Immunomodulatory α-galactoglycosphingolipids: Synthesis of 2′-fluoro-2′-deoxy-α-galactosylceramide and an evaluation of its immunostimulating properties

Article abstract of DOI:10.1002/ejoc.200500053

In the framework of a project to assess the immunomodulating properties of α-galactosyl glycosphingolipids, the total synthesis of 1-O-(2-fluoro-2-deoxy-α-D-galactopyranosyl)-2-docosanoylamino-1,3, 4-octadecanetriol (1), a 2′-fluoro analogue of the immunostimulating α-galactoglycosphingolipids, was accomplished. The glycosidation reaction of the azido precursor of sphingosine was performed using the Mukaiyama glycosidation reaction. The stimulation of mouse splenocyte proliferation by the 2′-fluoro analogue was highly reduced compared to that of the α-galactoglycosphingolipids, thereby confirming that a free galactose 2-OH group is essential for the immunostimulatory activity. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500053

FULL PAPER
Immunomodulatory α-Galactoglycosphingolipids: Synthesis of 2Ј-Fluoro-2Ј-
deoxy-α-galactosylceramide and an Evaluation of Its Immunostimulating
Properties[‡]
Lucia Barbieri,[a] Valeria Costantino,*[a] Ernesto Fattorusso,[a] Alfonso Mangoni,[a]
Nicoletta Basilico,[b] and Monica Mondani,[b] and Donatella Taramelli[b]
Keywords: Fluorine / Glycolipids / Glycosidation / Immunomodulatory activity / Mukaiyama reaction
In the framework of a project to assess the immunomodulat-
ing properties of α-galactosyl glycosphingolipids, the total
synthesis of 1-O-(2-fluoro-2-deoxy-α-D-galactopyranosyl)-2-
kaiyama glycosidation reaction. The stimulation of mouse
splenocyte proliferation by the 2Ј-fluoro analogue was highly
reduced compared to that of the α-galactoglycosphingolipids,
docosanoylamino-1,3,4-octadecanetriol (1), a 2Ј-fluoro ana- thereby confirming that a free galactose 2-OH group is es-
logue of the immunostimulating α-galactoglycosphingolip-
ids, was accomplished. The glycosidation reaction of the
azido precursor of sphingosine was performed using the Mu-
sential for the immunostimulatory activity.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
of α-Gal-GSLs, and raised the question of why the 2-O-
substitution makes α-Gal-GSLs inactive. Two hypotheses
can be proposed in this respect: (a) it is the mere steric
hindrance of the additional sugar that prevents the glyco-
lipid from binding to the TCR receptor, or (b) the 2-OH of
the first sugar is the very group involved in a specific inter-
action at the active site of the receptor.
Even though quite a large number of natural α-Gal-
GSLs are available, they are not the best compounds to be
used for this type of experiments. Natural α-Gal-GSLs dif-
fer from simple α-galactopyranosylceramide mostly in the
presence of additional sugars and, given the glycosidase
capability of APC cells, this prevents any sound structure–
activity relationship study. Therefore, we synthesized some
α-Gal-GSL analogues modified at the key position 2, i.e.
2Ј-deoxy-α-d-galactopyranosylceramide (3),[8] and 2Ј-O-
methyl-α-d-galactopyranosylceramide[9] (4). This synthetic
modification resulted in loss of the capacity to stimulate the
proliferation of murine NKT cells for compound 3, and in
a strong reduction of the activity for compound 4. These
results support the speculation that the galactose 2-OH
group is responsible for the binding to the TCR receptor,
most likely through a hydrogen bond.
Introduction
Agelas and Axinella sponges[1–4] have been shown to pro-
duce α-galactoglycosphingolipids (α-Gal-GSLs), which are
glycosphingolipids with an α-galactopyranose as the first
sugar attached to the ceramide head-group (e.g. agelasphin
2). They are immunostimulating agents that are capable of
activating the NKT cells’ response[5] when presented by An-
tigen Presenting Cells (APC).[6] The first event in this pro-
cess is recognition of α-Gal-GSL by the TCR receptor of
the NKT cells, which can only happen when the glycolipid
binds a protein called CD1d, located on the surface of the
APCs.
Some years ago, in a survey of the immunostimulatory
activity of several natural α-Gal-GSLs, we found that glyco-
sylation of the galactose 2-OH group results in complete
loss of activity.[4] More recently, it has been demonstrated
that α-Gal-GSLs with a glycosylated 2Ј-OH retain in vitro
activity following the intracellular hydrolysis of the intergly-
cosidic linkage by α-glycosidases identified in the lysosomes
of APCs.[7] These findings pointed to a crucial role of the
galactose 2-OH group for the immunostimulatory activity
[‡] Synthesis of Glycolipids, 3. Part 2: Ref.[8]
[a] Dipartimento di Chimica delle Sostanze Naturali, Università di
Napoli “Federico II”
In this paper, we attempt to gain further insight into the
TCR receptor binding nature by introducing a fluorine
atom in place of the galactose 2-OH group. The capacity of
organically bound fluorine to act as an OH mimic and enter
into hydrogen bonding as an acceptor has been widely dis-
cussed, and it is now generally acknowledged that the fluor-
ine atom can indeed act as hydrogen-bond acceptor, al-
though the resulting F···H hydrogen bonds are clearly
weaker (about half as much) than O···H hydrogen bonds.[10]
Via D. Montesano 49, 80131 Napoli, Italy
Fax: +39-081-678-552
E-mail: valeria.costantino@unina.it
[b] Istituto di Microbiologia, Università di Milano,
Via Pascal 36, 20133 Milano, Italy
Fax: +39-02-5031-5071
E-mail: donatella.taramelli@unimi.it
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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DOI: 10.1002/ejoc.200500053
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V. Costantino et al.
