Immunomodulating glycosphingolipids: An efficient synthesis of a 2′-deoxy-α-galactosyl-GSL

Article abstract of DOI:10.1016/S0040-4020(01)01163-2

A new and efficient approach to the total synthesis of 2′-deoxy-α-galactosyl glycosphingolipids was accomplished. Commercially available 3,4,6-tri-O-acetylgalactal was used as the chiral starting material for both the sugar and phytosphingosine building blocks required for the synthesis of 1-O-(2-deoxy-α-D-galactopyranosyl)-2-docosanoylamino-1,3, 4-octadecanetriol. The key step of the synthetic strategy was the stereoselective α-glycosidation of the azido precursor of sphingosine.

Full text of DOI:10.1016/S0040-4020(01)01163-2

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 369±375
Immunomodulating glycosphingolipids: an ef®cient synthesis
of a 20-deoxy-a-galactosyl-GSL
Valeria Costantino,p Ernesto Fattorusso, Concetta Imperatore and Alfonso Mangoni
Á
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli ªFederico IIº, via D. Montesano 49, 80131 Naples, Italy
Received 27 July 2001; accepted 22 November 2001
AbstractÐA new and ef®cient approach to the total synthesis of 20-deoxy-a-galactosyl glycosphingolipids was accomplished. Commer-
cially available 3,4,6-tri-O-acetylgalactal was used as the chiral starting material for both the sugar and phytosphingosine building blocks
required for the synthesis of 1-O-(2-deoxy-a-d-galactopyranosyl)-2-docosanoylamino-1,3,4-octadecanetriol. The key step of the synthetic
strategy was the stereoselective a-glycosidation of the azido precursor of sphingosine. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
is not present at all. The synthesis of a 20-deoxygalactosyl-
ceramide has never been performed before, except for a
report by Koezuka's group5 that described a procedure
involving the coupling of a bromide donor with a ceramide,
leading to the desired compound in unspeci®ed yield and
stereoselectivity.
As part of our continuing study of the chemistry of sponges
belonging to genera Agelas and Axinella, we discovered a
new class of glycolipids (a-galactosyl glycosphingolipids)
characterized by the presence of an a-galactose as the ®rst
sugar of the saccharide chain.1 The simplest a-galacto-
syl glycosphingolipid, agelasphin, was discovered by a
Japanese group.2 This compound showed an interesting
immunostimulating activity, which is related to its in vivo
antitumor properties. Further studies3,4 demonstrated that
(i) the stereochemistry of the glycosidic linkage strongly
affects the bioactivity of these compounds, a-glycosides
being more active than b-glycosides, and (ii) the bioactivity
is highest when the ®rst sugar linked to the ceramide chain is
a galactose.
2. Results and discussion
2.1. Synthetic strategy
From a retrosynthetic point of view, the target compound 1
is composed of two main building blocks: the saccharide
unit and the phytosphingosine unit (that is acylated with a
fatty acid to give the ceramide). Both these units can be
derived from the commercially available 3,4,6-tri-O-acetyl-
d-galactal (2) that possesses the d-arabino con®guration
required for the synthesis of natural phytosphingosine
(amination leads to inversion of con®guration at C-2). To
prepare the ceramide moiety, we based our strategy on the
synthesis of phytosphingosine proposed by Schmidt in
1995,6 starting from tri-O-benzyl-d-galactal, that was
suitably modi®ed to meet our needs.
Eight new a-galactosyl glycosphingolipids have been
discovered in our laboratory. Some of these compounds
showed immunostimulating properties, being able to induce
proliferation of T-cells in vitro, others resulted to be inac-
tive. By a comparison of the structure of active and inactive
glycolipids, we proposed a structure±activity relationship
for the immunomodulating activity: only glycosphingo-
lipids with a non-glycosylated OH at position 2 of the ®rst
sugar are immunostimulating agents.1
In our synthetic scheme, selective deprotection of the
primary OH of phytosphingosine was required before glyco-
sylation; therefore, the easily removable TIPS group was
introduced from the beginning of the synthesis to protect
the OH at position 6 of galactal (the incoming position 1of
phytosphingosine). Because the TIPS group is acid labile,
an alternative way was required for the preparation of the
protected 2-deoxygalactose that is usually obtained by acid-
catalyzed hydration of the corresponding galactal. For
this purpose, we used the recently reported7 reaction with
N-iodosuccinimide (NIS) in CH3CN±H2O and subsequent
removal of the iodine atom with Na2S2O4.
To perform more detailed structure±activity studies, we
planned to synthesize some analogues of natural a-Gal-
GSLs. Our ®rst target was the total synthesis of 1-O-(2-
deoxy-a-d-galactopyranosyl)-2-docosanoylamino-1,3,4-octa-
decanetriol (1), where the key hydroxyl group at position 20
Keywords: galactal; a-glycosidation; glycosphingolipids; immunomodulat-
ing.
Corresponding author. Tel.: 139-81-678-504; fax: 139-81-678-552;
e-mail: costanti@unina.it
p
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)01163-2

370
V. Costantino et al. / Tetrahedron 58 (2002) 369±375
Scheme 1. Structure and retrosynthetic analysis of (2S,3S,4R)-1-O-(2-deoxy-a-d-galactopyranosyl)-2-docosanoylamino-1,3,4-octadecanetriol (1).
The key point of our strategy was to ®nd out a suitable way
to couple the two building blocks with a highly selec-
tive a-glycosidation. Stereoselective a-glycosidation is a
dif®cult task in carbohydrate chemistry. The possibility of
utilizing glycals as glycosyl donors in the synthesis of
2-deoxyglycosides in an iodonium-mediated coupling with
a suitable acceptor has been exploited in the pioneering
research by Thiem.8 The stereochemistry of this reaction
is governed by trans-diaxial addition and the a-linked
glycoside is produced with high yield and stereoselectivity.
