Synthesis and evaluation of N-acetyl-2-amino-2-deoxy-α-D-galactosyl 1-thio-7-oxaceramide, a new analogue of α-D-galactosyl ceramide

Article abstract of DOI:10.1002/hlca.200800454

The N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl 1-thio-7-oxaceramide 1 was synthesized by substituting the 7-oxasphingosine triflate 3 with α-D-N-acetyl-1-thiogalactosamine (2). The triflate 3 was obtained from azide 4. Thiol 2 was prepared according to

Full text of DOI:10.1002/hlca.200800454

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Helvetica Chimica Acta – Vol. 92 (2009)
Synthesis and Evaluation of N-Acetyl-2-amino-2-deoxy-a-d-galactosyl
1-Thio-7-oxaceramide, a New Analogue of a-d-Galactosyl Ceramide
by Roshini Rajana), Thresen Mathewa), Radovan Buffaa), Fre´de´ric Bornancinb), Marco Cavallaric),
Peter Nussbaumerb), Gennaro De Liberoc), and Andrea Vasella*a)
a) Laboratorium fꢀr Organische Chemie, Departement Chemie und Angewandte Biowissenschaften,
ETH Zꢀrich, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich (e-mail: vasella@org.chem.ethz.ch)
b) Novartis Institute for BioMedical Research, Brunner Strasse 59, A-1235 Vienna
c) Department of Biomedicine, University Hospital Basel, CH-4031 Basel
The N-acetyl-2-amino-2-deoxy-a-d-galactopyranosyl 1-thio-7-oxaceramide 1 was synthesized by
substituting the 7-oxasphingosine triflate 3 with a-d-N-acetyl-1-thiogalactosamine (2). The triflate 3 was
obtained from azide 4. Thiol 2 was prepared according to a known procedure from a-d-galactosamine
hydrochloride. As compared to ceramide (Cer), 1 is neither a substrate of ceramide kinase (CerK),
consistent with the absence of the C(1)ꢀOH group, nor an inhibitor of Cer phosphorylation by CerK.
While 1 partially displaced CD1d-bound lipids, it failed to stimulate invariant natural killer T (iNKT)
cells when presented by human CD1d-transfected cells. These results suggest that 1 binds weakly to
recombinant CD1d, but does not form immunogenic complexes with CD1d.
Introduction. – Several a-d-galactopyranosyl ceramides [1 – 5], especially KRN-
7000 [6][7], OCH [8], and RCAI-611) [9], incorporating phytosphingosine or
sphingosine moieties, possess important biological properties [10 – 13], comprising
immunostimulating [14 – 16] and antitumor activities [17 – 24]. Several analogues of
these a-d-galactopyranosides were synthesized, and they also display notable bio-
logical activities [8][14][25 – 37]. The synthesis and evaluation of a thioglycoside
analogue of KRN7000 has recently been published [38], prompting us to report our
results on the synthesis and evaluation of N-acetyl-2-amino-2-deoxy-a-d-galactopyr-
anosyl 1-thio-7-oxaceramide 1. Replacing the galactosyl by an N-acetyl-2-amino-2-
deoxygalactosyl moiety was thought to contribute to elucidating the effect of
C(2)ꢀOH of the glycon on T-cell receptor (TCR) recognition, while the S – glycosyl
bond will confer stability against chemical and enzymatic cleavage [39 – 42]. 7-
Oxasphingosines behave very similarly to ceramides, and allow to independently
modify the head group and lipid moiety of sphingosine, an advantage that has since
been realized using olefin metathesis [43][44].
Conceivably, 1 may be obtained either by glycosidation [45] of a 1-thioceramide or
of a 1-thiosphingosine, or by nucleophilic substitution by an N-acetyl a-d-galactopyr-
anosyl thiol of a ceramide possessing a leaving group at C(1) and preferentially a non-
participating neighbouring group, such as an N3 substituent [46][47]. We opted for the
1
)
OCH is a truncated and RCAI-61 the 6’-O-methyl analogue of KRN7000.
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

Helvetica Chimica Acta – Vol. 92 (2009)
919
second strategy, assuming that nucleophilic substitution will prevail over single electron
transfer from the thiolate to the N3 group, and report on the synthesis of 1 and the
results of its evaluation as substrate of Cer kinase [48] and as T cell-stimulatory antigen
[49].
Synthesis. – The synthesis of the N-acetyl-2-amino-2-deoxy-a-d-galactopyranosyl 1-
thio-7-oxaceramide 1 is shown in the Scheme. The thiogalactoside 8 was obtained in
75% yield by the reaction between 2-acetamido-2-deoxy-a-d-galactopyranosyl thiol
(2) [50] and the 7-oxasphingosine azido triflate (¼trifluoromethylsulfonate) 3. The
thiol 2 was synthesized in three steps and in an overall yield of 99% from 2-acetamido-
1,3,4,6-tetra-O-acetyl-2-deoxy-a-d-galactopyranose, according to the procedure of
Knapp and Myers [50]. The azido triflate 3 was prepared from azide 4, derived from 7-
t
oxasphingosine [51], by first silylating the hydroxymethyl group with BuPh2SiCl
(TBDPSCl) to provide 5 in 81% yield and then protecting the HOꢀC(3) group by
treating it with MeOCH2Cl (MOMCl) in the presence of Hꢀnigꢂs base, to yield 91% of
6. The alcohol 7 was obtained in a yield of 95% by desilylating 6, and converted to the
triflate 3. The triflate, obtained as a pale yellow oil, was directly subjected to
substitution by the thiol 2 in the presence of Hꢀnigꢂs base, to provide 75% of the
protected S-galactosaminide 8 (J(1,2) ¼ 5.4). Reducing this galactosaminide by the
action of polymer-bound Ph3P [52], followed by N-acylation with stearic anhydride,
yielded 86% of the protected ceramide 9 that was deprotected. The MOM group was
removed by treating 9 with Tf2O in MeCN to provide 10 in 87% yield [53][54].