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We report below the first total synthesis of 2Ј-fluoro-2Ј-de- tion has been most often used to perform the stereoselective
oxy-α-d-galactopyranosylceramide (1), and a preliminary α-glycosidation of the synthetic precursor of ceramide.[9,13]
evaluation of its biological activity using lymphocyte prolif- This reaction can provide a very high α-selectivity, and of-
eration tests (Scheme 1).
fers the additional advantage that its stereoselectivity is not
dependent on the configuration of the glycosyl fluoride
used as substrate. However, the yields are in most cases only
moderate.
Over the years, several improved versions of the original
Mukaiyama glycosidation reaction have been proposed in
an attempt to improve the yield of the reaction. One inter-
esting modification,[14] involving a glycosyl acetate as glyco-
syl donor and an azidosphingosine activated with a trityl
group, has been used recently by our group to prepare 2Ј-O-
methyl-α-d-galactopyranosylceramide (4). In that case, the
reaction provided considerably better yields than the origi-
nal Mukaiyama reaction, while the stereoselectivity re-
mained excellent. Therefore, it was natural for us to use this
reaction as the key step for the synthesis of 1. The required
glycosyl donor 6 was prepared in only three steps from
commercial galactal as follows (Scheme 2). After ben-
zylation of the galactal (Sigma, 462233) by a standard pro-
cedure,[15] the obtained tri-O-benzylgalactal was converted
into the corresponding 2-deoxy-2-fluorotri-O-benzyl-d-gal-
actopyranose (5) by treatment with Selectfluor as the fluori-
nating agent.[16] Selectfluor reacts regioselectively with gly-
cals in the presence of water to give specific fluorination at
position 2. The reaction is remarkably stereoselective be-
cause of the directing effect of the axial protecting group at
position 4, and only the equatorially fluorinated products 5
was obtained as an α/β anomeric mixture. Finally, com-
pound 5 was acetylated to give 6, once again as an α/β an-
omeric mixture.
Scheme 1.
Results and Discussion
A glycosphingolipid is made by assembling three build-
ing blocks: a saccharide chain, a sphingoid base, and a fatty
acid. The crucial step of synthesis of an α-galactoglycos-
phingolipid is the glycosidation of a suitably protected
sphingoid base. To build up the target compound 1 we took
advantage of our previous experience in synthesizing α-Gal-
GSLs.[8,9] We used the azido precursor of the sphingosine,
rather than the sphingosine itself or the ceramide, as glyco-
syl acceptor because the former is a better nucleophile,[11]
and the appropriate glycosyl fluoride as glycosyl donor.
The use of glycosyl fluorides in oligosaccharide synthesis
Scheme 2. Synthesis of the glycosyl donors 6 and 7. Reagents: (a)
NaH, DMF, 0 °C, then BnBr (90%); (b) F-TEDA-BF4, DMF/H2O
(1:1), 12 h, room temp.; (c) Ac2O in py, room temp., quantitative;
(c) DAST in THF, –30 °C, (71%).
Compound 6 was used in the subsequent glycosylation
was pioneered by the Mukaiyama group.[12] They demon- reaction together with the tritylated azidosphingosine 9, in
strated that glycosyl fluorides can be activated for glycosid- the presence of AgClO4 and SnCl4 (Scheme 3). Unfortu-
ation of alcohols in the presence of silver perchlorate and nately, the outcome of the reaction was different from what
tin dichloride. If an ester protecting group is present at posi- we expected. We observed complete loss of stereoselectivity,
tion 2, the mechanism involves the participation of this with a ratio between the α isomer 10α and the β isomer 10β
group, and this directs the reaction to the β product. With- of about 1:1. In addition, the yield was low, only 32% over-
out a participating group at the 2-position, α-glycosides are all, and it was not possible to recover either the tritylated
formed preferentially. The Mukaiyama glycosidation reac- azide 9 or the unprotected azide 8 as unreacted material.
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on both faces of the cation, thus allowing the nucleophile
to attack both sides. On the other hand it is well known
that the strong electron-withdrawing effect of a fluoro sub-
stituent can significantly destabilize an oxocarbenium
ion,[19] so the observed loss of selectivity could be explained
by the higher reactivity of the oxocarbenium ion intermedi-
ate, which could prevent ion-pair formation.
Finally, the target compound 1 (29% overall yield based
on the azidosphingosine 6 used as starting material) was
obtained by reduction of the azide group with triphenyltin
hydride, amidation with docosanoyl chloride, and removal
of the benzyl protecting groups by hydrogenolysis over Pd/
C, as previously reported (Scheme 5).[8]
Scheme 3. Modified Mukaiyama reaction applied to the synthesis
of α-Gal-GSL’s.
At this point, we decided to go back to the original Mu-
kaiyama glycosidation reaction, which involves a glycosyl
fluoride as the glycosyl donor and AgClO4 and SnCl2 as a
Lewis acid catalytic system. The bis-fluorinated glycosyl
donor 7 was prepared by treatment of the 2-fluorosugar 5
with DAST (Et2NSF3), a well known reagent for mild and
direct transformation of glycosyl hemiacetals into glycosyl
fluorides, in THF.[17] Compound 7 was obtained in 71%
yield. The stereoselectivity of the glycosidation reaction
(Scheme 4), although slightly better (the 10α/10β ratio was
56:44), was still disappointing. However, the yield of the
reaction was much higher (49%, α + β), and, moreover, we
could recover as much as 41% of the azidosphingosine 8,
so that the overall yield of 10α and 10β based on the con-
sumed 8 was 84%, and the yield of 10α alone was 47%.
