Concise synthesis of α-galactosyl ceramide from d-galactosyl iodide and d-lyxose

Article abstract of DOI:10.1016/j.carres.2012.11.008

α-Galactosyl ceramide is synthesized from d-lyxose in 32% overall yield in five steps. The short and efficient protocol involves a one-pot protection and glycosidation of d-lyxose with d-galactosyl iodide as a key step. The α-linked disaccharide obtained was subsequently transformed into α-galactosyl ceramide in four steps involving Z-selective Wittig olefination at C-1, stereo-inversion at C-4 using azide, one-pot reduction of azide and amidation, and global deprotection.

Full text of DOI:10.1016/j.carres.2012.11.008

Carbohydrate Research 368 (2013) 35–39
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Concise synthesis of
and D-lyxose
a-galactosyl ceramide from D-galactosyl iodide
Yu-Fen Yen a, Suvarn S. Kulkarni b, Chun-Wei Chang a, Shun-Yuan Luo a,
⇑
a Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan
b Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 October 2012
Received in revised form 12 November 2012
Accepted 14 November 2012
Available online 21 November 2012
a
-Galactosyl ceramide is synthesized from
D
-lyxose in 32% overall yield in five steps. The short and effi-
cient protocol involves a one-pot protection and glycosidation of
key step. The -linked disaccharide obtained was subsequently transformed into
in four steps involving Z-selective Wittig olefination at C-1, stereo-inversion at C-4 using azide, one-pot
reduction of azide and amidation, and global deprotection.
D-lyxose with
D-galactosyl iodide as a
a
a
-galactosyl ceramide
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
a-Galactosyl ceramide
KRN7000
Glycosyl iodide
One-pot protection-glycosylation
Wittig reaction
1. Introduction
to a more reactive b-iodide and its SN2-like displacement by a phy-
tosphingosine acceptor. On the other hand, the preparation of cer-
Glycolipids play significant roles in numerous biological pro-
cesses.1 For example,
-galactosylceramide 1, also known as
amide acceptors is done mostly through chiron approach using
various starting materials such as amino acids, sugars, and other
chiral materials. However, some of the approaches involve multi-
a
KRN7000,2 a simplified glycolipid analog of Agelasphins originally
isolated from a marine sponge Agelas mauritianus,3 acts as a pow-
erful stimulator of the invariant Natural Killer T (iNKT) cells by
binding to the CD1d glycoprotein. The binding complex of CD1d
and 1 recognizes the T-cell receptor of iNKT cells to produce cyto-
kines that trigger an inflammatory response called T helper 1
(Th1), which governs the antitumor and antimicrobial properties,
and immunomodulatory response termed T helper 2 (Th2), which
controls various autoimmune diseases.4 Since these responses can-
cel out each other’s effect, there is an ongoing search for glycolipid
analogs, with varied lipid chain length, that selectively trigger
either Th1 or Th2 type responses.5
step sequences and result in moderate to low overall yields. D-Lyx-
ose that has all the requisite chiral centers in place is the most
suitable precursor for phytosphingosine.9 Based on this finding,
Panza and co-workers8c reported a novel synthesis of
a
-galactosyl
-galactosyl bromide with a -lyxose
-mannose. For the development of glycolip-
ids as vaccine adjuvants, we are interested in establishing a short
and general synthesis of -GalCer and related analogs. We envi-
ceramide via the coupling of
acceptor derived from
D
D
D
a
sioned that a merger of Panza’s approach and Gervay-Hague’s con-
venient protocol7a would offer a facile access to these glycolipids.
Herein, we report an efficient five-step route for the synthesis of
Structurally,
-linked -galactopyranoside with phytosphingosine-derived
ceramide. Numerous methods for the synthesis of -glactosyl cera-
a
-galactosyl ceramide 1 (Fig. 1), is composed of an
KRN7000 from commercially available
yields. To the best of our knowledge this is the shortest reported
sequence for the synthesis of -galactosyl ceramide till date. The
key feature of this synthesis lies in controlling the regio- and
stereoselectivities of glycosylation to obtain a [ -Galp? -lyxf]
disaccharide, the right hand sugar unit ( -lyxofuranose) of which
could be subsequently manipulated to install the required function-
alities, thus avoiding any complications and difficulties usually
encountered in coupling glycosyl donors with azido ceramide or
ceramide acceptors. Hung and co-workers10 have established
earlier a one-pot protection-glycosylation protocol starting from
D-lyxose, in 32% overall
a
D
a
a
mide are reported in the literature, mostly involving stereoselective
coupling of phytosphingosine derivatives with various glycosyl
donors.6–8 Among them, Gervay-Hague’s elegant protocol of glyco-
syl iodide mediated stereoselective glycosylation offers excellent
a
-D
D
D
selectivity and yields.7 The high
a-selectivity is attributed to the
in situ anomerization of the initially formed
a-galactosyl iodide
⇑
Corresponding author. Tel.: +886 4 22840411; fax: +886 4 22862547.
