CD1a-binding glycosphingolipids stimulating human autoreactive T-cells: Synthesis of a family of sulfatides differing in the acyl chain moiety

Article abstract of DOI:10.1016/S0040-4020(02)01092-X

Native sulfatide (a mixture of 3-sulfated β-D-galactopyranosylceramides with different fatty acids at the ceramide moiety) is an antigen presented by CD1a proteins. Herein the preparation of four sulfatides, which are constituents of the natural mixture a

Full text of DOI:10.1016/S0040-4020(02)01092-X

Tetrahedron 58 (2002) 8703–8708
CD1a-binding glycosphingolipids stimulating human
autoreactive T-cells: synthesis of a family of sulfatides
differing in the acyl chain moiety
a
Federica Compostella, Laura Franchini, Gennaro De Libero, Giovanni Palmisano,
a
b
c
a
Fiamma Ronchetti and Luigi Panza
d,
*
a
Dipartimento di Chimica e Biochimica Medica, Universit a` di Milano, Via Saldini 50, 20133 Milano, Italy
Experimental Immunology, University Hospital, 4031 Basel, Switzerland
b
c
Dipartimento di Scienze Chimiche, Fisiche e Matematiche, Universit a` dell’ Insubria, Via Valleggio 11, 22100 Como, Italy
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universit a` del Piemonte Orientale,
Viale Ferrucci 33, 28100 Novara, Italy
d
Received 9 May 2002; revised 6 August 2002; accepted 29 August 2002
Abstract—Native sulfatide (a mixture of 3-sulfated b-D-galactopyranosylceramides with different fatty acids at the ceramide moiety) is an
antigen presented by CD1a proteins. Herein the preparation of four sulfatides, which are constituents of the natural mixture and bear palmitic,
stearic, behenic or nervonic fatty acid chains, is described. Azidosphingosine was stereoselectively synthesized through a CuCN-catalyzed
allylic alkylation of a hexenitol dimesylate derived from D-xylose; b-glycosylation of azidosphingosine with a suitable D-galactosyl
trichloroacetimidate led, after reduction of the azido group, to the galactosylsphingosine skeleton, which was derivatized with the different
fatty acids. Final regioselective 3-sulfation gave the desired sulfatides, which were tested for activation of sulfatide-specific and CD1a-
restricted T-cell clones. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
mixture of sulfatides purified from bovine and porcine brain
and differing in the lipid moiety was used.
T lymphocytes recognize as antigens short peptides (8–12
aminoacids long) associated with the MHC class I and II
proteins. These molecules are characterized by a peptide-
binding groove and the complex formed by the presentation
molecule with the bound peptide is the antigenic complex
Herein is described an account of our studies which has led
to the synthesis of a family of 3-sulfated galactosylcer-
amides (sulfatide, 1), as antigens presented by CD1a; the
synthesized compounds differ in the chain length and the
structure of the fatty acid amide, and are constituents of
the natural mixture. These compounds have been shown
to be capable of stimulating sulfatide-specific and
CD1a-restricted T-cell clones.
1
stimulating T-cells.
Recently, a new family of antigen presenting molecules
(
presentation of lipid and glycolipid antigens to T-cells.
APM), the CD1 molecules, have been shown to mediate
2
–7
The CD1 family of APM in humans is composed of five
functional proteins: CD1a, CD1b, CD1c, CD1d and CD1e.
A number of lipids presented by CD1 molecules have been
characterized. The CD1a isoform was shown to present
mycobacterial lipid antigens to specific T-cells, although the
precise structure(s) of the presented lipid(s) was not
2
. Results and discussion
In order to obtain the sulfatide 1 (Fig. 1) by synthesis, it is
necessary to have access to the aglycone part, which is the
ceramide. In particular, we focused our attention on 3-OH
protected azidosphingosine as it represents a better substrate
8
reported. Only recently sulfatide 1 (a mixture of 3-sulfated
b-D-galactosylceramides containing fatty acid residues of
11
than ceramide in glycosylation reactions.
different structure and chain length) was demonstrated to
9
bind to the CD1a molecule, and to be presented by CD1a to
1
sulfatide-specific T-cell clones. In all these studies a
0
Keywords: azidosphingosine; antigens; glycolipids; biologically active
compounds.
*
Corresponding author. Tel.: þ39-0321-657651; fax: þ39-0321-657621;
e-mail: panza@pharm.unipmn.it
Figure 1.
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01092-X

8704
F. Compostella et al. / Tetrahedron 58 (2002) 8703–8708
Scheme 1. (i): (a) Me
iii) n-C12
Ref. 18.
3
SiCH
2
MgCl, THF, 708C, 3 h, (b) KH, THF, 508C, 3 h, 58% (two steps); (ii): MsCl, DMAP, collidine, 08C to rt, 3 h, 74%;
(
4 3 2
H25MgBr, CuCN, THF, 08C, 1 h, 41% trans, 22% cis; (iv): Bu NN , toluene, 808C, 30 h, 90%; (v): (a) HCl 6N, THF/H O, rt, 30 h, 80%; (vi): see
1
6
The synthesis of azidosphingosine requires the installation
of both the proper stereochemistry and the (E)-double bond,
and a considerable body of work has been reported in the
differently from the result described in the literature,
besides the expected product 5, a significant amount of the
cis isomer was also formed (E/Z 2:1). Although this result
is not easy to explain, it is apparent that changing the
protecting groups on position 1 and 3 from benzyl to
isopropylidene influences the stereochemistry of the double
bond formation during the allylic displacement. After
careful chromatographic separation of the double bond
isomers, compound 5 was treated with freshly prepared
tetrabutylammonium azide in toluene to allow substitution of
the second mesylate with an azido group. Compound 6 was
then deprotected at positions 1 and 3, and was transformed
into the desired 3-O-benzoylazidosphingosine 7 through
1
2–15
literature.
which exploits a starting material from the chiral pool,
We decided to use a synthetic approach
0
attachment of the long chain and introduction of the (E)-
namely D-xylose, through a S 2 -type reaction for the
N
1
6
double bond. The published procedure utilizes benzyl
ethers as protecting groups for the xylose hydroxyl groups at
positions 3 and 5. However, benzyl-protecting groups are
not suitable for our purposes as they are incompatible with
the azide and the alkene functions in deprotection steps. We
therefore decided to use the known 3,5-O-isopropylidene-D-
xylofuranose 2 as starting material, which can be easily
1
8
standard protection–deprotection reactions (Scheme 1).