Scheme 5. Final steps of the synthesis of compound 1. Reagents:
(a) Ph3SnH, AIBN, benzene, reflux (81%); (b) C21H43COCl, pyri-
dine/CH2Cl2 (84%); (c) H2, Pd(OH)2/C, EtOH/AcOH, 40 °C
(73%).
Preliminary evaluation of the biological activity of com-
pound 1 compared to α-Gal-GSL agelasphin (2) was per-
formed using the splenocyte proliferation test. As expected,
compound 2 stimulated spleen cell proliferation from
C57Bl/6 mice in a dose-dependent manner. The activity
peaked after 72 h of incubation at 37 °C. On the contrary,
when compound 1 was tested under the same experimental
conditions, no significant proliferation was seen at any of
the doses tested (Figure 1, panel A). Furthermore, different
doses of compound 1 were added to compound 2 and pro-
liferation evaluated after 72 h (Figure 1, panel B). No nega-
tive or positive interaction was observed between the two
Scheme 4. Original Mukaiyama reaction applied to the synthesis
of α-Gal-GSL’s.
The reason for the loss of selectivity of the Mukaiyama compounds, indicating that the stimulatory activity of 2 is
reaction when a 2-fluoroglycosyl donor is used can only be not modified by the presence of 1. These data also exclude
hypothesized at this stage. The mechanism postulated for any toxicity of compound 1 on normal splenocytes. These
this reaction is of the SN1 type, with the bulky perchlorate results indicate that compound 1 is unable to interact with
ion forming an ion pair with the oxocarbenium ion interme- the CD1d molecules and/or be appropriately presented by
diate. The perchlorate ion prefers to stay on the less-hin- APC.[7]
dered β face of the ion, and therefore the nucleophile at-
Overall, these results strongly confirm that a free galac-
tacks the α face preferentially.[18] The presence of the fluor- tose 2-OH group is essential for the activity of α-Gal-GSLs,
ine atom could affect the stereochemical outcome of the and suggest that this hydroxyl group is the key group in-
reaction in two (nonmutually exclusive) ways − steric and volved in the binding to the receptor. A more extensive ex-
electronic. On one hand, the limited steric hindrance of the amination of the biological activity of compound 2 and
fluorine atom (much smaller than any protected OH group, other modified α-Gal-GSLs is in progress and will be re-
including OCH3) would allow the perchlorate ion to stay ported elsewhere.
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to proceed for 12 h at room temperature. The reaction was
quenched with water (200 mL) and then extracted with EtOAc
(3×300 mL). The combined organic extracts were dried with
Na2SO4 and concentrated under reduced pressure. The crude fluo-
ride 5 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 compound 6 (761 mg, 1.54 mmol, quantitative) as a mixture
of anomers, α/β = 4:3; [α]D = +68 (c = 3.0, CHCl3), which was
used in the next step without further purification.
α-Anomer: Colorless oil. ESI MS (positive ion): m/z = 517 [M +
Na]+. 1H NMR (500 MHz, CDCl3): δ = 2.09 (s, 3 H, acetyl pro-
tons), 3.51 (dd, J = 9.6 and 6.4 Hz, 1 H, 6b-H), 3.56 (dd, J = 9.6
and 8.7 Hz, 1 H, 6a-H), 4.09–4.02 (m, 2 H, 3-H and 5-H), 4.12 (br.
t, J = 2.9 Hz, 1 H, 4-H), 4.48 and 4.41 (AB system, J = 11.7 Hz,
1 H each, geminal benzyl protons), 4.74 and 4.72 (AB system, J =
11.7 Hz, 1 H each, geminal benzyl protons), 4.84 and 4.51 (AB
system, J = 11.7 Hz, 1 H each, geminal benzyl protons), 4.97 (ddd,
J = 49.4, 9.8, 4.1 Hz, 1 H, 2-H), 6.34 (br. d, J = 4.1 Hz, 1 H, 1-
H), 7.41–7.21 (15 H, aromatic protons) ppm. 13C NMR (500 MHz,
CDCl3): δ = 69.6 (CH2, C-6), 73.1 (CH, C-5), 73.6 (d, JC,F = 2 Hz,
CH2, benzyl methylene group), 74.4 (CH2, benzyl methylene
group), 76.2 (CH2, benzyl methylene group), 76.5 (d, JC,F = 8 Hz,
CH, C-4), 78.2 (d, JC,F = 16 Hz, CH, C-3), 89.1 (d, JC,F = 187 Hz,
CH, C-2), 90.9 (d, JC,F = 23 Hz, CH, C-1), 129.5–128.7 (CH, aro-
matic C atoms), 139.7–139.2 (C, aromatic C atoms), 170.9 (C, ace-
tyl CO) ppm.
β-Anomer: Colorless oil. ESI MS (positive ions): m/z = 517 [M +
Na]+. 1H NMR (500 MHz, CDCl3): δ = 2.09 (s, 3 H, acetyl pro-
tons), 3.57–3.55 (m, 2 H, 6-H2), 3.89–3.81 (m, 2 H, 3-H and 5-H),
4.02 (m, 1 H, 4-H), 4.48 and 4.40 (AB system, J = 11.7 Hz, 1 H
each, geminal benzyl protons), 4.64 (ddd, J = 52.5, 9.6, 7.9 Hz, 1
H, 2-H), 4.72 and 4.70 (AB system, J = 11.7 Hz, 1 H each, geminal
benzyl protons), 4.83 and 4.52 (AB system, J = 11.7 Hz, 1 H each,
geminal benzyl protons), 5.71 (dd, J = 7.9 and 4.7 Hz, 1 H, 1-
H), 7.41–7.21 (15 H, aromatic protons) ppm. 13C NMR (500 MHz,
CDCl3): δ = 69.2 (CH2, C-6), 73.8 (d, JC,F = 2 Hz, CH2, benzyl
methylene group), 74.3 (CH2, benzyl methylene group), 75.4 (CH,
C-5), 75.7 (d, JC,F = 9 Hz, CH, C-4) 76.1 (CH2, benzyl methylene
group), 81.3 (d, JC,F = 16 Hz, CH, C-3), 91.7 (d, JC,F = 184 Hz,
CH, C-2), 93.3 (d, JC,F = 25 Hz, CH, C-1), 129.5–128.7 (CH, aro-
matic C atoms), 139.7–139.2 (C, aromatic C atoms), 170.8 (C, ace-
tyl CO) ppm.