E-mail address: syluo@dragon.nchu.edu.tw (S.-Y. Luo).
2,3-di-O-TMS protected
D-glucopyranosides to access various
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.11.008

36
Y.-F. Yen et al. / Carbohydrate Research 368 (2013) 35–39
boldfaced carbon framework. The azido group in precursor 2 could
be reduced to amine and the obtained galactosyl phytosphingosine
can be coupled with suitable fatty acids to give -galactosyl cera-
mide 1. The -galactosyl phytosphingosine derivative 2 is a key
HO
HO
OH
O
O
C25H51
a
NH OH
HO
D
O
C13H27
intermediate which could be in turn obtained by a Wittig olefin-
ation with the galactosyl-lyxose disaccharide 4, followed by SN2
displacement of the sulfonate activated hydroxyl group by an
azide. It was envisaged that, the disaccharide 4 can be obtained di-
OH
1
Figure 1. Structure of KRN7000 1.
rectly from unprotected D-lyxose 5 by coupling with an appropri-
ate galactosyl donor 6 via a one-pot protection–glycosylation
involving in situ anomerization of glycosyl iodides.
3-O-arylmethylated 1?2 linked disaccharides. However, a tandem
regioselective protection and glycosylation of free sugars is unprec-
edented in the literature.
The synthesis started with a regio- and stereoselective one-pot
preparation of the key disaccharide 88c (Scheme 2). First, a selec-
tive acetonide protection of 2,3-dihydroxyl groups of D-lyxose 5
was achieved using catalytic sulfuric acid and anhydrous ace-
tone.11 Upon completion of the reaction, as indicated by TLC
(4 h), DIPEA (1 equiv), TBAI (3 equiv) and 4 Å MS in toluene were
sequentially added to the reaction and the temperature was raised
to 65 °C. A preformed and well azeotroped solution of 2,3,4,6-tetra-
2. Results and discussion
Our retrosynthetic plan for the synthesis of
commercially available -lyxose 5 as a precursor (Scheme 1). The
stereochemical resemblance between the two is indicated by the
a-GalCer 1 entails
D
O-benzyl-
in the same pot, and the reaction was continued for an hour. This
one-pot operation afforded exclusively -linked disaccharide 8 in
63% overall yield. Thus, the coupling between glycosyl iodide and
ispropylidene -lyxofuranose 1,5-diol took place regio- and stere-
a-D
-galactosyl iodide 712 (1 equiv) was added dropwise
HO
HO
OH
O
a
O
C25H51
NH OH
D
HO
O
C13H27
oselectively at the primary 5-OH group leaving the hemiacetal
OH free for further reaction. The selectivity observed in this reac-
tion saved two steps which would have been otherwise necessary
for blocking the anomeric OH and removing it post-glycosylation.
Wittig olefination of hemiacetal 8 with Ph3P@CHC12H25 at 0 °C
gave the Z-olefin 9 as a single isomer in 92% yield. It should be
noted that, of the several conditions attempted for Wittig reaction,
the in situ generation of the phosphorane13 via a slow addition of
the base to the well-stirred suspension of 8 and the phosphonium
salt at 0 °C worked the best to afford the desired product 9, stere-
oselectively. Compound 9 was sequentially treated with trifluoro-
methanesulfonic anhydride (Tf2O) and tetramethylguanidinium
azide (TMGA)14 to produce the azido compound 10 (82% yield)
with the inversion of the reacting carbon center.15 After the reduc-
tion of the azido group in 10 with PPh3 and pyridine in ACS grade
quality of THF critical, hexacosanoic acid, EDC, HOBt and triethyl-
amine were directly added into reaction mixture to obtain the cor-
responding amide 11 in 72% yield in a one-pot manner. After
meticulous experimentation, we found the appropriate reaction
conditions to remove the acetonide and simultaneously reduce
the double bond and remove benzyl groups of 11 in a one-pot man-
ner. Thus, a solution of 11 in 0.8 N HCl (aq) in 3:1 MeOH/CHCl3
solution was stirred for 1 d at room temperature until the starting
material was consumed completely and then treated with a cata-
lytic amount of Pd(OH)2 under an atmosphere of hydrogen for
2 d at room temperature to obtain KRN7000 1 (94%).
OH
1
amide bond
formation
PO
PO
OP
O
N3 OP
C
12H25
PO
O
2
OP
PO
PO
OP
O
OH OP1
OP1
C
12H25
PO
O
3
Ph3P=CHC12H25
PO
PO
OP
O
PO
O
OP1
O
OP1
3. Conclusion
4
OH
In conclusion, we successfully synthesized biologically potent
a-galactosyl ceramide 1 from the commercially available D-lyxose
in 32% yield over five steps. The similarity between our approach
and the synthesis reported by Panza and co-workers is evident.
The key difference however lies in the regioselective one-pot pro-
PO
PO
OP
O
tection and glycosylation reaction on the unprotected
D-lyxose.
