Synthesis of sulfated galactocerebrosides from an orthogonal β-D-galactosylceramide scaffold for the study of CD1-antigen interactions

Article abstract of DOI:10.1002/chem.200501586

CD1a protein binds sulfatide (3-O-sulfo-β-D-galactosylceramide) to form an antigen complex that interacts with T cell receptors and activates T cells. To assess the role of the position of the sulfate in T cell activation, the synthesis of three β-D-galac

Full text of DOI:10.1002/chem.200501586

FULL PAPER
DOI: 10.1002/chem.200501586
Synthesis of Sulfated Galactocerebrosides from an Orthogonal b-d-
Galactosylceramide Scaffold for the Study of CD1–Antigen Interactions
Federica Compostella,*[a] Silvia Ronchi,[a] Luigi Panza,[b] Sabrina Mariotti,[c]
Lucia Mori,[c] Gennaro De Libero,[c] and Fiamma Ronchetti[a]
Abstract: CD1a protein binds sulfatide
(3-O-sulfo-b-d-galactosylceramide) to
form an antigen complex that interacts
with T cell receptors and activates
T cells. To assess the role of the posi-
tion of the sulfate in T cell activation,
the synthesis of three b-d-galactosylcer-
amides, variously bearing a sulfate at
position 2, 4, or 6 of galactose, has
been planned and carried out. The
compounds were synthesized by an or-
thogonal sulfation strategy from
a
zoylazidosphingosine. Immunological
evaluation of the three sulfated com-
pounds in CD1a-mediated T cell acti-
vation, in comparison with natural sul-
fatide, provided evidence of the influ-
ence of the sulfate position in the rec-
ognition event between the antigen,
the CD1 protein and the T cell recep-
tor.
common b-d-galactosylceramide scaf-
fold, which was in turn obtained
through an efficient glycosylation reac-
tion between a fully orthogonally pro-
tected galactosyl imidate and 3-O-ben-
Keywords: carbohydrate scaffold ·
galactosylceramides · glycolipids ·
protecting groups · sulfatide
Introduction
tives for biological tagging, such as with fluorescent probes.
In this context, we have recently been involved in the study
of sulfatide–protein interactions.[1] Sulfatide is a mammalian
glycolipid antigen that participates in intracellular signalling
mechanisms and is presented to the immune system by the
CD1 family of antigen-presenting surface proteins. The bio-
logical mechanism involving sulfatide consists of T cell acti-
vation after recognition between the T cell receptor and the
CD1–antigen complex; sulfatide is a particularly promiscu-
ous ligand that binds all human CD1 molecules and is pre-
sented by the different CD1 isoforms to specific T cells.[2]
Structurally it is a b-d-galactosylceramide bearing a sulfate
group at position 3 in galactose. The solved 3D structures of
CD1s reveal that these molecules each possess a narrow and
deep hydrophobic antigen-binding groove, specially tailored
to recognize and bind the long alkyl chains of lipid antigens
such as sulfatide.[3] In particular, the atomic structure of
CD1a with bound sulfatide shows that the protein has two
hydrophobic pockets that extend out of the centre of the
binding groove, each of which accommodates an alkyl chain,
The availability of biologically active molecules with well
defined chemical and structural properties is a fundamental
requirement for structure–function studies in biochemical
systems. The basic recognition motif of the ligand, identified
through structure elucidation and biological activity studies,
can in many cases be used as a template for the preparation
of a large number of related derivatives. This approach is
useful to assess different biological questions, such as the
identification of functionalities capable of modulating the
binding of the ligand to biological receptors, the study of po-
tential to induce specific responses of structurally different
antigen–receptor complexes, or the development of deriva-
[a]Dr. F. Compostella, Dr. S. Ronchi, Prof. F. Ronchetti
Dipartimento di Chimica, Biochimica e Biotecnologie
per la Medicina, Università di Milano, Via Saldini 50
20133-Milano (Italy)
Fax : (+39)02-5031-6036
E-mail: federica.compostella@unimi.it
[b]Prof. L. Panza
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e
Farmacologiche, Università del Piemonte Orientale
Via Bovio 6, 28100-Novara (Italy)
[c]Dr. S. Mariotti, Dr. L. Mori, Prof. G. De Libero
Experimental Immunology, Department of Research
Basel University Hospital, Hebelstrasse 20, 4031-Basel (Switzerland)
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while the charged and polar headgroups of the bound anti-
gen stick out from the middle of the lipid-binding groove of
CD1a for T cell receptor recognition. The galactose moiety
and the 3-sulfate group of sulfatide (that is, the polar part of
the antigen) form contacts with the charged and polar resi-
due of the protein, located at the intersection of the two hy-
drophobic pockets.[3c] From the consideration that these con-
tacts are important for the orientation of the bound antigen
and its presentation to T cell receptor, it is interesting to
evaluate how the position of the sulfate on the galactose in-
fluences the spatial arrangement of the antigen bound to the
CD1a protein and hence the potential of the complex pre-
sented to T cells.
phenylsilyl ether, at C-4 and C-6, respectively. The four pro-
tecting groups on galactose are orthogonal and are not con-
strained to any particular deprotection sequence for the in-
troduction of the desired functionalities. Furthermore, al-
though the three ether groups can be simultaneously
removed under acid conditions, they are at the same time
stable enough to acidic glycosidation procedures. In addi-
tion, we were not interested in having selective access to the
hydroxy group on ceramide, which was thus protected as a
benzoate. As a consequence, the protecting groups on galac-
tose are not only orthogonal between themselves, but also
towards the benzoate, which, in contrast, is not orthogonal
with respect to levulinate.
To address this problem, the availability of a b-d-galacto-
sylceramide scaffold with a set of mutually orthogonal pro-
tecting groups on galactose[4]—through selective deprotec-
tion at the desired hydroxy group, sulfation and final remov-
al of the remaining protections—could allow the preparation
of a family of sulfated galactosylceramides differing in the
position of the sulfate on galactose.
The preparation of compound 1 requires quantities of the
galactosyl imidate 2 and azidosphingosine 3, to drive the
glycosidation reaction to the production of the glycolipid
template (Scheme 1). To date the preparation of glycosylcer-
amides through the Schmidt “azidosphingosine glycosylation
procedure” remains the most efficient approach for their
synthesis.[6] This procedure has been widely explored in our
laboratories, where we have developed efficient large-scale
Here we report the synthesis of the b-d-galactosylcer-
A
syntheses
of
(2S,3R,4E)-3-O-benzoylazidosphingosine
sugar moiety. The versatility of this glycolipid scaffold has
been investigated through the synthesis of a family of posi-
(3).[1b,7]
Allyl a-d-galactopyranoside derivative 6 (Scheme 2) was
chosen as the precursor of imidate 2 to allow selective de-
protection of the anomeric position, thus making compound
6 a fully orthogonally protected derivative.
The synthesis started from allyl 3-O-p-methoxybenzyl-a-
d-galactopyranoside,[8] which was selectively protected at
the primary hydroxy group as the tert-butyldiphenylsilyl
ether to yield compound 4, which was in turn subsequently
acylated at position 2 by treatment with levulinic acid and a
condensing agent. Finally, protection of the free 4-OH
group was accomplished by use of [b-(trimethylsilyl)ethoxy]-
methyl (SEM) chloride and Hünig base to give galactopy-
tion isomers of sulfatide:[5] that is,
galactosylcer
or 4 or 6 in galactose. These compounds have been tested
for activation of a sulfatide-specific and CD1a-restricted
T cell clone to assess the role of the position of the sulfate
in T cell activation.
Synthesis: The design of the b-d-glycosylceramidic scaffold
1 involved the incorporation of a levulinate moiety at C-2, a
p-methoxybenzyloxy group at C-3, and a [b-(trimethylsil-
AHCTREUNG
A
gonality of this attractive set of protecting groups on this
Scheme 2. a) TBDPSCl, CH2Cl2/Py, 87%; b) Levulinic acid, EDCI,
DMAP,
CH2Cl2,
62%;
c) SEMCl,
DIPEA,
CH2Cl2,
95%;
d) NH2NH2·AcOH, CH2Cl2/MeOH, 89%; e) DDQ, CH2Cl2/H2O, 85%;
f) TBAF, THF/AcOH, 91%; g) MgBr2·Et2O, Et2O/CH3NO2, 87%; h) Ir
catalyst, THF, then NBS, THF/H2O, 85%; i) Cl3CCN, DBU, CH2Cl2,
85%. DBU= 1,8-diazabicyclo
A
=
levulinyl,
Scheme 1. a) TESOTf, CH2Cl2, m.s., À508C, 86%; b) nervonic acid, Bu3P,
NBS=N-bromosuccinimide, PMB = p-methoxybenzyl, SEM = [b-(tri-
methylsilyl)ethoxy]methyl, TBDPS = tert-butyldiphenylsilyl.
EDCI, CH2Cl2, 60%.
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Synthesis of Sulfated Galactocerebrosides
FULL PAPER
“model” compound (Scheme 2), so the Lev ester was re-
moved by treatment with hydrazine acetate in dichloro-
were prepared as described in Scheme 3. Glycolipid 1 was
first selectively deprotected in high yield at position 6 of gal-
actose and was then subjected to sulfation at the primary
hydroxy group by treatment with sulfur trioxide/trimethyl-
methane/methanol to give the 2-deprotected compound 7.
Oxidative cleavage of the 3-O-p-methoxybenzyl group of 6,
on the other hand, was easily accomplished by treatment
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in
dichloromethane/water to give 8. The tert-butyldiphenylsilyl
ether at C-6 was instead removed by treatment with tetrabu-
tylammonium fluoride buffered with acetic acid; these con-
ditions did not affect the levulinate at position 2, which was
otherwise partially hydrolyzed if acetic acid was not present.