Figure 1. Proliferative response of C57Bl/6 spleen cells to 2Ј-fluoro-
2Ј-deoxy-α-galactosylceramide (1) or α-galactosylceramide (2).
Panel A: Dose-dependent proliferation of C57Bl/6 spleen cells to
compound 1 or compound 2 after 72 h of culture. The results are
representative of three different experiments conducted in quadru-
plicate. Data are expressed as mean SD. Panel B: Lack of interac-
tions between compound 1 and compound 2 when tested in combi-
nation on C57Bl/6 spleen cells The results are representative of two
different experiments conducted in quadruplicate. Data are ex-
pressed as mean SD.
Experimental Section
3,4,6-Tri-O-benzyl-2-deoxy-2-fluoro-D-galactopyranosyl
Fluoride
General Remarks: High-resolution ESI mass spectra were recorded
on a Micromass QTOF Micro mass spectrometer, dissolving the
sample in MeCN/H2O (1:1) with 0.1% TFA. ESI MS/MS experi-
ments were performed on a Finnigan LCQ ion-trap mass spectrom-
eter. The spectra were recorded by infusion into the ESI source
using MeOH/CHCl3 (4:1) as the solvent. Optical rotations were
measured at 589 nm on a Perkin–Elmer 192 polarimeter using a
(7): The crude fluoride 5, prepared from tri-O-benzylgalactal
(640 mg, 1.54 mmol) as described above, was dissolved in dry THF
(17 mL) and cooled to –30 °C. DAST (0.24 mL, 1.8 mmol) was
rapidly added and the cooling bath removed immediately. After
stirring for 30 min at room temperature, the mixture was cooled
again to –30 °C and quenched with MeOH (2 mL). After evapora-
tion of solvent under reduced pressure, water (200 mL) was added
to the residue, which was extracted with DCM (3×300 mL). The
combined organic layers were dried with Na2SO4 and concentrated
under reduced pressure. The residue was purified by column
chromatography on SiO2 (CH2Cl2) to give 495 mg (1.09 mmol,
71%) of 7 (mixture of anomers, α/β = 2:1).
1
10-cm microcell. H and 13C NMR spectra were determined on a
Varian UnityInova 500 spectrometer at 500.13 and 125.77 MHz,
respectively; chemical shifts were referenced to the residual solvent
signal (CDCl3: δH = 7.26, δC = 77.0 ppm. CD5N: δH = 8.71, 7.56,
and 7.19, δC = 149.8, 135.3, and 123.4 ppm). The spectra of new
compounds were assigned thanks to COSY and HSQC two-dimen-
sional NMR experiments.
α-Anomer: Colorless oil, [α]D = +6 (c = 0.2, CHCl3). ESI MS (posi-
1
tive ions): m/z = 477 [M + Na]+. H NMR (500 MHz, CDCl3): δ
3,4,6-Tri-O-benzyl-2-deoxy-2-fluoro-
H2O (6 mL) and 1-chloromethyl-4-fluoro-1,4-diazoniabicy-
clo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor, 1.1 g,
3.08 mmol) were added to a solution of tri-O-benzylgalactal
(640 mg, 1.54 mmol) in DMF (6 mL) and the reaction was allowed
D
-galactopyranosyl Acetate (6):
= 3.63–3.59 (m, 2 H, 6-H2), 4.03 (ddd, J = 10.3, 10.3, 2.7 Hz, 1 H,
3-H), 4.09 (m, 1 H, 4-H), 4.16 (br. t, J = 6.6 Hz, 1 H, 5-H), 4.54
and 4.46 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl pro-
tons), 4.85 and 4.73 (AB system, J = 11.7 Hz, 1 H each, geminal
benzyl protons), 4.97 and 4.60 (AB system, J = 11.7 Hz, 1 H each,
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geminal benzyl protons), 5.01 (dddd, J = 49.2, 24.1, 9.7, 2.7 Hz, 1
H, 2-H), 5.81 (br. dd, J = 54.3 and 2.7 Hz, 1 H, 1-H), 7.45–7.28
(15 H, aromatic protons) ppm. 13C NMR (500 MHz, CDCl3): δ =
protons), 4.82 and 4.69 (AB system, J = 11.7 Hz, 1 H each, geminal
benzyl protons), 4.92 (m, JH,F = 49 Hz, 0.5 H, the high field part
of the 2Ј-H signal), 4.94 and 4.56 (AB system, J = 11.7 Hz, 1 H
each, geminal benzyl protons), 5.04–4.98 (m, 1.5 H, 1Ј-H and the
low field part of the 2Ј-H signal), 7.42–7.24 (25 H, aromatic pro-
tons) ppm. 13C NMR (500 MHz, CDCl3): δ = 14.1 (CH3, C-18),
22.6 (CH2, C-17), 25.5 (CH2, C-6), 29.8–29.6 (CH2, alkyl chain
67.8 (CH2, C-6), 71.9 (d, JC,F = 3 Hz, CH, C-5), 72.9 (d, JC,F
=
2 Hz, CH2, benzyl methylene group), 73.5 (CH2, benzyl methylene
group), 74.7 (d, JC,F = 9 Hz, CH, C-4), 75.0 (CH2, benzyl methyl-
ene group), 76.2 (d, JC,F = 16 Hz, CH, C-3), 88.8 (dd, JC,F = 188
and 25 Hz, CH, C-2), 104.8 (dd, JC,F = 227 and 24 Hz, CH, C-1), CH2 groups), 29.9 (CH2, C-5), 31.9 (CH2, C-16), 61.6 (CH, C-2),
128.5–127.5 (CH, aromatic C atoms), 138.0–137.5 (C, aromatic C 68.5 (CH2, C-6Ј), 68.8 (CH2, C-1), 69.8 (CH, C-5Ј), 72.1 (CH2,
atoms) ppm.