1
7
obtained from D-xylose, although only in moderate yield.
Attempts to perform the Wittig reaction on compound 2
with tetradecyliden-triphenylphosphorane gave quite low
and poorly reproducible yields. Thus, compound 2 was
converted into the protected hexenitol 3 (58% yield) through
a Peterson olefination, by condensation between 2 and the
Grignard reagent derived from chloromethyltrimethyl-
silane, followed by treatment of the b-silylalcohol with
potassium hydride. Treatment of diol 3 with methanesulfo-
nyl chloride smoothly afforded the dimesylate 4 in excellent
yield. Allylic displacement of the mesyl group in position 3
was effected with n-dodecylmagnesium bromide in the
presence of catalytic copper cyanide. To our surprise, and
Stereoselective b-glycosylation of acceptor 7 with 2,3,4,6-
tetra-O-pivaloyl-b-D-galactopyranosyl trichloroacetimidate
8 in the presence of boron trifluoride–diethyl ether
complex gave compound 9 in very good yield.
1
9
In order to introduce the fatty acid and, finally, perform the
sulfation of the position 3 of the sugar moiety, the galactosyl
1
9
sphingosine precursor 9 was transformed into compound
10 by Z e` mplen reaction followed by reduction of the azido
group.
The crude amine 10 was used directly for the subsequent
Scheme 2. (i): BF ·OEt
3
2
, CH
2
Cl
2
, 08C to rt, 88%; (ii): see Ref. 19; (iii): RCOCl, 50% aq. AcONa, THF, rt, 3 h (for 11a–c, 71–80%), or nervonic acid, EDCI,
SnO, MeOH, reflux, 2 h, (b) Me N·SO , THF, rt, 2–20 h, 70–75%.
CH Cl , reflux, 2 h (for 11d, 64%); (iv): (a) Bu
2
2
2
3
3

F. Compostella et al. / Tetrahedron 58 (2002) 8703–8708
8705
2
Table 1. Activation of T-cells clone by sulfatides
(ES ) as indicated. All NMR spectra were recorded at
303 K with a Bruker AM-500 spectrometer equipped with
an Aspect-3000 computer, a process controller, and an array
a
Compound
Fatty acid substituent
Released TNFa (pg/mL)
processor; chemical shifts of NMR spectra are reported as d
(ppm) relative to tetramethylsilane as internal standard.
1
1
1
1
a
b
c
16:0
18:0
22:0
24:1
Natural mixture
–
330
345
1425
3354
4580
298
Solvents were purified and dried in the usual way. All
reactions were monitored by TLC on Silica Gel 60 F-254
plates (Merck) with detection by spraying with 50% H SO
d
Bovine sulfatide
Medium
2
4
solution and heating at 1108C, or by dipping in an
ammonium molybdate solution followed by heating. Flash
column chromatography was performed on Silica Gel 60
(230–400 mesh, Merck). All evaporations were carried out
under reduced pressure at 408C. Compound 2 was obtained
from D-xylose as described in Ref. 17. EDCI: 1-(3-di-
methylaminopropyl)-3-ethylcarbodiimide hydrochloride.
Bovine sulfatide was purchased from Fluka (Buchs,
Switzerland).
a
T cell clone A124.
acylation step. The saturated fatty acid chains were
introduced by treatment of the amine with the appropriate
acid chloride in a 1:1 mixture of THF and 50% aqueous
sodium acetate, and the amides 11a–c, derived from
palmitoyl, stearoyl or behenoyl chlorides were obtained in
yields ranging from 60 to 70%. A different procedure was
used in the case of the nervonoyl derivative 11d which was
obtained by treatment of compound 10 with nervonic acid
and EDCI in dichloromethane.
1
9
4.1.1. (2R,3R,4S)-1,3-O-Isopropylidene-5-hexen-1,2,3,4-
tetrol (3). Mg (1.54 g, 63.8 mmol) was covered with a
13 mL portion of a solution of 8.8 mL (63.8 mmol) of
Finally, compounds 11a–d were selectively sulfated to the
desired sulfatides 1a–d in about 70% yield, according to
chloromethyltrimethylsilane in 53 mL of dry THF. To this
mixture were first added few drops of 1,2-dibromoethane
(evolution of ethylene), then, dropwise at slight reflux, the
rest of the chloromethyltrimethylsilane solution. The
mixture was stirred until complete consumption of Mg.
To this solution of the Grignard reagent, a solution of
compound 2 (2.42 g, 12.8 mmol) in 53 mL of dry THF was
added via cannula. The reaction was heated at 708C for 3 h,
and then quenched by the careful addition of a saturated
aqueous NH Cl solution; THF was removed under reduced
4
pressure, and the aqueous phase extracted with AcOEt
(5£50 mL). The combined organic phases were dried
(Na SO ) and concentrated to give the crude b-silylalcohol
2 4
as a white solid. This adduct, without further purification,
was diluted in 55 mL of dry THF. KH was slowly added to
this solution under Ar until the appearance of a precipitate.
The mixture was heated at 508C for 3 h, turning from yellow
to brick-red, and then quenched by the slow addition of
water. After evaporation of the THF, the aqueous phases
were extracted with AcOEt (6£50 mL), and the combined
organic phases were dried and concentrated. Flash chroma-
tography (hexane/AcOEt, from 4:6 to 2:8) of the residue
2
0
Flitsch et al., by activation of the sugar hydroxyls in
position 3 and 4 with dibutyltin oxide followed by treatment
of the dibutylstannylene acetal with Me N·SO as sulfation
3
3
reagent (Scheme 2).
The synthetic compounds 1a–d were tested for activation of
sulfatide-specific T-cells and compared to a commercial
sample of sulfatide isolated from bovine brain and contain-
2
1
ing the natural fatty acids mixture on ceramide moiety.
The activity of the synthetic and natural sulfatides was
studied through the release of TNFa as T-cell activation
1
0
read out. The results (Table 1) show that the analyzed T-
cells are differently activated by the synthetic sulfatides.
Thus, the tested synthetic compounds have a biological
activity, which is modulated by the type and the length of
acyl chain, as shown in Table 1.
