Development of a fluorescent probe for the study of the sponge-derived simplexide immunological properties

Article abstract of DOI:10.1016/j.carres.2011.11.017

Simplexide is a glycolipid of marine origin, endowed with immunological properties, composed of a long chain secondary alcohol glycosylated by an α-d-glucosyl-(1→4)-β-d-galactosyl disaccharide residue. Herein we describe the preparation of a fluorescent d

Full text of DOI:10.1016/j.carres.2011.11.017

Carbohydrate Research 348 (2012) 27–32
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Development of a fluorescent probe for the study of the sponge-derived
simplexide immunological properties
Riccardo Di Brisco a, Fiamma Ronchetti a, Alfonso Mangoni b, Valeria Costantino b, Federica Compostella a,
⇑
a Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Via Saldini 50, 20133 Milano, Italy
b Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli ‘Federico II’, Via D. Montesano 49, 80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Simplexide is a glycolipid of marine origin, endowed with immunological properties, composed of a long
chain secondary alcohol glycosylated by an -glucosyl-(1?4)-b- -galactosyl disaccharide residue.
Received 15 September 2011
Received in revised form 14 November 2011
Accepted 15 November 2011
Available online 25 November 2011
a
-D
D
Herein we describe the preparation of a fluorescent derivative of simplexide, labeled at position 6 of
the distal glucose with a dansyl group, as a probe for future studies on the mechanism by which simplex-
ide affects the immune system. Fluorescent simplexide was prepared from a 600-amino functionalized
compound, which in turn was obtained through a highly efficient glycosylation between the preformed
activated disaccharide and the long chain secondary alcohol 18-pentatriacontanol.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Glycolipids
Fluorescent probes
Marine compounds
1. Introduction
examination. Several glycolipids have been recognized as activa-
tors of the immune cell functions, and pathways and molecules
Sponges of the genus Plakortis are skilled in the production of
secondary metabolites,1 and the Caribbean species Plakortis simplex
is prolific in this regard providing a number of structurally unique
metabolites.2 Beside peroxides, that is, plakortin,3 which are espe-
cially prominent species produced by the sponge, and triterpenoid
compounds, presumably produced by the sponge microbial symbi-
onts, a number of glycolipids were also found in the total lipophilic
extract.2 Glycosphingolipids are the most common glycolipids
from sponge. They display a fairly conserved pattern of ceramide
aglycons, and saccharide chains composed of one to six sugar units.
Nevertheless, a number of glycolipids have been isolated from P.
simplex that show unusual features. These include simplexides, a
series of glycosylated long-chain secondary alcohols, which repre-
sent a new structural kind of glycolipids (Fig. 1).4 The polar portion
targeted to glycolipid recognition have been identified.5 As a con-
sequence, it could be interesting to have a deeper evaluation of
the immunological activity of simplexide, as well as to investigate
the precise relationship between the structure of the glycolipid and
its activity. The need to obtain larger amounts of pure and chemi-
cally homogeneous simplexide, has already stimulated the synthe-
sis of the three unbranched homologs of simplexide depicted in
Figure 1.6
Herein, we report the preparation of a fluorescently labeled
simplexide, to be used in cytofluorimetric assays, which can sup-
port the studies toward the identification of the biological receptor
of simplexide and/or its cellular trafficking. We have designed and
is conserved throughout the natural mixture and consists of an
a-
OH
O
D
-glucosyl-(1?4)-b- -galactosyl disaccharide residue. In contrast,
D
simplexides
HO
HO
the lipophilic part shows variability, the homologous species dif-
fering by the length and branching of the secondary alcohol alkyl
chains. The chains range from C33 to C37, with the hydroxyl group
in the middle of the alkyl skeleton.
HO
O
OH
R1
R2
O
O
HO
HO
At the time of its first isolation, simplexide was shown to inhibit
concanavalin-A induced proliferation of murine T-cells, without
being cytotoxic.4 The mechanism of this immunosuppressive effect
was not investigated at that time, but is surely worthy of further
R1, R2 = CH2(CH2)14CH3
CH2(CH2)13CH(CH3)2
CH2(CH2)15CH3
CH2(CH2)13CH(CH3)CH2CH3
CH2(CH2)16CH3
⇑
Corresponding author. Tel.: +39 02 50316045; fax: +39 02 50316036.
E-mail address: federica.compostella@unimi.it (F. Compostella).
Figure 1. Chemical structure of simplexide and composition of the natural mixture.
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.11.017

28
R. Di Brisco et al. / Carbohydrate Research 348 (2012) 27–32
N3
N
O
BnO
BnO
O
CCl3
NH
BnO
4
+
NHSO2
O
OH
OBz
HO
HO
O
OAll
HO
BzO
O
OH
OBz
5
O
C17H35
C17H35
O
O
i
HO
HO
N3
1
O
BnO
BnO
NH2
O
BnO
HO
HO
O
OBz
O
HO
O
OAll
OH
BzO
O
OBz
C17H35
C17H35
3
HO
HO
2
Scheme 1. The glycosylation reaction to disaccharide 3. Reagents and conditions:
(i) TMSOTf, Et2O, MS 4 Å, ꢀ40 °C, 53%.
Figure 2. Structure of fluorescently labeled simplexide 1 and its precursor 2.