Deacetylation of 10 was effected with 2m NH3 in MeOH to yield 96% of the desired N-
acetyl-a-d-galactopyranosyl 1-thio-7-oxaceramide 1. The a-d configuration is evi-
denced by J(1,2) ¼ 5.4 Hz.
Biological Studies. – 1. Phosphorylation by Ceramide Kinase (CerK). Phosphor-
ylation of 1 by ceramide kinase (CerK) was assayed using an established in vitro
radioassay [55]. As compared with C8-ceramide, 1 was not phosphorylated by CerK
(Fig. 1,a), in agreement with previous observations showing the requirement for a free
OH group on the first C-atom of ceramide to allow for phosphorylation by CerK [56].

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Scheme
a) tBuPh2SiCl (TBDPSCl), 1H-imidazole, 4-(dimethylamino)pyridine (DMAP), CH2Cl2; 81%. b)
MeOCH2Cl (MOMCl), Hꢀnigꢂs base, CH2Cl2; 91%. c) Bu4NF · 3 H2O, AcOH, THF; 95%. d) Tf2O, py,
CH2Cl2. e) Hꢀnigꢂs base, CH2Cl2; 75%. f) 1. Polymer-bound PPh3, 1,4-dioxane; 2. stearic anhydride;
86%. g) Tf2O, MeCN; 87%. h) 2m NH3 in MeOH; 96%.
In addition, 1 did not inhibit the phosphorylation of ceramide by CerK, suggesting that
1 does not compete with ceramide for binding to CerK (Fig. 1,b), in contrast to the
recently identified CerK inhibitor NVP-231 [57].
2. Human CD1d Binding and T-Cell Activation. The galactosaminide 1 is neither
cytotoxic to antigen-presenting cells (APC) nor to iNKT cells at the tested dosage (up
to 20 mg/ml), as assessed by flow cytometry (data not shown).
We first investigated whether 1 binds recombinant human CD1d and displaces the
strong agonist a-d-galactosyl ceramide (aGalCer) that activates iNKT cells and
induces release of large amounts of cytokines from this cell population. CD1d was
attached to the plastic and incubated for 4.5 h with a tenfold molar excess of 1 and then
with aGalCer (2 mg/ml). Compound 1 partially reduced the activation of iNKT cells
(Fig. 2,a), indicating a weak capacity to prevent binding of aGalCer to CD1d under
these experimental conditions. Next, we investigated whether 1 activates iNKT cells
when presented by living APC. Different APC were used to exclude cell type-specific
effects. In all experiments, there was no activation of iNKT cells (Fig. 2,b). Lack of
iNKT cell stimulatory activity may be ascribed to the replacement of the OH group at

Helvetica Chimica Acta – Vol. 92 (2009)
921
Fig. 1. Phosphorylation of ceramide and the ceramide analogue 1, assayed as described in the Method
section. a) Direct assay showing that 1 is not a substrate for CerK; b) indirect assay showing that 1 does
not prevent phosphorylation of ceramide by CerK.
C(2) of the glycon by the acetamido group. Replacements of OH at C(2) of the glycon
with an NH2 group also abolishes iNKT cell activation [7]. The OH group at C(2)
establishes H-bonding with two amino acids (R95 and G96) on the alpha chain of iNKT
TCR [58] that is mandatory for T-cell activation, as shown by the CD1d: aGalCer
binding footprint for the NKT TCR [59]. The observed weak displacement capacity of
1 may be ascribed to the different constitution and rigidity of the oxasphingosine
moiety of 1 as compared to the phytosphingosine moiety of aGalCer. The different
structure of oxasphingosine might impair the interaction between lipid 1 and the CD1d
Fꢂ pocket, and influence the position of the sugar head, preventing an optimal
interaction with the TCR.
We thank Novartis AG, Basel, the ETH-Zꢀrich, and the Swiss National Science Foundation (grant
3100A0-109918) for generous support, and Dr. Bruno Bernet for checking the analytical data.
Experimental Part
1. Synthesis. General. THF was distilled from Na and benzophenone, CH2Cl2 from P2O5, and MeOH
and MeCN from CaH2. Reactions were carried out under Ar, unless stated otherwise. Qual. TLC:
precoated silica-gel plates (Merck silica gel 60 F254); detection by heating with ꢃmostainꢂ (400 ml of 10%
H2SO4 soln., 20 g of (NH4)6Mo7O24 · 6 H2O, 0.4 g of Ce(SO4)2). Flash chromatography (FC): silica gel
Fluka 60 (0.04 – 0.063 mm). Optical rotations: 1-dm cell at 258, 589 nm. FT-IR spectra: KBr or ca. 2%
soln. in CHCl3, absorption in cmꢀ1. 1H- and 13C-NMR Spectra: chemical shifts d in ppm rel. to Me4Si as
external standard, and coupling constants J in Hz. HR-MALDI-MS: in gentisic acid (¼2,5-
dihydroxybenzoic acid, DHB) matrix.