3. Conclusion
In conclusion, a series of sulfatides bearing different fatty
acids have been synthesized and evaluated for T-cells
activation when presented by CD1a molecules. More
detailed studies to evaluate the marked influence of the
fatty acid in binding to CD1 molecules and in forming
immunogenic complexes recognized by T-cells, are in
progress and will be described in due course.
furnished compound 3 (1.39 g, 58%) as an oil. [a]
D
¼210.2
); 1.44 (s,
); 2.68 (br s, 1H, OH); 2.86–2.91 (m, 1H, OH);
3.44–3.48 (m, 1H, H-2); 3.61 (dd, 1H, J3,4¼7.5 Hz,
2,3¼1.5 Hz, H-3); 3.77 (dd, 1H, J1a,1b¼12.5 Hz, J1a,2
2.0 Hz, H-1a); 3.97 (dd, 1H, J1a,1b¼12.5 Hz, J1b,2¼1.5 Hz,
H-1b); 4.29–4.34 (m, 1H, H-4); 5.25 (ddd, 1H, J5,6a
1
(c 1, CHCl
3H, CH
). H NMR (CDCl ): 1.43 (s, 3H, CH
3 3
3
3
J
¼
¼
1
1
0.5 Hz, J6a,6b¼1.0 Hz, J ¼1.0 Hz, H-6a); 5.44 (ddd,
4,6a
H, J5,6b¼17.0 Hz, J
¼1.0 Hz, J ¼1.0 Hz, H-6b);
6a,6b 4,6b
4
. Experimental
5.82 (ddd, 1H, J5,6b¼17.0 Hz, J5,6a¼10.5 Hz, J4,5¼6.5 Hz,
1
3
H-5). C NMR (CDCl ): 19.1; 30.2; 64.4; 66.5; 73.3;
3
4
.1. General methods
75.9; 100.2; 119.0; 135.6. nmax (liquid film): 3400,
1
2
3
090, 2990, 2950, 1645, 1380 cm . CI-MS: m/z 206
: calcd C 57.43, H 8.57; found C
þ
57.21, H 8.45.
Optical rotations were determined on a Perkin–Elmer 241
polarimeter in a 1 dm cell at 208C. IR spectra were recorded
on a Perkin–Elmer 1420 spectrophotometer; NaCl crystal
windows. Mass spectrometry was carried out on a quadru-
polar Hewlett Packard HP 5988A or Thermo Quest
Finningan LCQeDECA mass spectrometers using
[MþNH
4
] . C
9
H
16
O
4
4.1.2. (2R,3R,4S)-1,3-O-Isopropylidene-2,4-di-O-methyl-
sulfonyl-5-hexen-1,2,3,4-tetrol (4). 2,4,6-Collidine
(3,05 mL, 23.07 mmol), DMAP (0.05 g) and methanesulfo-
nyl chloride (1.65 mL, 21.3 mmol) were added to a solution
chemical ionization (CI/NH ) and negative electrospray
3

8706
F. Compostella et al. / Tetrahedron 58 (2002) 8703–8708
at 08C of compound 3 (1.34 g, 7.1 mmol) in dry CH Cl
2
4.1.5. (2S,3R,4E)-2-Azido-3-benzoyloxy-4-octadecen-1-
ol (7). 6N HCl (2 mL) was added dropwise to a solution
of compound 6 (0.45 g, 1.23 mmol) in THF/H O 9:1
2
(
32 mL). The reaction mixture was slowly warmed to rt and
stirred for about 3 h at this temperature, then the solvent was
evaporated off. The residue was diluted with AcOEt
(30 mL) and washed with a saturated aqueous NaHCO3
solution (30 mL). The aqueous phase was separated and
2
(20 mL), and the reaction was stirred for 30 h. The solution
was neutralized with a saturated aqueous NaHCO solution
3
and extracted with chloroform (3£40 mL). The combined
organic extracts were washed with brine, dried and
concentrated. Purification by flash chromatography
(hexane/ AcOEt, 7:3) gave the known (2S,3R,4E)-2-azido-
extracted with AcOEt (3£25 mL). The combined organic
layers were washed with brine, dried (MgSO ) and
4
concentrated. Flash chromatography of the residue (hexane/
AcOEt, 6:4), afforded compound 4 (1.81 g, 74%) as a foam.
1
8
octadec-4-ene-1,3-diol (0.32 g, 80%) as an oil. The diol
was transformed into the title compound 7 (0.32 g, 75%
three steps) according to Ref. 18. [a] ¼246.5 (c 4, CHCl )
1
[
CH ); 1.45 (s, 3H, CH ); 3.02 (s, 3H, CH SO ); 3.12 (s, 3H,
a] ¼225.8 (c 1, CHCl ). H NMR (CDCl ): 1.44 (s, 3H,
D
3
3
3
3
3
2
D
3
1
8
CH SO ); 3.99–4.04 (m, 2H, H-1a,3); 4.23 (dd, 1H,
3
(Lit.: 246.0 (c 4, CHCl )).
3
2
J1a,1b¼13.8 Hz, J ¼2.0 Hz, H-1b); 4.48–4.51 (m, 1H,
1b,2
H-2); 5.08 (dd, 1H, J ¼7.0 Hz, J ¼6.5 Hz, H-4); 5.52
4.1.6. (2S,3R,4E)-2-Amino-1-(b-D-galactopyranosyloxy)-
3-hydroxy-4-octadecene (10). Boron trifluoride diethyl
ether in dry CH Cl (0.1 M solution, 4.7 mL) was added
dropwise to a solution at 08C under argon of imidate 8
(0.77 g, 1.16 mmol) and 7 (0.20 g, 0.47 mmol) in dry
3
,4
4,5
(
d, 1H, J ¼10.5 Hz, H-6a); 5.70 (d, 1H, J ¼17.0 Hz,
5,6b 5,6a
5
,6a
5,6b
H-6b); 5.91 (ddd, 1H, J ¼17.0 Hz, J ¼10.5 Hz,
2
2
1
3
19
J ¼6.5 Hz, H-5). C NMR (CDCl ): 19.5; 29.4; 39.4;
4
,5
3
4
0.5; 62.9; 71.7; 72.4; 82.7; 100.3; 124.0; 129.9. n
liquid film): 2995, 2940, 1640, 1380, 1310, 1210, 1070,
max
(
CH Cl (6 mL). The reaction was warmed to rt, and after
2
2
2
1
þ
C 38.36, H 5.85; found C 38.47, H 5.72.
