Immunomodulatory α-Galactoglycosphingolipids: Synthesis of a 2′-O-Methyl-α-Gal-GSL and Evaluation of Its Immunostimulating Capacity

Article abstract of DOI:10.1002/ejoc.200300512

The total synthesis of 1-2-docosanoylamino-O-(2-O-methyl-α -D-galactopyranosyl)-1,3,4-octadecanetriol (2), a 2′-methoxy analog of the immunostimulating α-galactoglycosphingolipid 1, is reported. Stereoselective α-glycosylation of the azido precursor of sp

Full text of DOI:10.1002/ejoc.200300512

FULL PAPER
Immunomodulatory α-Galactoglycosphingolipids: Synthesis of a 2Ј-O-Methyl-
α-Gal-GSL and Evaluation of Its Immunostimulating Capacity
Lucia Barbieri,[a] Valeria Costantino,*[a] Ernesto Fattorusso,[a] Alfonso Mangoni,[a]
Elisabetta Aru,[b] Silvia Parapini,[b] and Donatella Taramelli[b]
Keywords: Immunomodulatory activity / Glycolipids / Glycosylation / Sphingolipids / Total synthesis
The total synthesis of 1-2-docosanoylamino-O-(2-O-methyl-
sayed in a 72 h splenocyte proliferation test, compound 2 was
α- -galactopyranosyl)-1,3,4-octadecanetriol (2), a 2Ј-methoxy significantly less stimulatory than the non-methylated com-
D
analog of the immunostimulating α-galactoglycosphingolipid
1, is reported. Stereoselective α-glycosylation of the azido
precursor of sphingosine was successfully performed for the
pound 1, suggesting that the galactose 2-OH group is essen-
tial for the immunostimulatory activity of α-Gal-GSLs.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
first time using an improved Mukaiyama reaction. When as- Germany, 2004)
α-Galactoglycosphingolipids (α-Gal-GSLs) are glycos- tose 2-OH group, although some subsequent results ob-
phingolipids, found in Agelas and Axinella sponges,[1Ϫ4] tained by other researchers seemed to be in contrast to
having an α-galactose as the first sugar attached to the cera- these findings. This inconsistency was solved by a recent
mide headgroup. The simplest natural α-Gal-GSL, named study,[8] demonstrating that α-Gal-GSLs glycosylated at the
agelasphin, shows a considerable immunostimulating ac- 2Ј- or 3Ј-positions are not active themselves, but are pro-
tivity, being the target of the T-cells response.[5] A synthetic cessed by the antigen presenting cells (APCs) with removal
analog, KRN7000 (1; Scheme 1), is now in clinical trials of the sugars linked to the galactose. A lysosomal enzyme,
as an antitumor agent.[6] In the last few years, it has been α-galactosidase A, was found to be responsible for the gen-
demonstrated that the cell-mediated immune recognition of eration of the antigenic epitope recognized by T lympho-
α-Gal-GSLs involves lipid-binding molecules known as cytes.
CD1, a family of HLA-like, β2-microglobulin-associated
The lack of activity of glycosylated α-Gal-GSLs could be
ascribed to either the steric hindrance of the additional su-
gar preventing binding to the T-cell receptor, or direct par-
ticipation of the galactose 2-OH group in the binding to the
receptor. In an attempt to clarify this point, we undertook a
synthetic program directed to the preparation of a number
of analogs modified at the key 2Ј-position in a way that
cannot be affected by APC processing.
transmembrane proteins.[7]
In this paper, we report the stereoselective total synthesis
of the 2Ј-methoxy analog of the α-Gal-GSL, 2Ј-O-methyl-
α--galactopyranosylceramide (2; Scheme 1), and a prelimi-
nary evaluation of its biological activity using lymphocyte
proliferation tests.
Scheme 1. KRN7000 (1) and its 2Ј-methoxy analog 2
As part of our ongoing search for immunomodulating
compounds, we discovered a number of more complex α-
Gal-GSLs, which were tested on murine T-cell popu-
lations.[4] The obtained data suggested that the immunosti-
mulating activity is reduced by glycosylation of the galac-
Results and Discussion
[a]
`
Dipartimento di Chimica delle Sostanze Naturali, Universita di
The synthesis of glycosphingolipids involves the assembly
of three fragments: a saccharide chain, a sphingoid base,
and a fatty acid. A common strategy begins with the prep-
aration of the protected saccharide moiety, followed by at-
tachment of a sphingosine synthetic precursor, and N-acyl-
ation with the fatty acid to give the glycosyl ceramide. It is
well established that the O-glycosylation reaction can be
Napoli ‘‘Federico II’’,
via D. Montesano 49, 80131 Napoli, Italy
Fax: (internat.) ϩ39-081-7486-552
E-mail: valeria.costantino@unina.it
[b]
`
Istituto di Microbiologia, Universita di Milano,
Via Pascal 36, 20133 Milano, Italy
Fax: (internat.) ϩ39-02-5031-5076
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
468
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200300512
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Immunomodulatory α-Galactoglycosphingolipids
FULL PAPER
more suitably performed using the azido precursor of the anhydrous conditions. The reagent was added to a dichloro-
sphingosine, rather than the sphingosine itself or the ceram- methane solution containing the galactal 5 and an excess of
ide as glycosyl acceptor, because the former is a better nu- methanol. After SiO2 chromatography, the desired methyl
cleophile.[9]
galactoside 6 (deriving from the α-oriented oxirane ring)
In the synthesis of a GSL with an α-glycosidic bond, the was obtained in 56% yield as a mixture of anomers (β:α ϭ
need for a 1,2-cis glycosylation reaction with complete 5:1), along with minor amounts of the α-talo stereoisomer
stereoselectivity is the most important aspect. While forma- from the β-oriented oxirane ring.
tion of 1,2-trans glycosides (β for glucose and galactose, α
for mannose) can be easily achieved taking advantage of
the participation of a neighboring group, such as O-acetyl
or O-benzoyl, at C-2, no successful general method for 1,2-
cis glycosylation has yet emerged. Each particular case thus
requires a careful selection of the available techniques.