(4E)-2-Azido-1-O-[(tert-butyl)diphenylsilyl]-2,4,5-trideoxy-6-O-undecyl-d-erythro-hex-4-enitol (5).
A suspension of 4 [16] (570 mg, 1.74 mmol) and 1H-imidazole (154 mg, 2.26 mmol) in dry CH2Cl2

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Helvetica Chimica Acta – Vol. 92 (2009)
Fig. 2. Binding of 1 to CD1d and failure to activate iNKT cells. N-Acetyl-a-d-galactopyranosyl 1-thio-7-
oxaceramide (1) was tested for its capacity to prevent binding of aGalCer to plate-bound human CD1d
and subsequent T-cell response to aGalCer (a). Supernatants were taken after 24 h, and released human
GM-CSF and human IL-4 (data not shown) were measured by ELISA and expressed as pg/ml ꢃ SD of
triplicates. Weak but significant competition of 1 with aGalCer was seen as compared to complete
inhibition of aGalCer by ganglioside monosialic acid (GM1) used as control competitor. In contrast, 1
^
(
^
) failed to activate iNKT cells when titrated on CD1d transfectants and compared to aGalCer ( ) (b).
Supernatants were assessed for release of human IL-4, human IFN-g, and human TNF-a giving similar
results.
t
(10 ml) was treated with BuPh2SiCl (TBDPSCl; 0.58 ml, 2.26 mmol) and 4-(dimethylamino)pyridine
(DMAP; 21 mg, 0.174 mmol), stirred for 4 h, diluted with H2O, and extracted with CH2Cl2 (3 ꢁ 100 ml).
The combined org. layers were dried (Na2SO4) and evaporated. FC (AcOEt/hexane 1:9) gave 5 (797 mg,
81%). Colourless oil. Rf (AcOEt/hexane 1:9) 0.27. [a]2D5 ¼ þ13.1 (c ¼ 2.0, CHCl3). IR (CHCl3): 3451w
(br.), 3073w, 3012w, 2930s, 2857s, 2105s, 1601w, 1590w, 1471w, 1428w, 1363w, 1264w, 1220m, 1112s, 1007w,
973w, 862w. 1H-NMR (CDCl3, 300 MHz): 7.70 – 7.67 (m, 4 arom. H); 7.48 – 7.37 (m, 6 arom. H); 5.86 (dtd,
J ¼ 15.6, 5.1, 0.9, HꢀC(5)); 5.71 (ddt, J ¼ 15.6, 6.3, 1.2, HꢀC(4)); 4.30 (dtd, J ¼ 6.3, 5.1, 0.9, HꢀC(3));
3.94 (br. d, J ¼ 5.4, 2 HꢀC(6)); 3.85 – 3.78 (dd, J ¼ 10.8, 6.0, HꢀC(1)); 3.80 (dd, J ¼ 10.8, 4.5, H’ꢀC(1));
3.51 (dt, J ꢂ 5.7, 4.5, HꢀC(2)); 3.39 (t, J ¼ 6.3, 2 HꢀC(1’)); 2.54 (br. s, J ¼ 5.4, OH); 2.31 (br. s, J ¼ 5.4,
OH); 1.62 – 1.53 (m, 2 HꢀC(2’)); 1.35 – 1.26 (m, 16 H); 1.08 (s, Me3C); 0.88 (t, J ¼ 6.6, Me). 13C-NMR
(CDCl3, 75 MHz): 135.46 (4d); 132.53 (2s); 130.54 (d, C(4)); 130.08 (d, C(5)); 129.82 (2d); 127.73 (4d);
72.10 (d, C(3)); 70.70 (t, C(6)); 70.40 (t, C(1’)); 66.59 (d, C(2)); 64.06 (t, C(1)); 31.99, 29.82, 29.70, 29.60,
29.42, 26.26, 22.78 (several t); 26.73 (q, Me3C); 19.23 (s, Me3C); 14.32 (q, Me). HR-MALDI-MS:
588.3583 (100, [M þ Na]þ, C33H51N3NaO3Siþ; calc. 588.3592). Anal. calc. for C33H51N3O3Si (565.87): C
70.04, H 9.08, N 7.43; found: C 70.27, H 8.95, N 7.51.