Many kinds of a-2-deoxyglycosides have been prepared in
this way, but the reaction has never been used in the syn-
thesis of glycolipids. We found that the iodonium-mediated
coupling of glycals can be conveniently used for the syn-
thesis of glycosphingolipids, provided that a proper acceptor
is chosen.
was easily completed with reductive removal of the iodine
atom with simultaneous reduction to amine of the azido
group, followed by acylation and deprotection.
2.2. Synthetic procedure
As outlined in Scheme 2, the azidosphingosine triol 3 was
prepared starting from the commercially available 3,4,6-tri-
O-acetylgalactal. 3,4,6-Tri-O-acetylgalactal 2 was con-
verted into 3,4-di-O-benzyl-6-O-TIPS-galactal 3 in three
steps, and the corresponding 2-deoxysugar (4) was obtained
in 93% yield by treatment of 3 with NIS in CH3CN±H2O
95:5 followed by removal of the iodide group with
Na2S2O4.7
For the chain extension, the Wittig reaction was used.6 We
have recently demonstrated that the Wittig ylide causes
benzylic alcohol elimination from the benzyl-protected
2-deoxysugar, unless catalytic amounts of a triphenyl- or
trialkyltin halide are added.10 Therefore, treatment of
compound 4 with 4 equiv. of the Wittig ylide and
0.5 equiv. of triphenyltin chloride gave the desired
(2R,3S,4R)-3,4-di-O-benzyl-1-O-TIPS-6-octadecene-1,2,3,4-
tetrol 5 in 84% yield (E/Z mixture). This chiral precursor
was converted in the azidosphingosine 9 as follows.
The double bond was selectively reduced with diimine
(prepared in situ from p-toluenesulfonyl hydrazide and
As shown in Scheme 1, the glycosidation reaction
succeeded when the azidosphingosine triol 9 was used as
acceptor, while any attempt failed when the 2,3-O-dibenzyl-
ated ceramide was used. In fact, it has been reported that the
primary hydroxyl group of a ceramide shows a much lower
nucleophilicity than that expected for such a group;9 in
contrast, the corresponding group of the azidosphingosine
is a much better nucleophile.
After glycosylation, the synthesis of the target compound 1
Scheme 2. Synthesis of (2S,3S,4R)-2-azido-3,4-di-O-benzyl-1,3,4-octadecanetriol (9). Reagents: (a) MeONa, MeOH; (b) TIPSCl, DMF, imidazole (80%, 2
steps); (c) NaH, DMF, 08C, then BnBr (90%); (d) (i) N-iodosuccinimide, CH3CN±H2O 95:5, 08C, (ii) Na2S2O4, DMF±H2O 1:1, NaHCO3 (94%);
(e) C12H25PPh3Br, BuLi, Ph3SnCl, CH2Cl2±toluene (84%); (f) TsNHNH2, AcONa, EtOH (85%); (g) Tf2O, CHCl2±pyridine; (h) NaN3, DMF (71%, 2
steps); (i) TBAF, THF (82%).

V. Costantino et al. / Tetrahedron 58 (2002) 369±375
371
Scheme 3. Synthesis of compound 1. Reagents: (a) N-iodosuccinimide, CH2Cl2 (69% based on consumed 9); (b) Ph3SnH, AIBN, benzene, re¯ux (65%);
(c) C21H43COCl, pyridine±CH2Cl2 (82%); (d) H2, Pd(OH)2±C, EtOH±AcOH, 408C; (e) MeOH±Et3N 8:2, 408C (73%, 2 steps).
sodium acetate) giving compound 6 (85% yield). Com-
pound 6 was then transformed in the 2-azido derivative 8
in a one-pot reaction by treatment with tri¯uoromethan-
sulfonic anhydride, giving the 2-O tri¯ate derivative 7,
followed by reaction with sodium azide in DMF with inver-
sion of con®guration at C-2 (71% yield based on 6). Finally,
the TIPS protecting group was selectively removed by
treating compound 8 with a 1M solution of tetra-butyl-
ammonium¯uoride (TBAF) in THF to give the azido-
sphingosine 9 (82% yield).
EtOH±AcOH 9:1, while the acetyl groups were removed
in MeOH±Et3N 8:2 to give the target compound 1.
In conclusion, the total synthesis of compound 1 was
successfully achieved starting from commercially available
tri-O-acetylgalactal in only 12 steps. The overall yield was
7.6% based on tri-O-acetylgalactal 2 used for the prepa-
ration of the azidosphingosine 9. The biological activity of
this compound is currently being evaluated and will be
reported elsewhere.
Stereoselective 2-deoxygalactosidation of acceptor
9
3. Experimental
(Scheme 3) was then accomplished using 4 equiv. of tri-
O-acetylgalactal 2, and 5 equiv. of NIS in dry CH2Cl2.
The I1 addition to the double bond of the galactal 2 is
followed by the nucleophilic attack of the azidosphingosine
9 in a one-pot reaction that was allowed to proceed over-
night. This reaction con®rmed to have a high proclivity
for trans-diaxial addition and provided only the desired
a-galactoside 10, bearing an axial iodine atom at position
2 of galactose, in 59% yield and with 14% recovery of
unreacted compound 9 (69% yield based on consumed 9).
3.1. General methods
EI and FAB mass spectra were recorded on a VG Prospec-
Autospec (Fisons) mass spectrometer. FAB spectra were
performed in glycerol (positive ion mode) or triethanol-
amine (negative ion mode) matrices. ESI mass spectra
were recorded on a Finnigan LCQ ion-trap mass spectro-
meter, using CHCl3±MeOH (1:1) as solvent. Optical rota-
tions were measured at 589 nm on a Perkin±Elmer 192
polarimeter using a 10 cm microcell. H and 13C NMR
1
The axial orientation of the iodine atom was indicated by the
small coupling constant (4.9 Hz) of H-20 with H-30 in the 1H
NMR spectrum. Due to the equatorial orientation of H-2, the
small (,2 Hz) coupling constant between H-10 and H-20 was
not diagnostic for the a con®guration of the glycosidic bond.