R2
OH
prepared by total synthesis the fluorescent simplexide 1 (Fig. 2),
where a dansyl group is attached to the 6 position of the outer glu-
cose residue through a robust sulfonamide linkage. We decided to
attach the fluorophore to the distal part of the polar moiety rather
than to the lipid part in order to prevent any possible interference
on the association of the glycolipid with a receptor via the lipid
portion. Glycolipid antigen recognition by the immune system is,
in fact, mostly mediated by the CD1 antigen presenting molecules,
which bind the glycolipid antigen by loading its lipid part into a
hydrophobic pocket and generate an immunogenic complex that
is presented to the T-cell receptors,5 even thought some new in-
sights suggest the existence of a previously unrecognized popula-
tion of glycolipid antigen-specific, CD1-independent T cells.7 The
presence of a label on the sugar part of immunogenic glycolipid
is generally tolerated,8 and the biological activity preserved, pro-
vided that glycolipid processing does not occur before receptor
binding.9
O
R1
OAll
HO
OH
v
10: R1 = H, R2 = OH
5
6: R1 = OH, R2 = H
i
OTs
O
HO
HO
OAll
OH
7
ii
N3
O
R1O
R1O
OR2
OR1
8: R1 = H; R2 = All
9: R1 = Bn; R2 = All
iii
iv
In our synthetic scheme, the glycolipid probe 1 is derived from
‘amino-simplexide’ 2, bearing an amino functionality at the 6-posi-
tion of glucose (Fig. 2).8a,b This provides a quite general approach
for the development of simplexide probes for biological evaluation.
In fact, compound 2 could be, for example, the precursor of a more
soluble pegylated simplexide.10
4: R1 = Bn; R2 = OC(NH)CCl3
Scheme 2. Synthesis of glucosyl donor 4 and galactosyl acceptor 5. Reagents and
conditions: (i) tosyl chloride, Py, 0 °C, 80%; (ii) sodium azide, TBAI, DMF, 80 °C, 90%;
(iii) benzyl bromide, NaH, DMF, 55 °C, 90%; (iv) (a) Ir catalyst (2.6 mol %), THF, then
NBS, H2O, 70%, (b) Cl3CCN, K2CO3, CH2Cl2, 77%; (v) BzCl, Py, ꢀ30 °C, 50%.
2. Results and discussion
Glucosyl trichloroacetimidate 4 (Scheme 2) was obtained start-
ing from known allyl glucopyranoside 6,13 which was selectively
activated at the 6-hydroxyl group as its p-toluenesulfonyl ester
to give compound 7 in high yield. Tosylate 7 was subjected to a
nucleophilic displacement reaction with sodium azide to yield 6-
azido-glucoside 8, and the remaining hydroxyl groups were then
protected as benzyl ethers to give glucoside 9. Compound 9 was fi-
nally transformed into donor 4 by removal of the anomeric allyl
group through smooth isomerization of the double bond with the
bis (methyldiphenylphosphine)cyclooctadiene-iridium(I) hexa-
fluorophosphate/hydrogen catalyst system,14 followed by mild
hydrolysis of the enol ether, and activation as a b-trichloroacetim-
idate with trichloroacetonitrile in the presence of potassium car-
bonate. The known allyl b-galactopyranoside 1015 was the
starting material for the synthesis of acceptor 5. It was the sub-
strate for a selective limited benzoylation at low temperature
which gave directly in one step the desired 2,3,6-tri-O-benzoylated
galactoside 5 in more than satisfactory 50% yield. The reaction
The synthesis of ‘amino-simplexide’ 2 and, then, of fluorescent
simplexide 1, started with the preparation of allyl
a-D-glucosyl-
(1?4)-b- -galactoside 3. The construction of the disaccharide in
D
the early stage of the synthesis opens the possibility to use dif-
ferent alcohols in the glycosylation step, and, therefore, to syn-
thesize other members of the natural mixture besides the most
represented C-35 alkanol.4 It has indeed been reported that the
immunological properties of glycolipids are somehow modulated
by the length of the acyl chain.11 Furthermore, glycosylations
with very long alcohols are usually affected by low yields, mak-
ing it convenient to postpone the reaction to a late synthetic
step.12
6-Azido glucosyl donor 4, with a non-participating 2-O-Bn
group and the amino functionality already installed on the
monosaccharide, and 4-OH galactosyl acceptor 5 were the sub-
strates designed for the preparation of disaccharide 3 through a
Schmidt’s glycosylation procedure (Scheme 1).

R. Di Brisco et al. / Carbohydrate Research 348 (2012) 27–32
29
N3
simplexide which can be also used for the development of other
probes for biological studies. The synthetic scheme is suitable for
the synthesis of simplexide as such only by changing the glucosyl
donor in the formation of the disaccharide, that is, using perbenzy-
lated glucosyl trichloroacetimidate.
Fluorescent assays based on a fluorescent substrate analogue
require that the analogue is processed and incorporated. If the fluo-
rescent derivative 1 preserves the immunological activity of natu-
ral simplexide, it will represent a novel useful tool in the ongoing
studies on the mechanism of action of simplexide.
O
BnO
BnO
BnO
i
O
OBz
3
O
BzO
BzO
11
O
CCl3
NH
ii
R1
O
R2O
R2O
R2O
OR3
O
4. Experimental
O
C17H35
C17H35
R3O
O
OR3
4.1. General methods
12: R1 = N3, R2 = Bn, R3 = Bz
iii
1: R1 = NHdansyl, R2, R3 = H
All reagents were bought at highest commercial quality and
used without further purification except where noted. Air- and
moisture-sensitive liquids and solutions were transferred via
oven-dried syringe or stainless steel cannula through septa. Organ-
ic solutions were concentrated by rotary evaporation below 45 °C
at approximately 20 mmHg. All reactions were carried out under
anhydrous conditions using oven-dried glassware within an Argon
atmosphere. Dry solvents and liquid reagents were distilled prior
to use. Tetrahydrofuran (THF) was distilled from sodium; dichloro-
methane (CH2Cl2) and pyridine were distilled from calcium hy-
dride; dimethylformamide (DMF), methanol (MeOH) and diethyl
ether (Et2O) were dried on activated 4 Å molecular sieves (MS);
chloroform (CHCl3) was treated with calcium chloride overnight,
then it was filtered through an alumina column (neutral, Typ
507C, 150 mesh, Sigma–Aldrich), and finally dried on activated
4 Å MS; triethylamine (TEA) was distilled from calcium hydride.