Also, a highly Z-stereoselective Wittig reaction circumvents isomer
separation or hydrogenation at an early stage. Along with this, one-
pot global deprotection saves several steps. In Panza’s synthesis,
which although in itself is a marked improvement over the existing
routes, the protected lyxofuranose 5-OH acceptor was prepared in
PO
HO
I
OH OH
O
6
OH
5 : D-lyxose
four steps from
D-mannose and the low yields encountered during
Scheme 1. Retrosynthetic analysis of a-galactosyl ceramide 1.
the azide displacement of the E/Z olefin, possibly due to the con-

Y.-F. Yen et al. / Carbohydrate Research 368 (2013) 35–39
37
BnO
BnO
OBn
O
BnO
BnO
OBn
O
OH
C12H25
a
b
BnO
BnO
5
D-Lyxose
O
O
O
O
O
O
O
OH
8
9
BnO
BnO
BnO
OBn
O
OBn
O
O
C25H51
BnO
c
d
NH
C12H25
N3
C12H25
BnO
BnO
O
O
O
O
O
O
11
10
e
1
Scheme 2. Concise synthesis of
a-galactosyl ceramide 1. Reagent and conditions: (a) cat. H2SO4, acetone, room temp, 4 h; then TBAI, DIPEA, toluene, 65 °C, 10 min; then
2,3,4,6-tetra-O-benzyl- -galactopyranosyl iodide 7, 1 h, 63%; (b) LHMDS, C13H27PPh3Br, THF, 0 °C, 6 h, 92%; (c) Tf2O, pyridine, CH2Cl2, 0 °C, 2 h; then TMGA, CH2Cl2, 0 °C,
a-D
overnight, 82%; (d) PPh3, pyridine, wet THF, 60 °C, 12 h; then EDC, HOBt, C25H51COOH, Et3N, room temp, 12 h, 72%; (e) 0.8 N HCl, MeOH, CHCl3, room temp, 1 d; then Pd(OH)2/
C, H2, room temp, 2 d, 94%.
current reaction of olefin with the azido group, necessitated the ex-
tra step for double bond reduction. We employed TMGA instead of
sodium azide and observed that it does not react with the double
bond of the compound 9. The short and efficient route described
(7.0 mL) was added iodotrimethylsilane (224 lL, 1.511 mmol) at
0 °C under nitrogen. After 30 min, the mixture was evaporated in
vacuo. Toluene (10 mL) was added to the residue and evaporated
in vacuo three times to afford 7 as a pale yellowish residue. This
residue was dissolved in toluene and kept under dry nitrogen
here for the synthesis of a-galactosyl ceramide is expected to pro-
vide access to other structurally related glycolipids for exploring
their immunostimulating activities and other biological properties.
The phytosphingolipid chains can be easily modified by using a dif-
ferent Wittig salt, and the analogs can be altered by the use of dif-
ferent starting iodoglycosides.
atmosphere. To a solution of
anhydrous acetone (2 mL), prepared in a separate flask, was added
sulfuric acid (5.7 L, 0.106 mmol) and stirred for 4 h at rt. After the
complete disappearance of the starting material on TLC, diisopro-
pylethylamine (210 L, 1.209 mmol), tetrabutylammonium iodide
D-lyxose 5 (200 mg, 1.332 mmol) in
l
l
(1.33 g, 3.627 mmol) and 4 Å molecular sieves in anhydrous tolu-
ene (3 mL) were added to it and the mixture was stirred for
10 min at 65 °C under nitrogen. A solution of iodide 7 in toluene
was added into the reaction flask at this stage and the mixture
was kept stirring for 1 h. The reaction was stopped by adding ethyl
acetate and the suspension was filtered through celite to separate
the white precipitate and molecular sieves. The resulting solution
was washed with aqueous Na2S2O3 and brine, and the organic lay-
ers were dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography on
silica gel to afford disaccharide 8 (552 mg, 63%) as a colorless oil:
4. Experimental section
4.1. General remarks
Dichloromethane was purified and dried from a safe purifica-
tion system containing activated Al2O3. Anhydrous pyridine, and
methanol were used as purchased. All reagents obtained from
commercial sources were used without purification, unless other-
wise mentioned. Flash column chromatography was carried out
on Silica Gel 60 (230–400 mesh, E. Merck). TLC was performed
on pre-coated glass plates of Silica Gel 60 F254 (0.25 mm, E.
Merck); detection was executed by spraying with a solution of
Ce(NH4)2(NO3)6 (0.5 g), (NH4)6Mo7O24 (24 g) and H2SO4 (28 mL)
in water (500 mL) and subsequently heating on a hot plate. Melting
points were determined with a capillary apparatus and are uncor-
rected. Optical rotations were measured at 589 nm (Na) at ꢀ25 °C.
1H, 13C NMR, DEPT, 1H–1H COSY, 1H–13C COSY, and NOESY spectra
were recorded with 400 and 600 MHz instruments. Chemical shifts
are in ppm from Me4Si, generated from the CDCl3 lock signal at d
7.24. IR spectra were taken with a FT-IR spectrometer using KBr
plates. Mass spectra were analyzed on a Finnigan LTQ-Orbitrap
XL instrument with an ESI source.