The ether protection at C-4, the SEM group, was cleaved
with magnesium bromide in a diethyl ether/nitromethane
system, yielding compound 5. These mild cleavage condi-
tions did not affect the other ether groups on compound 6.
All the deprotection conditions, as tested on allyl galacto-
pyranoside 6, should also be compatible when the sugar is
linked to ceramide, in which there are no groups unstable to
these procedures. The allyl group of compound 6 was hydro-
lysed by smooth isomerization of the allyloxy group, fol-
lowed by hydrolysis of the enol ether. In conclusion, this
particular set of protecting groups should offer an exciting
opportunity to obtain a range of well defined structures in a
fast and facile manner.
A
ed by simultaneous removal of ether protections with 5%
trifluoroacetic acid in dichloromethane, followed by Zem-
plØn transesterification of the benzoate and levulinate. The
desired 6-O-sulfated 12 was recovered as its sodium salt
after loading onto a cation-exchange resin column, followed
by flash chromatography.
On the other hand, treatment of galactosylceramide 1
with magnesium bromide afforded a high yield of 4-O-un-
protected 13, which was then sulfated. The sodium salt of 4-
O-sulfated 14 was recovered after treatment with trifluoro-
acetic acid, followed by removal of the silyl ether with tetra-
butylammonium fluoride, ester deprotection and standard
purifications. Finally, compound 1 was selectively deprotect-
ed at the 2-position of galactose by hydrolysis of the Lev
ester with hydrazine acetate. The free hydroxy group was
smoothly sulfated under standard conditions, but a longer
reaction time was needed. The desired 2-O-sulfated 16 was
obtained in good yield after removal of the protecting
groups and final purification.
The 1-O-unprotected derivative was converted into the
trichloroacetimidate donor 2 by treatment with trichloroace-
tonitrile in dichloromethane in the presence of DBU as the
base (Scheme 2).
Although it should in principle also be possible to obtain
the 3-O-sulfate from scaffold 1, it was not synthesized as it
had been prepared by us previously.[1a]
The glycosylation reaction to give 10 (Scheme 1) was suc-
Biology: We evaluated the biological activity of the synthet-
ic sulfated galactosylceramides, differing in the positions of
their sulfate groups, by a T cell antigen presentation assay.
A CD1a-restricted human T cell clone reactive to natural b-
cessfully accomplished by treating imidate
2
with
(2S,3R,4E)-3-O-benzoylazidosphingosine (3)[1b,7] in dichloro-
methane at low temperature with catalysis by triethylsilyl
triflate. The conditions proved
to be compatible with the acid-
sensitive
ether
protecting
groups on galactose, provided
that the reaction was carried
out at À508C. The glycosylation
product was recovered in high
yield and with good stereoselec-
tivity, as confirmed by the char-
acteristic 8.0 Hz value of the
trans-diaxial J1’,2’ coupling con-
stant found in the 1H NMR
spectrum. The synthesis of b-d-
galactosylceramide derivative 1
was completed by reduction of
the azido functionality in
a
Staudinger reaction with con-
comitant formation of the de-
sired amide bond through con-
densation with nervonic acid[1a]
in the presence of soluble car-
bodiimide.
With scaffold 1 to hand, the
desired 6-O-, 4-O- and 2-O-
sulfo-b-d-galactosylceramides
Scheme 3. a) TBAF, THF/AcOH, 86%; b) SO3·Me3N, DMF, 408C, 73%; c) 1) CF3COOH 5% in CH2Cl2,
2) MeONa, MeOH/CH2Cl2, 3) Dowex Na+ form, 70%; d) MgBr2, Et2O/CH3NO2, 84%; e) SO3·Me3N, DMF,
408C, 68%; f) 1) CF3COOH 5% in CH2Cl2, 2) TBAF, THF, 3) MeONa, MeOH/CH2Cl2, 4) Dowex Na+ form,
66%; g) NH2NH2·AcOH, CH2Cl2/MeOH 82%; h) SO3·Me3N, DMF, 408C, 62%; i) see f), 75%.
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F. Compostella et al.
d-galactosylceramides bearing the sulfate group in position 3
of galactose was selected for these studies because desulfa-
tion experiments had clearly demonstrated the importance
of the sulfate group for TCR recognition.[9] The clone was
stimulated with two types of CD1a-expressing human anti-
gen-presenting cells (APCs): monocyte-derived dendritic
cells (DCs) (Figure 1A), or CD1a-transfected promyelocytic
Moreover, these data also confirm the discriminatory ca-
pacity of the T cell receptor, which is influenced by the pre-
cise position of the polar moiety of the glycolipid antigen.[10]
Indeed, other experimental models have shown the impor-
tance for immunogenicity of the anomeric form of the glyco-
sidic bond to the ceramide,[11] of the type of sugars present
in the CD1-associated ligand[12,13] and of the presence of ad-
ditional sugar moieties, which are not cleaved during antigen
processing and facilitate or inhibit antigen recognition.[13]
Conclusion
Activation of T cells is triggered by contact between the
CD1a–sulfatide antigen complex and specific T cell recep-
tors. Shedding light on the chemical requirements underly-
ing this process may be significant for understanding of the
molecular determinants of immune response.
Herein the synthesis of a series of sulfo-b-d-galactosylcer-
amides, each bearing a sulfate ester variously at position 2,
4, or 6 of galactose—that is, a family of position isomers of
mammalian sulfatide—through the use of an orthogonally
protected galactosylceramide scaffold has been described.
The isomers of the natural sulfatide were used to investigate
the role of the position of the sulfate ester in determining
the antigen–CD1a protein interaction. In this context, our
study has confirmed the discriminatory capacity of the T cell
receptor, which depends on a molecular recognition process,
influenced by the precise position of the polar moiety of the
glycolipid antigen.
Figure 1. The position of the sulfate group influences the glycolipid im-
munogenicity. Monocyte-derived DCs (A) or CD1a-transfected human
promyelocitic cell line THP-1 (B) were preincubated with (black bars) or
without (white bars) the indicated sulfated-b-d-galactosylceramides prior
to the addition of sulfatide-specific CD1a-restricted T cell clone K34B9.1.
IL-4 or TNF-a released were measured. Data are expressed as meansÆ
SDs (ngmLÀ1) of triplicates.
THP-1 cells (Figure 1B) preincubated with the various com-
pounds. T cell activation was detected by measuring released
IL-4 or TNF-a in the culture supernatant and was compared
with the optimal response obtained with the 3-O-sulfated
galactosylceramide. The 4-O-sulfated compound (14) and to
a lesser extent the 6-O-sulfated compound (12) were active
with both types of APC, whereas the 2-O-sulfated analogue
(16) did not stimulate T cells.
We chose CD1a-restricted presentation of sulfatide as our
experimental model in order to exclude the possibility that
presentation of sulfatide analogues might be influenced by
late-endosomal resident proteins. As CD1a recycles in early
endosomes and not in late endosomes,[2] its loading with gly-
colipid antigens is not influenced by lipid transfer proteins,
and nor by sulfatases, which reside in late endosomes. In ad-
dition, CD1a can be directly loaded on the surface of anti-
gen-presenting cells without internalization and recombinant
CD1a can associate in vitro with sulfatide in the absence of
other proteins.[9] These unique characteristics of CD1a load-
ing with sulfatide make it unlikely that differences in anti-
gen presentation would be responsible for the observed re-
duced responses to the tested analogues.
The published crystal structure of CD1a–sulfatide com-
plex shows the exact position of the galactose, partially pro-
truding out of the CD1a a helices.[3c] In this structure, the
sulfate in position 3 is in an optimal orientation to make
direct contact with the TCR, thus participating in the estab-
lishment of a high-affinity TCR–CD1a–sulfatide interaction,
sufficient for T cell stimulation. Our findings with sulfatide
analogues are in agreement with this hypothesis and show
that the exact position of sulfate is important for optimal
stimulation of tested sulfatide-specific T cells.
Moreover, to the best of our knowledge, this is the first
example of the preparation of a galactosylceramidic scaf-
fold. We believe that this scaffold and the approach we have
presented can be used to obtain new series of cerebroside
derivatives, bearing various functionalities at different posi-
tions.
Experimental Section
General: Optical rotations were measured with a Perkin–Elmer 241 po-
1
larimeter at 208C. H and 13C NMR spectra were recorded at 298 K with
a
Bruker AVANCE 500 spectrometer operating at 500.13 and
125.76 MHz for 1H and 13C, respectively. Chemical shifts are reported on
the d (ppm) scale and are relative to TMS as internal reference. MS spec-
tra were recorded in the negative or positive mode on a Thermo Quest
Finnigan LCQ deca spectrometer by electrospray ionization as indicat-
ed. All reactions were monitored by TLC on silica gel 60 F-254 plates
(Merck), spots being developed with 5% sulfuric acid in methanol/water
(1:1) or with phosphomolybdate-based reagent. Flash column chromatog-
raphy was performed on silica gel 60 (230–400 mesh, Merck). Organic
solutions were dried over sodium sulfate. All evaporations were carried
out under reduced pressure at 408C. Dry solvents and liquid reagents
were distilled prior to use: THF and diethyl ether were distilled from
sodium, whilst dichloromethane and pyridine were distilled from calcium
hydride. DMF and methanol were dried on molecular sieves (4 Š).
EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
Allyl 6-O-(tert-butyldiphenylsilyl)-3-O-(4-methoxybenzyl)- a-d-galacto-
pyranoside (4): TBDPSCl (4.60 mL, 17.69 mmol) was added at 08C to a
solution of allyl 3-O-(4-methoxybenzyl)-a-d-galactopyranoside[8] (3.00 g,
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Synthesis of Sulfated Galactocerebrosides
FULL PAPER
8.81 mmol) in CH2Cl2/Py (2:1, 150 mL). The reaction mixture was al-
lowed to warm slowly to room temperature, and stirring was continued
for 20 h. The solution was diluted with CH2Cl2 (300 mL), washed with
HCl (2n, 2”150 mL) and water (2”150 mL) and dried, and the solvent
was removed under reduced pressure. Flash chromatography (hexane/
2.52–2.77 (m, 4H; OCOCH2CH2), 3.43–3.54 (m, 2H; CH2OCH2O), 3.75–
3.83 (m, 6H; H-5, -6a, -6b and OCH3), 3.83 (dd, J2,3 = 10.5, J3,4
=
3.7 Hz, 1H; H-3), 3.89–3.94 (m, 1H; OCHaHbCH=CH2), 4.03–4.09 (m,
2H; H-4 and OCHaHbCH=CH2), 4.53 (d, J = 11.5 Hz, 1H; OCHaHbPh),
4.61 (d, J
= 11.5 Hz, 1H; OCHaHbPh), 4.70 (d, J = 7.0 Hz, 1H;
AcOEt 4:6) afforded 4 as an amorphous white solid (4.44 g, 87%). Rf
=
CH2OCHaHbO), 4.86 (d, J = 7.0 Hz, 1H; CH2OCHaHbO), 5.04 (d, J1,2
=
0.48 (hexane/AcOEt 4:6); [a]D20
= +67.7 (c = 1 in chloroform);
3.7 Hz, 1H; H-1), 5.11–5.23 (m, 3H; H-2 and CH=CH2), 5.78–5.87 (m,
1H; CH=CH2), 6.83–6.87 (m, 2H; arom.), 7.21–7.26 (m, 2H; arom.),
7.32–7.42 (m, 6H; arom.), 7.62–7.66 ppm (m, 4H; arom.); 13C NMR
(CDCl3): d = À0.8 (3”C), 18.4, 19.9, 27.5 (3”C), 28.7, 30.5, 38.6, 55.9,
64.0, 66.3, 68.8, 71.8, 72.1, 72.8, 73.2, 76.6, 95.8, 96.2, 114.5 (2”C), 118.1,
128.4–136.2 (16”C), 159.9, 172.8, 207.0 ppm; ESI-MS (positive-ion
mode): m/z (%): 829.3 (79) [M+Na]+, 1635.7 (100) [2M+Na]+; elemen-
tal analysis calcd (%) for C44H62O10Si2 (807.13): C 65.48, H 7.74; found:
C 65.39, H 7.80.
1H NMR (CDCl3): d = 1.04 (s, 9H; (CH3)3CSi), 2.05 (d, J2,OH = 8.0 Hz,
1H; OH), 2.41 (s, 1H; OH), 3.60 (dd, J2,3 = 9.5, J3,4 = 3.0 Hz, 1H; H-3),
3.74–3.85 (m, 5H; H-5, -6a and OCH3), 3.88 (dd, J5,6b = 5.5, J6a,6b
9.5 Hz, 1H; H-6b), 3.93–4.03 (m, 2H; H-2 and OCHaHbCH=CH2), 4.05
(brs, 1H; H-4), 4.13–4.19 (m, 1H; OCHaHbCH=CH2), 4.62 (d, J
=
=
11.7 Hz, 1H; OCHaHbPh), 4.68 (d, J = 11.7 Hz, 1H; OCHaHbPh), 4.94
(d, J1,2 = 3.7 Hz, 1H; H-1), 5.15–5.20 (m, 1H; CH=CHaHb), 5.21–5.29
(m, 1H; CH=CHaHb), 5.84–5.94 (m, 1H; CH=CHaHb), 6.85–6.89 (m, 2H;
arom.), 7.27–7.43 (m, 8H; arom.), 7.63–7.68 ppm (m, 4H; arom.);
13C NMR (CDCl3): d = 19.0, 26.6 (3”C), 55.1, 63.0, 66.7, 68.2, 68.4, 70.1,
71.6, 78.2, 97.3, 113.8 (2”C), 117.7, 127.5–135.4 (16”C), 159.2 ppm; ESI-
MS (positive-ion mode): m/z (%): 601.3 (30) [M+Na]+, 1179.0 (100)
[2M+Na]+; elemental analysis calcd (%) for C33H42O7Si (578.76): C
68.48, H 7.31; found: C 68.67, H 7.40.
Allyl
6-O-(tert-butyldiphenylsilyl)-3-O-(4-methoxybenzyl)-4-O-{[b-(tri-
methylsilyl)ethoxy]methyl}-a-d-galactopyranoside (7): A solution of hy-
drazine acetate (0.033 g, 0.36 mmol) in methanol (0.10 mL) was added to
a stirred solution of compound 6 (0.10 g, 0.12 mmol) in dichloromethane
(10 mL) and the reaction mixture was kept stirring at room temperature
for 3 h. Water (10 mL) was added, and after separation the aqueous layer
was extracted with dichloromethane (2”10 mL). The combined organic
layers were dried, concentrated and purified by flash chromatography
Allyl 6-O-(tert-butyldiphenylsilyl)-2-O-levulinyl-3-O-(4-methoxybenzyl)-
a-d-galactopyranoside (5)
From compound 4: EDCI (4.37 g, 22.80 mmol) and DMAP (1.86 g,
15.20 mmol) were added under argon to a solution of compound 4
(4.40 g, 7.60 mmol) and levulinic acid (1.32 g, 11.4 mmol) in dichlorome-
thane (100 mL). The reaction mixture was stirred at room temperature
overnight (TLC: dichloromethane/AcOEt 8:2; Rf of 5 = 0.60), and the
solvent was removed under reduced pressure. The residue was purified
by flash chromatography (hexane/AcOEt 6:4) to give 5 (3.20 g, 62%) as
an oil.
(dichloromethane/AcOEt 9:1) to yield 7 (0.076 g, 89%) as an oil. Rf
=
0.44 (dichloromethane/AcOEt 9:1); [a]D20
= +85.8 (c = 1 in chloro-
form); 1H NMR (CDCl3): d = À0.14 (s, 9H; (CH3)3Si), 0.68–0.79 (m,
2H; SiCH2), 1.04 (s, 9H; (CH3)3CSi), 2.07 (d, J2,OH = 7.5 Hz, 1H; OH),
3.47–3.55 (m, 3H; H-5 and CH2OCH2O), 3.59 (dd, J2,3 = 10.0, J3,4
=
2.7 Hz, 1H; H-3), 3.74–3.82 (m, 5H; H-6a, -6b and OCH3), 3.93–3.99 (m,
1H; OCHaHbCH=CH2), 3.99–4.05 (m, 1H; H-2), 4.08–4.14 (m, 2H; H-4
and OCHaHbCH=CH2), 4.57 (d, J = 11.5 Hz, 1H; OCHaHbPh), 4.69 (d,
J = 11.5 Hz, 1H; OCHaHbPh), 4.71 (d, J = 7.0 Hz, 1H; CH2OCHaHbO),
4.88 (d, J = 7.0 Hz, 1H; CH2OCHaHbO), 4.96 (d, J1,2 = 3.7 Hz, 1H; H-
1), 5.12–5.18 (m, 1H; CH=CHaHb), 5.19–5.25 (m, 1H; CH=CHaHb),
5.81–5.90 (m, 1H; CH=CH2), 6.84–6.89 (m, 2H; arom.), 7.26–7.43 (m,
8H; arom.), 7.62–7.68 ppm (m, 4H; arom.); 13C NMR (CDCl3): d = À0.8
(3”C), 18.4, 19.9, 27.5 (3”C), 56.0, 64.0, 66.3, 68.9, 69.6, 72.3 (3”C),
79.3, 96.1, 98.0, 114.6 (2”C), 118.5, 128.4–136.2 (16”C), 159.9 ppm; ESI-
MS (positive-ion mode): m/z (%): 709.2 (100) [M+H]+; elemental analy-
sis calcd (%) for C39H56O8Si2 (709.03): C 66.06, H 7.96; found: C 66.28, H
8.00.
From compound 6: MgBr2 (0.46 g, 1.80 mmol) was treated with dry dieth-
yl ether (0.50 mL), and MeNO2 (0.10 mL) was added to this two-phase
solution. The resulting (one-phase) solution was added under argon to a
stirred solution of compound 6 (0.10 g, 0.12 mmol) in diethyl ether
(1 mL). The reaction mixture was stirred for 3 h at room temperature
and was then diluted with AcOEt (10 mL) and washed with water
(10 mL). The aqueous layer was extracted with AcOEt (2”10 mL), and
the combined organic layers were washed with brine (2”10 mL), dried
and concentrated. The crude product was purified by flash chromatogra-
phy (hexane/AcOEt 1:1) to afford 5 (0.073 g, 87%) as an oil. Rf = 0.50
(hexane/AcOEt 1:1); [a]D20 = +59.0 (c = 1 in chloroform); 1H NMR
(CDCl3): d = 1.02 (s, 9H; (CH3)3CSi), 2.16 (s, 3H; CH3CO), 2.50 (brs,
1H; OH), 2.53–2.78 (m, 4H; OCOCH2CH2), 3.76–3.91 (m, 7H; H-3, -5,
-6a, -6b and OCH3), 3.92–3.98 (m, 1H; OCHaHbCH=CH2), 4.04 (brd,
J3,4 = 3.0 Hz, 1H; H-4), 4.07- 4.13 (m, 1H; OCHaHbCH=CH2), 4.56 (d,
J=11.7 Hz, 1H; OCHaCHbPh), 4.61 (d, J = 11.7 Hz, 1H; OCHaCHbPh),
5.01 (d, J1,2 = 3.5 Hz, 1H; H-1), 5.13 (dd, J1,2 = 3.5, J2,3 = 10.0 Hz, 1H;
H-2), 5.14–5.18 (m, 1H; CH=CHaHb), 5.20–5.27 (m, 1H; CH=CHaHb),
5.81–5.91 (m, 1H; CH=CHaHb), 6.84–6.88 (m, 2H; arom.), 7.23–7.27 (m,
2H; arom.), 7.32–7.43 (m, 6H; arom.), 7.63–7.69 ppm (m, 4H; arom.);
13C NMR (CDCl3): d = 19.9, 27.5 (3”C), 28.7, 30.5, 38.6, 55.9, 63.8, 68.1,
68.9, 70.7, 71.2, 72.8, 76.0, 95.9, 114.6 (2”C), 118.3, 128.4–136.3 (16”C),
160.1, 172.9, 207.0 ppm; ESI-MS (positive-ion mode): m/z (%): 699.2 (75)
[M+Na]+, 1375.0 (100) [2M+Na]+; elemental analysis calcd (%) for
C38H48O9Si (676.87): C 67.43, H 7.15; found: C 67.58, H 7.26.