(4E)-2-Azido-1-O-[(tert-butyl)diphenylsilyl]-2,4,5-trideoxy-3-O-(methoxymethyl)-6-O-undecyl-d-
erythro-hex-4-enitol (6). A soln. of 5 (790 mg, 1.39 mmol) and Hꢀnigꢂs base (0.69 ml, 4.19 mmol) in
ClCH2CH2Cl was treated with MOMCl (0.16 ml, 2.09 mmol), heated to 758, stirred for 6 h, after which
another 0.16 ml of MOMCl was added, stirred further for 16 h at 758, diluted with H2O, and extracted
with CH2Cl2 (2 ꢁ 50 ml). The combined org. layers were dried (Na2SO4) and evaporated. FC (AcOEt/
hexane 1:19) gave 6 (773 mg, 91%). Colourless oil. Rf (AcOEt/hexane 1:9) 0.30. [a]2D5 ¼ ꢀ36.5 (c ¼ 1.7,
CHCl3). IR (CHCl3): 3018m, 2930s, 2857s, 2103m, 1599w, 1471w, 1428w, 1362w, 1217m, 1152m, 1112s,
1
1005s, 1024m. H-NMR (CDCl3, 300 MHz): 7.70 – 7.65 (m, 4 arom. H); 7.48 – 7.36 (m, 6 arom. H); 5.78
(dtd, J ¼ 15.3, 5.3, 0.6, HꢀC(5)); 5.56 (ddt, J ¼ 15.6, 8.1, 1.5, HꢀC(4)); 4.65 (d, J ¼ 6.9, OꢀCHaꢀO); 4.50

Helvetica Chimica Acta – Vol. 92 (2009)
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(d, J ¼ 6.9, OꢀCHbꢀO); 4.18 (dd, J ¼ 7.8, 4.5, HꢀC(3)); 3.95 (dd, J ¼ 5.1, 1.2, 2 HꢀC(6)); 3.77 (dd, J ¼
10.5, 5.4, HꢀC(1)); 3.72 (dd, J ¼ 10.5, 6.6, H’ꢀC(1)); 3.63 (dt J ¼ 6.6, 5.1, HꢀC(2)); 3.37 (t, J ¼ 6.6,
2 HꢀC(1’)); 3.28 (s, MeO); 1.60 – 1.50 (m, 2 HꢀC(2’)); 1.34 – 1.25 (m, 16 H); 1.08 (s, Me3C); 0.88 (t, J ¼
6.3, Me). 13C-NMR (CDCl3, 75 MHz): 135.50 (4d); 133.27 (d, C(4)); 132.91, 132.86 (2s); 129.75 (2d);
127.69 (4d); 127.20 (d, C(5)); 93.71 (t, OCH2O); 75.45 (d, C(3)); 70.56 (t, C(6)); 70.35 (t, C(1’)); 66.59 (d,
C(2)); 63.73 (t, C(1)); 55.66 (q, MeO); 32.03, 29.85, 29.75, 29.64, 29.46, 26.31, 22.83 (several t); 26.86 (q,
Me3C); 19.27 (s, Me3C); 14.28 (q, Me)). HR-ESI-MS: 632.3848 (100, [M þ Na]þ, C35H55N3NaO4Siþ; calc.
632.3854).
(4E)-2-Azido-2,4,5-trideoxy-3-O-(methoxymethyl)-6-O-undecyl-d-erythro-hex-4-enitol (7). A soln.
of 6 (773 mg, 1.27 mmol) in THF (20 ml) was treated with Bu4NF · 3 H2O (801 mg, 2.54 mmol) and
AcOH (0.14 ml, 2.54 mmol) at 08, warmed to r.t., stirred for 4 h, diluted with H2O, and extracted with
CH2Cl2 (3 ꢁ 100 ml). The combined org. layers were dried (Na2SO4), and evaporated. FC (AcOEt/
hexane 3 :7) gave 7 (449 mg, 95%). Colourless oil. Rf (AcOEt/hexane 3 :7) 0.19. [a]2D5 ¼ ꢀ104.7 (c ¼ 2.0,
CHCl3). IR (CHCl3): 3448w (br.), 3018m, 2929s, 2856s, 2130m, 2106s, 1599w, 1466w, 1372w, 1354w,
1298w, 1274w, 1219m, 1152m, 1099m, 1068w, 1033m, 974w, 915w. 1H-NMR (CDCl3, 300 MHz): 5.87 (dtd,
J ¼ 15.6, 5.1, 0.6, HꢀC(5)); 5.64 (ddt, J ¼ 15.6, 8.1, 1.2, HꢀC(4)); 4.69 (d, J ¼ 6.9, 1 H, OCH2O); 4.55 (d,
J ¼ 6.6, 1 H, OCH2O); 4.21 (dd, J ¼ 7.8, 5.4, HꢀC(3)); 3.99 (dd, J ¼ 5.1, 1.5, 2 HꢀC(6)); 3.76 (dd, J ¼ 11.2,
4.2, HꢀC(1)); 3.71 (dd, J ¼ 11.2, 6.0, H’ꢀC(1)); 3.55 (td, J ¼ 5.7, 4.2, HꢀC(2)); 3.41 (t, J ¼ 6.6,
2 HꢀC(1’)); 3.38 (s, MeO); 2.40 (br. s, HOꢀC(1)); 1.62 – 1.52 (m, 2 HꢀC(2’)); 1.34 – 1.23 (m, 16 H); 0.87
(t, J ¼ 6.9, Me). 13C-NMR (CDCl3, 75 MHz): 133.48 (d, C(4)); 127.17 (d, C(5)); 93.80 (t, OCH2O); 76.29
(d, C(3)); 70.75 (t, C(6)); 70.22 (t, C(1’)); 66.16 (d, C(2)); 62.12 (t, C(1)); 55.79 (q, OMe); 31.96, 29.75,
29.67, 29.56, 29.40, 26.33, 22.76 (several t); 14.21 (q, Me). HR-MALDI-MS: 394.2669 (100, [M þ Na]þ,
C19H37N3NaOþ4 ; calc. 394.2676). Anal. calc. for C19H37N3O4 (371.52): C 61.43, H 10.04, N 11.31; found: C
61.60, H 10.05, N 11.35.