Among them, the glycosylation reaction first proposed by
Mukaiyama[10] in 1981 is worthy of note, as it produces α-
glycosides with high selectivity but only modest yields. This
reaction has previously been used in the synthesis of glycos-
phingolipids, giving the desired α-glycosyl ceramide with
36% yield.[11]
The original Mukayama glycosidation reaction involves
a glycosyl fluoride as the glycosyl donor, and AgClO4 and
SnCl2 as a Lewis acid catalytic system. An ion pair is postu-
lated[12] to be produced by the cationic anomeric center and
Scheme 3. Syntheses of 3,4,6-tri-O-benzyl-2-O-methyl--galactosyl
the perchlorate ion, with the perchlorate ion placed on the
less-hindered β face of the sugar so as to cause nucleophilic
attack at the α face. Over the years, several improved ver-
sions of the original Mukayama glycosidation reaction have
appeared in an attempt to improve the yield of the reaction.
acetate (3) and (2S,3S,4R)-2-azido-3,4-di-O-benzyl-1-O-trityl-1,3,4-
octadecanetriol (4); reagents: (a) NaH, DMF, 0 °C, then BnBr
(90%); (b) BzOOH/KF (1:2), CH2Cl2/MeOH, room temp. (56%);
(c) NaH, THF, 0 °C, then MeI (82%); (d) 1  aqueous TfOH/
AcOH, 80 °C (82%); (e) Ac2O, pyridine (quantitative); (f) TrCl,
DMAP, pyridine, 60 °C (85%)
One interesting modification has been proposed by Houdier
[13]
´
and Vottero,
involving the use of a glycosyl acetate as
Conversion of 6 into the corresponding 2-O-methyl de-
glycosyl donor, while the alcohol is activated with a trityl rivative 7 (82%) was accomplished by treatment with so-
group; the catalytic system is similar, but SnCl4 is used in- dium hydride and methyl iodide under the usual conditions.
stead of SnCl2.
Cleavage of the glycosidic bond to afford 8 (82% yield, mix-
Even though this method has never been used for glycos- ture of anomers) was promoted by treatment with trifluoro-
phingolipid preparation, we selected it for the crucial glyco- methanesulfonic acid (1  solution in water) and glacial
sylation step (Scheme 2) because the reported yields ap- acetic acid for two hours at 80 °C. Finally, the free 2-meth-
peared very interesting. In addition, the stereochemical out- oxysugar 8 was acetylated with acetic anhydride in pyridine
come of the reaction was not dependent on the anomeric to give the desired 3,4,6-tri-O-benzy-2-O-methylgalactopyr-
configuration of the glycosyl acetate used as substrate, and anosyl acetate 3 as a mixture of anomers (α:β ϭ 1:1) in
this allowed an easier preparation of the glycosyl donor 3.
The glycosyl donor 3,4,6-tri-O-benzyl-2-O-methylgalac-
quantitative yield.
The glycosyl acceptor 2-azido-3,4-O-benzyl-1-O-trityl-
topyranosyl acetate (3) was prepared as follows (Scheme 3). 1,3,4-octadecanetriol (4) was synthesized from the corre-
-Galactal was first converted into 3,4,6-tri-O-benzylgalac- sponding non-tritylated azide 9, prepared as described pre-
tal (5) and, after purification, converted into the 1,2-trans- viously.[15] Compound 9 was dissolved in pyridine and
methylglycoside 6 through a one-pot reaction involving treated with a 10-fold molar excess of trityl bromide in the
stereoselective α-epoxidation, followed by in situ opening of presence of p-(dimethylamino)pyridine at 60 °C giving,
the oxirane ring. The epoxidation was performed with after chromatography, compound 4 in 85% yield.
Camp’s reagent, i.e. m-chloroperoxybenzoic acid (MCPBA,
Having synthesized the two building blocks 3 and 4, we
3.6 equiv.) and KF (7.2 equiv.)[14] in dichloromethane under performed the coupling reaction by dissolving the glycosyl
Scheme 2. Retrosynthetic analysis of (2S,3S,4R)-2-docosanoylamino-1-O-(2-O-methyl-α--galactopyranosyl)-1,3,4-octadecanetriol (2)
Eur. J. Org. Chem. 2004, 468Ϫ473
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469

V. Costantino et al.
FULL PAPER
acceptor 4 (130 mg, 0.17 mmol) and the glycosyl donor 3 of the biological activity of compound 2 and other modified
(170 mg, 2.0 equiv.) in Et2O and adding the obtained solu- α-Gal-GSLs is in progress and will be reported elsewhere.
tion to the catalytic system, previously prepared by sus-
pending AgClO4 and SnCl4 in dry Et2O (Scheme 4). After
workup, isolation of the reaction product was performed
by preparative HPLC rather than column chromatography
because all the reaction products showed similar retention
times. The α-glycoside 10 was obtained in a satisfactory
69% yield, whereas only trace amounts of the β-isomer were
obtained. This is a significant improvement on the yields
reported so far for α-glycosylation reactions as applied to
the synthesis of glycosphingolipids.