Allyl 6-O-(tert-butyldiphenylsilyl)-2-O-levulinyl-4-O-{[b-(trimethylsilyl)-
AHCTREUNG
AHCTREUNG
vigorously for 3 h, diluted with dichloromethane (15 mL) and washed
successively with saturated Na2S2O3 (1”10 mL), NaHCO3 (1”10 mL)
and brine (1”10 mL). The organic phase was dried and concentrated
under reduced pressure. The residue was purified by flash chromatogra-
phy (hexane/AcOEt 7:3) to give compound 8 (0.070 g, 85%) as an oil. Rf
=
0.30 (hexane/AcOEt 7:3); [a]D20
1H NMR (CDCl3): d
À0.02 (s, 9H; (CH3)3Si), 0.88–0.96 (m, 2H;
= +41.2 (c = 1 in chloroform);
=
SiCH2), 1.03 (s, 9H; (CH3)3CSi), 2.15 (s, 3H; CH3CO), 2.60–2.82 (m, 4H;
OCOCH2CH2), 3.46–3.54 (m, 1H; CHaHbOCH2O), 3.71 (dd, J5,6a = 5.7,
J6a,6b = 10.0 Hz, 1H; H-6a), 3.80 (dd, J5,6b = 8.0, J6a,6b = 10.0 Hz, 1H;
H-6b), 3.82–3.89 (m, 1H; CHaHbOCH2O), 3.89–3.99 (m, 3H; H-3, -5 and
OCHaHbCH=CH2), 4.01 (brd, J3,4 = 3.0 Hz, 1H; H-4), 4.03–4.08 (m, 1H;
OCHaHbCH=CH2), 4.57 (d, J = 7.0 Hz, 1H; CH2OCHaHbO), 4.75 (d, J
= 7.0 Hz, 1H; CH2OCHaHbO), 4.92 (d, J1,2 = 3.7 Hz, 1H; H-1), 4.98
(dd, J1,2 = 3.7 Hz, J2,3 = 10.3 Hz, 1H; H-2), 5.12–5.17 (m, 1H; CH=
CHaHb), 5.21–5.27 (m, 1H; CH=CHaHb), 5.79–5.88 (m, 1H; CH=CH2),
7.33–7.44 (m, 6H; arom.), 7.60–7.65 ppm (m, 4H; arom.); 13C NMR
(CDCl3): d = À0.8 (3”C), 18.7, 19.9, 27.5 (3”C), 28.8, 30.5, 38.7, 62.4,
67.5, 68.1, 69.2, 71.1, 72.6, 81.5, 96.6, 97.8, 118.1, 128.5–136.2 (13”C),
173.1, 207.1 ppm; ESI-MS (positive-ion mode): m/z (%): 709.1 (41)
[M+Na]+, 1395.8 (100) [2M+Na]+; elemental analysis calcd (%) for
C36H54O9Si2 (686.98): C 62.94, H 7.92; found: C 62.85, H 7.78.
Allyl 6-O-(tert-butyldiphenylsilyl)-2-O-levulinyl-3-O-(4-methoxybenzyl)-
4-O-{[b-(trimethylsilyl)ethoxy]methyl}-a-d-galactopyranoside (6): Com-
pound
5 (3.15 g, 4.65 mmol) was dissolved in dry dichloromethane
(50 mL). N,N-Diisopropylethylamine (6.37 mL, 37.20 mmol) and [b-(tri-
methylsilyl)ethoxy]methyl chloride (3.28 mL, 18.60 mmol) were succes-
sively added to this solution, which was then heated at reflux for 15 h
under argon, during which an additional quantity of SEMCl (0.80 mL)
was added. The solvent was removed under reduced pressure and pure 6
(3.56 g, 95%) was recovered after flash chromatography (hexane/AcOEt
7.5:2.5) as an oil. Rf = 0.63 (hexane/AcOEt 6:4); [a]2D0 = +36.7 (c = 1
in chloroform); 1H NMR (CDCl3): d = À0.14 (s, 9H; (CH3)3Si), 0.64–
0.76 (m, 2H; SiCH2), 1.03 (s, 9H; (CH3)3CSi), 2.14 (s, 3H; CH3CO),
Chem. Eur. J. 2006, 12, 5587 – 5595
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5591

F. Compostella et al.
Allyl
2-O-levulinyl-3-O-(4-methoxybenzyl)-4-O-{[b-(trimethylsilyl)eth-
2H; 2”H-6), 2.14 (s, 3H; CH3CO), 2.54–2.85 (m, 4H; OCOCH2CH2),
3.33–3.39 (m, 2H; H-3’, -5’), 3.42 (dd, J1a,1b = 10.5, J1a,2 = 6.3 Hz, 1H;
H-1a), 3.46–3.57 (m, 2H; CH2OCH2O), 3.75–3.94 (m, 7H; H-1b, -2, -6a’,
ACHTREUNG
(0.18 mL, 1m in THF) was added to a solution of compound 6 (0.10 g,
0.12 mmol) in tetrahydrofuran/acetic acid (4:1, 2 mL) and the solution
was stirred at room temperature for five days, during which additional
aliquots of TBAF were added (overall 5 equiv). The solution was diluted
with ethyl acetate (20 mL), washed with NaHCO3 (2”10 mL) and NaCl
(1”10 mL), dried and concentrated. The residue was purified by flash
chromatography (hexane/AcOEt 1:1) to give compound 9 (0.062 g, 91%)
as an oil. Rf = 0.32 (hexane/AcOEt 1:1); [a]2D0 = +41.0 (c = 1 in chloro-
form); 1H NMR (CDCl3): d = À0.01 (s, 9H; (CH3)3Si), 0.86–0.98 (m,
2H; SiCH2), 2.14 (s, 3H; CH3CO), 2.52–2.77 (m, 4H; OCOCH2CH2),
3.02 (t, J6,OH = 7.5 Hz, 1H; OH), 3.47–3.53 (m, 1H; CHaHbOCH2O),
3.64–3.72 (m, 2H; H-6a, -6b), 3.74–3.82 (m, 4H; CHaHbOCH2O and
OCH3), 3.87–3.94 (m, 2H; H-3, -5), 3.96–4.01 (m, 1H; OCHaHbCH=
-6b’ and OCH3), 4.04 (brd, J3’,4’ = 2.5 Hz, 1H; H-4’), 4.26 (d, J1’,2’
=
8.0 Hz, 1H; H-1’), 4.47 (d, J = 12.0 Hz, 1H; OCHaHbPh), 4.62 (d, J =
12.0 Hz, 1H; OCHaHbPh), 4.69 (d, J = 7.0 Hz, 1H; CH2OCHaHbO), 4.85
(d, J = 7.0 Hz, 1H; CH2OCHaHbO), 5.23 (dd, J1’,2’ = 8.0, J2’,3’ = 10.0 Hz,
1H; H-2’), 5.44–5.54 (m, 2H; H-3, -4), 5.78–5.89 (m, 1H; H-5), 6.84–6.90
(m, 2H; arom.), 7.20–7.26 (m, 2H; arom.), 7.32–7.68 (m, 13H; arom.),
7.98–8.04 ppm (m, 2H; arom.); 13C NMR (CDCl3): d
=
À0.8 (3”C),
14.8, 18.5, 19.9, 23.4, 27.5 (3”C), 28.6–38.6 (14”C), 55.9, 63.6, 64.3, 66.3,
68.0, 71.2, 71.9, 72.0, 75.7, 76.5, 79.3, 96.1, 101.8, 114.5 (2”C), 123.4,
128.5–139.4 (22”C), 160.0, 165.8, 172.0, 207.3 ppm; ESI-MS (positive-ion
mode): m/z (%): 1200.3 (100) [M+Na]+; elemental analysis calcd (%) for
C66H95N3O12Si2 (1178.64): C 67.26, H 8.12, N 3.57; found: C 67.39, H 8.21,
N 3.62.
CH2), 4.06 (brd, J3,4
=
3.0 Hz, 1H; H-4), 4.10–4.15 (m, 1H;
OCHaHbCH=CH2), 4.56 (d, J = 11.5 Hz, 1H; OCHaHbPh), 4.60 (d, J =
11.5 Hz, 1H; OCHaHbPh), 4.76 (d, J = 7.0 Hz, 1H; CH2OCHaHbO), 4.81
(d, J = 7.0 Hz, 1H; CH2OCHaHbO), 5.04 (d, J1,2 = 3.5 Hz, 1H; H-1),
5.15 (dd, J1,2 = 3.5, J2,3 = 10.5 Hz, 1H; H-2), 5.15–5.20 (m, 1H; CH=
CHaHb), 5.22–5.29 (m, 1H; CH=CHaHb), 5.82–5.92 (m, 1H; CH=CH2),
6.83–6.88 (m, 2H; arom.), 7.21–7.26 ppm (m, 2H; arom.); 13C NMR
(CDCl3): d = À0.8 (3”C), 18.8, 28.7, 30.5, 38.6, 56.0, 61.3, 67.2, 69.3,
70.4, 71.8, 73.4, 74.5, 76.0, 96.3, 97.6, 114.6 (2”C), 118.3, 129.9 (2”C),
130.9, 134.5, 160.0, 172.8, 206.9 ppm; ESI-MS (positive-ion mode): m/z
(%): 591.2 (100) [M+Na]+, 1159.5 (40) [2M+Na]+; elemental analysis
calcd (%) for C28H44O10Si (568.73): C 59.13, H 7.80; found: C 58.90, 7.57.
