temperature, and
they are much more soluble compared to KRN7000 in the medium
for bioassay.
References and notes
1. Reviews: (a) Rossjohn, J.; Pellicci, D. G.; Patel, O.; Gapin, L.; Godfrey, D. I. Nat.
Rev. Immunol. 2012, 12, 845; (b) Banchet-Cadeddu, A.; Hénon, E.; Dauchez, M.;
Renault, J.-H.; Monneaux, F.; Haudrechy, A. Org. Biomol. Chem. 2011, 9, 3080; (c)
Cheng, J. M. H.; Khan, A. A.; Timmer, M. S. M.; Stocker, B. L. Int. J. Carbohydr.
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Akimoto, K.; Natori, T.; Morita, M. Tetrahedron Lett. 1994, 35, 5593; (c) Natori,
T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994, 50, 2771.
3. Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.;
Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176.
4. Taniguchi, M.; Harada, M.; Kojo, S.; Nakayama, T.; Wakao, H. Annu. Rev.
Immunol. 2003, 21, 483.
5. Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.;
Nakagawa, R.; Sato, H.; Kaneko, E.; Koseki, H.; Taniguchi, M. Science 1997, 278,
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6. Yu, K. O. A.; Porcelli, S. A. Immunol. Lett. 2005, 100, 42.
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Mori, K. Carbohydr. Res. 2013, 370, 46.
8. (a) Marusic, S.; Leach, M. W.; Pelker, J. W.; Azoitei, M. L.; Uozumi, N.; Cui, J.;
Shen, M. W. H.; DeClercq, C. M.; Miyashiro, J. S.; Carito, B. A.; Thakker, P.;
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Lehuen, A. J. Immunol. 2008, 181, 2321.
4.2. Methods for measurement of biological activity
4.2.1. Bioassay (mouse in vivo)
The stock solutions (1.0 mg/mL in DMSO) of KRN7000 and RCAI-
9. (a) The amounts of secreted cytokines induced by C6 epimers were fairly
different. RCAI-10 [(2S,3R,6R)-3,6-dihydroxy-2-(hexacosanoylamino)octadecyl
172 were diluted to 10
saline (Sigma, Product No. D8537) just before injection into mice.
Each glycolipid solution (10 g/mL, 200 L) was administered
lg/mL in Dulbecco’s phosphate buffered
a
-
D
-galactopyranoside]: 64% of IFN
3,6-dihydroxy-2-(hexacosanoylamino)octadecyl
of IFN and 75% of IL-4 compared to KRN7000 (unpublished results).; (b) Wun,
c
and 19% of IL-4 and RCAI-45 [(2S,3R,6S)-
a-D
-galactopyranoside]: 160%
l
l
c
intravenously. Peripheral blood was collected from the retro-orbi-
tal plexus of mice at indicated time points, using heparin-coated
capillary tubes (Funakoshi Pharmaceutical, Japan), and plasma
was prepared.
K. S.; Cameron, G.; Patel, O.; Pang, S. S.; Pellicci, D. G.; Sullivan, L. C.;
Keshipeddy, S.; Young, M. H.; Uldrich, A. P.; Thakur, M. S.; Richardson, S. K.;
Howell, A. R.; Illarionov, P. A.; Brooks, A. G.; Besra, G. S.; McCluskey, J.; Gapin, L.;
Porcelli, S. A.; Godfrey, D. I.; Rossjohn, J. Immunity 2011, 34, 327.
10. Shiozaki, M.; Tashiro, T.; Koshino, H.; Shigeura, T.; Watarai, H.; Taniguchi, M.;
Mori, K. Tetrahedron 2013, 69, 9710.
11. All biological experiments were in accordance with protocols approved by
RIKEN Animal Care and Use Committee.
4.2.2. Cytokine measurement
The cytokine concentrations in plasma were quantified by cyto-
metric bead array (CBA) system (BD Biosciences) for mouse IFN-
and IL-4 according to the manufacturer’s protocol.
12. Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413, 531.
13. (a) Trappeniers, M.; Goormans, S.; Van Beneden, K.; Decruy, T.; Linclau, B.; Al-
Shamkhani, A.; Elliott, T.; Ottensmeier, C.; Werner, J. M.; Elewaut, D.; Van
Calenbergh, S. ChemMedChem 2008, 3, 1061; (b) Park, J.-J.; Lee, J. H.; Ghosh, S.
C.; Bricard, G.; Venkataswamy, M. M.; Porcelli, S. A.; Chung, S.-K. Bioorg. Med.
Chem. Lett. 2008, 18, 3906.
14. (a) Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.; Pellicci, D. G.; Koh, R.;
Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.; McCluskey, J.; Rossjohn, J. Nature
2007, 448, 44; (b) Pellicci, D. G.; Patel, O.; Kjer-Nielsen, L.; Pang, S. S.; Sullivan,
L. C.; Kyparissoudis, K.; Brooks, A. G.; Reid, H. H.; Gras, S.; Lucet, I. S.; Koh, R.;
Smyth, M. J.; Mallevaey, T.; Matsuda, J. L.; Gapin, L.; McCluskey, J.; Godfrey, D.
I.; Rossjohn, J. Immunity 2009, 31, 47.
c
4.2.3. EAE induction by active immunization in C57BL/6 (B6)
mice, and Clinical Score8,12
EAE is induced in B6 female mice by immunization with an
emulsion of MOG35-55 peptide in complete Freund’s adjuvant
(CFA). MOG in CFA (Hooke Lab) solution (200
ously injected at 2 sites on lower back (one injection over each
hip/base of tail) with 100 L of emulsion at each site on day 1.
5 ng/mL pertussis toxin (200 L in PBS) was intraperitoneally in-
lL) was subcutane-
15. Dangerfield, E. M.; Cheng, J. M. H.; Knight, D. A.; Weinkove, R.; Dunbar, P. R.;
Hermans, I. F.; Timmer, M. S. M.; Stocker, B. L. ChemBioChem 2012, 13, 1349.
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