Figure 1. (a) Dose-dependent proliferation of C57Bl/6 spleen cells
to the non-methylated α-Gal-GSL KRN7000 (1) or its 2Ј-methoxy
analog (2) after 72 hours of culture; the results are representative
of three different experiments conducted in quadruplicate; data are
expressed as mean Ϯ SD; (b) kinetics of the proliferative response
of C57Bl/6 spleen cells to KRN7000 (1, 0.01 µg/mL) or its 2Ј-meth-
oxy analog (2, 0.01 µg/mL); data are expressed as mean Ϯ SD
Scheme 4. Synthesis of compound 2; reagents: (a) AgClO4, SnCl4,
Et2O (69%); (b) Ph3SnH, AIBN, benzene, reflux (81%); (c)
C21H43COCl, pyridine/CH2Cl2 (84%); (d) H2, Pd(OH)2/C, EtOH/
AcOH, 40 °C (70%)
Experimental Section
General Remarks: High resolution ESI-MS spectra were performed
on a Micromass QTOF Micro mass spectrometer, dissolving the
sample in MeCN/H2O (1:1) with 0.1% TFA. ESI MS/MS experi-
Completion of the synthesis[15] required reduction of the
azide group with triphenyltin hydride, amidation with doco-
sanoyl chloride, and removal of the benzyl protecting trometer. The spectra were recorded by infusion into the ESI source
ments were performed on a Finnigan LCQ ion-trap mass spec-
using MeOH/CHCl3 (4:1) as the solvent. Optical rotations were
groups by hydrogenolysis over Pd/C, to afford the target
compound 2 in 47% yield over the three steps, and 28%
overall yield based on the azidosphingosine 9 used as start-
ing material.
Preliminary evaluation of the biological activity of com-
pound 2 compared to the non-methylated α-Gal-GSL
KRN7000 (1) was performed using a splenocyte prolifer-
ation test. As expected, compound 1 stimulated spleen-cell
proliferation from C57Bl/6 mice in a dose- and time-depen-
measured at 589 nm on a PerkinϪElmer 192 polarimeter using a
1
10-cm microcell. H and 13C NMR spectra were determined on a
Bruker AMX-500 spectrometer at 500.13 and 125.77 MHz, respec-
tively; chemical shifts are referenced to the residual solvent signal
(CDCl3: δH ϭ 7.26 ppm; δC ϭ 77.0 ppm; [D5]pyridine: δH ϭ 8.71,
7.56, and 7.19 ppm; δC ϭ 149.8, 135.3, and 123.4 ppm). The spectra
of new compounds were assigned with the aid of COSY and
HMQC two-dimensional NMR experiments. The reverse multiple-
quantum heteronuclear correlation (HMQC) spectra were recorded
dent manner (Figure 1). The activity peaked after 72 h of by using a pulse sequence with a BIRD pulse 0.5 s before each scan
to suppress the signal originating from protons not directly bound
incubation at 37 °C. The proliferative response was signifi-
1
to 13C; the interpulse delays were adjusted for an average JC,H
cantly reduced when the 2Ј-methoxy analog 2 was used.
of 142 Hz.
These results indicate that a free galactose 2-OH group is
essential for the activity of α-Gal-GSLs, and suggest that
this hydroxyl group is probably directly involved in the
3,4,6-Tri-O-benzyl-
was dissolved in dry DMF (10 mL), cooled to 0 °C, and NaH
D-galactal (5): -Galactal (250 mg, 1.71 mmol)
binding to the TCR receptor. A more extensive examination (170 mg, 6.7 mmol) was slowly added. After a few minutes, benzyl
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Eur. J. Org. Chem. 2004, 468Ϫ473

Immunomodulatory α-Galactoglycosphingolipids
FULL PAPER
bromide (0.70 mL, 5.9 mmol) was added dropwise. After stirring 4.58 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons),
for 12 h at room temperature, water (100 mL) was added and the
reaction mixture was extracted three times with EtOAc (150 mL).
7.41Ϫ7.25 (15 H, aromatic protons) ppm. 13C NMR (125 MHz,
CDCl3, β anomer): δ ϭ 56.9 (CH3, OMe), 60.9 (CH3, OMe), 68.7
The combined organic layers were dried with Na2SO4 and concen- (CH2, C-6), 72.8 (CH2, benzyl methylene group), 73.2 (CH, C-5),
trated under reduced pressure. The residue was purified by chroma-
tography on silica gel to give 640 mg of 5 (1.54 mmol, 90%) as a
colorless oil, identified by comparison of its 1H and 13C NMR
spectra with those reported previously.[16]
73.4 (CH, C-4), 73.5 (CH2, benzyl methylene group), 74.3 (CH2,
benzyl methylene group), 81.5 (CH, C-3), 104.5 (CH, C-1),
128.7Ϫ127.7 (CH, aromatic carbons) ppm.
3,4,6-Tri-O-benzyl-2-O-methyl-D-galactose (8): Compound
7
Methyl 3,4,6-Tri-O-benzyl-
D
-galactoside (6): Anhydrous KF
(340 mg, 0.71 mmol) was dissolved in glacial AcOH (2.5 mL),
heated to 80 °C, and an aqueous 1  solution of CF3SO3H (0.5
mL) was added. The reaction mixture was stirred at 80 °C for 2 h.
After cooling, a saturated aqueous NaHCO3 solution was added
until effervescence disappeared, and the reaction mixture was par-
titioned between water (30 mL) and DCM (50 mL). The organic
(450 mg, 7.7 mmol) was added to a DCM solution (25 mL) of
freshly recrystallized m-chloroperoxybenzoic acid (640 mg,
3.7 mmol), previously dried over Na2SO4 and CaSO4 (Sikkon,
Fluka) for 20 min, and the suspension was stirred at room tempera-
ture for 30 min. After this time, a solution of compound 5 (640 mg,
1.54 mmol) in dichloromethane (DCM; 5 mL) and MeOH (5 mL) layer was washed with water (2 ϫ 30 mL), and the combined aque-
was added, and the reaction was allowed to proceed for 12 h at
room temperature. Water (200 mL) was added to the reaction mix-
ous layers were in turn washed with EtOAc (100 mL). All the or-
ganic layers were combined, dried over Na2SO4, and concentrated
ture, which was extracted with DCM (3 ϫ 300 mL). The combined under vacuum, Column chromatography of the residue (n-hexane/
organic extracts were dried over Na2SO4, concentrated under re-
duced pressure, and purified by column chromatography on SiO2
(n-hexane/EtOAc, 8:2) to give 400 mg (0.86 mmol, 56%) of 6 (mix-
EtOAc, 7:3) yielded 270 mg (0.58 mmol, 82%) of 8 (mixture of ano-
mers, α:β ϭ 3:1) as a colorless oil. [α] ϭ ϩ20 (CHCl3, c ϭ 0.2).