7
90 cm . CI-MS: m/z 362 [MþNH ] . C H O S : calcd
30 min an additional amount of compound 8 (0.30 g) was
added. After 1 h the reaction mixture was diluted with
CH Cl and treated with sat. NaHCO solution (40 mL).
4
11 20 8 2
2
2
3
4.1.3. (2R,3R,4E)-1,3-O-Isopropylidene-2-O-methylsul-
fonyl-4-octadecen-1,2,3-triol (5). A flame-dried flask was
After separation, the aqueous layer was extracted with
CH Cl (3£30 mL). The combined organic layers were
2
2
2
2
charged with freshly prepared CuCN
.49 mmol). After the flask had been flushed with argon,
dry THF (15 mL) was added, and the mixture was cooled to
8C. A solution of freshly prepared n-C H MgBr
(0.044 g,
dried and concentrated. Purification by flash chromato-
graphy (hexane/AcOEt, 9:1) gave the known (2S,3R,4E)-2-
azido-3-benzoyloxy-1-(2,3,4,6-tetra-O-pivaloyl-b-D-galacto-
0
1
9
0
pyranosyloxy)-octadec-4-ene (9) (0.38 g, 88%) as an oil.
Compound 9 was transformed, according to Ref. 19, into
the title amino derivative 10 (0.14 g, 76%), by Z e` mplen
reaction followed by reduction of the azido group with
hydrogen sulfide in pyridine/water. Crude 10 was used
directly in the next step.
1
2 25
(
9.88 mmol) in dry Et O (60 mL) was added, and the
2
mixture stirred for 10 min. A solution of compound 4
1.70 g, 4.94 mmol) in dry THF (60 mL) was then slowly
added via cannula, and stirring was continued for 1 h. The
(
reaction was quenched with saturated aqueous NH Cl
4
(
40 mL), and the resulting mixture extracted with Et O
2
(
3£30 mL). The combined organic phases were washed
4.1.7. (2S,3R,4E)-1-(b-D-Galactopyranosyloxy)-2-(hexa-
decanoylamino)-3-hydroxy-4-octadecene (11a). Com-
pound 10 (0.030 g, 0.063 mmol) was dissolved in THF
(7.5 mL) and treated with aqueous sodium acetate
(50%, 6.5 mL) and hexadecanoyl chloride (0.023 mL,
0.076 mmol); the mixture was vigorously stirred for 3 h.
The aqueous phase was extracted with THF (2£10 mL). The
combined organic layers were dried and concentrated. The
residue was purified by flash chromatography (CH Cl /
with brine, dried and concentrated. Purification by flash
chromatography (hexane/AcOEt, 9:1) gave first the cis-
adduct (0.43 g, 20%), then compound 5 (0.848 g, 41%) as a
1
foam. [a] ¼233.9 (c 1, CHCl ). H NMR (CDCl ): 0.86 (t,
D
3
3
3
CH ); 1.45 (s, 3H, CH ); 1.47 (s, 3H, CH ); 2.01–2.09 (m,
H, CH ); 1.16–1.32 (m, 20H, 10 CH ); 1.33–1.41 (m, 2H,
3 2
2
3
3
2
H, 2 H-6); 3.04 (s, 3H, CH SO ); 3.98–4.13 (m, 2H, 2
3 2
H-1); 4.41–4.47 (m, 2H, H-2,3); 5.51 (dd, 1H, J ¼6.5 Hz,
3
,4
2
2
J ¼15.5 Hz, H-4); 5.82 (dt, 1H, J ¼15.5 Hz, J ¼
MeOH, 9:1) to give compound 11a (0.035 g, 80%) as a
19
4
,5
4,5
5,6
1
3
6
3
1
9
.7 Hz, H-5). C NMR (CDCl ): 14.8; 19.6; 23.4; 29.5–
3
foam. [a] ¼25.0 (c 1, pyridine) (Lit.: 25.2 (c 1,
D
0.3 (10C); 32.6; 33.0; 39.5; 63.7; 72.1; 75.2; 99.7; 125.8;
36.8. n
pyridine)). Physical data of compound 11a were in
agreement with those reported in Ref. 19.
(liquid film): 2990, 2950, 1460, 1380, 1180,
þ
max
1
2
60 cm . CI-MS: m/z 436 [MþNH ] . C H O S: calcd
4
22 42 5
C 63.12, H 10.11; found C 65.30, H 11.92.
4.1.8. (2S,3R,4E)-1-(b-D-Galactopyranosyloxy)-2-(octa-
decanoylamino)-3-hydroxy-4-octadecene (11b). Com-
pound 11b was obtained from the amino derivative 10
(0.027 g, 0.059 mmol) as described for compound 11a. The
reaction mixture was purified by flash-chromatography
(CH Cl /MeOH, 85:15) to yield compound 11b (0.030 g,
4.1.4. (2S,3R,4E)-2-Azido-1,3-O-isopropylidene-4-octa-
decen-1,3-diol (6). Tetrabutylammonium azide (0.97 g,
4
1
.41 mmol) was added to a solution of 5 (0.57 g,
.36 mmol) in dry toluene (30 mL). The reaction was
2
2
1
stirred at 808C for 30 h. Water (100 mL) was added, the
mixture was extracted with AcOEt (3£80 mL), and the
combined organic layers were dried and concentrated to
dryness. Purification by flash chromatography (hexane/
AcOEt, 97:3) afforded pure 6 (0.45 g, 90%) as an oil.
71%) as a foam. [a] ¼24.9 (c 1, Py). H NMR (CDCl /
3 3
D
3
CD OD, 1:1): 0.86 (t, 6H, J¼7.5 Hz, 2 CH ); 1.20–1.40 (m,
50H, 25 CH ); 1.52–1.62 (m, 2H, CH ); 1.96–2.02 (m, 2H,
2
2
2 H-6); 2.14 (t, 2H, J¼7.5 Hz, COCH ); 3.44–3.50 (m, 2H,
2
0
0
0
H-3 ,5 ); 3.53 (dd, 1H, J
0
0
¼7.5 Hz, J
0 0
2 ,3
¼9.0 Hz, H-2 );
1
,2
1
8
[
a] ¼241.5 (c 1.5, CHCl ) (Lit.: 242.6 (c 1.5, CHCl ))
3.56 (dd, 1H, J1a,1b¼10.0 Hz, J ¼3.0 Hz, H-1a); 3.71 (dd,
D
3
3
1a,2
0
All the physical data were in agreement with those reported
in Ref. 18.