½
a 2D8
ꢁ
+34.5 (c 1.3, CHCl3); IR (thin film):
v 3436, 3030, 1454,
1096 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 7.40–7.28 (m, 20H,
;
ArH), 5.32 (d, J = 2.8 Hz, 1H, H-1), 4.94 (d, J = 11.6 Hz, 1H, CH2Ph),
4.90 (d, J = 3.6 Hz, 1H, H-10), 4.84 (d, J = 11.6 Hz, 1H, CH2Ph), 4.82
(d, J = 12.0 Hz, 1H, CH2Ph), 4.76–4.74 (m, 1H, H-2), 4.74 (d,
J = 11.2 Hz, 1H, CH2Ph), 4.68 (d, J = 12.0 Hz, 1H, CH2Ph), 4.57 (d,
J = 11.6 Hz, 1H, CH2Ph), 4.56 (d, J = 5.6 Hz, 1H, H-3), 4.45
(d, J = 11.6 Hz, 1H, CH2Ph), 4.41–4.38 (m, 1H, H-4), 4.38 (d,
J = 12.0 Hz, 1H, CH2Ph), 4.06–3.96 (m, 4H, H-20, H-30, H-40, H-50),
3.85–3.83 (m, 2H, H-5a, H-5b), 3.52–3.51 (m, 2H, H-6a0, H-6b0),
2.5 (s, 1H, OH), 1.40 (s, 3H, CH3), 1.28 (s, 3H, CH3); 13C NMR (CDCl3,
150 MHz): d 138.8 (C), 138.6 (C), 138.5 (C), 137.8 (C), 128.33
(CH ꢃ 2), 128.28 (CH ꢃ 2), 128.25 (CH ꢃ 2), 128.2 (CH), 128.16
(CH ꢃ 2), 128.1 (CH ꢃ 2), 127.9 (CH), 127.8 (CH ꢃ 2), 127.66 (CH),
127.61 (CH), 127.5 (CH), 127.37 (CH), 127.35 (CH ꢃ 2), 112.4 (C),
101.0 (CH), 97.9 (CH), 85.4 (CH), 79.9 (CH), 78.9 (CH), 78.6 (CH),
76.4 (CH), 74.9 (CH), 74.7 (CH2), 73.3 (CH2), 73.2 (CH2), 72.9
(CH2), 69.2 (CH), 68.9 (CH2), 66.3 (CH2), 26.0 (CH3), 24.8 (CH3);
4.2. 2,3-O-isopropylidene-5-O-(2,3,4,6-tetra-O-benzyl-
a-D-
galactopyranosyl)- -lyxofuranose (8)
D
To a solution of 1-O-acetyl-2,3,4,6-tetra-O-benzyl-b-
pyranoside (704 mg, 1.209 mmol) in anhydrous dichloromethane
D-galacto-

38
Y.-F. Yen et al. / Carbohydrate Research 368 (2013) 35–39
HRMS (FAB): m/z Calcd for C42H48O10Na ([M+Ma]+): 735.3145.
Found: 735.3129.
4.04 (m, 2H, H-3, H-20), 4.02 (dd, J = 10.8, 3.0 Hz, 1H, H-1a), 3.99–
3.97 (m, 2H, H-30, H-40), 3.94 (m, 1H, H-50), 3.65 (dd, J = 10.8,
7.2 Hz, 1H, H-1b), 3.53–3.48 (m, 3H, H-2, H-6a0, H-6b0), 2.18–2.06
(m, 2H, H-7a, H-7b), 1.42 (s, 3H, CH3), 1.41–1.36 (m, 2H, CH2),
1.23 (s, 3H, CH3), 1.29–1.24 (m, 18H, CH2), 0.88 (t, J = 6.6 Hz, 3H,
CH3); 13C NMR (CDCl3, 150 MHz) d 138.8 (C ꢃ 2), 138.6 (C), 137.9
(C), 135.8 (CH), 128.33 (CH ꢃ 2), 128.27 (CH ꢃ 2), 128.24
(CH ꢃ 2), 128.18 (CH ꢃ 2), 128.17 (CH ꢃ 2), 127.7 (CH ꢃ 2),
127.64 (CH), 127.57 (CH ꢃ 2), 127.53 (CH), 127.49 (CH ꢃ 2),
127.38 (CH ꢃ 2), 123.9 (CH), 108.9 (C), 98.7 (CH), 78.6 (CH), 76.5
(CH), 76.1 (CH), 75.1 (CH), 74.7 (CH2), 73.4 (CH2), 73.3 (CH), 73.2
(CH2), 72.8 (CH2), 69.7 (CH), 69.5 (CH2), 69.0 (CH2), 60.5 (CH),
31.9 (CH2), 29.7 (CH2), 29.64 (CH2), 29.62 (CH2), 29.57 (CH2), 29.5
(CH2), 29.4 (CH2), 29.3 (CH2), 29.28 (CH2), 27.9 (CH3), 27.7 (CH2),
25.4 (CH3), 22.7 (CH2), 14.1 (CH3); HRMS (ESI): m/z Calcd for
4.3. (2R,3S,4R)-3,4-O-isopropylidene-1-O-(2,3,4,6-tetra-O-
benzyl-a-D-galactopy-ranosyl)-octadec-5-en-1,2,3,4-tetraol (9)
A mixture of disaccharide 8 (147.6 mg, 0.207 mmol) and tri-
decanyltriphenylphosphonium bromide (490 mg, 0.932 mmol) in
anhydrous tetrahydrofuran (1.5 mL) was cooled down to 0 °C un-
der nitrogen. A 1.0 M solution of lithium hexamethyldisilylamide
in THF (932 lL, 0.932 mmol) was added to the mixture and the
reaction solution was kept stirring for another 6 h at 0 °C. Water
(1.5 mL) was added to quench the reaction and the mixture was
extracted with EtOAc (3 ꢃ 2 mL). The combined organic layers
were washed with brine, dried over anhydrous MgSO4, filtered,
and concentrated in vacuo to give a residue. The residue was puri-
fied by column chromatography to give the olefin 9 (167.2 mg,
C
55H73O8N3Na ([M+Na]+): 926.5290. Found: 926.5275.