benzyl methylene group), 73.0 (CH2, benzyl methylene group), 73.5
(CH2, benzyl methylene group), 73.8 (CH2, benzyl methylene
group), 74.9 (CH2, benzyl methylene group), 75.2 (d, J = 7 Hz, CH,
C-4Ј), 76.7 (d, J = 15 Hz, CH, C-3Ј), 78.5 (CH, C-3), 79.5 (CH, C-
4), 89.1 (d, J = 189 Hz, CH, C-2Ј), 97.6 (d, J = 21 Hz, CH, C-1Ј),
128.4–127.6 (CH, aromatic C atoms) ppm.
β-Anomer: Colorless oil, [α]D = +3 (c = 0.2, CHCl3). ESI MS (posi-
tive ions): m/z = 477 [M + Na]+. H NMR (500 MHz, CDCl3): δ
1
= 3.71–3.64 (m, 3 H, 3-H and 6-H2), 3.74 (br. t, J = 6.7 Hz, 1 H,
5-H), 3.99 (m, 1 H, 4-H), 4.52 and 4.46 (AB system, J = 11.7 Hz,
1 H each, geminal benzyl protons), 4.79 and 4.72 (AB system, J =
11.7 Hz, 1 H each, geminal benzyl protons), 4.83 (dddd, J = 52.2,
15.8, 9.3, 6.6 Hz, 1 H, 2-H), 4.96 and 4.63 (AB system, J = 11.7
Hz, 1 H each, geminal benzyl protons), 5.27 (ddd, J = 53.4, 6.6,
5.7 Hz, 1 H, 1-H), 7.46–7.29 (15 H, aromatic protons) ppm. 13C
(2S,3S,4R)-2-Azido-3,4-di-O-benzyl-1-O-(3,4,6-tri-O-benzyl-2-de-
oxy-2-fluoro-β-D-galactopyranosyl)-1,3,4-octadecanetriol (10β):
Colorless oil, [α]D = +9 (c = 1.1, CHCl3). ESI MS (positive ions):
1
m/z = 981 [M + Na]+. H NMR (500 MHz, CDCl3): δ = 0.88 (t, J
NMR (500 MHz, CDCl3): δ = 67.9 (CH2, C-6), 72.8 (d, JC,F
=
= 6.6 Hz, 3 H, H3-18), 1.26 (alkyl chain CH2 protons), 1.32 (m, 1
H, 6b-H), 1.39 (m, 1 H, 6a-H), 1.56 (m, 1 H, 5b-H), 1.65 (m, 1 H,
5a-H), 3.49 (m, 1 H, 3-H) 3.53 (d, J = 5.3 Hz, 1 H, 6Јb-H), 3.61–
3.54 (m, 2 H, 6Јa-H and 5Ј-H), 3.63 (m, 1 H, 3Ј-H), 3.71 (t, J =
4.5 Hz, 1 H, 4-H), 3.77 (m, 1 H, 2-H), 3.84 (dd, J = 10.7 and
3.6 Hz, 1 H, 1b-H), 3.94 (br. t, J = 2.9 Hz, 1 H, 4Ј-H), 4.09 (dd, J
= 10.7 and 6.8 Hz, 1 H, 1a-H), 4.43 and 4.38 (AB system, J = 11.7
Hz, 1 H each, geminal benzyl protons), 4.44–4.38 (m, 3 H, 1Ј-H
and two benzylic protons), 4.49 and 4.57 (AB system, J = 11.7 Hz,
1 H each, geminal benzyl protons), 4.68 and 4.63 (AB system, J =
11.7 Hz, 1 H each, geminal benzyl protons), 4.76 and 4.66 (AB
system, J = 11.7 Hz, 1 H each, geminal benzyl protons), 4.77–4.64
(2Ј-H submerged by another signal), 4.92 and 4.59 (AB system, J
= 11.7 Hz, 1 H each, geminal benzyl protons), 7.39–7.20 (25 H,
aromatic protons) ppm. 13C NMR (500 MHz, CDCl3): δ = 14.1
(CH3, C-18), 20.8 (CH2, C-17), 24.8 (CH2, C-6), 29.3–29.1 (CH2,
alkyl chain CH2 groups), 29.7 (CH2, C-5), 30.9 (CH2, C-16), 61.6
(CH, C-2), 68.0 (CH2, C-6Ј), 68.7 (CH2, C-1), 71.6 (CH2, benzyl
methylene group), 72.4 (CH2, benzyl methylene group), 72.6 (CH2,
benzyl methylene group), 73.2 (CH2, benzyl methylene group), 73.4
(CH, C-3), 73.8 (CH, C-4Ј), 74.5 (CH2, benzyl methylene group),
78.6 (CH, C-4), 79.2 (CH, C-3Ј), 80.0 (CH, C-5Ј), 91.4 (CH, d, J
= 60 Hz, C-2Ј), 100.5 (CH, C-1Ј), 127.8–127.2 (CH, aromatic C
atoms) ppm.