1H, J5
0
0
0
¼5.0 Hz, J
0 0
6 a,6 b
¼11.5 Hz, H-6 a); 3.78 (dd, 1H,
,6 a
0
J6
0
¼11.5 Hz, J
0 0
¼6.6 Hz, H-6 b); 3.84 (br d, 1H,
a,6 b
5 ,6 b

F. Compostella et al. / Tetrahedron 58 (2002) 8703–8708
8707
0
J3
0
0
¼3.0 Hz, H-4 ); 3.93–3.99 (m, 1H, H-2); 4.08 (dd, 1H,
J ¼15.5 Hz, J ¼7.0 Hz, H-5); 7.67 (d, 1H, J
¼
,4
4,5
5,6
2,NH
1
3
J ¼J ¼7.5 Hz, H-3); 4.17 (dd, 1H, J
¼10.0 Hz,
9.2 Hz, NH). C NMR (CDCl /CD OD, 1:2): 14.9 (2C);
3
2
,3
3,4
1a,1b
3
0
J
¼4.3 Hz, H-1b); 4.19 (d, 1H, J
0 0
1 ,2
¼7.5 Hz, H-1 );
23.9 (2C); 27.2; 28.3 (2C); 30.6–31.3 (27C); 33.1 (2C);
33.6; 37.6; 54.8; 62.6; 70.0; 70.3; 72.7; 73.1; 74.8; 76.6;
1
b,2
5
.42 (ddt, 1H, J ¼15.5 Hz, J ¼7.5 Hz, J ¼1.0 Hz,
4,5 5,6
4
,5
3,4
4,6
H-4); 5.66 (dt, J ¼15.5 Hz, J ¼7.0 Hz, H-5); 7.57 (d,
105.2; 131.0 (3C); 135.3; 176.0. n
21
(nujol): 3445,
max
1650 cm . ESI/MS (negative-ion mode): m/z 808.7
2
1
3
1
1
(
H, J
¼9.0 Hz, NH). C NMR (CDCl /CD OD, 1:1):
2
,NH
3
3
5.0 (2C); 23.9 (2C); 26.3; 27.3; 30.6–30.9 (20C); 33.2
2C); 33.6; 37.7; 54.7; 62.7; 70.0; 70.3; 72.7; 73.2; 74.8;
[M2H] . C H NO : calcd C 71.15, H 11.32, N 1.73;
8
4
8
91
found C 70.90, H 11.10, N 1.85.
7
1
6.5; 105.1; 130.8; 135.5; 176.0. n
2
(nujol): 3450,
max
645 cm . ESI/MS (negative-ion mode): m/z 726.7
2
1
4.1.11. (2S,3R,4E)-1-[3-O-(Sodium oxysulfonyl)-b-D-
galactopyranosyloxy]-2-(hexadecanoylamino)-3-hydroxy-
4-octadecene (1a). Compound 11a (0.035 g, 0.049 mmol)
[M2H] . C H NO : calcd C 69.28, H 11.21, N 1.92;
42 81 8
found C 68.95, H 11.35, N 2.03.
and Bu SnO (0.018 g, 0.073 mmol) were stirred in MeOH
2
4.1.9. (2S,3R,4E)-1-(b-D-Galactopyranosyloxy)-2-(doco-
sanoylamino)-3-hydroxy-4-octadecene (11c). Compound
(1 mL) at reflux under argon for 2 h. The solvent was
evaporated off under reduce pressure and the dibutyl-
stannylene complex was treated with Me N·SO (0.013 g,
1
1c was obtained from the amino derivative 10 (0.027 g,
.059 mmol) as described for compound 11a. The reaction
3
3
0
mixture was purified by flash-chromatography (CH Cl /
0.098 mmol) in THF at rt for 6 h. The solvent was removed
under reduced pressure, than the residue was dissolved
in CHCl /MeOH 1:1 (1 mL) and loaded onto a cation
2
2
MeOH, 9:1) to yield compound 11c (0.032 g, 71%) as a
1
3
þ
foam. [a] ¼27.2 (c 1, Py). H NMR (CDCl /CD OD, 1:1):
exchange resin column (Dowex 50X8, Na form, 0.5£
D
3
3
0
CH ); 1.52–1.62 (m, 2H, CH ); 1.96–2.02 (m, 2H, 2 H-6);
.86 (t, 6H, J¼7.5 Hz, 2 CH ); 1.20–1.40 (m, 58H, 29
4 cm). The mixture was eluted with CHCl /MeOH 1:1,
3
3
concentrated in vacuo and subjected to flash chroma-
tography (CH Cl /MeOH, 85:15) to give compound 1a
2
2
2
H-3 ,5 ); 3.53 (dd, 1H, J
.14 (t, 2H, J¼7.5 Hz, COCH ); 3.44–3.50 (m, 2H,
2
2
2
0
0
0
1
0
0
¼7.5 Hz, J
0
0
¼9.0 Hz, H-2 );
(0.029 g, 74%) as a foam. [a] ¼þ2.5 (c 1, CHCl ). H
1
,2
2 ,3
D
3
3
1
J6
J3
.56 (dd, 1H, J1a,1b¼10.0 Hz, J ¼3.0 Hz, H-1a); 3.71 (dd,
NMR (CDCl /CD OD, 1:1): 0.86 (t, 6H, J¼6.5 Hz, 2 CH );
1.15–1.40 (m, 46H, 23 CH ); 1.51–1.60 (m, 2H, CH );
2 2
1a,2
3 3 3
0
H, J5
0
0