A
O-(4-methoxybenzyl)-4-O-{[b-(trimethylsilyl)ethoxy]methyl}-b-d-galacto-
pyranosyloxy]-2-((Z)-tetracos-15-enoylamino)-octadec-4-ene (1): Bu3P
(0.47 mL, 1.91 mmol) was added under argon to a solution of compound
10 (1.5 g, 1.27 mmol) and nervonic acid (0.70 g, 1.91 mmol) in dry di-
chloromethane (25 mL). After consumption of the starting material (3 h),
as shown by TLC (hexane/AcOEt 7:3), EDCI (0.73 g, 3.81 mmol) was
added and the reaction mixture was stirred overnight at room tempera-
ture. The solvent was evaporated off and pure 1 (1.14 g, 60%) was recov-
ered as a colourless oil after purification of the crude product by flash
chromatography (hexane/AcOEt from 8:2 to 7:3). Rf = 0.50 (hexane/
6-O-(tert-Butyldiphenylsilyl)-2-O-levulinyl-3-O-(4-methoxybenzyl)-4-O-
{[b-(trimethylsilyl)ethoxy]methyl}-a,b-d-galactopyranosyl trichloroacet-
AcOEt 7:3); [a]2D0 = À13.9 (c = 1 in chloroform); H NMR (CDCl3): d=
À0.12 (s, 9H; (CH3)3Si), 0.70–0.76 (m, 2H; SiCH2), 0.86 (t, J = 6.5 Hz,
6H; 2”CH3), 1.01 (s, 9H; (CH3)3CSi), 1.12–1.36 (m, 54H; 27”CH2),
1.51–1.60 (m, 2H; CH2), 1.88–2.04 (m, 9H; 3”CH2C=C and CH3CO),
2.04–2.18 (m, 2H; CH2CONH), 2.34–2.42 and 2.47–2.59 and 2.73–2.82
(3”m, 4H; OCOCH2CH2), 3.29–3.35 (m, 2H; H-3’, -5’), 3.46–3.59 (m,
3H; H-1a and CH2OCH2O), 3.73–3.78 (m, 2H; 2”H-6’), 3.78 (s, 3H;
ACHTREUNG
ACHTREUNG
drofuran (25 mL) under Ar, and hydrogen was bubbled through the mix-
ture for 15 min. The resulting clear solution was then added dropwise to
a stirred solution of 6 (3.10 g, 3.84 mmol) in dry tetrahydrofuran (40 mL)
under Ar. After 3 h, additional tetrahydrofuran (200 mL), water (15 mL),
and N-bromosuccinimide (1.03 g, 5.77 mmol) were added. After 15 min,
the mixture was concentrated, dichloromethane was added (200 mL), and
the organic layer was washed with saturated NaHCO3 solution (2”
100 mL), dried and evaporated. The galactopyranose product (2.51 g,
85%) was recovered after flash chromatography (hexane/AcOEt 6:4;
Rf =0.22), and dissolved in dry dichloromethane (45 mL). Trichloroaceto-
OCH3), 4.03 (brd, J3’,4’ = 2.5 Hz, 1H; H-4’), 4.07 (dd, J1a,1b = 10.5, J1b,2
3.7 Hz, 1H; H-1b), 4.21 (d, J1’,2’ = 8.0 Hz, 1H; H-1’), 4.36–4.45 (m, 2H;
H-2 and OCHaHbPh), 4.61 (d, J = 12.0 Hz, 1H; OCHaHbPh), 4.70 (d, J
=
CH2OCHaHbO), 5.23 (dd, J1’,2’ = 8.0, J2’,3’ = 10.0 Hz, 1H; H-2’), 5.27–
5.37 (m, 2H; CH=CH), 5.38–5.48 (m, 2H; H-3, -4), 5.78 (dt, J4,5 = 15.0,
J5,6 = 6.5 Hz, 1H; H-5), 6.60 (d, J2,NH = 9.3 Hz, 1H; NH), 6.84–6.87 (m,
2H; arom.), 7.18–7.22 (m, 2H; arom.), 7.28–7.64 (m, 13H; arom.), 7.93–
7.97 ppm (m, 2H; arom.); 13C NMR (CDCl3): d = À0.8 (3”C), 14.8 (2”
C), 18.4, 19.8, 23.4–38.5 (38”C), 51.5, 55.9, 63.3, 66.3, 67.8, 71.1, 71.9 (2”
C), 75.3, 76.2, 79.5, 96.1, 102.3, 114.5 (2”C), 125.6–137.5 (25”C), 159.9,
166.0, 171.9, 173.8, 207.5 ppm; ESI-MS (positive-ion mode): m/z (%):
1522.6 (100) [M+Na]+; elemental analysis calcd (%) for C90H141NO13Si2
(1501.25): C 72.00, H 9.47, N 0.93; found: C 72.15, H 9.25, N 0.96.
nitrile (3.28 mL, 32.70 mmol) and 1,8-diazabicyclo[5,4,0]undec-7-ene
G
(0.10 mL) were added and the mixture was stirred for 2 h at room tem-
perature. The solvent was evaporated, and the residue was purified by
flash chromatography (hexane/AcOEt 7:3, 1% triethylamine) to yield 2
(2.53 g, 85%) as an oil. Rf = 0.30 (hexane/AcOEt 7:3); [a]2D0 =+20.9
1
(c= 1 in chloroform); H NMR (CDCl3) (selected signals): d = 3.48 (dd,
J2,3 = 10.0, J3,4 = 3.0 Hz, 0.3H; H-3b), 3.91 (dd, J2,3 = 10.5, J3,4 = 2.5 Hz,
0.7H; H-3a), 5.38 (dd, J1,2 = 3.5, J2,3 = 10.5 Hz, 0.7H; H-2a), 5.47 (dd,
J1,2 = 8.5, J2,3 = 10.0 Hz, 0.3H; H-2b), 5.67 (d, J1,2 = 8.5 Hz, 0.3H;
H-1b), 6.47 (d, J1,2 = 3.5 Hz, 0.7H; H-1a), 8.47 (s, 0.7H; a-NH), 8.56 ppm
(s, 0.3H; b-NH); ESI-MS (positive-ion mode): m/z (%): 932.0 (100)
[M+Na]+; elemental analysis calcd (%) for C43H58Cl3NO10Si2 (911.45): C
56.66, H 6.41; found: C 56.49, H 6.36.
A
{[b-(trimethylsilyl)ethoxy]methyl}-b-d-galactopyranosyloxy]-2-((Z)-tetra-
cos-15-enoylamino)-octadec-4-ene (11): Tetrabutylammonium fluoride
(1.33 mL, 1m in THF) was added to a solution of compound 1 (0.20 g,
0.13 mmol) in tetrahydrofuran/acetic acid (4:1, 7 mL) and the solution
was stirred at room temperature for 5 days, during which additional ali-
quots of TBAF were added (overall 5 equiv). The solution was diluted
with ethyl acetate (20 mL), washed with saturated NaHCO3 (2”10 mL)
and NaCl (1”10 mL), dried and concentrated. The residue was purified
by flash chromatography (hexane/AcOEt 1:1) to give compound 11
A
levulinyl-3-O-(4-methoxybenzyl)-4-O-{[b-(trimethylsilyl)ethoxy]methyl}-
b-d-galactopyranosyloxy]-octadec-4-ene (10): Triethylsilyl trifluorometha-
nesulfonate in dry CH2Cl2 (0.1m solution, 2.1 mL) was added dropwise at
À508C under argon to a mixture of imidate 2 (2.29 g, 2.52 mmol), 3-O-
benzoylazidosphingosine (3,[7] 0.90 g, 2.10 mmol) and molecular sieves
(4 Š, 1.80 g) in dry CH2Cl2 (70 mL). After 30 min the reaction mixture
was quenched by addition of saturated NaHCO3 solution and filtered
over a pad of celite. After separation, the aqueous layer was extracted
with CH2Cl2 (3”50 mL) and the combined organic layers were dried and
concentrated. Purification by flash chromatography (hexane/AcOEt 8:2)
gave pure 10 (2.12 g, 86%) as a colourless oil. Rf = 0.38 (hexane/AcOEt
(0.14 g, 86%) as a colourless oil. Rf = 0.29 (hexane/AcOEt 1:1); [a]D20
À21.9 (c 0.00 (s, 9H;
1 in chloroform); 1H NMR (CDCl3): d
(CH3)3Si), 0.82–0.98 (m, 8H; 2”CH3 and SiCH2), 1.15–1.38 (m, 54H;
27”CH2), 1.52–1.61 (m, 2H; CH2), 1.94–2.04 (m, 9H; 3”CH2C=C and
CH3CO), 2.06–2.18 (m, 2H; CH2CONH), 2.36–2.44 and 2.46–2.54 and
2.56–2.64 and 2.71–2.80 (4 m, 4H; OCOCH2CH2), 3.18 (brs, 1H; OH),
3.40–3.53 (m, 3H; H-3’, 5’ and CHaHbOCH2O), 3.56–3.70 (m, 3H; H-1a
and 2”H-6’), 3.72–3.80 (m, 4H; OCH3 and CHaHbOCH2O), 3.97–4.02
(m, 2H; H-1b, 4’), 4.27 (d, J1’,2’ = 8.0 Hz, 1H; H-1’), 4.39–4.47 (m, 2H;
H-2 and OCHaHbPh), 4.56 (d, J = 12.0 Hz, 1H; OCHaHbPh), 4.76 (d, J=
7.0 Hz, 1H; CH2OCHaHbO), 4.81 (d, J = 7.0 Hz, 1H; CH2OCHaHbO),
1
8:2); [a]2D0 = À21.0 (c = 1 in chloroform); H NMR (CDCl3): d = À0.12
(s, 9H; (CH3)3Si), 0.70–0.76 (m, 2H; SiCH2), 0.86 (t, J = 6.0 Hz, 3H;
CH3), 1.03 (s, 9H; (CH3)3CSi), 1.11–1.38 (m, 22H; CH2), 1.96–2.04 (m,
5592
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 5587 – 5595

Synthesis of Sulfated Galactocerebrosides
FULL PAPER
5.20 (dd, J1’,2’ = 8.0, J2’,3’ = 10.3 Hz, 1H; H-2’), 5.27–5.37 (m, 2H; CH=
and was then diluted with AcOEt (20 mL) and washed with water
(20 mL). The aqueous layer was extracted with AcOEt (3”15 mL), and
the combined organic layers were washed with brine (2”20 mL), dried
and concentrated. The crude product was purified by flash chromatogra-
phy (hexane/AcOEt 7:3) to afford 13 (0.15 g, 84%) as an oil. Rf = 0.35
CH), 5.45 (brdd, J3,4 = 7.5, J4,5 = 15.0 Hz, 1H; H-4), 5.53 (t, J2,3 = J3,4
=
7.5 Hz, 1H; H-3), 5.83 (dt, J4,5 = 15.0, J5,6 = 6.5 Hz, 1H; H-5), 6.44 (d,
J2,NH = 9.0 Hz, 1H; NH), 6.83–6.87 (m, 2H; arom.), 7.17–7.21 (m, 2H;
arom.), 7.38–7.44 (m, 2H; arom.), 7.49–7.55 (m, 1H; arom.), 7.97–
8.02 ppm (m, 2H; arom.); 13C NMR (CDCl3): d = À0.8 (3”C), 14.8 (2”
C), 18.8, 23.4–38.5 (35”C), 51.5, 55.9, 61.3, 67.1, 68.2, 71.9, 72.4, 72.6,
75.2 (2”C), 79.2, 97.2, 102.2, 114.5 (2”C), 125.6–137.9 (13”C), 160.1,
166.1, 172.0, 173.8, 207.3 ppm; ESI-MS (positive-ion mode):
m/z (%): 1284.5 (100) [M+Na]+; elemental analysis calcd (%) for
C74H123NO13Si (1262.85): C 70.38, H 9.82, N 1.11; found: C 70.49, H 9.91,
N 1.18.