92%) as a colorless oil: Rf 0.50 (EtOAc/Hex = 1:3); ½a D29
ꢁ
+9.5 (c 1.3,
4.5. (2S,3S,4R)-2-Hexacosanoylamino-3,4-O-isopropylidene-1-
O-(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-octadec-5-en-
1,3,4-triol (11)
CHCl3); IR (thin film):
v
3492, 2925, 1605, 1454, 1099 cmꢂ1
;
1H
NMR (CDCl3, 600 MHz): d 7.39–7.24 (m, 20H, ArH), 5.69–5.62 (m,
2H, H-5, H-6), 4.95 (t, J = 7.8 Hz, 1H, H-4), 4.92 (d, J = 11.4 Hz, 1H,
CH2Ph), 4.82 (d, J = 12.0 Hz, 1H, CH2Ph), 4.81 (d, J = 3.6 Hz, 1H, H-
10), 4.80 (d, J = 12.0 Hz, 1H, CH2Ph), 4.73 (d, J = 11.4 Hz, 1H, CH2Ph),
4.67 (d, J = 11.4 Hz, 1H, CH2Ph), 4.55 (d, J = 11.4 Hz, 1H, CH2Ph),
4.47 (d, J = 12.0 Hz, 1H, CH2Ph), 4.39 (d, J = 12.0 Hz, 1H, CH2Ph),
4.12 (dd, J = 6.6, 3.0 Hz, 1H, H-3), 4.05–4.02 (m, 2H, H-20, H-50),
3.96–3.93 (m, 2H, H-30, H-40), 3.77 (br s, 1H, H-2), 3.65 (dd,
J = 10.8, 4.2 Hz, 1H, H-1a), 3.53–3.48 (m, 3H, H-1b, H-6a0, H-6b0),
2.73 (d, J = 5.4 Hz, 1H, 2-OH), 2.13–1.98 (m, 2H, H-7a, H-7b), 1.50
(s, 3H, CH3), 1.36 (s, 3H, CH3), 1.30–1.24 (m, 20H, CH2), 0.88
(t, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3, 150 MHz) d 138.7 (C),
138.5 (C), 138.4 (C), 137.9 (C), 135.4 (CH), 128.33 (CH ꢃ 3),
128.31 (CH ꢃ 2), 128.2 (CH ꢃ 3), 127.9 (CH ꢃ 2), 127.72 (CH ꢃ 2),
127.65 (CH ꢃ 2), 127.54 (CH), 127.45 (CH), 127.4 (CH ꢃ 2), 125.0
(CH), 108.4 (C), 98.0 (CH), 79.0 (CH), 77.4 (CH), 76.3 (CH), 74.8
(CH), 74.7 (CH2), 73.42 (CH2), 73.37 (CH2), 73.0 (CH), 72.9 (CH2),
70.0 (CH2), 69.4 (CH), 68.9 (CH2), 68.6 (CH), 31.9 (CH2), 29.64
(CH2 ꢃ 3), 29.62 (CH2 ꢃ 2), 29.58 (CH2), 29.49 (CH2), 29.47 (CH2),
29.32 (CH2), 29.25 (CH2), 27.7 (CH2), 27.1 (CH3), 25.0 (CH3), 22.7
(CH2), 14.1 (CH3); HRMS (FAB): m/z Calcd for C55H74O9Na
([M+Na]+): 901.5231. Found: 901.5220.