2 Hz, CH2, benzyl methylene group), 73.5 (d, JC,F = 9 Hz, CH, C-
4), 73.6 (CH2, benzyl methylene group), 73.9 (d, JC,F = 4 Hz, CH,
C-5), 74.8 (CH2, benzyl methylene group), 78.9 (dd, JC,F = 16 and
9 Hz, CH, C-3), 91.5 (dd, JC,F = 184 and 24 Hz, CH, C-2), 107.1
(dd, JC,F = 216 and 27 Hz, CH, C-1), 128.5–127.6 (CH, aromatic
C atoms), 138.0–137.5 (C, aromatic C atoms) ppm.
Modified Mukaiyama Glycosidation Reaction: SnCl4 (50 μL of a
0.5 m toluene solution, 0.025 mmol) was added to a suspension of
AgClO4 (7.0 mg, 0.034 mmol) in anhydrous Et2O (2 mL). The mix-
ture was stirred for 4 h, and compound 6 (170 mg, 0.34 mmol) and
compound 9 (130 mg, 0.17 mmol)[8] were added simultaneously in
an anhydrous Et2O solution (2 mL). After 14 h, the reaction mix-
ture was diluted with DCM (30 mL), washed with a saturated
NaHCO3 solution (20 mL), dried with Na2SO4, and taken to dry-
ness. The residue was chromatographed by HPLC (n-hexane/
EtOAc, 9:1) to give 26 mg (0.027 mmol, 16%) of the α-glycoside
10α and 26 mg (0.027 mmol, 16%) of β-glycoside 10β.
Original Mukaiyama Glycosidation Reaction: A solution of the azi-
dosphingosine 8 (190 mg, 0.36 mmol)[9] and the fluoride 7 (495 mg,
1.09 mmol) in dry THF (3 mL) was added at –15 °C to a flask
containing solid AgClO4 (225 mg, 1.09 mmol) and SnCl2 (205 mg,
1.09 mmol). After stirring for 12 h at room temperature, the mix-
ture was filtered through Celite and the solvents evaporated. The
reaction mixture was diluted with DCM (30 mL), washed with a
saturated NaHCO3 solution (20 mL), dried with Na2SO4, and
taken to dryness. The residue was chromatographed by HPLC (n-
hexane/EtOAc, 9:1) to give 97 mg (0.101 mmol, 28%) of the α-gly-
coside 10α and 72 mg (0.076 mmol, 21%) of the β-glycoside 10β
(α/β = 56:44), along with 77 mg (0.147 mmol, 41%) of unreacted
8.
(2S,3S,4R)-2-Amino-3,4-di-O-benzyl-1-O-(3,4,6-tri-O-benzyl-2-de-
oxy-2-fluoro-α-D-galactopyranosyl)-1,3,4-octadecanetriol (11):
Ph3SnH (434 μL, 597 mg, 1.72 mmol) and a small amount of
AIBN were added to a solution of azide 10α (165 mg, 0.172 mmol)
in dry benzene (10 mL), and the resulting solution was allowed to
react for 2 h under reflux at 80 °C. The solution was cooled to
room temperature and concentrated under reduced pressure. Col-
umn chromatography on SiO2 (n-hexane/EtOAc, 6:4 with 0.1%
pyridine) gave 142 mg (0.152 mmol, 88%) of 11 as a colorless oil.
[α]D = +33 (c = 1.1, CHCl3). ESI MS (positive ions): m/z = 933 [M
(2S,3S,4R)-2-Azido-3,4-di-O-benzyl-1-O-(3,4,6-tri-O-benzyl-2-de-
oxy-2-fluoro-α-D-galactopyranosyl)-1,3,4-octadecanetriol (10α):
1
Colorless oil, [α]D = +53 (c = 1.1, CHCl3). ESI MS (positive ions):
+ H]+. H NMR (500 MHz, CDCl3): δ = 0.88 (t, J = 6.6 Hz, 3 H,
m/z = 981 [M + Na]+. H NMR (500 MHz, CDCl3): δ = 0.88 (t, J H3-18), 1.26 (alkyl chain CH2 protons), 1.35 (m, 1 H, 6b-H), 1.45
1
= 6.6 Hz, 3 H, H3-18), 1.26 (alkyl chain CH2 protons), 1.35 (m, 1 (m, 1 H, 6a-H), 1.59 (m, 1 H, 5b-H), 1.69 (m, 1 H, 5a-H), 3.14 (m,
H, 6b-H), 1.45 (m, 1 H, 6a-H), 1.59 (m, 1 H, 5b-H), 1.69 (m, 1 H,
5a-H), 3.55–3.46 (m, 2 H, 6Ј-H2), 3.65–3.59 (m, 2 H, 2-H and 4- H, 6Јa-H and 3-H), 3.73 (m, 1 H, 4-H), 4.03–3.94 (m, 4 H, 1a-H,
H), 3.76–3.70 (m, 2 H, 1b-H and 3-H), 3.98–3.95 (m, 2 H, 5Ј-H 4Ј-H, 3Ј-H, and 5Ј-H), 4.46 and 4.39 (AB system, J = 11.7 Hz, 1
and 4Ј-H), 4.08–3.99 (m, 2 H, 1a-H and 3Ј-H), 4.47 and 4.39 (AB H each, geminal benzyl protons), 4.64 and 4.54 (AB system, J =
1 H, 2-H), 3.52–3.43 (m, 2 H, 6Јb-H and 1b-H), 3.60–3.53 (m, 2
system, J = 11.7 Hz, 1 H each, geminal benzyl protons), 4.62 and
4.52 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl protons),
4.74 and 4.62 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl
11.7 Hz, 1 H each, geminal benzyl protons), 4.76 and 4.57 (AB
system, J = 11.7 Hz, 1 H each, geminal benzyl protons), 4.81 and
4.70 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl protons),
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V. Costantino et al.