(hexane/AcOEt 7:3); [a]D20
=
À1.4 (c
=
1 in chloroform); 1H NMR
(CDCl3): d = 0.86 (t, J = 6.5 Hz, 6H; 2”CH3), 1.00 (s, 9H; (CH3)3CSi),
1.12–1.36 (m, 54H; 27”CH2), 1.51–1.64 (m, 2H; CH2), 1.88–2.21 (m,
11H; 3”CH2C=C, CH3CO and CH2CONH), 2.35–2.43 and 2.46–2.63 and
2.75–2.84 (3”m, 5H; OCOCH2CH2 and OH), 3.36–3.43 (m, 2H; H-3’,
-5’), 3.53 (dd, J1a,1b = 10.5, J1a,2 = 3.5 Hz, 1H; H-1a), 3.67 (dd, J6a’,6b’
=
10.3, J5’,6a’ = 5.5 Hz, 1H; H-6a’), 3.76–3.83 (m, 4H; H-6b’ and OCH3),
4.00 (brd, J3’,4’ = 3.0 Hz, 1H; H-4’), 4.06 (dd, J1a,1b = 10.5, J1b,2 = 3.7 Hz,
1H; H-1b), 4.26 (d, J1’,2’ = 8.0 Hz, 1H; H-1’), 4.38–4.44 (m, 1H; H-2),
A
oxysulfonyl)-b-d-galactopyranosyloxy]-2-
solution of com-
A
4.50 (d, J
= 12.0 Hz, 1H; OCHaHbPh), 4.57 (d, J = 12.0 Hz, 1H;
pound 11 (0.13 g, 0.10 mmol) in dry DMF (2 mL) was treated with sulfur
trioxide/trimethylamine complex (0.042 g, 0.30 mmol), and the resulting
mixture was stirred at 408C for 1 h. The mixture was concentrated to a
few microlitres under reduced pressure (high vacuum pump) and purified
by flash chromatography (CH2Cl2/MeOH 9:1) to afford the 6-sulfated
product (0.098 g, 73%) as a colourless oil. Rf = 0.34 (CH2Cl2/MeOH
9:1); 1H NMR (CDCl3/CD3OD 2:1): d = À0.05 (s, 9H; (CH3)3Si), 0.75–
0.92 (m, 8H; 2”CH3 and SiCH2), 1.16–1.36 (m, 54H; 27”CH2), 1.50–
1.60 (m, 2H; CH2), 1.94–2.22 (m, 11H; 3”CH2C=C and CH3CO and
CH2CONH), 2.38–2.82 (m, 4H; OCOCH2CH2), 3.48 (dd, J2’,3’ = 10.0,
OCHaHbPh), 5.17 (dd, J1’,2’ = 8.0, J2’,3’ = 10.0 Hz, 1H; H-2’), 5.29–5.36
(m, 2H; CH=CH), 5.38–5.50 (m, 2H; H-3, 4), 5.78 (brdt, J4,5 = 15.0, J5,6
= 6.5 Hz, 1H; H-5), 6.63 (d, J2,NH = 9.3 Hz, 1H; NH), 6.83–6.87 (m, 2H;
arom.), 7.18–7.64 (m, 15H; arom.), 7.91–7.95 ppm (m, 2H; arom.);
13C NMR (CDCl3): d = 14.8 (2”C), 19.8–38.5 (39”C), 51.5, 55.9, 62.6,
66.4, 67.4, 71.6, 72.0, 75.1, 75.3, 79.0, 101.7, 114.6 (2”C), 125.6–137.6
(25”C), 165.5, 166.0, 172.1, 173.9, 207.6 ppm; ESI-MS (positive-ion
mode): m/z (%): 1392.7 (100) [M+Na]+; elemental analysis calcd (%) for
C84H127NO12Si (1370.99): C 73.59, H 9.34, N 1.02; found: C 73.70, H 9.40,
N 1.05.
J3’,4’
= 3.0 Hz, 1H; H-3’), 3.56–3.68 (m, 3H; H-1a and CH2OCH2O),
A
oxysulfonyl)-b-d-galactopyranosyloxy]-2-
solution of com-
3.75–3.85 (m, 4H; H-5’ and OCH3), 3.98–4.18 (m, 4H; H-1b, 4’, 6a’, 6b’),
4.28–4.42 (m, 3H; H-1’, -2 and OCHaHbPh), 4.62 (d, J = 11.5 Hz, 1H;
A
pound 13 (0.14 g, 0.10 mmol) in dry DMF (2 mL) was treated with sulfur
trioxide/trimethylamine complex (0.042 g, 0.30 mmol), and the resulting
mixture was stirred at 408C for 3 h. The mixture was concentrated to a
few microlitres under reduced pressure (high vacuum pump) and purified
by flash chromatography (CH2Cl2/MeOH 9.5:0.5) to afford the 4-sulfated
product (0.098 g, 68%) as a colourless oil. Rf = 0.15 (CH2Cl2/MeOH
OCHaHbPh), 4.72 (d, J
= 7.0 Hz, 1H; CH2OCHaHbO), 4.85 (d, J=
7.0 Hz, 1H; CH2OCHaHbO), 5.17 (dd, J1’,2’ = 8.0, J2’,3’ = 10.0 Hz, 1H;
H-2’), 5.27–5.36 (m, 2H; CH=CH), 5.41–5.52 (m, 2H; H-3, -4), 5.81 (dt,
J4,5 = 15.0, J5,6 = 7.0 Hz, 1H; H-5), 6.81–6.89 (m, 2H; arom.), 7.16–7.25
(m, 2H; arom.), 7.38–7.58 (m, 4H; 3”H arom. and NH), 7.93–8.02 ppm
(m, 2H; arom.); ESI-MS (negative-ion mode): m/z (%): 1340.8 (100)
[MÀH]À.
The 6-sulfated compound (0.080 g, 0.060 mmol) was treated with tri-
fluoroacetic acid in dichloromethane (5% 2 mL) at room temperature
for 10 min., and the solvent was then evaporated under reduced pressure.