(4E)-2-Azido-2,4,5-trideoxy-3-O-(methoxymethyl)-1-O-[(trifluoromethyl)sulfonyl]-6-O-undecyl-d-
erythro-hex-4-enitol (3). Tf2O (60 ml, 0.37 mmol) was added dropwise to a soln. of 7 (113 mg, 0.31 mmol)
and pyridine (60 ml, 0.92 mmol) in dry CH2Cl2 (8 ml), cooled to ꢀ 788. The mixture was warmed to ꢀ 208
within 3.5 h, diluted with NH4Cl soln., and extracted with CH2Cl2 (3 ꢁ 50 ml). The combined org. layers
were dried (Na2SO4) and evaporated to give pale yellow oily 3, which was used for the next step without
further purification. Rf (AcOEt/hexane 2 :8) 0.46. 1H-NMR (CDCl3, 300 MHz): 5.94 (dtd, J ¼ 15.6, 5.1,
0.9, HꢀC(5)); 5.62 (ddt, J ¼ 15.6, 8.1, 1.5, HꢀC(4)); 4.71 (d, J ¼ 6.6, 1 H, OCH2O); 4.63 (dd, J ¼ 10.6, 3.5,
HꢀC(1)); 4.53 (d, J ¼ 6.9, 1 H, OCH2O); 4.48 (dd, J ¼ 10.5, 7.8, H’ꢀC(1)); 4.22 (dd, J ¼ 7.8, 5.7, HꢀC(3));
4.02 (dd, J ¼ 5.1, 1.8, 2 HꢀC(6)); 3.80 (ddd, J ¼ 7.8, 6.0, 3.6, HꢀC(2)); 3.43 (t, J ¼ 6.6, 2 HꢀC(1’)); 3.38 (s,
MeO); 1.61 – 1.54 (m, 2 HꢀC(2’)); 1.38 – 1.20 (m, 16 H); 0.88 (t, J ¼ 6.3, Me). 13C-NMR (CDCl3,
75 MHz): 135.23 (d, C(4)); 125.43 (d, C(5)); 93.67 (t, OCH2O); 75.24 (d, C(3)); 74.44 (t, C(1)); 70.95 (t,
C(6)); 69.92 (t, C(1’)); 63.17 (d, C(2)); 56.00 (q, MeO); 31.96, 29.75, 29.67, 29.56, 29.40, 26.33, 22.76
(several t); 14.21 (q, Me). 19F-NMR (CDCl3): ꢀ 74.22 (s, CF3).
(4E)-1-S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-d-galactopyranosyl)-2-azido-2,4,5-trideoxy-3-
O-(methoxymethyl)-1-thio-6-O-undecyl-d-erythro-hex-4-enitol (8). A soln. of 2 (95 mg, 0.25 mmol) and
crude 3 in dry CH2Cl2 (10 ml) was treated with Hꢀnigꢂs base (63 ml, 0.38 mmol), stirred at r.t. for 2.5 h,
diluted with NH4Cl, and extracted with CH2Cl2. The combined org. layers were dried (Na2SO4) and
evaporated. FC (AcOEt/hexane 1:1) gave 8 (136 mg, 75%). Colourless oil. Rf (AcOEt/hexane 7:3) 0.20.
[a]2D5 ¼ þ38.5 (c ¼ 1.75, CHCl3). IR (CHCl3): 3018w, 2955w, 2929m, 2855m, 2120m, 1747s, 1680m, 1601w,
1511m, 1466w, 1371m, 1262s, 1250s, 1169w, 1151w, 1085s, 1053m, 1029m, 978w, 914w, 862w, 821w.
1H-NMR (CDCl3, 300 MHz): 5.85 (dtd, J ¼ 15.6, 5.1, 0.9, HꢀC(5)); 5.73 (d, J ¼ 8.7, NH); 5.59 (d, J ¼ 5.4,
HꢀC(1’’’)); 5.58 (ddt, J ¼ 15.6, 8.1, 1.5, HꢀC(4)); 5.38 (dd, J ¼ 3.3, 1.2, HꢀC(4’’’)); 5.03 (dd, J ¼ 12.0, 3.3,
HꢀC(3’’’)); 4.76 (ddd, J ¼ 12.0, 8.7, 5.4, HꢀC(2’’’)); 4.68 (d, J ¼ 6.9, 1 H, OCH2O); 4.56 (td, J ¼ 6.6, 0.9,
HꢀC(5’’’)); 4.53 (d, J ¼ 6.3, 1 H, OCH2O); 4.13 (dd, J ¼ 11.4, 6.0, HꢀC(6’’’)); 4.15 (dd, J ¼ 8.1, 4.5,
HꢀC(3)); 4.04 (dd, J ¼ 11.4, 6.9, H’ꢀC(6’’’)); 3.98 (dd, J ¼ 5.4, 1.2, 2 HꢀC(6)); 3.68 (dt, J ¼ 9.6, 4.2,
HꢀC(2)); 3.39 (t, J ¼ 6.6, 2 HꢀC(1’)); 3.36 (s, MeO); 2.79 (dd, J ¼ 13.8, 4.2, HꢀC(1)); 2.67 (dd, J ¼ 13.8,
9.6, H’ꢀC(1)); 2.14 (s, AcNH); 2.04, 1.99, 1.97 (3s, 3 AcO); 1.60 – 1.51 (m, 2 HꢀC(2’)); 1.35 – 1.24 (m,
16 H); 0.86 (t, J ¼ 6.3, Me). 13C-NMR (CDCl3, 75 MHz): 170.82 (s, NHAc); 170.21, 170.04, (2s, 3 AcO);
134.38 (d, C(4)); 126.19 (d, C(5)); 93.66 (t, OCH2O); 86.46 (d, C(1’’’)); 77.83 (d, C(3)); 70.85, 70.20 (2t,

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Helvetica Chimica Acta – Vol. 92 (2009)
C(6), C(1’)); 68.29, 67.59, 67.28 (3d, C(3’’’), C(4’’’), C(5’’’)); 65.81 (d, C(2)); 61.93 (t, C(6’’’)); 55.86 (q,
OMe); 48.43 (d, C(2’’’)); 32.82, 31.99, 29.78, 29.70, 29.59, 29.43, 26.26, 22.78 (several t); 23.39 (q, AcNH);
20.84, 20.80, 20.77 (3q, 3 AcO); 14.24 (q, Me). HR-ESI-MS: 739.3548 (100, [Mþ Na]þ, C33H56N4NaO11Sþ;
calc. 739.3559). Anal. calc. for C33H56N4O11S (716.89): C 55.29, H 7.87, N 7.82; S 4.47; found: C 55.14, H
8.07, N 7.70; S 4.44.