This was later demonstrated by the proton spectrum of the
deiodinated compound 11 (described later) whose anomeric
proton resonated as a broad doublet (Jˆ2.7 Hz) at d 4.91.
spectra were determined on a Bruker AMX-500 spectro-
meter at 500.13 and 125.77 MHz, respectively; chemical
shifts were referenced to the residual solvent signal
(CDCl3: dHˆ7.26, dCˆ77.0; CD5N: dHˆ8.73, 7.58, and
7.21, dCˆ149.8, 135.3, and 123.4). The spectra of new
compounds were assigned thanks to COSY, HMQC, and,
when needed, HMBC two-dimensional NMR experiments.
The reverse multiple-quantum heteronuclear correlation
(HMQC) spectra were recorded 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 to 13C;
After SiO2 chromatography, compound 10 was subjected to
reduction to remove the iodine atom and to convert the
azido group to amine. This was obtained in one step using
Ph3SnH and AIBN in benzene for 20 h at room temperature
followed by 1h under re¯ux. The resulting compound 11
was puri®ed on SiO2 column using eluents containing 1%
of pyridine, and then treated with docosanoyl chloride in
pyridine±CH2Cl2 at room temperature for 18 h, affording
1-O-(3,4,6-tri-O-acetyl-2-deoxy-a-d-galactopyranosyl)-2-
docosanoylamino-3,4-di-O-benzyl-1,3,4-octadecanetriol
(12) in 82% yield. Finally, the benzyl protecting groups
were removed by hydrogenolysis using Pd(OH)2 in
1
the interpulse delays were adjusted for an average JCH of
142 Hz. The gradient-enhanced multiple-bond hetero-
nuclear correlation (HMBC) experiment was optimized
for a JCH of 8.3 Hz.
3
3.2. Synthesis
3.2.1. 3,4-Di-O-benzyl-6-O-TIPS-d-galactal (3). 3,4,6-Tri-
O-acetyl-d-galactal (1.37 g, 5.0 mmol) was dissolved in a

372
V. Costantino et al. / Tetrahedron 58 (2002) 369±375
solution of MeONa in MeOH (0.01M, 20 ml), and kept
stirring for 24 h at room temperature. After removal of the
solvent, 0.73 g of d-galactal was obtained, used in the subse-
quent reaction without any further puri®cation. d-Galactal
was dissolved in dry DMF (5 ml), and imidazole (1.0 g,
14.7 mmol) and triisopropylsilyl chloride (TIPSCl, 0.96
ml, 1.06 g, 5.5 mmol) were added. After stirring for 12 h,
water (100 ml) was added and the reaction mixture was
extracted with EtOAc (3£200 ml). The combined organic
layers were washed with water and brine, dried with MgSO4
and concentrated under reduced pressure. The residue was
puri®ed by chromatography on silica gel (solvent n-hex-
ane±EtOAc 6:4) to give 1.2 g (4.0 mmol, 80%) of 6-O-
TIPS-d-galactal as a colorless oil. It was dissolved in dry
DMF (50 ml) and cooled to 08C, and NaH (238 mg,
9.9 mmol) was slowly added. After a few minutes, benzyl
bromide (0.99 ml, 7.9 mmol) was added dropwise. After
stirring for 12 h at room temperature, water (100 ml) was
added and the reaction mixture was extracted three times
with EtOAc (300 ml). The combined organic layers were
washed with water and brine, dried with MgSO4 and
concentrated under reduced pressure. The residue was
puri®ed by chromatography on silica gel (solvent n-hex-
ane±EtOAc 9:1) to give 1.72 g (3.57 mmol, 90%) of
compound 3: colorless oil, [a]Dˆ228.4 (CHCl3, cˆ2.8);
ESIMS (positive ions): m/z 505 ([M1Na]1; 1H NMR
(CDCl3): d 7.36±7.33 (10H, m, aromatic protons), 6.40
(1H, d, Jˆ6.2 Hz, H-1), 4.87 (1H, d, Jˆ11.4 Hz, benzylic
proton), 4.85 (1H, dd, Jˆ6.2, 2.7 Hz, H-2), 4.77 (1H, d,
Jˆ11.4 Hz, benzylic proton), 4.65 (1H, d, Jˆ11.4 Hz,
benzylic proton), 4.59 (1H, d, Jˆ11.4 Hz, benzylic proton),
4.22 (1H, m, H-5), 4.05 (1H, dd, Jˆ11.3, 3.4 Hz, H-6a),
4.01(1H, dd, Jˆ11.3, 2.5 Hz, H-6b), 3.94 (2H, H-3 and
H-4 overlapped), 1.10 (3H, m, TIPS methine protons),
1.07 (18H, d, Jˆ6.5 Hz, TIPS methyl protons); 13C NMR
(125 MHz, CDCl3): d 144.2 (CH, C-1), 138.7 (C, aromatic
carbon), 138.6 (C, aromatic carbon), 128.3±127.4 (CH,
aromatic carbons), 100.1 (CH, C-2), 77.7 (CH, C-3 or
C-4), 73.7 (CH2, benzylic methylene group), 71.4 (CH,
C-5), 71.1 (CH, C-4 or C-3), 70.8 (CH2, benzylic methylene
group), 61.6 (CH2, C-6), 18.0 (CH3, TIPS methyl groups),
11.9 (CH, TIPS methine groups).