The residue was dissolved in dry dichloromethane/methanol (1:1, 1.5 mL)
and sodium methoxide in dry methanol (0.05m solution, 1.48 mL) was
added. The reaction mixture was stirred overnight at room temperature
and was then neutralized with an ion-exchange resin (Dowex 50”8, H+
form), filtered and concentrated. The solvent was removed under re-
duced pressure, and the residue was then dissolved in CHCl3/MeOH (1:1,
1 mL) and loaded onto a cation-exchange resin column (Dowex 50X8,
Na+ form, 0.5”4 cm). The mixture was eluted with CHCl3/MeOH 1:1,
concentrated and subjected to flash chromatography (CH2Cl2/MeOH 8:2)
1
9.5:0.5); H NMR (CDCl3/CD3OD 2:1): d = 0.84 (t, J = 6.5 Hz, 6H; 2”
CH3), 0.97 (s, 9H; (CH3)3CSi), 1.12–1.36 (m, 54H; 27”CH2), 1.48–1.62
(m, 2H; CH2), 1.90–2.15 (m, 11H; 3”CH2C=C, CH3CO and
CH2CONH), 2.35–2.43 and 2.46–2.55 and 2.59–2.68 and 2.75–2.84 (4”m,
4H; OCOCH2CH2), 3.39–3.43 (m, 2H; H-3’, -5’), 3.48 (dd, J1a,1b = 10.0,
J1a,2 = 3.5 Hz, 1H; H-1a), 3.76 (s, 3H; OCH3), 3.84 (dd, J6a’,6b’ = 11.5,
J5’,6a’ = 7.4 Hz, 1H; H-6a’), 3.99 (dd, J6a’,6b’ = 11.5, J5’,6b’ = 4.0 Hz, 1H;
H-6b’), 4.07 (dd, J1a,1b = 10.0, J1b,2 = 4.5 Hz, 1H; H-1b), 4.25 (d, J1’,2’
=
8.0 Hz, 1H; H-1’), 4.31–4.39 (m, 2H; H-2 and OCHaHbPh), 4.72 (d, J =
12.0 Hz, 1H; OCHaHbPh), 4.79 (d, J3’,4’ = 3.0 Hz, 1H; H-4’), 4.94 (dd,
J1’,2’ = 8.0, J2’,3’ = 10.0 Hz, 1H; H-2’), 5.26–5.34 (m, 2H; CH=CH), 5.35–
5.45 (m, 2H; H-3, -4), 5.79 (dt, J4,5 = 15.0, J5,6 = 6.5 Hz, 1H; H-5), 6.80–
6.86 (m, 2H; arom.), 7.18–7.65 (m, 15H; arom.), 7.89–7.95 (m, 3H; arom.
and NH) ppm; ESI-MS (negative-ion mode): m/z (%): 1448.8 (100)
[MÀH]À.
The 4-sulfated compound (0.080 g, 0.055 mmol) was treated with tri-
fluoroacetic acid in dichloromethane (5%, 2 mL) at room temperature
for 10 min., and the solvent was then evaporated under reduced pressure.
Tetrabutylammonium fluoride (0.21 mL, 1m in THF) was added to a sol-
ution of the residue in dry THF (2 mL) and the solution was stirred at
room temperature overnight. After evaporation of the solvent, the resi-
due was dissolved in dry dichloromethane/methanol (1:1, 1.5 mL) and
sodium methoxide in dry methanol (0.05m solution, 1.6 mL) was added.
The reaction mixture was stirred overnight at room temperature and was
then neutralized with an ion-exchange resin (Dowex 50”8, H+ form), fil-
tered and concentrated. The solvent was removed under reduced pres-
sure, and the residue was then dissolved in CHCl3/MeOH (1:1, 1 mL)
and loaded onto a cation-exchange resin column (Dowex 50X8, Na+
form, 0.5”4 cm). The mixture was eluted with CHCl3/MeOH 1:1, concen-
trated and subjected to flash chromatography (CH2Cl2/MeOH 9.5:0.5) to
to give compound 12 (0.038 g, 70%) as an amorphous white solid. Rf
=
0.20 (CH2Cl2/MeOH 8:2); [a]2D0 = À4.4 (c = 0.5 in chloroform/methanol
1:1); 1H NMR (CDCl3/CD3OD 1:1): d = 0.88 (t, J = 6.5 Hz, 6H; 2”
CH3), 1.20–1.45 (m, 54H; 27”CH2), 1.53–1.63 (m, 2H; CH2), 1.97–2.08
(m, 6H; 3”CH2C=C), 2.17 (t, J = 7.5 Hz, 2H; CH2CO), 3.50–3.60 (m,
3H; H-1a, -2’, -3’), 3.75–3.80 (m, 1H; H-5’), 3.94 (d, J3’,4’ = 2.5 Hz, 1H;
H-4’), 3.98–4.02 (m, 1H; H-2), 4.09 (t, J2,3 = J3,4 = 7.5 Hz, 1H; H-3),
4.14 (dd, J1a,1b = 10.5, J1b,2 = 4.7 Hz, 1H; H-1b), 4.16–4.24 (m, 3H; H-1’,
-6a’, -6b’), 5.29–5.37 (m, 2H; CH=CH), 5.43 (ddt, J3,4 = 7.5, J4,5 = 15.0,
Jall = 1 Hz, 1H; H-4), 5.69 ppm (dt, J4,5 = 15.0, J5,6 = 6.5 Hz, 1H; H-5);
13C NMR (CDCl3/CD3OD 1:1): d = 13.6 (2”C), 22.5–36.3 (32”C), 53.4,
65.8, 68.2, 69.0, 71.2, 71.9, 72.8, 73.0, 103.8, 129.3, 129.7 (2”C), 134.1,
174.8 ppm; ESI-MS (negative-ion mode): m/z (%): 888.9 (100) [MÀNa]À;
elemental analysis calcd (%) for C48H90NNaO11S (912.28): C 63.19, H
9.94, N 1.54; found: C 63.03, H 9.80, N 1.50.
ACHTREUNG
give compound 14 (0.033 g, 66%) as an amorphous white solid. Rf
=
0.26 (CH2Cl2/MeOH 8:2); [a]2D0 = À1.6 (c = 0.75 in chloroform/metha-
ACHTREUNG
1
dry diethyl ether (1 mL), and MeNO2 (0.20 mL) was added to this bipha-
sic solution. The resulting (one-phase) solution was added under argon to
a stirred solution of compound 1 (0.20 g, 0.13 mmol) in diethyl ether
(2 mL). The reaction mixture was stirred for 3 h at room temperature
nol 1:1); H NMR (CDCl3/CD3OD 1:1): d = 0.87 (t, J = 6.5 Hz, 6H; 2”
CH3), 1.18–1.43 (m, 54H; 27”CH2), 1.53–1.63 (m, 2H; CH2), 1.97–2.06
(m, 6H; 3”CH2C=C), 2.18 (t, J = 7.5 Hz, 2H; CH2CO), 3.54–3.60 (m,
2H; H-1a, -2’), 3.62–3.68 (m, 2H; H-3’, -5’), 3.72 (dd, J5’,6a’ = 6.5, J6a’,6b’
Chem. Eur. J. 2006, 12, 5587 – 5595
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5593

F. Compostella et al.
= 11.0 Hz, 1H; H-6a’), 3.83 (dd, J5’,6b’ = 7.5 Hz, J6a’,6b’ = 11.0 Hz, 1H;
H-6b’), 3.95–4.00 (m, 1H; H-2), 4.08 (t, J2,3 = J3,4 = 7.5 Hz, 1H; H-3),
4.15 (dd, J1a,1b = 10.0, J1b,2 = 4.5 Hz, 1H; H-1b), 4.24 (d, J1’,2’ = 7.5 Hz,
1H; H-1’), 4.73 (brd, J3’,4’ = 3.0 Hz, 1H; H-4’), 5.29–5.37 (m, 2H; CH=
CH), 5.43 (ddt, J3,4 = 7.5, J4,5 = 15.0, Jall = 1 Hz, 1H; H-4), 5.69 ppm
(dt, J4,5 = 15.0, J5,6 = 6.7 Hz, 1H; H-5); 13C NMR (CDCl3/CD3OD 1:1):
d = 13.7 (2”C), 22.5–36.3 (32”C), 53.3, 60.0, 68.9, 71.6, 71.7, 72.5, 74.0,
74.8, 103.6, 129.3, 129.7 (2”C), 134.1, 174.7 ppm; ESI-MS (negative-ion
mode): m/z (%): 888.9 (100) [MÀNa]À; elemental analysis calcd (%) for
C48H90NNaO11S (912.28): C 63.19, H 9.94, N 1.54; found: C 63.30, H 9.99,
N 1.58.
pound (0.037 g, 75%) as a white amorphous solid after purification by
flash chromatography (CH2Cl2/MeOH 9:1). Rf = 0.05 (CH2Cl2/MeOH
9:1); [a]2D0
=
À2.2 (c
=
0.5 in chloroform/methanol 1:1); 1H NMR
(CDCl3/CD3OD 1:1): d = 0.87 (t, J = 6.5 Hz, 6H; 2”CH3), 1.17–1.47
(m, 54H; 27”CH2), 1.51–1.63 (m, 2H; CH2), 1.93–2.08 (m, 6H; 3”
CH2C=C), 2.16–2.34 (m, 2H; CH2CO), 3.46 (dd, J1a,1b
= 9.5, J1a,2 =
3 Hz, 1H; H-1a), 3.50 (t, J5’,6a’ = J5’,6b’ = 5.8 Hz, 1H; H-5’), 3.67–3.86 (m,
3H; H-3’, -6a’, -6b’), 3.88–3.93 (m, 1H; H-2), 3.95 (d, J3’,4’ = 3.0 Hz, 1H;
H-4’), 4.12 (t, J2,3 = J3,4 = 7.5 Hz, 1H; H-3), 4.32–4.41 (m, 3H; H-1b, -1’,
-2’), 5.29–5.37 (m, 2H; CH=CH), 5.42 (ddt, J3,4 = 7.5, J4,5 = 15.0, Jall
=
1 Hz, 1H; H-4), 5.69 ppm (dt, J4,5 15.0, J5,6 6.5 Hz, 1H; H-5);
=
=
13C NMR (CDCl3/CD3OD 1:1): d = 13.5 (2”C), 22.3–36.3 (32”C), 52.8,
61.2, 68.8, 68.9, 71.1, 72.5, 74.3, 77.8, 101.9, 129.3, 129.7 (2”C), 133.8,
174.7 ppm; ESI-MS (negative-ion mode): m/z (%): 888.9 (100) [MÀNa]À
; elemental analysis calcd (%) for C48H90NNaO11S (912.28): C 63.19, H
9.94, N 1.54; found: C 63.30, H 9.98, N 1.56.