To a solution of compound 10 (106 mg, 0.12 mmol) and tri-
phenylphosphine (62 mg, 0.24 mmol) in ACS grade THF (1.5 mL)
was added pyridine (0.5 mL). The reaction flask was warmed up
to 60 °C, and the mixture was kept stirring for 12 h. The reaction
was gradually cooled down to room temperature, hexaeicosanoic
acid (60 mg, 0.15 mmol), HOBt (29 mg, 0.21 mmol), 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbo-diimide
hydro-chloride
(EDC,
41 mg, 0.21 mmol), and triethylamine (17
lL, 0.12 mmol) were
sequentially added to the solution, and the mixture was continu-
ously stirred for 12 h. The reaction solution was diluted with
EtOAc, and the resulting mixture was washed with water. The or-
ganic layer was dried over anhydrous MgSO4, filtered, and concen-
trated in vacuo. Purification of this residue via column
chromatography afforded amide 11 (105 mg, 72%): Rf 0.43
(EtOAc/Hex = 1:4); ½a D29
ꢁ
+19.6 (c 1.6, CHCl3); IR (thin film)
;
v 3316,
2919, 2850, 1644 cmꢂ1
1H NMR (CDCl3, 500 MHz): d 7.39–7.23
(m, 20H, ArH), 6.06 (d, J = 9.0 Hz, 1H, NH), 5.58–5.53 (m, 1H, H-
6), 5.43–5.39 (m, 1H, H-5), 4.93 (d, J = 4.2 Hz, 1H, H-10), 4.91 (d,
J = 11.5 Hz, 1H, CH2Ph), 4.83 (dd, J = 9.0, 5.5 Hz, 1H, H-4), 4.81 (d,
J = 12.0 Hz, 1H, CH2Ph), 4.79 (d, J = 11.0 Hz, 1H, CH2Ph), 4.73 (d,
J = 11.5 Hz, 1H, CH2Ph), 4.66 (d, J = 11.5 Hz, 1H, CH2Ph), 4.56 (d,
J = 11.5 Hz, 1H, CH2Ph), 4.49 (d, J = 12.0 Hz, 1H, CH2Ph), 4.37 (d,
J = 12.0 Hz, 1H, CH2Ph), 4.23 (dd, J = 9.0, 6.0 Hz, 1H, H-3), 4.09–
4.02 (m, 2H, H-2, H-20), 3.98–3.88 (m, 4H, H-1a, H-30, H-40, H-50),
3.65 (dd, J = 11.5, 2.5 Hz, 1H, H-1b), 3.50 (dd, J = 9.0, 6.5 Hz, 1H,
H-6a0), 3.41 (dd, J = 9.5, 6.5 Hz, 1H, H-6b0), 2.08–1.84 (m, 4H,
CH2), 1.56–1.47 (m, 2H), 1.44 (s, 3H, CH3), 1.34 (s, 3H, CH3),
1.30–1.23 (m, 64H, CH2), 0.87 (t, J = 7.0 Hz, 6H, CH3 ꢃ 2); 13C
NMR (CDCl3, 150 MHz): d 172.3 (C), 138.7 (C), 138.5 (C), 138.4
(C), 137.6 (C), 134.9 (CH), 128.4 (CH ꢃ 2), 128.36 (CH ꢃ 2),
128.35 (CH ꢃ 2), 128.3 (CH ꢃ 2), 128.2 (CH ꢃ 2), 127.9 (CH ꢃ 2),
127.8 (CH ꢃ 3), 127.76 (CH), 127.6 (CH), 127.5 (CH), 127.4
(CH ꢃ 2), 124.3 (CH), 108.3 (C), 99.3 (CH), 78.9 (CH), 76.8 (CH),
76.1 (CH), 74.7 (CH2), 74.67 (CH), 73.5 (CH2), 73.4 (CH2), 73.1
(CH), 72.9 (CH2), 69.7 (CH), 69.6 (CH2), 69.1 (CH2), 49.1 (CH), 36.7
(CH2), 31.9 (CH2 ꢃ 2), 29.7 (CH2 ꢃ 20), 29.6 (CH2 ꢃ 2), 29.5 (CH2),
29.46 (CH2 ꢃ 2), 29.3 (CH2 ꢃ 3), 27.9 (CH3), 26.7 (CH2), 25.6
(CH3), 25.4 (CH2), 22.7 (CH2 ꢃ 2), 14.1 (CH3 ꢃ 2); HRMS (ESI): m/z
Calcd for C81H126O9N ([M+H]+): 1256.9427. Found: 1256.9472.