ESIMS (positive ions): m/z ϭ 508 [M Ϫ Hϩ2Na]ϩ, 487 [M ϩ
1
ture of anomers, α:β ϭ 1:5) as a colorless oil. [α] ϭ ϩ2.9 (CHCl3, Na]ϩ. H NMR (500 MHz, CDCl3, α anomer): δ ϭ 3.43 (dd, J ϭ
1
c ϭ 0.6). ESIMS (positive ions): m/z ϭ 487 [M ϩ Na]ϩ. H NMR
9.4, 5.9 Hz, 1 H, 6-Hb), 3.55 (m, 1 H, 6-Ha), 3.57 (s, 3 H, OMe),
(500 MHz, CDCl3, β anomer): δ ϭ 3.44 (dd, J ϭ 9.8, 2.7 Hz, 1 H, 3.82 (dd, J ϭ 9.6, 3.6 Hz, 1 H, 2-H), 3.88 (m, 1 H, 3-H), 3.90 (br.
3-H), 3.54 (s, 3 H, OMe), 3.65Ϫ3.67 (overlapping signals, 3 H, 5-
H and 6-H2), 3.97Ϫ3.91 (overlapping signals, 2 H, 2-H and 4-H),
4.19 (d, J ϭ 7.6 Hz, 1 H, 1-H), 4.48 and 4.44 (AB system, J ϭ
11.7 Hz, 1 H each, geminal benzyl protons), 4.73 and 4.64 (AB
system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 4.88 and
4.61 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons),
7.38Ϫ7.23 (15 H, aromatic protons) ppm. 13C NMR (125 MHz,
CDCl3, β anomer): δ ϭ 56.9 (CH3, OMe), 68.7 (CH2, C-6), 71.2
(CH, C-2), 72.3 (CH2, benzyl methylene group), 72.7 (CH, C-4),
73.6 (CH2, benzyl methylene group), 73.7 (CH, C-5), 74.6 (CH2,
benzyl methylene group), 82.0 (CH, C-3), 104.0 (CH, C-1),
128.5Ϫ127.7 (CH, aromatic carbons) ppm.
s, 1 H, 4-H), 4.17 (t, J ϭ 6.3 Hz, 1 H, 5-H), 4.50 and 4.41 (AB
system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 4.81 and
4.73 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons),
4.95 and 4.59 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl
protons), 5.42 (d, J ϭ 3.1 Hz, 1 H, 1-H), 7.43Ϫ7.26 (15 H, aromatic
protons) ppm.
3,4,6-Tri-O-benzyl-2-O-methyl-D-galactosyl Acetate (3): Com-
pound 8 (270 mg, 0.58 mmol) was dissolved in pyridine (2.0 mL)
and Ac2O (0.2 mL) was added. After 12 h, the reaction was
quenched with MeOH, and after a further 30 min the mixture was
dried under reduced pressure, to give 295 mg (0.58 mmol, quanti-
tative) of 3 (mixture of anomers, α:β ϭ 1:1) as a colorless oil. [α]D ϭ
ϩ22 (CHCl3, c ϭ 1.4). ESIMS (positive ions): m/z ϭ 529 ([M ϩ
Na]ϩ. 1H NMR (CDCl3, α anomer): δ ϭ 2.12 (s, 3 H, Ac), 3.58 (s,
3 H, OMe), 3.63Ϫ3.50 (overlapping signals, 3 H, 3-H and 6-H2),
3.69Ϫ3.65 (overlapping signals, 2 H, 2-H and 5-H), 3.94 (br. d, J ϭ
2.7 Hz, 1 H, 4-H), 4.43 and 4.39 (AB system, J ϭ 11.7 Hz, 1 H
each, geminal benzyl protons), 4.74 and 4.72 (AB system, J ϭ
11.7 Hz, 1 H each, geminal benzyl protons), 4.93 and 4.61 (AB
system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 5.48 (d,
J ϭ 8.1 Hz, 1 H, 1-H), 7.41Ϫ7.24 (m, 15 H, aromatic protons)
Methyl 3,4,6-Tri-O-benzyl-2-O-methyl-D-galactoside (7): Com-
pound 6 (400 mg, 0.86 mmol) was dissolved in dry THF (15 mL),
cooled to 0 °C, and NaH (100 mg, 4.2 mmol) was added. After a
few minutes methyl iodide (0.25 mL, 4.0 mmol) was added drop-
wise. After stirring for 12 h at room temperature, water (100 mL)
was added and the reaction mixture was extracted three times with
EtOAc (150 mL). The combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure. The residue was
purified by column chromatography on SiO2 (n-hexane/EtOAc, 9:1)
to give 340 mg (0.71 mmol, 82%) of 7 (mixture of anomers, α:β ϭ
1:5) as a colorless oil. [α] ϭ ϩ6.5 (CHCl3, c ϭ 0.6). ESIMS
(positive ions): m/z ϭ 501 [M ϩ Na]ϩ. 1H NMR (500 MHz,
1
ppm. H NMR (CDCl3, β anomer): δ ϭ 2.12 (s, 3 H, Ac), 3.50 (s,
3 H, OMe), 3.55 (m, 2 H, 6-H2), 3.81 (dd, J ϭ 10.1, 2.7 Hz, 1 H,
3-H), 3.92 (dd, J ϭ 10.1, 3.7 Hz, 1 H, 2-H), 4.00 (m, 1 H, 5-H),