(4E)-1-S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-d-galactopyranosyl)-2,4,5-trideoxy-3-O-(meth-
oxymethyl)-2-(octadecanoylamino)-1-thio-6-O-undecyl-d-erythro-hex-4-enitol (9). A suspension of 8
(100 mg, 0.14 mmol) and polymer-bound PPh3 (352 mg, 0.56 mmol) in dioxane (5 ml) was heated at 808
for 1 h. The temp. was then reduced to 608, stearic anhydride (154 mg, 0.28 mmol) was added, the mixture
was stirred for 48 h, filtered, and the filtrate was evaporated. FC (AcOEt/hexane 6 :4) gave 9 (115 mg,
86%). White crystals. Rf (AcOEt/hexane 7:3) 0.31. M.p. 66 – 68.58. [a]2D5 ¼ þ30.8 (c ¼ 1.0, CHCl3). IR
(CHCl3): 3426w, 3019m, 2927s, 2855s, 1747s, 1676m, 1602w, 1511m, 1466w, 1371m, 1239m, 1151w, 1084m,
1050m, 1031m, 977w, 914w. 1H-NMR (CDCl3, 300 MHz): 6.01 (d, J ¼ 9.0, AcNH); 5.83 (dtd, J ¼ 15.6, 5.1,
0.9, HꢀC(5)); 5.60 (d, J ¼ 8.7, HNꢀC(2)); 5.58 (ddt, J ¼ 15.6, 6.9, 1.8, HꢀC(4)); 5.45 (d, J ¼ 5.1,
HꢀC(1’’’)); 5.38 (dd, J ¼ 3.0, 1.2, HꢀC(4’’’)); 5.01 (dd, J ¼ 12.0, 3.3, HꢀC(3’’’)); 4.77 (ddd, J ¼ 12.0, 9.0,
5.4, HꢀC(2’’’)); 4.65 (d, J ¼ 6.6, 1 H, OCH2O); 4.56 (td, J ꢂ 6.6, 0.9, HꢀC(5’’’)); 4.54 (d, J ¼ 6.9, 1 H,
OCH2O); 4.29 – 4.21 (m, HꢀC(2)); 4.17 (dd, J ¼ 11.4, 6.0, HꢀC(6’’’)); 4.14 (t, J ꢂ 6.2, HꢀC(3)); 4.07 (dd,
J ¼ 11.4, 6.6, H’ꢀC(6’’’)); 3.95 (br. d, J ¼ 5.1, 2 HꢀC(6)); 3.38 (t, J ¼ 6.9, 2 HꢀC(1’)); 3.37 (s, MeO); 3.00
(dd, J ¼ 14.1, 7.5, HꢀC(1)); 2.83 (dd, J ¼ 13.8, 3.9, H’ꢀC(1)); 2.20 – 2.08 (AB, 2 HꢀC(2’’)); 2.16 (s,
AcNH); 2.04, 2.00, 1.97 (3s, 3 AcO); 1.62 – 1.51 (m, 2 HꢀC(2’), 2 HꢀC(3’’)); 1.38 – 1.19 (m, 44 H); 0.87
(t, J ¼ 6.6, 2 Me). 13C-NMR (CDCl3, 75 MHz): 172.66 (s, C(1’’)); 170.82, 170.19, 170.05 (3s, 3 AcO);
169.92 (s, AcNH); 132.65 (d, C(4)); 128.01 (d, C(5)); 94.56 (t, OCH2O); 86.79 (d, C(1’’’)); 78.01 (d, C(3));
70.83, 70.35 (2t, C(6) C(1’)); 68.35, 67.69, 67.25 (3d, C(3’’’), C(4’’’), C(5’’’)); 61.79 (t, C(6’’’)); 56.02 (q,
MeO); 52.21 (d, C(2)); 48.41 (d, C(2’’’)); 36.89, 33.08, 32.02, 29.82, 29.75, 29.67, 29.51, 29.47, 26.29, 25.75,
22.81 (several t); 23.47 (q, AcNH); 20.86 (q, 3 AcO); 14.24 (q, 2 Me). HR-MALDI-MS: 979.6244 (84,
[M þ Na]þ, C51H92N2NaO12Sþ; calc. 979.6263). Anal. calc. for C51H92N2O12S (957.36): C 63.98, H 9.69, N
2.93; found: C 64.19, H 9.68, N 2.96.