¼5.0 Hz, J
0
0
¼11.5 Hz, H-6 a); 3.78 (dd, 1H,
,6 a
6 a,6 b
0
0
0
¼11.5 Hz, J
0
0
¼6.8 Hz, H-6 b); 3.84 (br d, 1H,
1.96–2.02 (m, 2H, 2 H-6); 2.16 (t, 2H, J¼7.5 Hz, COCH );
a,6 b
5 ,6 b
2
0
0
0
0
¼2.8 Hz, H-4 ); 3.93–3.98 (m, 1H, H-2); 4.08 (dd, 1H,
3.55 (dd, 1H, J
0
¼5.5 Hz, J
0
5 ,6b
¼6.5 Hz, H-5 ); 3.61 (dd,
,4
5 ,6a
J ¼J ¼7.5 Hz, H-3); 4.17 (dd, 1H, J
¼10.0 Hz,
1H, J1a,1b¼10.5 Hz, J ¼3.0 Hz, H-1a); 3.73 (dd, 1H,
2
,3
3,4
1a,1b
1a,2
0
0
J
¼4.0 Hz, H-1b); 4.19 (d, 1H, J
0
0
¼7.5 Hz, H-1 );
J
7.7 Hz, J
0
0
¼11.5 Hz, J
0
¼5.5 Hz, H-6 a); 3.74 (dd, 1H, J
0 0
1 ,2
¼
1
b,2
1 ,2
6 a,6 b
5 ,6a
0
5
.42 (ddt, 1H, J ¼15.5 Hz, J ¼7.5 Hz, J ¼1.0 Hz,
0
0
¼9.5 Hz, H-2 ); 3.79 (dd, 1H, J
0 0
6 a,6 b
¼11.5 Hz,
4
,5
3,4
4,6
2 ,3
0
H-4); 5.66 (dt, J ¼15.5 Hz, J ¼6.5 Hz, H-5); 7.57–7.59
J
0 0
¼6.5 Hz, H-6 b); 3.95–3.99 (m, 1H, H-2); 4.08 (dd,
4
,5
5,6
5 ,6 b
1
3
(
m, 1H, NH). C NMR (CDCl /CD OD, 1:1): 15.0 (2C);
3
1H, J ¼J ¼7.5 Hz, H-3); 4.15 (dd, 1H, J
¼10.5 Hz,
3
2,3
3,4
1a,1b
0
2
3
1
3.9 (2C); 26.0; 27.3; 30.6–30.9 (24C); 33.2 (2C); 33.6;
7.7; 54.7; 62.7; 70.0; 70.3; 72.7; 73.2; 74.8; 76.5; 105.1;
J
4.26 (dd, 1H, J
¼4.5 Hz, H-1b); 4.24 (br d, 1H, J
0 0
3 ,4
¼3.3 Hz, H-4 );
1
b,2
0
0
0
¼9.5 Hz, J
0 0
3 ,4
¼3.3 Hz, H-3 ); 4.33 (d, 1H,
2
,3
30.8; 135.5; 176.0. nmax (nujol): 3400, 1650 cm2
1
.
J
¼7.7 Hz, H-1 ); 5.42 (ddt, 1H, J ¼15.5 Hz, J ¼
0
0
0
1 ,2
4,5
3,4
2
ESI/MS (negative-ion mode): m/z 782.7 [M2H] .
C H NO : calcd C 70.45, H 11.44, N 1.79; found C
7.5 Hz, J ¼1.0 Hz, H-4); 5.67 (dt, 1H, J ¼15.5 Hz,
C
5,6 2,NH
4
,6
4,5
1
3
J ¼6.5 Hz, H-5); 7.69 (d, 1H, J ¼11.5 Hz, NH).
NMR (CDCl /CD OD, 1:1): 15.0 (2C); 23.9 (2C); 27.3;
3 3
4
6
89
8
7
0.17, H 11.53, N 1.85.
30.6–31.0 (18C); 33.2 (3C); 33.6; 37.7; 54.7; 62.6; 68.7;
70.2; 70.9; 73.1; 76.1; 81.7; 104.7; 130.7; 135.5; 176.2. n
4
.1.10. (2S,3R,4E)-1-(b-D-Galactopyranosyloxy)-2-
max
2
1
(15(Z)-tetracosenoylamino)-3-hydroxy-4-octadecene
(11d). Nervonic acid (0.036 g, 0.097 mmol) and EDCI
(0.025 g, 0.13 mmol) were added to a solution of compound
(nujol): 3440, 1650, 1550, 1250, 1090 cm . ESI/MS
2
(negative-ion mode): m/z 779.0 [M2Na] . C H NNaO S:
4
0
76
11
calcd C 59.90, H 9.55, N 1.75; found C 60.05, H 9.38, N
1.82.
1
0 (0.030 g, 0.065 mmol) in dry CH Cl2 (4 mL) under
2
argon. The reaction mixture was heated at reflux tempera-
ture for 2 h, then the solvent was removed under reduced
pressure. The crude was purified by flash chromatography
4.1.12. (2S,3R,4E)-1-[3-O-(Sodium oxysulfonyl)-b-D-
galactopyranosyloxy]-2-(octadecanoylamino)-3-hydroxy-
4-octadecene (1b). Sulfatide 1b was obtained as a foam
(0.017 g, 70%) from compound 11b (0.021 g, 0.029 mmol)
as described for 1a. After the addition of Me N·SO the
(
6
CH Cl /MeOH, 9:1) affording compound 11d (0.034 g,
2 2
1
4%) as a foam. [a] ¼23.0 (c 1, Py). H NMR (CDCl /
D
3
CD OD, 1:2): 0.87 (t, 6H, J¼7.5 Hz, 2 CH ); 1.20–1.40 (m,
3
3
3
3
5
3
1
7
4H, 27 CH ); 1.52–1.61 (m, 2H, CH ); 1.97–2.09 (m, 6H,
2
reaction was stirred at rt for 4 h. [a] ¼þ2.2 (c 1, MeOH).