NaH was washed three times with hexane prior to use. All reac-
tions were monitored by TLC on Silica Gel 60 F-254 plates (Merck),
spots being developed with 5% H2SO4 in MeOH/H2O (1:1) or with a
phosphomolybdate-based reagent and subsequently heated at
110 °C. Flash column chromatography was performed on Silica
Gel 60 (230–400 mesh, Merck). Optical rotations were measured
with a Perkin–Elmer 241 polarimeter. 1H NMR and 13C NMR spec-
tra were recorded at 298 K with a Bruker FT-NMR AVANCE™
DRX500 spectrometer using a 5 mm z-PFG (pulsed field gradient)
broadband reverse probe. Chemical shifts are reported on the d
(ppm) scale and are relative to TMS as internal reference. The sig-
nals were unambiguously assigned by 2D COSY and HSQC experi-
ments (standard Bruker pulse program). Scalar coupling constants
are reported in Hertz. The mass spectrometric analyses were per-
formed in positive or negative electrospray mode (ESI-MS). MS
spectra were recorded on a Thermo Quest Finnigan LCQ™deca
ion trap mass spectrometer; the mass spectrometer was equipped
with a Finnigan ESI interface. Data were processed by Finnigan
Xcalibur software system. TLC. High resolution MS spectra were re-
corded on a FT-ICR mass spectrometer APEX II & Xmass software
(Bruker Daltonics)—4.7 T Magnet (Magnex) equipped with an ESI
source. 18-Pentatriacontanol was purchased from Sigma–Aldrich.
Scheme 3. Synthesis of fluorescently labeled simplexide 1. Reagents and condi-
tions: (i) (a) Ir catalyst, THF, then NBS, H2O, 73%, (b) Cl3CCN, DBU, CH2Cl2, 80%; (ii)
18-pentatriacontanol, TMSOTf, MS AW300, CHCl3, 83%; (iii) (a) MeONa, MeOH, (b)
Ph3P, THF/H2O 58%, (c) Pd black, HCOOH, MeOH, (d) dansylCl, TEA, CHCl3/MeOH,
43% for two steps.
worked in the same way as reported in the literature on allyl
a-
galactopyranoside,16 despite the different anomeric configuration
which is known to influence the outcome of these regioselective
reactions.17
Donor 4 and acceptor 5 were then subjected to glycosylation to
give the thermodynamically favored 1,2-cis-
conditions were investigated in order to increase the yield and
the -selectivity of the reaction. Finally, disaccharide 3 was recov-
a-linkage. Different
a
ered in a reasonably satisfactory yield performing the glycosylation
in diethyl ether and under the catalysis of trimethylsilyl trifluoro-
methansulfonate. The
a-configuration of the newly formed glyco-
sidic linkage was confirmed by the value of the cis coupling
0
0
constant between H-1 and H-2 (J1 ,2 = 3.3 Hz).
Disaccharide 3 (Scheme 3) was first converted into donor 11 by
removal of the anomeric allyl group and activation as trichloroace-
timidate, and then subjected to the glycosylation reaction with 18-
pentatriacontanol. The choice of the solvent was essential for the
outcome of the reaction, and we finally decided to use chloroform
because, in our hands, the reaction system was not homogeneous
in dichloromethane due to the insolubility of the double-tailed
alcohol.18,7 Using chloroform as the solvent, the reaction of donor
11 with 18-pentatriacontanol under the catalysis of trimethylsilyl
trifluoromethanesulfonate provided glycoconjugate 12 in high
yield. The formation of the 1,2-trans-b-galactopyranoside linkage
was ensured by the glycosylation with a donor installed with a
neighboring participating group, that is, trichloroacetimidate 11
bearing 2-O-Bz.
A series of subsequent reactions allowed the transformation of
compound 12 into the target compound 1. Benzoates were first re-
moved through transesterification by sodium methoxide in meth-
anol, then the azido group was selectively reduced by Staudinger
reduction, which was followed by hydrogenolysis of the benzyl
ethers in the presence of formic acid as hydrogen donor. The
deprotected amine 2 was immediately derivatized with dansyl
chloride to give the desired fluorescently labeled simplexide 1 in
good overall yields.