ACHTREUNG
ACHTREUNG
A
A
hydrazine acetate (0.060 g, 0.65 mmol) in methanol (0.39 mL) was added
to a stirred solution of compound 1 (0.20 g, 0.13 mmol) in dichlorome-
thane (13 mL) and the reaction mixture was kept stirring at room tem-
perature for 6 h (TLC: dichloromethane/AcOEt 9:1). Water (15 mL) was
added and, after separation, the aqueous layer was extracted with
Biological assay: All experiments were performed in RPMI 1640 medium
containing glutamine (2 mm), Na pyruvate (1 mm) and non-essential
amino acids (1%) (all from Cambrex, Verviers, Belgium), Kanamycin
(Gibco, 100 mgmLÀ1) and AB human serum (5%, Swiss Red Cross,
Bern).
dichloromethane (2”15 mL). The combined organic layers were dried,
G
concentrated and purified by flash chromatography (hexane/AcOEt
7.5:2.5) to yield 15 (0.15 g, 82%) as an oil. Rf = 0.25 (hexane/AcOEt
7.5:2.5); [a]2D0 = À8.0 (c = 1 in chloroform); 1H NMR (CDCl3): d=
À0.09 (s, 9H; (CH3)3Si), 0.74–0.81 (m, 2H; SiCH2), 0.86 (t, J = 6.5 Hz,
6H; 2”CH3), 1.02 (s, 9H; (CH3)3CSi), 1.17–1.37 (m, 54H; 27”CH2),
1.50–1.60 (m, 2H; CH2), 1.92–2.04 (m, 6H; 3”CH2C=C), 2.04–2.14 (m,
2H; CH2CONH), 3.23 (dd, J2’,3’ = 9.5, J3’,4’ = 2.5 Hz, 1H; H-3’), 3.32 (t,
J5’,6a’ = J5’,6b’ = 6.5 Hz, 1H; H-5’), 3.46–3.53 (m, 1H; CHaHbOCH2O),
3.55–3.64 (m, 2H; H-1a and CHaHbOCH2O), 3.74–3.82 (m, 6H; H-2’,
-6a’, -6b’ and OCH3), 4.03–4.08 (m, 2H; H-1b, -4’), 4.13 (d, J1’,2’ =7.7 Hz,
The sulfatide-specific CD1a-restricted K34B9.1 T cell clone was establish-
ed and maintained as previously described.[9]
Human promyelocytic cell line THP-1 expressing CD1a was obtained by
transfection with human CD1a cDNA. The surface expression of CD1a
molecules was tested with a specific mAb (OKT6, ATCC CRL8019,
American Type Culture Collection).
DCs from healthy donors were isolated from peripheral blood mononu-
clear cells by culturing in the presence of GM-CSF and IL-4. Each prepa-
ration of DC was tested for the surface expression of CD1a molecules
with the OKT6 mAb.
1H; H-1’), 4.39–4.46 (m, 1H; H-2), 4.55 (d,
OCHaHbPh), 4.69 (d, J 7.0 Hz, 1H; CH2OCHaHbO), 4.73 (d, J=
12.0 Hz, 1H; OCHaHbPh), 4.84 (d, J 7.0 Hz, 1H; CH2OCHaHbO),
J = 12.0 Hz, 1H;
=
Synthetic compounds for antigen presentation assays were firstly dis-
solved in a chloroform/methanol 1:1 mixture and then dried under
stream of nitrogen. The dried material was re-dissolved in water and so-
nicated.
=
5.29–5.37 (m, 2H; CH=CH), 5.39–5.46 (brdd, J3,4 = 7.5, J4,5 = 15.5 Hz,
1H; H-4), 5.49 (t, J2,3 = J3,4 = 7.5 Hz, 1H; H-3), 5.80 (dt, J4,5 = 15.5,
J
5,6 =6.5 Hz, 1H; H-5), 6.26 (d, J2,NH = 9.5 Hz, 1H; NH), 6.84–6.88 (m,
DCs (2”104 per well) or CD1a-transfected THP-1 cells (4”104 per well)
2H; arom.), 7.25–7.42 (m, 10H; arom.), 7.47–7.53 (m, 1H; arom.), 7.57–
7.64 (m, 4H; arom.), 7.95–8.00 ppm (m, 2H; arom.); 13C NMR (CDCl3):
d = À0.8 (3”C), 14.8 (2”C), 18.5–37.6 (37”C), 52.1, 55.9, 63.4, 66.1,
69.7, 71.0, 71.8, 72.1, 75.4, 76.0, 81.4, 96.0, 105.4, 114.6 (2”C), 125.4–137.7
(25”C), 160.0, 166.1, 174.0 ppm; ESI-MS (positive-ion mode): m/z (%):
1424.7 (100) [M+Na]+; elemental analysis calcd (%) for C85H135NO11Si2
(1403.15): C 72.76, H 9.70, N 1.00; found: C 72.90, H 9.78, N 1.03.
were preincubated for 2 h at 378C with sonicated antigens (15 mgmLÀ1
)
before addition of K34B9.1 T cell clone (5”104 per well in triplicate). Su-
pernatants were harvested after 36 h and released cytokines were meas-
ured by ELISA. TNF-a was detected by use of sandwich ELISA kits ac-
cording to manufacturerꢁs instruction (Instrumentation Laboratory,
Schlieren, CH) and IL-4 was detected by use of anti-IL-4 mAbs (BD
PharMingen). Data are expressed as meansÆSDs (ngmLÀ1) of triplicates.
All experiments were repeated at least three times.
A
oxysulfonyl)-b-d-galactopyranosyloxy]-2-
solution of com-
A
pound 15 (0.14 g, 0.10 mmol) in dry DMF (2 mL) was treated with sulfur
trioxide/trimethylamine complex (0.042 g, 0.30 mmol), and the resulting
mixture was stirred at 408C for 4 days. The mixture was concentrated to
a few microlitres under reduced pressure (high vacuum pump) and puri-
fied by flash chromatography (CH2Cl2/MeOH 9.5:0.5) to afford the 2-sul-
Acknowledgements
fated product (0.092 g, 62%) as
CD3OD 2:1):
À0.16 (s, 6H; (CH3)2SiCH3), 0.07 (s, 3H;
a
colourless oil. 1H NMR (CDCl3/
This work was supported by MIUR-Italy (COFIN 2004: “Structure and
synthesis of glycolipids”; FIRB 2001: “Development of new immune re-
sponse modifiers and of peptide and DNA vaccines for tuberculosis im-
munotherapy”), the Swiss National Foundation (grant No. 3100 A0–
109918/1) and the Swiss Multiple Sclerosis Society.
d
=
(CH3)2SiCH3), 0.60–0.74 (m, 2H; SiCH2), 0.87 (t, J = 6.5 Hz, 6H; 2”
CH3), 0.96 (s, 9H; (CH3)3CSi), 1.15–1.36 (m, 54H; 27”CH2), 1.55–1.65
(m, 2H; CH2), 1.86–1.96 (m, 2H; CH2C=C), 1.97–2.05 (m, 4H; 2”
CH2C=C), 2.22–2.32 (m, 2H; CH2CONH), 3.28–3.46 (m, 5H; H-1a, -3’,
-5’ and CH2OCH2O), 3.65–3.73 (m, 2H; H-6a’, 6b’), 3.77 (s, 3H; OCH3),
3.82 (brd, J3’,4’ = 3.0 Hz, 1H; H-4’), 4.16–4.23 (m, 2H; H-1b, -1’), 4.33–
4.38 (m, 1H; H-2), 4.48 (dd, J1’,2’ = 8.0, J2’,3’ = 10.0 Hz, 1H; H-2’), 4.59
[1]a) F. Compostella, L. Franchini, G. De Libero, G. Palmisano, F. Ron-
chetti, L. Panza, Tetrahedron 2002, 58, 8703–8708; b) L. Franchini,
F. Compostella, A. Donda, L. Mori, D. Colombo, G. De Libero, P.
Matto, F. Ronchetti, L. Panza, Eur. J. Org. Chem. 2004, 4755–4761.
[2]G. De Libero, L. Mori, Nat. Rev. Immunol. 2005, 5, 485–496.
[3]a) Z. Zeng, A. R. Castano, B. W. Segelke, E. A. Stura, P. A. Peter-
son, I. A. Wilson, Science 1997, 277, 339–345; b) S. D. Gadola, N. R.
Zaccai, K. Harlos, D. Shepherd, J. C. Castro-Palomino, G. Ritter,
R. R. Schmidt, E. Y. Jones, V. Cerundolo, Nat. Immunol. 2002, 3,
721–726; c) D. M. Zajonc, M. A. Elsliger, L. Teyton, I. A. Wilson,
Nat. Immunol. 2003, 4, 808–815; d) T. Batuwangala, D. Shepherd,
(d,
J
=
7.0 Hz, 1H; CH2OCHaHbO), 4.62 (d,
J = 12.0 Hz, 1H;
OCHaHbPh), 4.75 (d, J
=
7.0 Hz, 1H; CH2OCHaHbO), 4.79 (d, J
=
12.0 Hz, 1H; OCHaHbPh), 5.29–5.42 (m, 3H; CH=CH and H-4), 5.58 (t,
J2,3 = J3,4 = 7.7 Hz, 1H; H-3), 5.83 (dt, J4,5 = 15.0, J5,6 = 6.5 Hz, 1H; H-
5), 6.83–6.88 (m, 2H; arom.), 7.16–7.60 (m, 15H; arom.), 7.90 (d, J2,NH
=
9.5 Hz, 1H; NH), 7.99–8.04 (m, 2H; arom.) ppm; ESI-MS (negative-ion
mode): m/z (%): 1480.9 (100) [MÀH]À.
The 2-sulfated compound (0.080 g, 0.054 mmol) was deprotected by the
procedure used to synthesize compound 14 and afforded the title com-
5594
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Chem. Eur. J. 2006, 12, 5587 – 5595

Synthesis of Sulfated Galactocerebrosides
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