FULL PAPER
4.92 (m, JH,F = 49 Hz, 0.5 H, the high field part of the 2Ј-H signal),
4.93 and 4.57 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl
protons), 5.04–4.98 (m, 1.5 H, 1Ј-H and the low field part of the
2Ј-H signal), 7.42–7.23 (25 H, aromatic protons) ppm. 13C NMR
7.1 Hz, 6 H, 18-H3 and 22ЈЈ-H3), 1.33–1.16 (alkyl chain CH2 pro-
tons), 1.50–1.34 (m, 3 H, 7a-H and 4ЈЈ-H2), 1.69 (m, 1 H, 6b-H),
1.85 (m, 2 H, 3ЈЈ-H2), 1.99–1.88 (m, 2 H, 5b-H and 6a-H), 2.22 (m,
1 H, 5a-H), 2.50 (m, 2 H, 2ЈЈ-H2), 4.23 (br. t, J = 6.8 Hz, 1 H, H-
(500 MHz, CDCl3): δ = 14.1 (CH3, C-18), 22.7 (CH2, C-17), 25.8 4), 4.35 (dd, J = 10.9 and 5.3 Hz, 1 H, 6Јb-H), 4.47–4.39 (m, 3 H,
(CH2, C-6), 29.8–29.6 (CH2, alkyl chain CH2 groups), 30.5 (CH2,
C-5), 31.9 (CH2, C-16), 52.6 (CH, C-2), 68.5 (CH2, C-6Ј), 69.5 (CH,
1b-H, 6Јa-H, and 3-H), 4.56–4.51 (m, 2 H, 5Ј-H and 4Ј-H), 4.61
(m, 1 H, 3Ј-H), 4.64 (dd, J = 10.9 and 3.2 Hz, 1 H, 1a-H), 5.17 (m,
C-5Ј), 71.8 (CH2, C-1), 72.0 (CH2, benzyl methylene group), 72.8 1 H, 2-H), 5.32 (ddd, J = 50.4, 9.6, 3.8 Hz, 1 H, 2Ј-H), 5.49 (d, J
(CH2, benzyl methylene group), 73.5 (CH2, benzyl methylene
group), 73.6 (CH2, benzyl methylene group), 74.9 (CH2, benzyl
methylene group), 75.4 (d, J = 8 Hz, CH, C-4Ј), 77.0 (CH, C-3Ј),
79.9 (CH, C-4), 81.6 (CH, C-3), 89.3 (d, J = 187 Hz, CH, C-2Ј),
97.7 (d, J = 21 Hz, CH, C-1Ј), 128.4–127.5 (CH, aromatic C
atoms), 138.7–137.8 (C, aromatic C atoms) ppm.
= 3.8 Hz, 1 H, 1Ј-H), 8.78 (d, J = 8.6 Hz, 1 H, 2-NH) ppm. 13C
NMR (500 MHz, [D5]pyridine): δ = 14.2 (CH3, C-18 and C-22ЈЈ),
22.9 (CH2, C-17 and C-21ЈЈ), 26.4 (CH2, C-3ЈЈ), 26.5 (CH2, C-6),
30.2–29.5 (CH2, alkyl chain CH2 groups), 32.1 (CH2, C-16 and C-
20ЈЈ), 33.7 (CH2, C-5), 36.7 (CH2, C-2ЈЈ), 52.6 (CH, C-2), 62.2
(CH2, C-6Ј), 69.1 (CH2, C-1), 69.3 (CH, C-3Ј), 71.5 (CH, C-4Ј),
72.6 (CH, C-4), 72.8 (CH, C-5Ј), 76.1 (CH, C-3), 91.1 (d, J =
188 Hz, CH, C-2Ј), 98.5 (d, J = 21 Hz, CH, C-1Ј), 173.6 (C, C-1ЈЈ)
ppm.