4.2. Allyl 6-O-tosyl-b-
D-glucopyranoside (7)
To a solution of allyl b-
D
-glucopyranoside 613 (2.00 g, 9.08
mmol) in dry pyridine (60 mL) 4-toluenesulfonyl chloride (2.94 g,
15.4 mmol) was added at 0 °C. The reaction was stirred at 0 °C
overnight. The reaction mixture was quenched with MeOH and
the solvents were removed under reduced pressure. The residue
was purified by flash chromatography (EtOAc/MeOH, 9:1) to afford
3. Conclusions
compound 7 (2.72 g, 80%) as an amorphous solid: ½a D20
ꢀ21.0 (c 1,
ꢁ
We have described the synthesis of the first fluorescently la-
beled derivative of simplexide. This straightforward protocol is
general, and involves the preparation of a 600-amino functionalized
MeOH); 1H NMR (CD3OD): d 2.47 (s, 3H, CH3), 3.16 (dd, 1H, J1,2 8.1,
J2,3 9.2 Hz, 2-H), 3.22 (t, 1H, J3,4 = J4,5 9.2 Hz, 4-H), 3.31 (t, 1H,

30
R. Di Brisco et al. / Carbohydrate Research 348 (2012) 27–32
J2,3 = J3,4 9.2 Hz, 3-H), 3.39–3.45 (m, 1H, 5-H), 4.01–4.10 (m, 1H,
OCHaHbCH@CH2), 4,17 (dd, 1H, J5,6a 6.1 Hz, J6a,6b 10.8 Hz, 6a-H),
4.18–4.28 (m, 2H, 1-H and OCHaHbCH@CH2), 4.36 (dd, 1H, J5,6a
1.7 Hz, J6a,6b 10.8 Hz, 6b-H), 5.14–5.21 (m, 1H, CH@CHaHb), 5.30–
5.36 (m, 1H, CH@CHaHb), 5.88–5.99 (m, 1H, CH@CH2), 7.42–7.50
(m, 2H, arom), 7.79–7.85 (m, 2H, arom); 13C NMR (CD3OD): d
20.2, 69.4, 69.7, 69.8, 73.4, 73.6, 76.5, 101.9, 116.2, 127.7 (2C),
129.6 (2C), 133.0, 134.4, 145.1; ESI-MS (positive-ion mode): m/z
397.1 (100) [M+Na]+, 770.7 (40) [2M+Na]+. Anal. Calcd for
additional tetrahydrofuran (150 mL), water (15 mL), and N-bromo-
succinimide (1.35 g, 7.56 mmol) were added. After 30 min, the
mixture was concentrated, ethyl acetate was added (200 mL),
and the organic layer was washed with satd NaHCO3 solution
(200 mL) and brine (100 mL). Drying (Na2SO4) and evaporation of
the solvent gave a crude residue which was purified through
flash-chromatography (petroleum ether/EtOAc, 6:4) to afford glu-
copyranose (1.68 g, 70%) as an amorphous solid. The identity of
the compound was assessed through MS experiment, and it was
used in the next step without further characterization. ESI-MS (po-
sitive-ion mode): m/z 498.2 (100) [M+Na]+, 972.8 (33) [2M+Na]+.
The glucopyranose was dissolved in dry CH2Cl2 (30 mL). Trichloro-
acetonitrile (3.36 mL, 33.5 mmol) and K2CO3 (1.45 g, 10.5 mmol)
were added and the mixture was stirred under Argon overnight be-
fore being filtered through a Celite pad and concentrated. The res-
idue was purified by flash-chromatography (petroleum ether/
EtOAc, 9:1 + 1% TEA) to afford compound 4 (1.66 g, 77%) as a color-
C16H22O8S (374.4): C, 51.33; H, 5.92. Found: C, 51.12; H, 5.99.
4.3. Allyl 6-azido-6-deoxy-b- -glucopyranoside (8)
D
A mixture of compound 7 (2.66 g, 7.11 mmol), sodium azide
(1.38 g, 21.3 mmol) and a catalytic amount of tetra-n-butyl-ammo-
nium iodide (0.520 g, 0.140 mmol) in dry DMF (50 mL) was put un-
der stirring at 80 °C. After 6 h, DMF was removed under high
vacuum and the residue was purified by flash-chromatography
(CH2Cl2/MeOH, 9:1) to afford compound 8 (1.57 g, 90%) as an
less oil: ½a 2D0
ꢁ
+35.7 (c 1, CHCl3); 1H NMR (CDCl3): d 3.35 (dd, 1H, J5,6a
5.3 Hz, 6a-H), 3.50 (dd, 1H, J5,6b 2.2 Hz, 6b-H), 3.60–3.84 (m, 4H, 2-
H, 3-H, 4-H, 5-H), 4.59–5.03 (m, 6H, CH2Ph), 5.87 (d, 1H, J1,2 7.5 Hz,
1-H), 7.23–7.40 (m, 15H, arom.), 8.78 (s, 1H, NH); 13C NMR (CDCl3):
d 50.87, 74.90, 75.12, 75.29, 75.60, 77.57, 80.84, 84.36, 97.80,
127.57–128.91 (15C), 137.65, 137.80, 138.24, 160.94; ESI-MS
(positive-ion mode): m/z 641.0 (95) [M+Na]+, 642.9 (100)
[M+2+Na]+. Anal. Calcd for C29H29O5N4Cl3 (619.9): C, 56.19; H,
4.72; N, 9.04. Found: C, 56.06; H, 4.93; N, 8.91.
amorphous solid: ½a D20
ꢁ
ꢀ81.5 (c 1, MeOH); 1H NMR (CD3OD): d
3.19–3.54 (m, 6H, 2-H, 3-H, 4-H, 5-H and 2 6-H), 4.14–4.21 (m,
1H, OCHaHbCH@CH2), 4.31–4.39 (m, 2H, 1-H and OCHaHbCH@CH2),
5.16–5.23 (m, 1H, CH@CHaHb), 5.30–5.38 (m, 1H, CH@CHaHb),
5.92–6.01 (m, 1H, CH@CH2); 13C NMR (CD3OD): d 51.4, 69.7, 71.2,
73.7, 75.7, 76.5, 101.9, 116.2, 134.2; ESI-MS (negative-ion mode):
m/z 243.9 (30) [Mꢀ1]ꢀ, 498.0 (100) [2Mꢀ1]ꢀ. Anal. Calcd for
C9H15O5N3 (245.2): C, 44.08; H, 6.17; N, 17.13. Found: C, 44.32;
H, 6.11; N, 17.08.