(1H, br s, H-1), 4.96 (1H, d, Jˆ11.4 Hz, benzylic proton),
4.70 (1H, d, Jˆ11.4 Hz, benzylic proton), 4.62 (2H, s,
benzylic protons), 4.01(1H, br s, H-4), 3.99 (2H, H-3 and
H-5 overlapped), 3.84 (1H, dd, Jˆ9.7, 8.0 Hz, H-6a), 3.72
(1H, dd, Jˆ9.7, 5.9 Hz, H-6b), 2.21(1H, m, H-2eq), 2.02
(1H, m, H-2ax), 1.10 (3H, m, TIPS methine protons), 1.05
(18H, d, Jˆ6.5 Hz, TIPS methyl protons).
3.2.3. (2R,3S,4R)-1-O-TIPS-3,4-di-O-benzyl-6-octadecene-
1,2,3,4-tetrol (5). Ph3SnCl (0.9 mmol, 347 mg) was dis-
solved in dry CH2Cl2 (15 ml) and added to the deoxysugar
4 (940 mg, 1.88 mmol). Then, the ylide was prepared from
n-BuLi (4.7 ml of a 1.6 M solution in n-hexane, 7.52 mmol)
and n-dodecyltriphenylphosphonium bromide (4.81g,
9.40 mmol) in dry toluene (15 ml). The solution of 2-deoxy-
sugar was then added dropwise to the ylide solution, and
allowed to react at room temperature for 2 h. The reaction
mixture was then diluted with CH2Cl2 (400 ml) and washed
with water (3 times, 400 ml each), followed by brine
(400 ml). The organic layer was puri®ed by column chroma-
tography (n-hexane±EtOAc 8:2) to give 1.02 g of com-
pound 5 (84% yield) as mixture of E/Z stereoisomers,
1
identi®ed by comparison of its H, 13C NMR spectra and
optical rotation with literature data:10 [a]Dˆ23.7 (CHCl3,
cˆ0.9); 1H NMR (500 MHz, CDCl3) d 7.35±7.28 (10H, m,
aromatic protons), 5.56±5.38 (2H, m, H-6 and H-7), 4.70
(1H, d, Jˆ11.8 Hz, benzylic proton), 4.61 (2H, s, benzylic
protons), 4.60 (1H, d, Jˆ11.8 Hz, benzylic protons), 3.97
(1H, m, H-2), 3.79±3.70 (4H, m, H2-1, H-2, and H-4), 3.11
(1H, br s, 2-OH), 2.51±2.37 (2H, m, H2-5), 2.03±1.96 (2H,
m, H2-8), 1.25 (alkyl chain CH2 protons), 1.08 (TIPS
methine protons), 1.05 (TIPS methyl protons), 0.88 (3H, t,
Jˆ7.0 Hz, H3-18); 13C NMR (125 MHz, CDCl3): d 132.2
(CH, C-7), 128.7±127.6 (CH, aromatic carbons), 124.4
IR: nmax (CHCl3) 2951, 2872, 1646, 1455, 1092 cm21
;
(CH, C-6), 80.1(CH, C-3), 78.1(CH, C-4), 73.7 (CH
,
2
benzylic methylene group), 72.5 (CH2, benzylic methylene
group), 71.3 (CH, C-2), 63.8 (CH2, C-1), 34.2 (CH2, C-5, E
isomer), 32.7 (CH2, C-8, E isomer), 29.7±29.2 (CH2, alkyl
chains CH2 groups), 29.0 (CH2, C-5, Z isomer), 27.6 (CH2,
C-8, Z isomer), 18.0 (CH3, TIPS methyl groups), 14.1 (CH3,
C-18), 11.8 (CH, TIPS methine groups).
3.2.4. (2R,3S,4R)-1-O-TIPS-3,4-di-O-benzyl-1,2,3,4-octa-
decanetetrol (6). The alkene 5 (1.30 g, 1.99 mmol) was
dissolved in 95% EtOH (40 ml) and p-toluenesulfonyl
hydrazide (4.1g, 22 mmol). The reaction mixture was
re¯uxed for 5 h, and during this time, a solution of sodium
acetate (2.9 g, 35.6 mmol) in water (12 ml) was added drop-
wise. After this, H2O (100 ml) was added to the reaction
mixture, that was subsequently extracted with CH2Cl2
(3£200 ml). The combined organic extracts were dried
over Na2SO4, and the solvent removed. Puri®cation of the
residue by column chromatography on SiO2 (n-hexane±
EtOAc 95:5) gave 1.11 g (1.69 mmol, 85%) of compound
6: colorless oil, [a]Dˆ26.9 (CHCl3, cˆ2.4); IR: nmax
3.2.2. 3,4-Di-O-benzyl-6-O-TIPS-2-deoxy-d-galactose (4).
The protected galactal 3 (965 mg, 2 mmol) was dissolved in
CH3CN m/z H2O (95:5, 15 ml) and cooled to 08C. Then, the
solution was treated with N-iodosuccinimide (495 mg,
2.2 mmol), allowed to warm to room temperature and left
to stir for 15 min. The solvent was then removed in vacuo.