(4E)-1-S-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-d-galactopyranosyl)-2,4,5-trideoxy-2-(octadeca-
noylamino)-1-thio-6-O-undecyl-d-erythro-hex-4-enitol (10). An ice-cold soln. of 9 (90 mg, 0.09 mmol) in
dry MeCN was treated with Tf2O (32 ml, 0.19 mmol), slowly warmed to r.t. in 2 h, diluted with sat. NH4Cl
soln., and extracted with CH2Cl2 (3 ꢁ 50 ml). The combined org. layers were dried (Na2SO4) and
evaporated. FC (AcOEt/hexane 9 :1) gave 10 (75 mg, 87%). White crystals. Rf (AcOEt/hexane 9 :1) 0.23.
M.p. 95 – 988. [a]D25 ¼ þ55.9 (c ¼ 2.25, CHCl3). IR (CHCl3): 3573w, 3485w, 3419w, 3021w, 2928m, 2855w,
1747m, 1675w, 1604w, 1512w, 1466w, 1372w, 1266s, 1170m, 1085w, 1044m, 1037m, 978 w, 929w. 1H-NMR
(CDCl3, 300 MHz): 6.14 (d, J ¼ 8.7, AcNH); 5.87 (br. dt, J ¼ 15.6, 5.3, HꢀC(5)); 5.75 (d, J ¼ 8.7,
HNꢀC(2)); 5.71 (br. dd, J ¼ 15.6, 5.7, HꢀC(4)); 5.50 (d, J ¼ 5.4, HꢀC(1’’’)); 5.37 (dd, J ¼ 3.0, 0.9,
HꢀC(4’’’)); 5.00 (dd, J ¼ 11.7, 3.3, HꢀC(3’’’)); 4.74 (ddd, J ¼ 11.8, 8.6, 5.1, HꢀC(2’’’)); 4.53 (br. t, J ¼ 6.6,
HꢀC(5’’’)); 4.29 (br. t, J ꢂ 4.9, HꢀC(3)); 4.15 (dd, J ¼ 11.6, 6.2, HꢀC(6’’’)); 4.15 – 4.05 (m, HꢀC(2));
4.08 (dd, J ¼ 11.3, 6.8, H’ꢀC(6’’’)); 3.95 (br. d, J ¼ 5.4, 2 HꢀC(6)); 3.39 (t, J ¼ 6.9, 2 HꢀC(1’)); 3.00 (dd,
J ¼ 14.0, 8.0, HꢀC(1)); 2.80 (dd, J ¼ 14.0, 4.1, H’ꢀC(1)); 2.22 – 2.12 (AB, 2 HꢀC(2’’)); 2.15 (s, AcNH);
2.05, 1.99, 1.97 (3s, AcO); 1.64 – 1.50 (m, 2 HꢀC(2’), 2 HꢀC(3’’)); 1.36 – 1.18 (m, 44 H); 0.87 (t, J ¼ 6.8,
2 Me). 13C-NMR (CDCl3, 75 MHz): 173.86 (s, C(1’’)); 170.80 (s, AcNH); 170.33, 170.07, 169.99 (3s, 3
AcO); 132.54 (d, C(4)); 130.05 (d, C(5)); 86.75 (d, C(1’’’)); 73.51 (d, C(3)); 70.87, 70.45 (2t, C(6) C(1’));
68.28, 67.63, 67.15 (3d, C(3’’’), C(5’’’), C(4’’’)); 61.89 (t, C(6’’’)); 54.30 (d, C(2)); 48.47 (d, C(2’’’)); 36.73 (t);
32.65 (t); 31.98 (2t); 29.78 – 29.42 (several t); 26.25 (t); 25.78 (t); 23.39 (q, AcNH); 22.77 (2t); 20.90, 20.82,
20.77 (3q, 3 AcO); 14.21 (q, 2 Me). HR-MALDI-MS: 935.5984 (100, [M þ Na]þ, C49H88N2NaO11Sþ; calc.
935.6001). Anal. calc. for C49H88N2O11S (913.29): C 64.44, H 9.71, N 3.07, S 3.51; found: C 64.18, H 9.74, N
3.11, S 3.36.
(4E)-1-S-(2-Acetamido-2-deoxy-a-d-galactopyranosyl)-2,4,5-trideoxy-2-(octadecanoylamino)-1-
thio-6-O-undecyl-d-erythro-hex-4-enitol (1). A soln. of 10 (70 mg, 0.076 mmol) in 6 ml of 2m NH3 in
MeOH was heated to 408 for 4.5 h, and then evaporated. FC (MeOH/CH2Cl2 3 :17) gave 1 (58 mg, 96%).