CDCl3, β anomer): δ ϭ 3.44 (dd, J ϭ 9.7, 2.7 Hz, 1 H, 3-H), 3.53 4.02 (br. d, J ϭ 2.7 Hz, 1 H, 4-H), 4.46 and 4.40 (AB system, J ϭ
(s, 3 H, OMe), 3.53Ϫ3.50 (overlapping signals, 2 H, 2-H and 5-H), 11.7 Hz, 1 H each, geminal benzyl protons), 4.82 and 4.72 (AB
3.59 (m, 2 H, 6-H2), 3.64 (s, 3 H, OMe), 3.87 (br. d, J ϭ 2.1 Hz, 1
H, 4-H), 4.18 (d, J ϭ 7.6 Hz, 1 H, 1-H), 4.45 and 4.42 (AB system,
system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 4.95 and
4.58 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons),
J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 4.76 and 4.72 (AB 6.39 (d, J ϭ 3.7 Hz, 1 H, 1-H), 7.41Ϫ7.24 (m, 15 H, aromatic
system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 4.94 and
4.62 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons),
7.41Ϫ7.25 (15 H, aromatic protons) ppm. 1H NMR (500 MHz,
CDCl3, α anomer): δ ϭ 3.42 (s, 3 H, OMe), 3.55 (m, 2 H, 6-H2),
protons) ppm. 13C NMR (125 MHz, CDCl3, α anomer): δ ϭ 21.2
(CH3, Ac), 60.9 (CH3, OMe), 67.9 (CH2, C-6), 72.8 (CH2, benzyl
methylene group), 73.1 (CH, C-4), 73.4 (CH2, benzyl methylene
group), 74.0 (CH, C-5), 74.6 (CH2, benzyl methylene group), 79.8
3.56 (s, 3 H, OMe), 3.84 (dd, J ϭ 9.7, 3.1 Hz, 1 H, 2-H), 3.87 (dd, (CH, C-2), 82.1 (CH, C-3), 94.3 (CH, C-1), 128.4Ϫ127.2 (CH, aro-
J ϭ 10.0, 2.4 Hz, 1 H, 3-H), 3.90 (m, 1 H, 5-H), 3.93 (br. s, 1 H,
matic carbons) ppm. 13C NMR (125 MHz, CDCl3, β anomer): δ ϭ
4-H), 4.50 and 4.42 (AB system, J ϭ 11.7 Hz, 1 H each, geminal 21.2 (CH3, Ac), 59.3 (CH3, OMe), 68.3 (CH2, C-6), 71.8 (CH, C-5),
benzyl protons), 4.82 and 4.71 (AB system, J ϭ 11.7 Hz, 1 H each,
geminal benzyl protons), 4.91 (d, J ϭ 3.1 Hz, 1 H, 1-H), 4.96 and
73.0 (CH2, benzyl methylene group), 73.5 (CH2, benzyl methylene
group), 74.5 (CH, C-4), 74.9 (CH2, benzyl methylene group), 77.5
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V. Costantino et al.
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(CH, C-2), 78.5 (CH, C-3), 90.3 (CH, C-1), 128.4Ϫ127.2 (CH, aro-
matic carbons) ppm.
CH2 group), 73.0 (CH2, benzyl methylene group), 73.4 (CH2,
benzyl methylene group), 73.7 (CH2, benzyl methylene group), 74.7
(CH2, benzyl methylene group), 75.2 (CH, C-4Ј), 78.5 (CH, C-2Ј),
78.7 (CH, C-3Ј), 79.2 (CH, C-3), 79.5 (CH, C-4), 98.2 (CH, C-1Ј),
128.5Ϫ127.7 (CH, aromatic carbons), 138.8Ϫ138.1 (C, aromatic
carbons) ppm.
(2S,3S,4R)-2-Azido-3,4-di-O-benzyl-1-O-trityl-1,3,4-octadecanetriol
(4): Compound 9 (104 mg, 0.20 mmol), prepared as described pre-
viously,[15] was dissolved in 3.0 mL of dry pyridine. 4-Dimethylami-
nopyridine (DMAP, 2.0 mg, 0.016 mmol) and triphenylbromo-
methane (trityl bromide, 650 mg, 2.0 mmol) were added, and the (2S,3S,4R)-2-Amino-3,4-di-O-benzyl-1-O-(2-O-methyl-3,4,6-tri-O-
reaction mixture was kept at 60 °C for 12 h. The reaction was
quenched with water (30 mL), and after 15 min the reaction mix-
benzyl-α-
D-galactopyranosyl)-1,3,4-octadecanetriol (11): Ph3SnH
(300 µL, 412 mg, 1.2 mmol) and a small amount of AIBN were
ture was extracted with DCM (3 ϫ 50 mL). The organic layers added to a solution of the azide 10 (114 mg, 0.12 mmol) in dry
were dried over Na2SO4, concentrated under vacuum and chroma-
tographed on SiO2 (n-hexane/EtOAc, 99:1), to gave 130 mg
benzene (10 mL), and the resulting solution was allowed to react
at room temperature for 24 h and subsequently for 1 h under reflux.