(2S,3S,4R)-3,4-Di-O-benzyl-2-docosanoylamino-1-O-(3,4,6-tri-O-
benzyl-2-deoxy-2-fluoro-α-D-galactopyranosyl)-1,3,4-octadecane-
triol (12): A solution of docosanoic acid (156 mg, 0.46 mmol) in
SOCl2 (1.0 mL, 1.64 g, 13.8 mmol) was refluxed for 90 min, and
the excess of SOCl2 was then removed under reduced pressure. A
solution of amine 11 (142 mg, 0.152 mmol) in dry pyridine (3 mL)
and dry CH2Cl2 (3 mL) was added to the obtained docosanoyl
chloride. After 12 h, the solvents were removed under vacuum, and
the residue was partitioned between CH2Cl2 (100 mL) and a satu-
rated NaHCO3 aqueous solution (50 mL). The organic phase was
dried with Na2SO4 and concentrated under reduced pressure. It
was then subjected to column chromatography on SiO2 (n-hexane/
EtOAc, 8:2) to give 183 mg (0.146 mmol, 96%) of 12 as a colorless
oil. [α]D = +21 (c = 1.1, CHCl3). ESI MS (positive ions): m/z =
1255 [M + H]+. 1H NMR (500 MHz, CDCl3): δ = 0.88 (t, J =
6.8 Hz, 6 H, 18-H3 and 22ЈЈ-H3), 1.26 (alkyl chain CH2 protons),
1.58–1.49 (m, 3 H, 3ЈЈ-H2 and 5b-H), 1.69 (m, 1 H, 5a-H), 2.01 (m,
2 H, 2ЈЈ-H2), 3.55–3.44 (m, 3 H, 6Јb-H, 6Јa-H, and 4-H), 3.84–3.76
(m, 2 H, 1b-H and 3-H), 3.97–3.89 (m, 4 H, 1a-H, 5Ј-H, 3Ј-H, and
4Ј-H), 4.24 (m, 1 H, 2-H), 4.48 and 4.39 (AB system, J = 11.7 Hz,
1 H each, geminal benzyl protons), 4.61 and 4.51 (AB system, J =
11.7 Hz, 1 H each, geminal benzyl protons), 4.79 and 4.69 (AB
system, J = 11.7 Hz, 1 H each, geminal benzyl protons), 4.80 and
4.53 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl protons),
4.91 and 4.56 (AB system, J = 11.7 Hz, 1 H each, geminal benzyl
protons), 4.95 (1 H, double multiplet, J = 51 Hz, 2Ј-H), 4.96 (d, J
= 3.6 Hz, 1 H, 1Ј-H), 5.87 (d, J = 8.9 Hz, 1 H, 2-NH), 7.42–7.20
(25 H, aromatic protons) ppm. 13C NMR (500 MHz, CDCl3): δ =
14.1 (CH3, C-18 and C-22ЈЈ), 22.6 (CH2, C-17 and C-21ЈЈ), 24.7
(CH2, C-3ЈЈ), 29.8–29.1 (CH2, alkyl chain CH2 groups), 30.3 (CH2,
C-5), 31.9 (CH2, C-16 and C-20ЈЈ), 34.1 (CH2, C-2ЈЈ), 50.1 (CH,
C-2), 68.8 (CH2, C-6Ј), 69.0 (CH2, C-1), 69.9 (CH, C-5Ј), 71.9
(CH2, benzyl methylene group), 72.8 (CH2, benzyl methylene
group), 73.4 (CH2, benzyl methylene group), 73.6 (CH2, benzyl
methylene group), 74.9 (CH2, benzyl methylene group), 75.3 (CH,
d, J = 8 Hz, C-4Ј) 77.4 (CH, C-3Ј), 78.8 (CH, C-3), 80.1 (CH, C-
4), 89.2 (CH, d, J = 188 Hz, C-2Ј), 98.1 (CH, d, J = 21 Hz, C-1Ј),
128.4–127.5 (CH, aromatic C atoms), 138.6–137.5 (C, aromatic C
atoms), 172.9 (C, C-1ЈЈ) ppm.
Lymphocyte Proliferation Test: Spleens from C57Bl/6 mice (Charles
River, Italia, Calco, Como) were aseptically removed, minced, and
cell suspensions were incubated at 5×105 per well in 96-well micro-
titer plates (3799 Costar, Italia; Milan, Italy) using RPMI 1640
medium supplemented with 100 UmL–1 of penicillin, 2 mm gluta-
mine, and 100 μgmL–1 streptomycin, 20 mm Hepes buffer (Euro-
clone, Celbio, Milan, Italy), and 10% FCS (Euroclone, Celbio, Mi-
lan, Italy). All reagents were free of endotoxin contamination by
the LAL assay. Various doses of the α-Gal-GSL, either agelasphin
(2) or the 2Ј-fluoro analog 1, were added. Concanavaline A
(1 μgmL–1) was used as positive control. Cell proliferation was
measured using the already described MTT assay.[20] Plates were
incubated for 72 h at 37 °C in 5% CO2, then 20 μL of a 5 mgmL–1
solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT; M-2128 Sigma) in PBS were added for an ad-
ditional 3 h at 37 °C. The plates were then centrifuged, the superna-
tants discarded, and the dark-blue formazan crystals dissolved in
100 μL of lysing buffer consisting of a solution 20% (w/v) of SDS
(Sigma), 40% of N,N-dimethylformamide (Merck) in H2O at pH
4.7 adjusted with 80% acetic acid. The plates were then read on a
microplate reader (Molecular Devices Co., Menlo Park, CA, USA)
using a test wavelength of 550 nm and a reference wavelength of
650 nm. The results are expressed as OD 550/OD650 which mean
that the values at OD 650 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.
Supporting Information: 1H NMR of compound 1a; 1D and 2D
1
NMR spectra of compound 1b; H NMR and CD spectra of the
degradation products 2–5 (see also the footnote on the first page
of this article).
Acknowledgments
This work is the result of research supported by MIUR PRIN (It-
aly). Mass and NMR spectra were recorded at the “Centro di Servi-
zio Interdipartimentale di Analisi Strumentale” (CSIAS), Uni-
versità di Napoli “Federico II”. The assistance of the staff there is
gratefully acknowledged.
(2S,3S,4R)-1-O-(2-Deoxy-2-fluoro-α-d-galactopyranosyl)-2-docos-
anoylamino-1,3,4-octadecanetriol (1): Compound 12 (80 mg,
0.064 mmol) was subjected to hydrogenolysis over Pd(OH)2/C
(50 mg, 20% w/w) as previously reported.[8] The reaction mixture
was purified by reversed-phase HPLC chromatography on RP-18
silica gel (MeOH 100%) to give 38 mg (0.047 mmol, 73%) of com-
pound 1 as an amorphous solid. [α]D = +4 (c = 0.4, CHCl3).
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HRESI MS (positive ions): m/z
= 804.6745 ([M +
H]+,
C46H91FNO8 gives 804.6729). ESI MS (positive ions): m/z = 804
[M + H]+. 1H NMR (500 MHz, [D5]pyridine): δ = 0.86 (t, J =
3284
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Immunomodulatory α-Galactoglycosphingolipids
FULL PAPER
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Received: January 23, 2005
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