The residue was dissolved in DMF (20 ml) and a solution of
2
H2O±HCO3 (10 mol equiv., 20 ml) was then added
followed by solid Na2S2O4 (1.537 g, 8 mmol). The reaction
was allowed to proceed under stirring for 5 h at room
temperature. After that, the reaction mixture was diluted
with 800 ml of AcOEt and washed with water (3 times,
400 ml each), followed by brine (400 ml). The organic
layer was puri®ed by column chromatography (n-hexane±
EtOAc 7:3) to give 940 mg of compound 4 (1.88 mmol,
94% yield) as mixture of anomers, identi®ed by comparison
of its 1H NMR spectrum and optical rotation with literature
(CHCl3) 3498, 2932, 2872, 1466, 1391, 1114, 1071 cm21
;
HREIMS: m/z 611.4525 ([M2i-Pr]1, C38H63O4Si gives
611.4496); EIMS: m/z 611 ([M2i-Pr]1, 10), 396 (85), 395
(91), 277 (41), 246 (67), 173 (100); H NMR (500 MHz,
1
CDCl3): d 7.37±7.27 (10H, aromatic protons), 4.74 (1H, d,
Jˆ11.5 Hz, benzylic proton), 4.70 (1H, d, Jˆ11.5 Hz,
benzylic proton), 4.66 (1H, d, Jˆ11.5 Hz, benzylic proton),
4.59 (1H, d, Jˆ11.5 Hz, benzylic proton), 3.94 (1H, m,
1
data:7 [a]Dˆ132 (CHCl3, cˆ0.2); H NMR (CDCl3, a
anomer): d 7.42±7.22 (10H, m, aromatic protons), 5.45

V. Costantino et al. / Tetrahedron 58 (2002) 369±375
373
H-2), 3.78±3.70 (4H, overlapping signals, H2-1, H-3, and
H-4), 3.19 (1H, d, Jˆ4.2 Hz, OH-2), 1.68 (1H, m, H-5a),
1.60 (1H, m, H-5b), 1.42 (1H, m, H-6a), 1.33±1.23 (alkyl
chain CH2 protons), 1.10 (TIPS methine protons), 1.08
(TIPS methyl protons), 0.90 (3H, t, Jˆ7.0 Hz, H3-18); 13C
NMR (125 MHz, CDCl3): d 137.3 (C, aromatic carbons),
128.7±127.6 (CH, aromatic carbons), 80.5 (CH, C-3), 78.4
(CH, C-4), 73.7 (CH2, benzylic methylene group), 72.9
(CH2, benzylic methylene group), 71.5 (CH, C-2), 63.9
(CH2, C-1), 31.9 (CH2, C-16), 31.3 (CH2, C-5), 29.7±29.2
(CH2, alkyl chains CH2 groups), 25.8 (CH2, C-6), 22.7 (CH2,
C-17), 18.0 (CH3, TIPS methyl groups), 14.1 (CH3, C-18),
11.8 (CH, TIPS methine groups).
tography (n-hexane±EtOAc 95:5) column to give 661mg
(1.26 mmol, 82%) of compound 9: colorless oil, [a]Dˆ13.4
(CHCl3, cˆ0.35); IR: nmax (CHCl3) 3332, 2936, 2865, 2118,
1104, 1021 cm21; HRFABMS (positive ions): m/z 524.3848
([M1H]1, C32H50N3O3 gives 524.3852); 1H NMR
(500 MHz, CDCl3): d 7.37±7.29 (10H, aromatic protons),
4.71(1H, d, Jˆ11.5 Hz, benzylic proton), 4.68 (1H, d, Jˆ
11.5 Hz, benzylic proton), 4.63 (1H, d, Jˆ11.5 Hz, benzylic
proton), 4.58 (1H, d, Jˆ11.5 Hz, benzylic proton), 3.90 (1H,
ddd, Jˆ11.5, 5.2, 5.2 Hz, H-1a), 3.80 (1H, m, H-1b), 3.71
(1H, m, H-3), 3.68 (1H, m, H-4), 3.65 (1H, m, H-2), 2.54
(1H, t, Jˆ6.1 Hz, OH-1), 1.68 (1H, m, H-5a), 1.57 (1H, m,
H-5b), 1.42 (1H, m, H-6a), 1.33±1.23 (alkyl chain CH2
protons), 0.89 (3H, t, Jˆ6.8 Hz, H3-18); 13C NMR
(125 MHz, CDCl3): d 137.6 and 136.8 (C, aromatic
carbons), 128.5±127.9 (CH, aromatic carbons), 80.5 (CH,
C-3), 79.0 (CH, C-4), 73.7 (CH2, benzylic CH2 groups), 72.6
3.2.5. (2S,3S,4R)-1-O-TIPS-2-azido-3,4-di-O-benzyl-1,3,
4-octadecanetriol (8). Compound 6 (1.41 g, 2.15 mmol)
was dissolved in dry CH2Cl2 (20 ml) and dry pyridine
(2 ml). The solution was cooled to 2158C, tri¯ic anhydride
(0.44 ml, 732 mg, 2.59 mmol) was slowly added, and the
mixture was stirred for 30 min. After this, dry DMF
(100 ml) and solid NaN3 (750 mg, 11.5 mmol) were
added, and the reaction mixture was allowed to react
under stirring for 12 h at room temperature. Water
(200 ml) was added to the reaction mixture, that was
extracted with n-hexane (2£300 ml) and EtOAc (2£
300 ml). The combined organic extracts were dried over
Na2SO4, concentrated under reduced pressure, and puri®ed
by column chromatography on SiO2 (n-hexane±EtOAc
95:5) to give 1.04 g (1.53 mmol, 71%) of compound 8:
colorless oil, [a]Dˆ14.7 (CHCl3, cˆ0.3); IR: nmax
(CH2, benzylic CH2 groups), 63.1(CH, C-2), 62.3 (CH ,
2
C-1), 32.0 (CH2, C-16), 30.2 (CH2, C-5), 29.7±29.4 (CH2,
alkyl chain CH2 groups), 25.5 (CH2, C-6), 22.7 (CH2, C-17),
14.2 (CH3, C-18).