4.6. Allyl 2,3,6-tri-O-benzoyl-b-D-galactopyranoside (5)
4.4. Allyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-b-
glucopyranoside (9)
D
-
A solution of benzoyl chloride (2.37 mL, 20.4 mmol) in dry pyr-
idine (30 mL) was added dropwise to a solution under Argon at
ꢀ30 °C of allyl-b-
D
-galactopyranoside 1015 (1.00 g, 4.54 mmol) in
Sodium hydride (60% in mineral oil, 1,64 g, 42.8 mmol), previ-
ously washed three times with n-hexane, was added under Argon
at 0 °C to a solution of compound 8 (1.50 g, 6.12 mmol) in dry DMF
(50 mL), and the foaming gray mixture was then allowed to warm
to room temperature. Benzyl bromide (6.55 mL, 55.1 mmol) was
slowly added, and the mixture was then heated and vigorously
stirred at 55 °C for 6 h. MeOH was added to destroy the excess of
NaH, then the mixture was concentrated under reduced pressure,
and diluted with EtOAc (100 mL). The organic layer was washed
with water (200 mL). After separation, the aqueous layer was fur-
ther extracted with EtOAc (2 ꢂ 50 mL). The combined organic ex-
tracts were dried (Na2SO4) and concentrated. The residue was
purified by flash-chromatography (petroleum ether/EtOAc, 9:1)
dry pyridine (30 mL). After 0.5 h the reaction mixture was
quenched with an aq satd NaHCO3 solution (100 mL), and ex-
tracted with CH2Cl2 (3 ꢂ 80 mL). The combined organics were
washed with brine (100 mL), dried and the solvent evaporated un-
der reduced pressure. The crude was purified twice by flash-chro-
matography and eluted first with toluene/EtOAc, 8:2, to remove
allyl 2,3,4,6-tetra-O-benzoyl-b-D-galactopyranoside, and then with
hexane/EtOAc, 75:25 to afford compound 5 (1.21 g, 50%) as an
amorphous solid ½a D20
ꢁ
+56.5 (c 1, CHCl3); 1H NMR (CDCl3): d 2.60
(d, 1H, J 5.4 Hz, OH), 4.09 (dd, 1H, J4,5 = J5,6 6.5 Hz, 5-H), 4.16–
4.23 (m, 1H, OCHaHbCH@CH2), 4.34–4.43 (m, 2H, OCHaHbCH@CH2
and 4-H), 4.65 (dd, 1H, J5,6a 6.5, J6a,6b 11.4 Hz, 6a-H), 4.72 (dd, 1H,
J5,6b 6.5, J6a,6b 11.4 Hz, 6b-H), 4.81 (d, 1H, J1,2 7.9 Hz, 1-H), 5.11–
5.17 (m, 1H, CH@CHaHb), 5.21–5.27 (m, 1H, CH@CHaHb), 5.38 (dd,
1H, J2,3 10.3, J3,4 3.2 Hz, 3-H), 5.77–5.89 (m, 2H, CH@CH2 and 2-
H), 7.36–7.64 (m, 10H, arom.), 7.96–8.12 (m, 5H, arom.); 13C
NMR (CDCl3): d 62.7, 67.3, 69.6, 69.9, 72.3, 74.2, 100.1, 117.7,
128.3–133.5 (19C), 165.3, 165.9, 166.4; ESI-MS (positive-ion
mode): m/z = 555.1 (52) [M+Na]+, 1087.0 (100) [2M+Na]+. Anal.
Calcd for C30H28O9 (532.5): C, 67.66; H, 5.30. Found: C, 67.45; H,
5.47.
to afford compound 9 (2.84 g, 90%) as an amorphous solid: ½a D20
ꢁ
+7.1 (c 1, CHCl3); 1H NMR (CDCl3): d 3.31–3.74 (m, 6H, 2-H, 3-H,
4-H, 5-H and 2 6-H), 4.16–4.24 (m, 1H, OCHaHbCH@CH2), 4.42–
4.50 (m, 1H, OCHaHbCH@CH2), 4.54 (d, 1H, J1,2 7.8 Hz, 1-H), 4.52–
5.06 (m, 6H, CH2Ph), 5.22–5.30 (m, 1H, CH@CHaHb), 5.35–5.43
(m, 1H, CH@CHaHb), 5.93–6.05 (m, 1H, CH@CH2), 7.23–7,45 (m,
15H arom.); 13C NMR (CDCl3): d 51.5, 70.3, 74.7, 74.9, 75.0, 75.7,
78.4, 82.3, 84.5, 102.5, 117.6, 127.7–128.5 (15C), 133.8, 137.7,
138.3, 138.4; ESI-MS (positive-ion mode): m/z 538.1 (100)