(0.17 mmol, 85%) of 4 as a colorless oil. [α]D ϭ ϩ12 (CHCl3, c ϭ The solution was cooled to room temperature and concentrated
1.1). ESIMS (positive ions): m/z ϭ 788 [M ϩ Na]ϩ, 760 [M ϩ Na
Ϫ N2]ϩ, 243 [trityl]ϩ. ESI MS/MS (parent ion: m/z ϭ 788): m/z ϭ
under reduced pressure. Column chromatography on SiO2 (n-hex-
ane/EtOAc, 6:4, with 0.1% pyridine) gave 90 mg (0.095 mmol, 81%)
760 [M ϩ Na Ϫ N2]ϩ; (parent ion: m/z ϭ 760): m/z ϭ 654 [M ϩ of 11 as a colorless oil. [α] ϭ ϩ20 (CHCl3, c ϭ 0.2). HRESIMS
1
Na Ϫ N2 Ϫ PhCHO]ϩ, m/z ϭ 654 [M ϩ Na Ϫ N2 Ϫ TrH]ϩ. H (positive ions): m/z ϭ 944.6071 ([M ϩ H]ϩ, C60H82NO8 gives
NMR (CDCl3): δ ϭ 0.88 (t, J ϭ 6.9 Hz, 3 H, 18-H3), 1.25 (alkyl
chain CH2 protons), 1.40 (m, 1 H, 6a-H), 1.50 (m, 1 H, 5b-H), 1.59 ϩ H]ϩ. ESI MS/MS (parent ion: m/z ϭ 966): m/z ϭ 874 [M ϩ Na
(m, 1 H, 5a-H), 3.36 (dd, J ϭ 10.0, 8.4 Hz, 1 H, 1b-H), 3.49 (dd,
Ϫ PhCH3]ϩ, 858 [M ϩ Na Ϫ PhCHOH]ϩ, 487 [sugar ϩ Na]ϩ. 1H
944.6040). ESIMS (positive ions): m/z ϭ 966 [M ϩ Na]ϩ, 944 [M
J ϭ 10.0, 2.8 Hz, 1 H, 1a-H), 3.56Ϫ3.51 (overlapping signals, 2 H, NMR (500 MHz, CDCl3): δ ϭ 0.88 (t, J ϭ 6.9 Hz, 3 H, H3Ϫ18),
3-H and 4-H), 3.76 (m, 1 H, 2-H), 4.41 (s, 2 H, benzyl protons), 1.25 (alkyl chain CH2 protons), 1.45 (m, 1 H, 6a-H), 1.59 (m, 1 H,
4.57 and 4.47 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl
5b-H), 1.69 (m, 1 H, 5a-H), 3.31 (m, 1 H, 2-H), 3.52 (s, 3 H, OMe),
protons), 7.46Ϫ7.06 (m, 25 H, aromatic protons) ppm. 13C NMR 3.55Ϫ3.45 (overlapping signals, 3 H, 1b-H, 6-H2), 3.67 (m, 1 H, 3-
(125 MHz, CDCl3): δ ϭ 14.0 (CH3, C-18), 22.6 (CH2, C-17), 25.4 H), 3.71 (m, 1 H, 4-H), 3.83 (dd, J ϭ 10.1, 3.4 Hz, 1 H, 2Ј-H), 3.89
(CH2, C-6), 29.7Ϫ29.4 (CH2, alkyl chain CH2 groups), 30.0 (CH2,
C-5), 31.9 (CH2, C-16), 63.2 (CH, C-2), 64.3 (CH2, C-1), 72.0 (CH2,
benzylic CH2 group), 73.5 (CH2, benzyl methylene group), 79.1
(CH, C-3), 79.4 (CH, C-4), 128.7Ϫ126.9 (CH, aromatic carbons)
ppm.
(dd, J ϭ 10.1, 2.5 Hz, 1 H, 3Ј-H), 3.97Ϫ3.92 (overlapping signals,
2 H, 4Ј-H and 5Ј-H), 4.08 (dd, J ϭ 9.9, 2.5 Hz, 1 H, 1a-H), 4.46
and 4.38 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl pro-
tons), 4.64 and 4.53 (AB system, J ϭ 11.7 Hz, 1 H each, geminal
benzyl protons), 4.73 and 4.63 (AB system, J ϭ 11.7 Hz, 1 H each,
geminal benzyl protons), 4.79 and 4.72 (AB system, J ϭ 11.7 Hz,
1 H each, geminal benzyl protons), 4.94 and 4.57 (AB system, J ϭ
11.7 Hz, 1 H each, geminal benzyl protons), 5.01 (d, J ϭ 3.4 Hz,
1 H, 1Ј-H), 7.42Ϫ7.25 (25 H, aromatic protons) ppm.
(2S,3S,4R)-2-Azido-3,4-di-O-benzyl-1-O-(2-O-methyl-3,4,6-tri-O-
benzyl-α-D-galactopyranosyl)-1,3,4-octadecanetriol (10): SnCl4 (50
µL from a 0.5  toluene solution, 0.025 mmol) was added to a
suspension of AgClO4 (7.0 mg, 0.034 mmol) in anhydrous Et2O (2
mL). The mixture was stirred for 4 h, and compound 3 (170 mg, (2S,3S,4R)-2-Docosanoylamino-3,4-di-O-benzyl-1-O-(2-O-methyl-
0.34 mmol) and compound 4 (130 mg, 0.17 mmol) were added sim- 3,4,6-tri-O-benzyl-α- -galactopyranosyl)-1,3,4-octadecanetriol (12):
ultaneously in an anhydrous Et2O solution (2 mL). After 14 h, the Docosanoic acid (100 mg, 0.29 mmol) dissolved in SOCl2 (0.63 mL,
D
reaction mixture was diluted with DCM (30 mL), washed with a
saturated NaHCO3 solution (20 mL), dried over Na2SO4, and
taken to dryness. The residue was chromatographed by HPLC (n-