3.2.7. (2S,3S,4R)-1-O-(3,4,6-Tri-O-acetyl-2-iodo-2-deoxy-
a-d-galactopyranosyl)-2-azido-3,4-di-O-benzyl-1,3,4-
octadecanetriol (10). To tri-O-acetylgalactal 2 (677 mg,
2.49 mmol) in dry CH2Cl2 (5 ml) were added the azido-
sphingosine 9 (322 mg, 0.62 mmol) in dry CH2Cl2 (4 ml)
and N-iodosuccinimide (698 mg, 3.12 mmol) at 08C. The
reaction mixture was re¯uxed for 90 min, then left at
room temperature for 12 h under stirring. The reaction
mixture was treated with a 10% aqueous solution of
Na2S2O3 until the purple color of the solution disappeared
and then with water (50 ml), and extracted with CH2Cl2
(4£100 ml). The organic extracts, dried over Na2SO4 and
concentrated under reduced pressure, were subjected to
column chromatography on SiO2 (n-hexane±EtOAc 9:1),
giving 45 mg of unreacted 9 (0.08 mmol) and 337 mg
(0.37 mmol, 69% based on consumed 9) of compound 10:
colorless oil, [a]Dˆ16.5 (CHCl3, cˆ0.3); IR: nmax (CHCl3)
2928, 2856, 2099, 1747, 1374, 1111 cm21; HREIMS: m/z
921.3602 (M1, C44H64IN3O10 gives 922.3636); EIMS: m/z
(relative intensity) 921(M 1, 2), 814 (4), 683 (10), 527 (12),
399 (53), 239 (88), 204 (100), 188 (48), 169 (57); 1H NMR
(500 MHz, CDCl3): d 7.37±7.28 (10H, aromatic protons),
5.36 (1H, br s, H-40), 5.27 (1H, br s, H-10), 4.90 (1H, dd,
Jˆ4.7, 3.7 Hz, H-30), 4.69 (1H, d, Jˆ11.5 Hz, benzylic
proton), 4.62 (1H, benzylic proton), 4.57 (2H, s, benzylic
protons), 4.22 (1H, br d, Jˆ4.9 Hz, H-20), 4.20 (1H, m,
H-50), 4.10 (1H, m, H-60a), 4.09 (1H, m, H-60b), 3.99 (1H,
dd, Jˆ10.5, 2.6 Hz, H-1a), 3.77 (1H, m, H-2), 3.63 (1H, m,
H-1b), 3.61 (2H, overlapped signals, H-3 and H-4), 2.18
(3H, s, acetyl protons), 2.08 (3H, s, acetyl protons), 1.95
(3H, s, acetyl protons), 1.67 (1H, m, H-5a), 1.57 (1H, m,
H-5b), 1.40 (1H, m, H-5a), 1.25 (alkyl chain CH2 protons),
0.88 (6H, t, Jˆ6.9 Hz, H3-18); 13C NMR (125 MHz,
CDCl3): d 171.4 (C, C-100), 170.5±169.5 (C, acetyl CO),
138.1 ad 137.7 (C, aromatic carbons), 128.5±127.8 (CH,
aromatic carbons), 103.11 (CH, C-10), 79.0 (CH, C-4),
78.8 (CH, C-3), 73.7 (CH2, benzylic CH2 group), 72.1
(CH2, benzylic CH2 group), 68.7 (CH2, C-1), 66.9 (CH,
C-50), 65.2 (CH, C-40), 65.0 (CH, C-30), 62.0 (CH2, C-60),
61.7 (CH, C-2), 32.0 (CH2, C-16), 30.0 (CH2, C-5), 29.7±
29.4 (CH2, alkyl chain CH2 groups), 25.2 (CH2, C-6), 22.7
(CHCl3) 2936, 2872, 2118, 1114 cm21
; HRFABMS
(positive ions): m/z 702.4988 ([M1Na]1, C41H69N3NaO3Si
gives 702.5006); EIMS: m/z 636 ([M2i-Pr]1, 5), 608 (3),
530 (7), 502 (8), 395 (22), 351 (100), 319 (13), 274 (28), 197
1
(39), 173 (26); H NMR (500 MHz, CDCl3): d 7.35±7.27
(10H, aromatic protons), 4.74 (1H, d, Jˆ11.5 Hz, benzylic
proton), 4.60 (1H, d, Jˆ11.5 Hz, benzylic proton), 4.59 (1H,
d, Jˆ11.5 Hz, benzylic proton), 4.53 (1H, d, Jˆ11.5 Hz,
benzylic proton), 4.07 (1H, dd, Jˆ10.5, 2.8 Hz, H-1a),
3.84 (1H, dd, Jˆ10.5, 7.5 Hz, H-1b), 3.62 (2H, overlapping
signal, H-3 and H-4), 3.58 (1H, m, H-2), 1.68 (1H, m, H-5a),
1.56 (1H, m, H-5b), 1.44 (1H, m, H-6a), 1.33±1.23 (alkyl
chain CH2 protons), 1.10 (TIPS methine protons), 1.08
(TIPS methyl protons), 0.88 (3H, t, Jˆ6.9 Hz, H3-18); 13C
NMR (125 MHz, CDCl3): d 137.3 (C, aromatic carbons),
128.6±127.5 (CH, aromatic carbons), 79.9 (CH, C-3), 78.4
(CH, C-4), 73.7 (CH2, benzylic methylene groups), 72.6
(CH2, benzylic methylene groups), 64.7 (CH2, C-1and
CH, C-2), 31.9 (CH2, C-16), 31.6 (CH2, C-5), 30.0±29.4
(CH2, alkyl chain methylene groups), 25.6 (CH2, C-6),
22.7 (CH2, C-17),18.0 (CH3, TIPS methyl groups), 14.2
(CH3, C-18), 11.8 (CH, TIPS methine groups).
3.2.6.
(2S,3S,4R)-2-Azido-3,4-di-O-benzyl-1,3,4-octa-
decanetriol (9). To the TIPS-protected azidosphingosine
8 (1.04 g, 1.53 mmol) dissolved in dry THF (40 ml), a
solution of TBAF (1M in THF, 3.1ml, 3.1mmol) was
added at 08C. After 10 min, the solution was allowed to
warm at room temperature, and stirred for 90 min. The
reaction mixture was treated with water (100 ml) and
extracted with EtOAc (4£200 ml). The organic phases
were combined, dried, and concentrated under reduced
pressure. The crude product was puri®ed by SiO2 chroma-

374
V. Costantino et al. / Tetrahedron 58 (2002) 369±375
(CH2, C-17), 21.1±20.7 (CH3, acetyl methyl groups), 20.6
(CH, C-20), 14.2 (CH3, C-18).