[M+Na]+. Anal. Calcd for C30H33O5N3 (515.6): C, 69.88; H, 6.45; N,
8.15. Found: C, 69.97; H, 6.39; N, 8.02.
4.7. Allyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyr-
anosyl-(1?4)-2,3,6-tri-O-benzoyl-b- -galactopyranoside (3)
D
4.5. 6-Azido-2,3,4-tri-O-benzyl-6-deoxy-b-
trichloroacetimidate (4)
D
-glucopyranosyl
Trimethylsilyl trifluoromethansulfonate (2.62 mL, 0.1 M sol in
Et2O) was added dropwise at ꢀ40 °C to a mixture under Argon of
donor 4 (1.63 g, 2.63 mmol), acceptor 5 (0.70 g, 1.31 mmol) and
4 Å MS (1.63 g) in dry Et2O (40 mL). The reaction mixture was
quenched after 0.5 h with solid NaHCO3 before being filtered
through a pad of Celite and concentrated. The residue was purified
by flash-chromatography (hexane/EtOAc, 85:15) to afford com-
Bis(methyldiphenylphosphine)cyclooctadiene-iridium(I) hexa-
fluorophosphate (0.130 g, 0.154 mmol) was suspended in dry tet-
rahydrofuran (25 mL) under Ar, and hydrogen was bubbled for
5 min through the mixture. The resulting clear solution was then
added dropwise to a stirred solution of compound 9 (2.60 g,
5.04 mmol) in dry tetrahydrofuran (60 mL) under Ar. After 3 h,
pound 3 (0.69 g, 53%) as an oil: ½a D20
ꢁ
+94.3 (c 1, CHCl3); 1H NMR
(CDCl3): d 3.14–3.25 (m, 2H, 2 60-H), 3.52 (dd, 1H, J1 ,2 3.4, J2 ,3
0
0
0
0

R. Di Brisco et al. / Carbohydrate Research 348 (2012) 27–32
31
9.8 Hz, 20-H), 3.61 (dd, 1H, J3 ,4 = J4 ,5 9.6 Hz, 40-H), 4.05 (t, 1H,
the reaction was warmed to room temperature and stirred for
0.5 h. The reaction was quenched with TEA and the molecular
sieves were removed through filtration. After evaporation of the
solvent, 18-pentatriacontanol was precipitated by adding Et2O
and separated by filtration. The organic solution was concentrated
and the crude was purified by flash-chromatography (hexane/
CH2Cl2/EtOAc, 8.5:1:0.5) to afford compound 12 (0.36 g, 83 %) as
0
0
0
0
J4 ,5 = J5 ,6 6.6 Hz, 50-H), 4.13–4.25 (m, 3H, 30-H, 50-H and
OCHaHbCH@CH2), 4.36–4.43 (m, 2H, 4-H and OCHaHbCH@CH2),
0
0
0
0
0
0
4.58–4.78 (m, 6H, 2 6-H, 1-H, 3CH2Ph), 4.88 (d, 1H, J1 ,2 3.4 Hz,
10-H), 4.92–5.06 (m, 3H, 3CH2Ph), 5.10–5.15 (m, 1H, CH@CHaHb),
5.20–5.27 (m, 1H, CH@CHaHb), 5.29 (dd, 1H, J2,3 10.6, J3,4 2.9 Hz,
3-H), 5.73 (dd, 1H, J1,2 7.8, J2,3 10.6 Hz, 2-H), 5.78–5.88 (m, 1H,
CH@CH2), 7.11–7.65 (m, 24H, arom), 7.93–8.09 (m, 6H, arom);
13C NMR (CDCl3): d 50.68, 63.0, 69.7, 70.0, 71.1, 72.8, 73.8, 74.0,
75.1, 75.6, 76.0, 77.9, 80.2, 80.2, 81.4, 99.9, 100.1, 117.8, 127.6–
133.6 (33C), 137.9, 138.3, 138.7, 165.4, 166.1, 166.2; ESI-MS (posi-
tive-ion mode): m/z 1012.3 (100) [M+Na]+. Anal. Calcd for
an oil: ½a 2D0
ꢁ
+79.1 (c 1, CHCl3); 1H NMR (CDCl3): d 0.73–1.76 (m,
70H, aliphatic), 3.12–3.26 (m, 2H, 2, 60-H), 3.47–3.66 (m, 3H, 20-
H, 40-H and OCH), 4.03 (t, 1H, J4,5 = J5,6 6.3 Hz, 5-H), 4.14–4.26 (m,
2H, 30-H, 50-H), 4.37 (d, 1H, J3,4 2.6 Hz, 4-H), 4.57–4.82 (m, 6H, 1-
H, 2, 6-H and 3CH2Ph), 4.87 (d, 1H, J1 ,2 3.4 Hz, 10-H), 4.90–5.07
(m, 3H, 3CH2Ph), 5.29 (dd, 1H, J2,3 10.7, J3,4 2.6 Hz, 3-H), 5.67 (dd,
1H, J2,3 10.7, J1,2 7.8 Hz, 2-H), 7.10–7.64 (m, 24H, arom), 7.91–
8.09 (m, 6H, arom); 13C NMR (CDCl3): d 14.1, 22.7–35.0 (33C),
50.7, 63.3, 70.0, 71.0, 72.7, 73.9, 74.0, 75.1, 75.6, 76.2, 78.0, 80.3,
81.4, 82.4, 99.9, 101.7, 127.6–129.9 (30C), 133.0, 133.1, 133.5,
138.0, 138.3, 138.7, 165.2, 166.1, 166.2; ESI-MS (positive-ion
mode): m/z 1463.7 (100) [M+Na]+, 1478.7 (33) [M+K]+. Anal. Calcd
for C89H121O13N3 (1440.93): C, 74.19; H, 8.46; N, 2.92. Found: C,
74.03; H, 8.66; N, 2.81.
0
0
C
57H55O13N3 (990.1): C, 69.15; H, 5.60; N, 4.24. Found: C, 68.88;
H, 5.73; N, 4.14.