hexane/EtOAc, 9:1) to gave 114 mg (0.12 mmol, 69%) of 10 as a
1.0 g, 8.6 mmol) was refluxed for 90 min, and the excess of SOCl2
removed under reduced pressure. A solution of the amine 11
(90 mg, 0.095 mmol) in dry pyridine (3 mL) and dry CH2Cl2 (3
mL) was added to the obtained docosanoyl chloride. After 12 h,
colorless oil. [α] ϭ ϩ26 (CHCl3, c ϭ 0.3). ESIMS (positive ions): the solvents were removed under vacuum, and the residue was par-
m/z ϭ 992 [M ϩ Na]ϩ, 964 [M ϩ Na Ϫ N2]ϩ. ESI MS/MS (parent titioned between CH2Cl2 (100 mL) and a saturated NaHCO3 aque-
ion: m/z ϭ 992): m/z ϭ 964 [M ϩ Na Ϫ N2]ϩ; (parent ion: m/z ϭ ous solution (50 mL). The organic phase, dried over Na2SO4 and
964): m/z ϭ 858 [M ϩ Na Ϫ N2 Ϫ PhCHO]ϩ. 1H NMR (500 MHz, concentrated under reduced pressure, was subjected to column
CDCl3): δ ϭ 0.88 (t, J ϭ 6.9 Hz, 3 H, H3Ϫ18)), 1.25 (alkyl chain chromatography on SiO2 (n-hexane/EtOAc, 8:2), giving 101 mg
CH2 protons), 1.40 (m, 1 H, 6a-H), 1.57 (m, 1 H, 5b-H), 1.67 (m, (0.080 mmol, 84%) of 12 as a colorless oil. [α] ϭ ϩ23 (CHCl3,
1 H, 5a-H), 3.49 (m, 2 H, 6-H2), 3.51 (s, 3 H, OMe), 3.60 (m, 1 H,
c ϭ 0.3). ESIMS (positive ions): m/z ϭ 1288 [M ϩ Na]ϩ. ESI MS/
4-H), 3.76Ϫ3.69 (overlapping signals, 3 H, 1b-H, 2-H, and 3-H), MS (parent ion: m/z ϭ 1288): m/z ϭ 824 [M ϩ Na Ϫ sugar]ϩ, 487
3.82 (dd, J ϭ 9.6, 3.5 Hz, 1 H, 2Ј-H), 3.95Ϫ3.86 (overlapping sig- [sugar ϩ Na]ϩ. H NMR (500 MHz, CDCl3): δ ϭ 0.88 (t, J ϭ 6.9
1
nals, 3 H, 3Ј-H, 4Ј-H, and 5Ј-H), 4.03 (m, 1 H, 1a-H), 4.44 and Hz, 6 H, 18-H3 and 22ЈЈЈ-H3), 1.25 (alkyl chain CH2 protons), 1.63
4.36 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons),
(overlapping signals, 3 H, 3ЈЈ-H2 and 5b-H), 1.69 (m, 1 H, 5a-H),
4.59 and 4.50 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl 2.34 (t, J ϭ 7.5 Hz, 2 H, 2ЈЈ-H2), 3.44 (dd, J ϭ 9.1, 6.8 Hz, 1 H,
protons), 4.71 and 4.66 (AB system, J ϭ 11.7 Hz, 1 H each, gemi-
nal benzyl protons), 4.81 and 4.70 (AB system, J ϭ 11.7 Hz, 1 H
each, geminal benzyl protons), 4.93 and 4.55 (AB system, J ϭ
6Јb-H), 3.50 (s, 3 H, OMe), 3.53Ϫ3.48 (overlapping signals, 2 H,
4-H and 6Јa-H), 3.77 (dd, J ϭ 10.9, 3.5 Hz, 1 H, 1b-H), 3.83Ϫ3.79
(overlapping signals, 2 H, 2Ј-H and 3Ј-H), 3.87 (dd, J ϭ 7.1, 2.0
11.7 Hz, 1 H each, geminal benzyl protons), 5.02 (d, J ϭ 3.5 Hz, Hz, 1 H, 3-H), 3.93Ϫ3.89 (overlapping signals, 2 H, 4Ј-H and 5Ј-
1 H, 1Ј-H), 7.40Ϫ7.23 (25 H, aromatic protons) ppm. 13C NMR
H), 4.01 (dd, J ϭ 10.9, 5.2 Hz, 1 H, 1a-H), 4.18 (m, 1 H, 2-H),
(125 MHz, CDCl3): δ ϭ 14.0 (CH3, C-18), 22.6 (CH2, C-17), 25.4 4.47 and 4.39 (AB system, J ϭ 11.7 Hz, 1 H each, geminal benzyl
(CH2, C-6), 29.7Ϫ29.4 (CH2, alkyl chain CH2 groups), 30.0 (CH2, protons), 4.62 and 4.47 (AB system, J ϭ 11.7 Hz, 1 H each,
C-5), 31.9 (CH2, C-16), 58.9 (CH3, OMe), 62.1 (CH, C-2), 68.6 geminal benzyl protons), 4.76 and 4.60 (AB system, J ϭ 11.7 Hz,
(CH2, C-1), 69.1 (CH2, C-6Ј), 69.8 (CH, C-5Ј), 72.1 (CH2, benzylic 1 H each, geminal benzyl protons), 4.82 and 4.55 (AB system, J ϭ
472
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 468Ϫ473

Immunomodulatory α-Galactoglycosphingolipids
FULL PAPER
11.7 Hz, 1 H each, geminal benzyl protons), 4.91 and 4.55 (AB
using the MTT assay already described.[17] Plates were incubated
system, J ϭ 11.7 Hz, 1 H each, geminal benzyl protons), 4.94 (br.