m, H-20a), 2.01(3H, s, acetyl protons), 2.00 (3H, s, acetyl
protons), 1.72 (2H, overlapping signals, H-20b and H-5a),
1.63 (2H, overlapping signals, H2-300 and H-5b), 1.25 (alkyl
chain CH2 protons), 0.88 (6H, t, Jˆ6.9 Hz, H3-18 and
3.2.8. (2S,3S,4R)-1-O-(3,4,6-Tri-O-acetyl-2-deoxy-a-d-
galactopyranosyl)-2-amino-3,4-di-O-benzyl-1,3,4-octa-
decanetriol (11). To a solution of the azide 10 (337 mg,
0.37 mmol) in benzene (10 ml), Ph3SnH (950 ml, 1.31 g,
3.72 mmol) and a small amount of AIBN were added, and
the resulting solution was allowed to react at room tempera-
ture for 24 h and subsequently for 1h under re¯ux. The
solution was cooled to room temperature and concentrated
under reduced pressure. Column chromatography on SiO2
(n-hexane±EtOAc±pyridine 66:39:1) gave 183 mg (0.24
mmol, 65%) of 11: colorless oil, [a]Dˆ125.3 (CHCl3,
cˆ0.75); IR: nmax (CHCl3) 3390, 2917, 2856, 1746, 1431,
1383, 1372, 1111, 1075 cm21; HRFABMS (positive ions):
m/z 770.4855 ([M1H]1, C44H68NO10 gives 770.4843);
13
H3-22000); C NMR (125 MHz, CDCl3): d 172.5 (C, C-100),
170.4±170.0 (C, acetyl CO), 138.4 (C, aromatic carbon),
128.5±127.9 (CH, aromatic carbons), 97.9 (CH, C-10),
80.1(CH, C-4), 78.4 (CH, C-3), 73.2 (CH , benzylic CH2
2
group), 72.1(CH , benzylic CH2 group), 67.2 (CH2, C-1),
2
66.8 (CH, C-50), 66.4 (CH, C-40), 66.0 (CH, C-30), 62.2
(CH2, C-60), 49.4 (CH, C-2), 34.1(CH , C-200), 31.9 (CH2,
2
C-16 and C-2000), 30.2 (CH2, C-5 and C-20), 30.0±29.1
(CH2, alkyl chain CH2 groups), 24.9 (CH2, C-300), 22.6
(CH2, C-17 and C-2100), 20.8±20.6 (CH3, acetyl methyl
groups), 14.1 (CH3, C-18 and C-2200).
3.2.10. (2S,3S,4R)-1-O-(2-Deoxy-a-d-galactopyranosyl)-
2-docosanoylamino-1,3,4-octadecanetriol (1). The pro-
tected glycosphingolipid 12 (197 mg, 0.18 mmol) and
Pd(OH)2±C (50 mg, 20% w/w) were suspended in 95%
EtOH (45 ml) and AcOH (5 ml). The obtained mixture
was hydrogenated at a pressure of 3 bar in a Parr reactor
for 48 h at 408C. The reaction mixture was ®ltered over
celite and the ®ltrate washed with 95% EtOH, MeOH, and
CHCl3. The organic extracts were combined and concen-
trated to dryness under reduced pressure, dissolved in
MeOH (16 ml) and Et3N (4 ml), and kept at 408C for 18 h.
After removal of the solvents, the crude reaction product
was puri®ed by reversed-phase column chromatography on
RP-18 silica gel (MeOH±EtOAc 95:5) to give 103 mg
(0.13 mmol, 73%) of compound 1: amorphous solid,
[a]Dˆ127.3 (CHCl3±MeOH 1:1, cˆ0.8); IR: nmax (KBr)
1
FABMS: m/z 770 [M1H]1, 498 [M1H-galactosyl]1; H
NMR (500 MHz, CDCl3): d 7.35±7.27 (10H, aromatic
protons), 5.30 (1H, br s, H-40), 5.27 (1H, ddd, Jˆ12.5,
4.8, 2.9 Hz, H-30), 4.91(1H, br d, Jˆ2.7 Hz, H-10), 4.76
(1H, d, Jˆ11.6 Hz, benzylic proton), 4.64 (1H, d, Jˆ
11.6 Hz, benzylic proton), 4.56 (1H, d, Jˆ11.6 Hz, benzylic
proton), 4.54 (1H, d, Jˆ11.6 Hz, benzylic proton), 4.09 (1H,
m, H-50), 4.04 (1H, m, H-60a), 3.99 (1H, m, H-60b), 3.88
(1H, dd, Jˆ9.5, 2.8 Hz, H-1a), 3.74 (1H, ddd, Jˆ7.8, 3.4,
3.4 Hz, H-4), 3.51(1H, dd, Jˆ6.7, 3.4 Hz, H-3), 3.36 (1H,
dd, Jˆ9.5, 7.4 Hz, H-1b), 3.12 (1H, ddd, Jˆ7.0, 7.0, 2.7 Hz,
H-2), 2.13 (3H, s, acetyl protons), 2.05 (1H, ddd, Jˆ12.5,
12.5, 3.4 Hz, H-20a), 1.99 (3H, s, acetyl protons), 1.95 (3H,
s, acetyl protons), 1.82 (1H, br dd, Jˆ12.5, 5.0 Hz, H-20b),
1.71 (1H, m, H-5a), 1.59 (1H, m, H-5b), 1.25 (alkyl chain
CH2 protons), 0.88 (3H, t, Jˆ6.9 Hz, H3-18).