2
D
1
CHvCHCH ); 2.15 (t, 2H, J¼7.5 Hz, COCH ); 3.47 (dd,
H NMR (CDCl /CD OD, 1:1): 0.86 (t, 6H, J¼7.0 Hz, 2
2
2
3
3
0
H, J
.0 Hz, J
0
0
¼9.5 Hz, J
0
0
¼3.5 Hz, H-3 ); 3.49 (dd, 1H, J
0
0
¼
CH ); 1.20–1.40 (m, 50H, 25 CH ); 1.54–1.62 (m, 2H,
2
2
,3
3 ,4
5 ,6a
3
0
0
0
¼5.2 Hz, H-5 ); 3.54 (dd, 1H, J
0
0
¼9.5 Hz,
CH ); 1.96–2.02 (m, 2H, 2 H-6); 2.16 (t, 2H, J¼7.5 Hz,
5
,6b
2 ,3
2
0
.2 Hz, H-1a); 3.71 (dd, 1H, J
0
J1
0
0
¼7.5 Hz, H-2 ); 3.57 (dd, 1H, J
¼10.5 Hz, J
¼
COCH ); 3.56 (dd, 1H, J
2
0
0
¼5.0 Hz, J
0 0
5 ,6 b
¼6.5 Hz, H-5 );
,2
1a,1b
1a,2
5 ,6 a
3
H-6 a); 3.78 (dd, 1H, J
H-6 b); 3.84 (br d, 1H, H-4 ); 3.95–3.99 (m, 1H, H-2);
4
J
7
1
0
0
¼11.5 Hz, J
0
0
0
¼7.0 Hz,
¼5.2 Hz,
3.62 (dd, 1H, J1a,1b¼10.5 Hz, J ¼3.0 Hz, H-1a); 3.70–
6
a,6 b
5 ,6a
1a,2
0
0
0
0
0
0
¼11.5 Hz, J
0
3.76 (m, 2H, H-2 ,6 a); 3.79 (dd, 1H, J
0
0
0
¼6.5 Hz,
6
a,6 b
0
5 ,6b
5 ,6 b
0
J6
0
¼11.5 Hz, H-6 b); 3.99 (m, 1H, H-2); 4.07 (dd, 1H,
a,6 b
.08 (dd, 1H, J ¼8.0 Hz, J ¼7.5 Hz, H-3); 4.17 (dd, 1H,
J ¼J ¼7.5 Hz, H-3); 4.12 (dd, 1H, J
¼10.5 Hz,
2
,3
3,4
2,3
3,4
1a,1b
0
¼10.5 Hz, J ¼4.5 Hz, H-1b); 4.20 (d, 1H, J
1b,2
0
0
¼
J
4.28 (dd, 1H, J
¼4.5 Hz, H-1b); 4.25 (br d, 1H, J
0 0
3 ,4
¼3.3 Hz, H-4 );
1
a,1b
1 ,2
1b,2
0
0
.5 Hz, H-1 ); 5.31 (m, 2H, CH CHvCHCH );5.43 (ddt,
2
0
0
¼9.5 Hz, J
0 0
3 ,4
¼3.3 Hz, H-3 ); 4.33 (d,
2
2 ,3
0
H, J ¼15.5 Hz, J ¼7.5 Hz, J ¼1.0 Hz, H-4); 5.67 (dt,
1H, J
0
0
¼7.7 Hz, H-1 ); 5.41 (ddt, 1H, J ¼15.5 Hz,
4,5
4
,5
3,4
4,6
1 ,2

8708
F. Compostella et al. / Tetrahedron 58 (2002) 8703–8708
J ¼7.5 Hz, J ¼1.0 Hz, H-4); 5.67 (dt, J ¼15.5 Hz,
C
Frontier Science Program (grant RG0168/2000-M), and
The Swiss Multiple Sclerosis Society.
3
,4
4,6
4,5
1
3
J ¼6.5 Hz, H-5); 7.69 (d, 1H, J
¼9.0 Hz, NH).
5
,6
2,NH
NMR (CDCl /CD OD, 1:1): 15.0 (2C); 23.9 (2C); 27.3;
3
3
3
7
0.6–31.0 (21C); 33.2 (2C); 33.7; 37.7; 54.8; 62.6; 68.8;
0.3; 70.9; 73.2; 76.0; 81.6; 104.7; 130.6; 135.6; 176.2. n
max
References
2
1
(
nujol): 3390, 1640, 1550, 1250, 1085 cm . ESI/MS
2
(
calcd C 60.77, H 9.71, N 1.69; found C 60.92, H 9.93, N
negative-ion mode): m/z 806.9 [M2Na] . C H NNaO S:
4
2
80
11
1. See e.g. (a) Garcia, K. C.; Degano, M.; Pease, L. R.; Huang,
M. D.; Peterson, P. A.; Teyton, L.; Wilson, I. A. Science 1998,
1
.75.
2
79, 1166–1172. (b) Ding, Y. H.; Smith, K. J.; Garboczi,
D. N.; Utz, U.; Biddison, W. E.; Wiley, D. C. Immunity 1998,
, 403–411.
4
.1.13. (2S,3R,4E)-1-[3-O-(Sodium oxysulfonyl)-b-D-
8
galactopyranosyloxy]-2-(docosanoylamino)-3-hydroxy-
-octadecene (1c). Sulfatide 1c (0.022 g, 75%) was
obtained as a foam from compound 11c (0.026 g,
.033 mmol) as described for 1a. After the addition of
Me N·SO the reaction was stirred at rt for 2 h. [a] ¼þ2.2
2
3
. Moody, D. B.; Besra, G. S.; Wilson, I. A.; Porcelli, S. A.
Immunol. Rev. 1999, 172, 285–296.
4
. Zeng, Z.; Castano, A. R.; Segelke, B. W.; Stura, E. A.;
Peterson, P. A.; Wilson, I. A. Science 1997, 277, 339–345.
0
3
3
D
4. Shamshiev, A.; Donda, A.; Prigozy, T. I.; Mori, L.; Chigorno,
V.; Benedict, C. A.; Kappos, L.; Sonnino, S.; Kronenberg, M.;
De Libero, G. Immunity 2000, 13, 255–264.
1
(
c 1, MeOH). H NMR (CDCl /CD OD, 1:1): 0.86 (t, 6H,
3 3
J¼7.0 Hz, 2 CH ); 1.20–1.40 (m, 58H, 29 CH ); 1.54–1.62
3
2
(
m, 2H, CH ); 1.96–2.02 (m, 2H, 2 H-6); 2.15 (t, 2H,
2
5. Burdin, N.; Brossay, L.; Degano, M.; Iijima, H.; Gui, M.;
Wilson, I. A.; Kronenberg, M. Proc. Natl Acad. Sci. USA
2000, 97, 10156–10161.