for 72 h at 37 °C in 5% CO2, then 20 µL of a 5 mg/mL solution of
s, 1 H, 1Ј-H), 6.22 (d, J ϭ 8.7 Hz, 1 H, 2-NH), 7.41Ϫ7.22 (25 H, 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium
bromide
aromatic protons) ppm. 13C NMR (125 MHz, CDCl3): δ ϭ 14.1
(MTT) (M-2128 Sigma) in PBS were added for an additional 3
(CH3, C-18 and C-22ЈЈ), 172.4 (C, C-1ЈЈ), 22.6 (CH2, C-17 and C- hours at 37 °C. The plates were then centrifuged, the supernatants
21ЈЈ), 24.9 (CH2, C-3ЈЈ), 30.0Ϫ29.1 (CH2, alkyl chain CH2 groups), discarded and the dark blue formazan crystals dissolved using 100
30.2 (CH2, C-5), 31.9 (CH2, C-16 and C-20ЈЈ), 34.1 (CH2, C-2ЈЈ), µL of lysing buffer consisting of 20% (w/v) of a solution of SDS
50.3 (CH, C-2), 59.2 (CH3, OMe), 69.1 (CH2, C-1), 69.2 (CH2, C- (Sigma), 40% of N,N-dimethyl formamide (Merck) in H2O, at pH
6Ј), 69.8 (CH, C-5Ј), 71.7 (CH2, benzylic CH2 group), 72.7 (CH2, 4.7 adjusted with 80% acetic acid. The plates were then read on a
benzylic CH2 group), 73.5 (CH2, benzylic CH2 group), 74.7 (CH,
microplate reader (Molecular Devices Co., Menlo Park, CA, USA)
C-4Ј), 74.7 (CH2, benzylic CH2 group), 78.4 (CH, C-3), 78.5 (CH, at a test wavelength of 550 nm and a reference wavelength of
C-2Ј or C-3Ј), 78.6 (CH, C-3Ј or C-2Ј), 80.2 (CH, C-4), 98.8 (CH, 690 nm. The results are expressed as OD 550/OD690 which mean
C-1Ј), 128.4Ϫ127.3 (CH, aromatic carbons), 138.8Ϫ138.1 (C, aro-
matic carbons) ppm.
that the values at OD 690 have been subtracted from the values
at OD 550. All the tests were performed at least three times in
quadruplicate and the statistical analysis was performed by one-
way ANOVA with Scheffe F-test post hoc; p values less than 0.05
were considered to be significant.
(2S,3S,4R)-2-Docosanoylamino-1-O-(2-O-methyl-α-D-galacto-
pyranosyl)-1,3,4-octadecanetriol (2): The protected glycosphingoli-
pid 12 (101 mg, 0.080 mmol) and Pd(OH)2/C (50 mg, 20% w/w)
were suspended in 95% EtOH (18 mL) and AcOH (2 mL). The
obtained mixture was hydrogenated at a pressure of 3 bar in a Parr
reactor for 48 h at 40 °C. The reaction mixture was filtered through
Celite and the filtrate washed with 95% EtOH and CHCl3. After
removal of the solvents, the combined organic extracts were puri-
fied by reversed-phase column chromatography on RP-18 silica gel
(MeOH/EtOAc, 95:5) to give 46 mg (0.056 mmol, 70%) of com-
pound 2 as an amorphous solid. [α] ϭ ϩ52 (CHCl3/MeOH, 1:1,
c ϭ 0.3). HRESIMS (positive ions): m/z ϭ 838.6777 ([M ϩ Na]ϩ,
C47H93NNaO9 gives 838.6748). ESI MS/MS (parent ion: m/z ϭ
838): m/z ϭ 820 [M ϩ Na Ϫ H2O]ϩ, 662 [M ϩ Na Ϫ (sugar Ϫ
H2O)]ϩ, 644 [M ϩ Na Ϫ sugar]ϩ, 512 [M ϩ Na Ϫ docosanoyl]ϩ.
1H NMR (500 MHz, [D5]pyridine): δ ϭ 0.83 (t, J ϭ 7.3 Hz, 6 H,
18-H3 and 22ЈЈ-H3), 1.31Ϫ1.20 (alkyl chain CH2 protons),
1.47Ϫ1.33 (overlapping signals, 3 H, 7a-H and 4ЈЈ-H2), 1.66 (m, 1
H, 6b-H), 1.81 (quintet, J ϭ 7.3 Hz, 2 H, 3ЈЈ-H2), 1.91 (m, 2 H,
5b-H and 6a-H), 2.21 (m, 1 H, 5a-H), 2.45 (t, J ϭ 7.3 Hz, 2 H,
2ЈЈ-H2), 3.58 (s, 3 H, OMe), 4.11 (dd, J ϭ 9.7, 3.3 Hz, 1 H, 2Ј-H),
4.24 (m, 1 H, 4-H), 4.33 (m, 1 H, 3-H), 4.43Ϫ4.37 (overlapping
signals, 4 H, 1b-H, 6-H2, and 3Ј-H, and 6Јa-H), 4.46 (m, 1 H, 5Ј-
H), 4.49 (br. s, 1 H, 4Ј-H), 4.63 (dd, J ϭ 10.5, 4.1 Hz, 1 H, 1a-H),
5.17 (m, 1 H, 2-H), 5.55 (d, J ϭ 3.3 Hz, 1 H, 1Ј-H), 8.60 (d, J ϭ
8.5 Hz, 1 H, 2-NH) ppm. 13C NMR (125 MHz,[D5]pyridine): δ ϭ
14.2 (CH3, C-18 and C-22ЈЈ), 23.0 (CH2, C-17 and C-21ЈЈ), 26.5
(CH2, C-3ЈЈ), 26.6 (CH2, C-6), 30.3Ϫ29.5 (CH2, alkyl chain CH2
groups), 32.1 (CH2, C-16 and C-20ЈЈ), 33.7 (CH2, C-5), 36.8 (CH2,
C-2ЈЈ), 51.8 (CH, C-2), 58.5 (CH3, OMe), 62.6 (CH2, C-6Ј), 68.7
(CH2, C-1), 70.5 (CH, C-3Ј), 71.0 (CH, C-4Ј), 72.7 (CH, C-4), 72.8
(CH, C-5Ј), 76.7 (CH, C-3), 80.0 (CH, C-2Ј), 98.6 (CH, C-1Ј), 173.5
(C, C-1ЈЈ) ppm.
Acknowledgments
This work is the result of research supported by MIUR PRIN
(Italy). Mass and NMR spectra were recorded at the ‘‘Centro Inter-
dipartimentale di Analisi Strumentale’’, Universita di Napoli ‘‘Fed-
erico II’’. The assistance of the staff there is gratefully acknowl-
edged.
`
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Received August 8, 2003
Eur. J. Org. Chem. 2004, 468Ϫ473
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
473