3385, 2926, 2850, 1643, 1542, 1365, 1078 cm21
;
HRFABMS (negative ions): m/z 784.6703 ([M2H]2,
C46H90NO8 gives 784.6666); FABMS: m/z 784 [M2H]2,
3.2.9. (2S,3S,4R)-1-O-(3,4,6-Tri-O-acetyl-2-deoxy-a-d-
galactopyranosyl)-2-docosanoylamino-3,4-di-O-benzyl-
1,3,4-octadecanetriol (12). Docosanoic acid (232 mg,
0.68 mmol) dissolved in SOCl2 (1.5 ml, 2.39 g, 20.1
mmol) was re¯uxed for 90 min, and the excess of SOCl2
removed under reduced pressure. A solution of the amine
11 (171 mg, 0.22 mmol) in dry pyridine (6 ml) and dry
CH2Cl2 (8 ml) was added to the obtained docosanoyl
chloride. After 12 h, the solvents were removed under
vacuum, and residue was partitioned between CH2Cl2 and
a saturated NaHCO3 aqueous solution. The organic phase,
dried over Na2SO4 and concentrated under reduced pres-
sure, was subjected to column chromatography on SiO2
(n-hexane±EtOAc 7:3), giving 197 mg (0.18 mmol, 82%)
of 12: colorless oil, [a]Dˆ16.5 (CHCl3, cˆ0.4); IR: nmax
(CHCl3) 3210, 2930, 2860, 1745, 1671, 1508, 1463, 1377,
1114 cm21; HRFABMS (negative ions): m/z 1090.7969
([M2H]2, C66H108NO11 gives 1090.7922); FABMS: m/z
1090 [M2H]2, 81 8 [M2galactosyl]2; 1H NMR (500
MHz, CDCl3): d 7.36±7.27 (10H, aromatic protons), 5.61
(1H, d, Jˆ9.3 Hz, NH-2), 5.30 (1H, br d, Jˆ3.0 Hz, H-40),
5.22 (1H, ddd, Jˆ12.3, 4.9, 3.0 Hz, H-30), 4.82 (1H, br d,
Jˆ3.0 Hz, H-10), 4.82 (1H, d, Jˆ11.7 Hz, benzylic proton),
4.61(1H, d, Jˆ11.7 Hz, benzylic proton), 4.58 (1H, d, Jˆ
11.7 Hz, benzylic proton), 4.51 (1H, d, Jˆ11.7 Hz, benzylic
proton), 4.24 (1H, m, H-2), 4.08 (1H, m, H-50), 4.04 (1H, m,
H-60a), 3.99 (1H, m, H-60b), 3.73±3.67 (3H, overlapping
signals, H2-1and H-3), 3.54 (H1, m, H-4), 2.37 (2H,
Jˆ7.1Hz, H 2-200), 2.13 (3H, s, acetyl protons), 2.02 (1H,
1
638 [M2galactosyl]2; H NMR (500 MHz, pyridine-d5):
d 8.78 (1H, d, Jˆ3.0 Hz, NH-2), 6.64 (1H, d, Jˆ6.0 Hz,
OH-3), 6.55 (1H, t, Jˆ5.3 Hz, OH-60), 6.24 (1H, d, Jˆ
4.6 Hz, OH-40), 6.20 (1H, d, Jˆ6.9 Hz, OH-30), 6.05 (1H,
d, Jˆ6.8 Hz, OH-4), 5.24 (1H, br d, Jˆ3.0 Hz, H-10), 5.15
(1H, m, H-2), 4.59 (1H, dd, Jˆ10.3, 3.5 Hz, H-1a), 5.15
(1H, m, H-30), 4.42±4.37 (4H, overlapping signals, H-3,
H-40, H-50, and H-60a), 4.35 (1H, m, H-60b), 4.30 (1H, dd,
Jˆ10.3, 7.3 Hz, H-1b), 4.26 (1H, m, H-4), 2.49 (3H, over-
lapping signals, H2-200 and H-20a), 2.24 (1H, m, H-5a), 2.18
(1H, br dd, Jˆ12.5, 4.8 Hz, H-20b), 1.94 (2H, m, H-5b and
H-6a), 1.84 (2H, quintet, Jˆ7.3 Hz, H2-300), 1.69 (1H, m,
H-6b), 1.47±1.33 (3H, overlapping signals, H-7a and
H2-400), 1.31±1.20 (alkyl chain CH2 protons), 0.85 (6H, t,
Jˆ7.3 Hz, H3-18 and H3-22000); 13C NMR (125 MHz,
pyridine-d5): d 173.6 (C, C-100), 99.4 (CH, C-10), 76.2
(CH, C-3), 72.8 (CH, C-50), 72.7 (CH, C-4), 69.7 (CH,
C-40), 67.7 (CH2, C-1), 66.2 (CH, C-30), 63.2 (CH2, C-60),
52.9 (CH, C-2), 36.9 (CH2, C-200), 34.3 (CH2, C-20), 33.7
(CH2, C-5), 32.1(CH , C-16 and C-2000), 30.3±29.5 (CH2,
2
alkyl chain CH2 groups), 26.6 (CH2, C-6), 26.5 (CH2, C-300),
23.0 (CH2, C-17 and C-2100), 14.3 (CH3, C-18 and C-2200).
Acknowledgements
This work is the result of research supported by MURST
(PRIN 1999 and `Progetto Giovani Ricercatori' 2000) and

V. Costantino et al. / Tetrahedron 58 (2002) 369±375
375
4. Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.;
Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H.
J. Med. Chem. 1995, 38, 2176±2187.
CNR, Italy. Mass and NMR spectra were recorded at the
`Centro Interdipartimentale di Analisi Strumentale',
Á
Universita di Napoli `Federico II'. The assistance of the
staff is gratefully acknowledged.
5. Motoki, K.; Morita, M.; Kobayashi, E.; Uchida, T.; Akimoto,
K.; Fukushima, H.; Koezuka, Y. Biol. Pharm. Bull. 1995, 18,
1487±1491.
6. Wild, R.; Schmidt, R. Liebigs Ann. 1995, 755±764.
7. Costantino, V.; Fattorusso, E.; Imperatore, C.; Mangoni, A.
Tetrahedron Lett. 2000, 41, 9177±9180.
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