4.4. (2S,3S,4R)-2-azido-3,4-O-isopropylidene-1-O-(2,3,4,6-tetra-
O-benzyl-a-D-galactopyranosyl)-octadec-5-en-1,3,4-triol (10)
A mixture of compound 9 (239 mg, 0.27 mmol) and pyridine
(55 L, 0.68 mmol) was dissolved in anhydrous dichloromethane
(2.5 mL) under nitrogen, the reaction flask was immersed in an
ice bath, and trifluoromethanesulfonic anhydride (126 L,
l
l
0.41 mmol) was added dropwise to the solution. After stirring for
2 h at 0 °C, the solution of TMGA (171 mg, 1.08 mmol) in dichloro-
methane (1 mL) was injected into the reaction mixture and the
reaction was stirred overnight at the same temperature. Water
(5 mL) was added to the solution, the mixture was extracted with
dichloromethane (3 ꢃ 5 mL), and the combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo to pro-
vide a residue, which was purified by column chromatography to
give azido-compound 10 (201 mg, 82%) as a colorless oil: Rf 0.60
(EtOAc/Hex = 1:6); ½a D29
ꢁ
+1.80 (c 1.1, CHCl3); IR (thin film):
v
2924, 2854, 2100, 1455, 1100 cmꢂ1
;
1H NMR (CDCl3, 600 MHz): d
7.39–7.23 (m, 20H, ArH), 5.75–5.70 (m, 1H, H-6), 5.46–5.42 (m,
1H, H-5), 4.98–4.96 (m, 1H, H-4), 4.94 (d, J = 11.4 Hz, 1H, CH2Ph),
4.92 (d, J = 3.6 Hz, 1H, H-10), 4.84 (d, J = 12.0 Hz, 1H, CH2Ph), 4.80
(d, J = 12.0 Hz, 1H, CH2Ph), 4.72 (d, J = 12.0 Hz, 1H, CH2Ph), 4.71
(d, J = 12.0 Hz, 1H, CH2Ph), 4.56 (d, J = 11.4 Hz, 1H, CH2Ph), 4.48
(d, J = 12.0 Hz, 1H, CH2Ph), 4.40 (d, J = 12.0 Hz, 1H, CH2Ph), 4.09–
4.6. a-Galactosyl ceramide (1)
To a solution of compound 11 (104 mg) in a mixed solvent of
MeOH/CHCl3 (3:1 ratio, 1 mL) was added 8 N HCl (100 L) and
the reaction was stirred for 1 d. Pd(OH)2/C (200 mg, Degussa type)
l

Y.-F. Yen et al. / Carbohydrate Research 368 (2013) 35–39
39
4. (a) Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.;
Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi, M. Science 1997, 278,
1626–1629; (b) Matsuda, J. L.; Kronenberg, M. J. Curr. Opin. Immunol. 2001, 13,
19–25; (c) Hong, S.; Wilson, M. T.; Serizawa, I.; Wu, L.; Nagendra, S.; Naidenko,
O.; Miura, T.; Haba, T.; Scherer, D. C.; Wie, J.; Kronenberg, M.; Koezuka, Y.; van
Kaer, L. Nat. Med. 2001, 7, 1052–1056; (d) Sharif, S.; Arreaza, G. A.; Zucker, P.;
Mi, Q.-S.; Sondhi, J.; Naidenko, O. V.; Kronenberg, M.; Koezuka, Y.; Delovitch, T.
L. Nat. Med. 2001, 7, 1057–1062; (e) Kaer, L. V. Nat. Rev. Immunol. 2005, 5, 31–
42; (f) Salio, M.; Ghadbane, H.; Shepherd, D.; Cypen, J.; Aichinger, M.; Jervis, P.;
Besra, G. S.; Cresswell, P.; Cerundolo, V. Mol. Immunol. 2012, 51, 38–39.
5. (a) De Libero, G.; Mori, L. Nat. Rev. Immunol. 2005, 5, 485; (b) Tsuji, M. Cell. Mol.
Life Sci. 2006, 1889; (c) Wu, D.; Zajonc, D. M.; Fujio, M.; Sullivan, B. A.; Kinjo, Y.;
Kronenberg, M.; Wilson, I. A.; Wong, C. H. Proc. Natl. Acad. Sci. U.S.A. 2006, 11,
3972; (d) Chang, Y. J.; Huang, J. R.; Tsai, Y. C.; Hung, J. T.; Wu, D.; Fujio, M.;
Wong, C.-H.; Yu, A. L. Proc. Natl. Acad. Sci. U.S.A. 2007, 25, 10299.
6. For a review on synthesis of GalCer see— Morales-Serna, J. A.; Boutureira, O.;
Díaz, Y.; Matheu, M. I.; Castillón, S. Carbohydr. Res. 2007, 342, 1595–1612.
7. (a) Du, W.; Gervay-Hague, J. Org. Lett. 2005, 7, 2063–2065; (b) Du, W.; Kulkarni,
S. S.; Gervay-Hague, J. Chem. Commun. 2007, 2336–2338; (c) Schombs, M.; Park,
F. E.; Kulkarni, S. S.; Gervay-Hague, J. J. Org. Chem. 2010, 75, 4891–4898; (d)
Kulkarni, S. S.; Gervay-Hague, J. In Handbook of Chemical Glycosylation: Advances
in Stereoselectivity and Therapeutic Relevance; Demchenko, A. V., Ed.; Wiley-
VCH, 2008; pp 59–93. and references therein.