Rf (CH2Cl2/MeOH 9 :1) 0.26. M.p. 207 – 2098. [a]2D5 ¼ þ56.3 (c ¼ 1.08, pyridine). IR (CHCl3): 3407m
(br.), 3289s, 3092w, 2953m, 2919s, 2850s, 1647s, 1551s, 1468m, 1435m, 1376m, 1298w, 1213w, 1168w,

Helvetica Chimica Acta – Vol. 92 (2009)
925
1116m, 1084m, 1049m, 1020w, 977w, 960w. 1H-NMR (CD3OD, 300 MHz): 7.98 (d, J ¼ 7.5, 0.4 H, AcNH);
7.88 (d, J ¼ 9.0, 0.6 H, HNꢀC(2)); 5.78 (br. dt, J ¼ 15.3, 5.2, HꢀC(5)); 5.71 (br. dd, J ¼ 15.8, 5.9,
HꢀC(4)); 5.52 (d, J ¼ 5.4, HꢀC(1’’’)); 4.40 (dd, J ¼ 11.4, 5.4, HꢀC(2’’’)); 4.21 (ddd, J ¼ 6.0, 4.8, 0.9,
HꢀC(3)); 4.05 (br. t, J ¼ 6.5, HꢀC(5’’’)); 4.03 – 3.97 (m, HꢀC(2)); 3.94 (d, J ¼ 4.5, 2 HꢀC(6)); 3.86 (d,
J ¼ 2.4, 2 HꢀC(4’’’)); 3.79 (dd, J ¼ 11.7, 7.2, HꢀC(6’’’)); 3.73 (dd, J ¼ 11.7, 5.1, H’ꢀC(6’’’)); 3.65 (dd, J ¼
11.6, 3.2, HꢀC(3’’’)); 3.41 (t, J ¼ 6.6, 2 HꢀC(1’)); 2.91 (dd, J ¼ 13.8, 3.3, HꢀC(1)); 2.82 (dd, J ¼ 13.7, 8.6,
H’ꢀC(1)); 2.18 (t, J ¼ 7.4, 2 HꢀC(2’’)); 1.96 (s, AcNH); 1.64 – 1.50 (m, 2 HꢀC(2’), 2 HꢀC(3’’)); 1.36 –
1.26 (m, 44 H); 0.89 (t, J ¼ 6.8, 2 Me). 13C-NMR ((D5)pyridine, 75 MHz): 173.46 (s, C(1’’)); 170.44 (s,
AcNH); 134.08 (d, C(4)); 128.69 (d, C(5)); 87.77 (d, C(1’’’)); 73.61 (d, C(5’’’)); 73.35 (d, C(3)); 71.08,
70.29 (2t, C(6), C(1’)); 70.17, 69.41 (2d, C(3’’’), C(4’’’)); 62.75 (t, C(6’’’)); 55.26 (d, C(2)); 52.24 (d, C(2’’’));
36.76 (t); 33.46 (t); 32.07 (2t); 30.21 – 29.56 (several t); 26.57 (t); 25.34 (t); 23.14 (q, AcNH); 22.89 (2t);
14.26 (2q, Me). HR-MALDI-MS: 809.5669 (100, [M þ Na]þ, C43H82N2NaO8Sþ; calc. 809.5684). Anal.
calc. for C43H82N2O8S · H2O (805.2001): C 64.14, H 10.52, N 3.48; found: C 64.10, H 10.69, N 3.45.
2. Biological Tests. Phosphorylation of Ceramide Analogue by CerK. Activity of CerK on C8-
ceramide (Cayman) and the ceramide analogue 1 was measured according to published procedures [55]
using 180 mm of either C8 ceramide or analogue 1 in the direct assay (Fig. 1,a), and 20 mm C8-ceramide
and 100 mm 1 in the competitive assay (Fig. 1,b).
T-Cell Clones and APC. Human CD1d-restricted iNKT cell clones were derived and cultivated as
described in [60]. Human CD1d-transfected C1R or THP1 cells were used as APC in the experiments.
Antigen-Presentation Assays. Human CD1d transfectants (2.5 ꢁ 104/well) in RPMI-1640 medium
containing 10% FCS were incubated at 378 with sonicated compounds at the concentrations indicated in
the Figures in the presence of T cells (7.5 ꢁ 104/well). Supernatants were harvested after 24 h, and
released cytokines were measured by ELISA.
Plate-Bound Human CD1d Competition Assay. Plates were coated overnight at r.t. with 10 mg/ml
BIR1.4 monoclonal antibody (mAb). Soluble recombinant human CD1d purified by isoelectric focusing
was incubated overnight at r.t. on BIR1.4-coated plates at twofold molar concentration of BIR1.4 mAb.
Sonicated compounds were added 4.5 h before aGalCer to plate-bound human CD1d and left overnight
at r.t. to allow competition. Then, T cells (1.5 ꢁ 105/well) were plated in RPMI-1640 medium containing
5% human serum and 100 U/ml human IL-2 after extensive washing. Supernatants were harvested after
24 h of incubation at 378, and released cytokines were measured by ELISA.
ELISA. Plates coated with 8D4-8 (anti-human IL-4 mAb, BD), MAb1 (anti-human TNF-a mAb,
BD), 6804 (anti-human GM-CSF mAb, R&D), or HB-8700 (anti-human IFN-g mAb, ATCC) were
blocked and then incubated with the supernatants of the antigen presentation assays.
Detection of human IL-4, human TNF-a, or human IFN-g was performed using MP4-25D2 (anti-
human IL-4 biotin-labelled mAb, BD), MAb11 (anti-human TNF-a biotin-labelled mAb, BD), 3209
(anti-human GM-CSF biotin-labelled mAb, R&D), or g69 [61] (anti-human IFN-g biotin-labelled mAb,
provided by G. Garotta) with streptavidin-HRP (Zymed, Invitrogen), and o-phenylenediamine
dihydrochloride (Sigma, according to the manufacturerꢂs instructions) as substrate. OD490 nm was read
on a spectrophotometer (Molecular Devices, Sunnyvale, CA) and converted to concentrations using the
SoftMax Pro 5 program by comparison to standards of human IL-4 (human IL-4 secreting X63, kind gift
of U. Grawunder), recombinant human TNF-a (Immunokontact), and recombinant human IFN-g
(BenderMedSystems).
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Received December 23, 2008