J¼7.5 Hz, COCH ); 3.55 (dd, 1H, J
0
0
¼5.0 Hz, J
0 0
5 ,6 b
¼
2
5 ,6 a
0
H-1a); 3.69–3.76 (m, 2H, H-2 ,6 a); 3.79 (dd, 1H,
6
.5 Hz, H-5 ); 3.59 (dd, 1H, J
¼10.5 Hz, J ¼3.0 Hz,
1
a,1b
1a,2
0
0
6
. Melian, A.; Watts, G. F.; Shamshiev, A.; De Libero, G.;
Clatworthy, A.; Vincent, M.; Brenner, M. B.; Behar, S.; Niazi,
K.; Modlin, R. L.; Almo, S.; Ostrov, D.; Nathenson, S. G.;
Porcelli, S. A. J. Immunol. 2000, 165, 4494–4504.
0
J6
0
a,6 b
0
¼11.5 Hz, J
5 ,6 b
0 0
¼6.5 Hz, H-6 b); 3.94–3.99 (m, 1H,
H-2); 4.08 (dd, 1H, J ¼J ¼7.5 Hz, H-3); 4.17 (dd, 1H,
2
,3
3,4
J
H-3 ,4 ); 4.32 (d, 1H, J
¼10.5 Hz, J ¼4.5 Hz, H-1b); 4.21–4.27 (m, 2H,
1
a,1b
1b,2
0
0
0
0
0
¼7.7 Hz, H-1 ); 5.41 (ddt, 1H,
1
,2
7. Niazi, K.; Chiu, M.; Mendoza, R.; Degano, M.; Khurana, S.;
Moody, D.; Melian, A.; Wilson, I.; Kronenberg, M.; Porcelli,
S.; Modlin, R. J. Immunol. 2001, 166, 2562–2570.
J ¼15.5 Hz, J ¼7.5 Hz, J ¼1.0 Hz, H-4); 5.67 (dt,
4
,5
3,4
4,6
1
3
J ¼15.5 Hz, J ¼6.5 Hz, H-5); 7.59 (br s, 1H, NH).
C
NMR (CDCl /CD OD, 1:1): 15.0 (2C); 23.9 (2C); 27.3;
4
,5
5,6
3
3
8. Rosat, J.-P.; Grant, E. P.; Beckman, E. M.; Dascher, C. C.;
Sieling, P. A.; Frederique, D.; Modlin, R. L.; Porcelli, S. A.;
Furlong, S. T.; Brenner, M. B. J. Immunol. 1999, 162,
3
7
0.6–31.0 (25C); 33.2 (2C); 33.7; 37.7; 54.7; 62.6; 68.7;
0.2; 70.9; 73.1; 76.1; 81.6; 104.7; 130.7; 135.6; 176.1. n
max
2
1
(
nujol): 3440, 1645, 1540, 1245, 1080 cm . ESI/MS
2
3
66–371.
(
calcd C 62.34, H 10.01, N 1.58; found C 62.00, H 9.90, N
negative-ion mode): m/z 862.9 [M2Na] . C H NNaO S:
4
6
88
11
9. Altamirano, M. M.; Woolfson, A.; Donda, A.; Shamshiev, A.;
Brise n˜ o-Roa, L.; Foster, N. W.; Veprintsev, D. B.; De Libero,
G.; Fersht, A. R.; Milstein, C. Proc. Natl Acad. Sci. USA 2001,
1
.31.
9
0. Shamshiev, A.; Gober, H.-J.; Donda, A.; Mazorra, Z.; Mori,
8, 3288–3293.
4
.1.14. (2S,3R,4E)-1-[3-O-(Sodium oxysulfonyl)-b-D-
1
1
1
galactopyranosyloxy]-2-(15(Z)-tetracosenoylamino)-3-
hydroxy-4-octadecene (1d). Sulfatide 1d (0.016 g, 70%)
was obtained as a foam from compound 11d (0.021 g,
0.026 mmol) as described for 1a. After the addition of
Me N·SO the reaction was stirred at rt for 20 h. Compound
L.; De Libero, G. J. Exp. Med. 2002, 195, 1013–1021.
1. Schmidt, R. R.; Zimmermann, P. Angew. Chem. Int. Ed. Engl.
1986, 25, 725–726.
2. Koskinen, P. M.; Koskinen, A. M. P. Synthesis 1998,
3
3
1075–1091.
13. Vankar, Y. D.; Schmidt, R. R. Chem. Soc. Rev. 2000, 29,
201–216.
1
d was purified by flash chromatography (CH Cl /MeOH,
2 2
2
0
9
Physical data of compound 1d were in agreement with those
reported in Ref. 20.
:1). [a] ¼þ2.7 (c 1, MeOH) (Lit.: þ2.6 (c 1, MeOH)).
D
1
1
4. Ohlsson, J.; Magnusson, G. Carbohydr. Res. 2001, 331,
1–94.
5. Gargano, J. M.; Lees, W. J. Tetrahedron Lett. 2001, 42,
845–5847.
9
4.1.15. Evaluation of the biological activity. The T-cell
clone A124 was isolated from peripheral blood. The assays
5
1
1
6. Li, Y.-L.; Wu, Y.-L. Liebigs Ann. 1996, 2079–2082.
7. Ko o´ s, M.; Mosher, H. S. Carbohydr. Res. 1986, 146,
were performed in triplicates using 10 mg/ml sulfatide,
4
4
5
antigen-presenting cells, and TNFa was measured by
£10 /well T-cells and 3£10 /well dendritic cells as
3
35–341.
8. Zimmermann, P.; Schmidt, R. R. Liebigs Ann. Chem. 1988,
63–667.
1
1
ELISA as described in Ref. 10.
6
9. Zimmermann, P.; Bommer, R.; B a¨ r, T.; Schmidt, R. R.
J. Carbohydr. Chem. 1988, 7, 435–452.
Acknowledgements
20. Guilbert, B.; Davis, N. J.; Pearce, M.; Aplin, R. T.; Flitsch,
S. L. Tetrahedron: Asymmetry 1994, 5, 2163–2178.
21. Hsu, F.-F.; Bohrer, A.; Turk, J. Biochem. Biophys. Acta 1998,
1392, 202–216.
This work was supported by the University of Milano, the
University of Piemonte Orientale, the MIUR, the Swiss
National Found (grant NF 3100-055698.98), Human
22. Barber, H. J. J. Chem. Soc. 1943, 79.