was added to the same flask and nitrogen was bubbled through the
solution for 5 min. The reaction flask was equipped with a hydro-
gen balloon and the suspension was kept stirring for 2 d at room
temperature. After restoring nitrogen, the mixture was filtered
through celite and the filtrate was concentrated in vacuo, and the
residue was purified by flash column chromatography on sephadex
G-10 to afford the target molecule 1 (67 mg, 94%): Rf 0.60 (MeOH/
CHCl3 = 1:4); ½a 2D9
ꢁ
+38.2 (c 0.05, CHCl3/MeOH); mp 182–185 °C; IR
(KBr) v
3426, 2918, 2851, 1645 cmꢂ1; 1H NMR (C5D5N, 600 MHz): d
8.55 (d, J = 8.4 Hz, 1H), 5.54 (d, J = 3.6 Hz, 1H), 5.23 (m, 1H), 4.65–
4.25 (m, 10H), 2.42 (t, 2H), 2.32–2.21 (m, 1H), 1.92–1.57 (m,5H),
1.29–1.22 (m, 66H), 0.84 (t, 6H); 13C NMR (C5D5N, 150 MHz): d
173.3 (C), 101.4 (CH), 76.5 (CH), 73.0 (CH), 72.4 (CH), 71.5 (CH),
70.9 (CH), 70.2 (CH), 68.5 (CH2), 62.6 (CH2), 51.4 (CH), 36.8 (CH2),
34.2 (CH2), 32.1 (CH2), 30.3–29.3 (CH2), 26.5 (CH2), 26.4 (CH2),
22.9 (CH2), 14.3 (CH3); HRMS (ESI) Calcd for C50H100O9N
([M+H]+) 858.7398. Found: 858.7408. Our NMR data of compound
1 matched well with the reported one.8c
8. Selected recent synthesis of
a-GalCer analogs: (a) Facciotti, F.; Mori, L.; De
Libero, G.; Lombardi, G.; Fallarini, S.; Panza, L.; Compostella, F.; Ronchetti, F. J.
Org. Chem. 2007, 72, 7757–7760; (b) Tsujimoto, T.; Ito, Y. Tetrahedron Lett. 2007,
48, 5513–5516; (c) Michieletti, M.; Bracci, A.; Compostella, F.; Libero, G. D.;
Mori, L.; Fallarini, S.; Lombardi, L. G.; Panza, L. J. Org. Chem. 2008, 73, 9192–
9195; (d) Park, J.; Lee, J. H.; Ghosh, S. C.; Bricard, G.; Venkataswamy, M.;
Porcelli, S. A.; Chung, S. Bioorg. Med. Chem. Lett. 2008, 18, 3906; (e) Boututeira,
O.; Morales-Serna, J. A.; Diaz, Y.; Matheu, M. I.; Castillón, S. Eur. J. Org. Chem.
1851, 2008; (f) Veerapen, N.; Brigl, M.; Garg, S.; Cerundolo, V.; Cox, L. R.;
Brenner, M. B.; Besra, G. S. Bioorg. Med. Chem. Lett. 2009, 19, 4288; (g) Enders,
Acknowledgments
The authors thank the National Science Council in Taiwan (NSC
99–2113-M-005–013-MY2 and NSC 101–2113-M-005–006-MY2)
and National Chung Hsing University for financial support.
ˇ
D.; Terteryan, V.; Palecek, J. Synthesis 2010, 2979–2984; (h) Khaja, S. D.; Kumar,
Supplementary data
V.; Ahmad, M.; Xue, J.; Matta, K. L. Tetrahedron Lett. 2010, 51, 4411–4414.
9. (a) Morita, M.; Sawa, E.; Yamaji, K.; Sakai, T.; Natori, T.; Koezuka, Y.; Fukushima,
H.; Akimoto, K. Biosci. Biotechnol. Biochem. 1996, 60, 288–292; (b) Lin, C. -C.;
Fan, G. -T.; Fang, J. -M. Tetrahedron Lett. 2003, 44, 5281–5283 and references
cited therein.
10. (a) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Fan, H.-F.; Pai, C.-L.; Yang, W.-C.; Lu, L.-D.;
Hung, S.-C. Angew. Chem., Int. Ed. 2002, 41, 2360–2362; (b) Wang, C.-C.;
Zulueta, M. M. L.; Hung, S.-C. Chimia 2011, 65, 54–58.
11. (a) Tsai, Y.-F.; Shih, C.-H.; Su, Y.-T.; Yao, C.-H.; Lian, J.-F.; Liao, C.-C.; Hsia, C.-W.;
Shui, H.-A.; Rani, R. Org. Biomol. Chem. 2012, 10, 931–934; (b) Pearson, M. S. M.;
Floquet, N.; Bello, C.; Vogel, P.; Plantier-Royon, R.; Szymoniak, J.; Bertus, P.;
Behr, J.-B. Bioorg. Med. Chem. 2009, 17, 8020–8026.
12. Kulkarni, S. S.; Gervay-Hague, J. Org. Lett. 2006, 8, 5765–5768.
13. Luo, S.-Y.; Kulkarni, S. S.; Chou, C.-H.; Liao, W.-M.; Hung, S.-C. J. Org. Chem.
2006, 71, 1226–1229.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.
11.008.
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