4.8. 6-Azido-2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyranosyl-
(1?4)-2,3,6-tri-O-benzoyl-a-D-galactopyranosyl
trichloroacetimidate (11)
Bis(methyldiphenylphosphine)cyclooctadiene-iridium(I) hexa-
fluorophosphate (0.015 g, 0.018 mmol) was suspended in dry tetra-
hydrofuran (4 mL) under Ar, and hydrogen was bubbled for 5 min
through the mixture. The resulting clear solution was then added
dropwise under stirring to a solution under Ar of compound 3
(0.67 g, 0.68 mmol) in dry tetrahydrofuran (15 mL). After 3 h, addi-
tional tetrahydrofuran (35 mL), water (3 mL), and N-bromosuccini-
mide (0.18 g, 1.0 mmol) were added. After 30 min, tetrahydrofuran
was removed under reduced pressure and ethyl acetate was added
(100 mL). The organic layer was washed with satd NaHCO3 solution
(100 mL) and brine (80 mL). Drying (Na2SO4) and evaporation of the
solvent gave a crude residue which was purified through flash-
chromatography (petroleum ether/EtOAc, 8:2) to afford the depro-
tected disaccharide (0.46 g, 73%). The identity of the compound was
assessed through MS experiment, and it was used in the next step
without further characterization. ESI-MS (positive-ion mode): m/z
972.3 (100) [M+Na]+, 1921.3 (12) [2M+Na]+. The de-allylated prod-
uct was dissolved under Argon in dry dichloromethane (5 mL). Tri-
4.10. 18-Pentatriacontanyl 6-O-[5-(N,N-dimethylamino)-naph
thalene-1-sulfonylamino)-6-deoxy-a-D-glucopyranosyl-(1?4)-
b-D-galactopyranoside (1)
MeONa (0.70 mL, 0.5 M solution in MeOH) was added to a solu-
tion under Argon of compound 12 (0.35 g, 0.24 mmol) in dry
CH2Cl2/MeOH, 1:1.5 (15 ml). The reaction was stirred at room tem-
perature overnight, then it was neutralized with an ion-exchange
resin (Dowex Marathon C–H+ form). The resin was filtered off
and the solution was concentrated under reduced pressure to give
the de-benzoylated product, which was reacted in the next step
without further purification. ESI-MS (positive-ion mode): m/z
1551.5 (100) [M+Na]+. Ph3P (0.13 g, 0.49 mmol) was added under
Argon to a solution of the de-benzoylated disaccharide in THF/
H2O, 9:1 (10 mL) and the reaction mixture was stirred overnight
at room temperature. The solvent was evaporated off, and the 600-
aminoderivative (0.15 g, 58%) was recovered as a colorless oil after
flash chromatography (CH2Cl2/MeOH/NH4OH, 95:5:0.5). ESI-MS
(positive-ion mode): m/z 1102.4 (78) [M+1]+, 1074.5 (100)
[M+1ꢀ28]+. The 600-amino-benzylated disaccharide (0.15 g,
0.14 mmol) was dissolved in 10% formic acid–MeOH (8 mL). Pd
black (0.075 g) was added and the reaction was stirred under an
Argon atmosphere at room temperature for 24 h. Filtration and
evaporation of the solvent under reduced pressure afforded the
corresponding deprotected crude 600-aminoderivative, which was
directly subjected to dansylation. ESI-MS (positive-ion mode): m/
z 854.9 (58) [M+Na]+, 826.7 (100) [M+Naꢀ28]+.
chloroacetonitrile
(0.48 mL,
4.8 mmol)
and
1,8-
diazabicyclo[5,4,0]undec-7-ene (two drops) were added and the
reaction was stirred for 3 h at room temperature. After evaporation
of the solvent, the residue was purified by flash-chromatography
(petroleum ether/EtOAc, 8:2 + 1% TEA) to afford trichloroacetimi-
date 11 (0.42 g, 80%) as an oil: ½a D20
ꢁ
+121.1 (c 1, CHCl3); 1H NMR
(CDCl3): d 3.07–3.17 (m, 2H, 2 60-H), 3.55 (dd, 1H, J1 ,2 3.3, J2 ,3
0
0
0
0
9.8 Hz, 20-H), 3.61 (dd, 1H, J3 ,4 = J4 ,5 9.5 Hz, 40-H), 4.11–4.23 (m,
2H, 30-H and 50-H), 4.56 (d, 1H, J3,4 2.6 Hz, 4-H), 4.58–4.83 (m, 6H,
0
0
0
0
H-5, 2 6-H, 3CH2Ph), 4.89 (d, 1H, J1 ,2 3.4 Hz, 10-H), 4.92–5.11 (m,
3H, 3CH2Ph), 5.82 (dd, 1H, J2,3 10.9, J3,4 2.6 Hz, 3-H), 5.94 (dd, 1H,
J1,2 3.7, J2,3 10.9 Hz, 2-H), 6.80 (d, 1H, J1,2 3.7 Hz, 1-H), 7.10–7.65
(m, 24H, arom), 7.92–8.06 (m, 6H, arom), 8.59 (s, 1H, NH); 13C
NMR (CDCl3): d 50.7, 63.1, 67.5, 70.5, 71.2, 71.6, 74.3, 75.2, 75.6,
76.2, 77.2, 78.0, 80.1, 81.4, 94.0, 99.7, 127.6–130.0 (30C), 133.2,
133.4, 133.6, 137.7, 138.1, 138.5, 160.6, 165.5, 165.9, 166.0; ESI-
MS (positive-ion mode): m/z 1115.0 (100) and 1116.9 (96)
[M+Na]+. Anal. Calcd for C56H51O13N4Cl3 (1094.4): C, 61.46; H,
4.70; N, 5.12. Found: C, 61.28; H, 4.87; N, 5.04.
0
0
To a solution under Argon at 0 °C of the crude amine and TEA
(0.045 mL, 0.32 mmol) in dry CHCl3/MeOH, 1:1 (15 mL), dansyl
chloride (0.049 g, 0.18 mmol) was added. The reaction was stirred
for 3 h at room temperature, then the solvent was removed under
reduced pressure. The product was purified by flash-chromatogra-
phy (CHCl3/MeOH, 95:5) affording the dansyl-derivative 1 (0.064 g,
43%) as an amorphous solid: 1H NMR (CDCl3/CD3OD, 1:1): d 0.87 (t,
6H, 2CH3); 1.18–1.66 (m, 64H, 32CH2); 2.89 (s, 6H, NMe2); 2.96
(dd, 1H, J5 ,6 a 7.6 Hz, J6 a,6 b 13.1 Hz, 60a-H); 3.09 (t, 1H, J3 ,4 = J4 ,5
0
0
0
0
0
0
0
0
4.9. 18-Pentatriacontanyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-
-glucopyranosyl-(1?4)-2,3,6-tri-O-benzoyl-b-
galactopyranoside (12)
a-
9.9 Hz, 40-H); 3.20 (dd, 1H, J1 ,2 3.8, J2 ,3 9.8 Hz, 20-H); 3.30–3.36
(m, 1H, 60b-H); 3.49 (dd, 1H, J1,2 = 7.5 Hz, J2,3 = 9.9 Hz, 2-H); 3.52–
3.69 (m, 5H, OCH(C17H35)2, 3-H, 30-H, 5-H, 6a-H); 3.75 (dd, 1H,
J5,6b 8.4, J6a,6b 10.8 Hz, 6b-H); 3.85 (br d, 1H, J3,4 2.5 Hz, 4-H);
4.06–4.13 (m, 1H, 50-H); 4.29 (d, 1H, J1,2 7.5 Hz, 1-H); 4.61–4.73
(m, 1H, 10-H); 7.20–7.25 (m, 1H, arom.); 7.51–7.61 (m, 2H, arom.);
8.16–8.21 (m, 1H, arom.); 8.30–8.35 (m, 1H, arom.); 8.50–8.56 (m,
1H, arom.); 13C NMR (CDCl3/CD3OD, 1:1): d 13.6 (2C), 22.5 (2C),
0
0
0
0
D
D-
A solution of trichloroacetimidate 11 (0.33 g, 0.30 mmol), 18-
pentatriacontanol (0.23 g, 0.45 mmol) and MS AW300 (2.4 g) in
dry CHCl3 (15 mL) was stirred under Argon for 0.5 h at room tem-
perature, and then cooled to 0 °C. Trimethylsilyl trifluoromethan-
sulfonate (0.60 mL, 0.1 M sol. in CHCl3) was added dropwise and

32
R. Di Brisco et al. / Carbohydrate Research 348 (2012) 27–32
M.; Wilson, I. A. Nat. Rev. Immunol. 2005, 5, 387–399; (f) Cheng, J. M. H.; Khan,
A. A.; Timmer, M. S. M.; Stocker, B. L. J. Carb. Chem. 2011. Article ID 749591, 13
pp.
24.7, 24.9, 29.2–29.7 (24C), 31.8 (2C), 33.5, 34.5, 44.0, 45.0 (2C),
59.1, 70.5, 71.4 (2C), 72.5, 73.4, 73.5, 74.3, 77.0, 80.7, 100.4,
103.0, 115.1, 119.1, 123.1, 128.0, 128.9, 129.6, 129.8, 130.0,
135.0, 151.7; ESI-MS (negative-ion mode): m/z 1063.7 (78)
[Mꢀ1]ꢀ, 1035.7 (100) [Mꢀ1ꢀ28]ꢀ. HRESIMS (positive-ion mode;
6. Lü, G.; Wang, P.; Liu, Q.; Zhang, Z.; Zhang, W.; Li, Y. Chi. J. Chem. 2009, 27, 2217–
2222.
7. Christiansen, D.; Vaughan, H. A.; Milland, J.; Dodge, N.; Mouhtouris, E.; Smyth,
M. J.; Godfrey, D. I.; Sandrin, M. S. Immunol. Cell Biol. 2011, 89, 502–510.
8. (a) Zou, X.-T.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.; Bendelac, A.; Savage, P.
B. Org. Lett. 2002, 4, 1267–1270; (b) Cheng, J. M. H.; Chee, S. H.; Knight, D. A.;
Acha-Orbea, H.; Hermans, I. F.; Timmer, M. S. M.; Stocker, B. L. Carbohydr. Res.
2011, 346, 914–926; (c) Franchini, L.; Compostella, F.; Donda, A.; Mori, L.;
Colombo, D.; De Libero, G.; Matto, P.; Ronchetti, F.; Panza, L. Eur. J. Org. Chem.
2004, 4755–4761.
calcd for
C59H104N2O12S + Na = 1087.72022): 1087.71919 (70)
[M+23]+, 1059.68805 (100) [M+23ꢀ28]+.
Acknowledgment
9. Zhou, D.; Mattner, J.; Cantu, C., III; Schrantz, N.; Yin, N.; Gao, Y.; Sagiv, Y.;
Hudspeth, K.; Wu, Y.-P.; Yamashita, T.; Teneberg, S.; Wang, D.; Proia, R. L.;
Levery, S. B.; Savage, P. B.; Teyton, L.; Bendelac, A. Science 2004, 306, 1786–
1789.
The Italian Ministry of University and Research (MIUR-Italy) is
gratefully acknowledged for financial support.
10. Ebensen, T.; Link, C.; Riese, P.; Shulze, K.; Morr, M.; Guzmán, C. A. J. Immunol.
2007, 179, 2065–2073.
Supplementary data
11. Yu, K. O. A.; Im, J. S.; Molano, A.; Dutronc, Y.; Illarionov, P. A.; Forestier, C.;
Fujiwara, N.; Arias, I.; Miyake, S.; Yamamura, T.; Chang, Y.-T.; Besra, G. S.;
Porcelli, S. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3383–3388.
12. Hada, N.; Hayashi, E.; Takeda, T. Carbohydr. Res. 1999, 316, 58–70.
13. Lin, Y. A.; Chalker, J. M.; Floyd, N.; Bernardes, G. J. L.; Davis, B. G. J. Am. Chem.
Soc. 2008, 130, 9642–9643.
Supplementary data (13C and 1H spectra of compounds 1, 3–9,
11, 12) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.carres.2011.11.017.
14. Oltvoort, J. J.; van Boeckel, C. A. A.; de Koning, J. H.; van Boom, J. H. Synthesis
1981, 305–308.
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