Divergent synthetic approach to 6′′-modified α-GalCer analogues

Article abstract of DOI:10.1039/c1ob06235b

A synthetic approach is presented for the synthesis of galacturonic acid and d-fucosyl modified KRN7000. The approach allows for late-stage functionalisation of both the sugar 6′′-OH and the sphingosine amino groups, which enables convenient synthesis of promising 6′′- modified KRN7000 analogues. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob06235b

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PAPER
Divergent synthetic approach to 6¢¢-modified a-GalCer analogues†
Nora Pauwels,a Sandrine Aspeslagh,b Gerd Vanhoenacker,c Koen Sandra,c Esther D. Yu,d Dirk M. Zajonc,d
Dirk Elewaut,b Bruno Linclaue and Serge Van Calenbergh*a
Received 23rd July 2011, Accepted 2nd September 2011
DOI: 10.1039/c1ob06235b
A synthetic approach is presented for the synthesis of galacturonic acid and D-fucosyl modified
KRN7000. The approach allows for late-stage functionalisation of both the sugar 6¢¢-OH and the sphin-
gosine amino groups, which enables convenient synthesis of promising 6¢¢-modified KRN7000 analogues.
Introduction
Similar to major histocompatibility complex (MHC) class I or
class II molecules that present peptide antigens to the classic
CD8+ and CD4+ T cells of the immune system, the evolutionary
related CD1 molecules are specialized in presenting lipid and
glycolipid antigens to non-MHC-restricted T lymphocytes.1 To
accommodate these glycolipids, CD1 proteins have evolved a
unique hydrophobic pocket in which the lipid chains are buried.2
From the five CD1 proteins present in humans, only a single
isoform is present in mice, namely mCD1d, which was found
homologous to the human isoform CD1d. CD1d presents glyco-
lipids to invariant natural killer T (NKT) cells that express a semi-
invariant T cell receptor (TCR) with a conserved TCR a-chain.
Like natural killer (NK) cells, NKT cells are activated in the first
line of immune response leading to secretion of proinflammatory
T helper 1 (Th1) and immunomodulatory Th2 cytokines that
initiate the proliferation of lymphocytes for inflammation and
immunoregulation activities.3 NKT cell activation propagates
rapidly to other cell types, such as NK cells, dendritic cells and
subsets of B and conventional T cells.
Fig. 1 Structures of KRN7000, OCH and a-C-GalCer.
with mouse melanoma cells.4 An unusual structural feature of
KRN7000, which is uncommon in mammalian glycolipids but
critical for NKT-cell activation, is the a-linkage between the sugar
and the ceramide.
Although KRN7000 appears promising for a broad range of
therapeutic applications, the concomitant stimulation of both
Th1-type (INF-g) and Th2-type (IL-4) cytokines, which coun-
teract each other’s effects, is believed to be responsible for the
limited therapeutic outcome in the clinic.5 It is suggested that
a-GalCer analogues capable of inducing a biased Th1 or Th2
response are required for superior clinical effectiveness.6 Hence,
several attempts have been undertaken to modify a-GalCer to
alter its stimulatory profile (recently reviewed in ref. 7). The best
documented examples are OCH 2, an a-GalCer analogue with
a shortened sphingosine moiety resulting in a Th2-biased NKT
cell activation profile,8 and a-C-GalCer 3, which shifts the profile
towards Th1 responses.9
Several studies have been performed to assess the S.A.R. of the
galactose part of a-GalCer. The 2¢¢-OH group of the galactose was
found critical for binding to CD1d. Upon removal of this hydroxyl
group10 or its replacement by a methoxy,11 or acetamide group,12
the cytokine response was dramatically decreased. Howell and
coworkers recently demonstrated that 3¢¢- or 4¢¢-deoxy or -fluoro
analogues of KRN7000 retained antigenic activity,13 in contrast to
similar modification at the 2¢¢-position.14 Inversion of the 4¢¢-OH
(resulting in a GluCer analogue of KRN7000) also affected the
activity,15 while 3¢¢-O-sulfo-a-GalCer gave comparable activity to
the parent compound.16
The archetypal CD1d ligand is a-galactosylceramide (a-
GalCer, aka KRN7000, 1, Fig. 1). KRN7000 was derived from
the Agelasphins, a group of galactosylceramides isolated from
the marine sponge Agelas mauritianus that showed potent activity
in prolonging the life span of mice intraperitoneally inoculated
aLaboratory for Medicinal Chemistry (FFW), Faculty of Pharma-
ceutical Sciences, UGent, Harelbekestraat 72, B-9000 Gent, Belgium.
E-mail: Serge.vancalenbergh@ugent.be; Fax: +32 9 264 81 46; Tel: +32
9 264 81 24
bLaboratory for Molecular Immunology and Inflammation, Ghent University
Hospital, UGent, De Pintelaan 185, 9000 Gent, Belgium
cResearch Institute for Chromatography (RIC), President Kennedypark 26,
B-8500 Kortrijk, Belgium
dDivision of Cell Biology, La Jolla Institute for Allergy and Immunology,
9420 Athena Circle, La Jolla, CA 92037, USA
eSchool of Chemistry, University of Southampton, Highfield, Southampton,
SO17 1BJ, UK
In contrast to the galactose secondary alcohol groups, modifi-
cations at the primary 6¢¢-OH group are generally well-tolerated.
Savage et al. demonstrated that attachment of small fluorophores
at that position resulted in modified a-GalCers that retained the
† Electronic supplementary information (ESI) available: 1H-spectra and
13C-spectra and chromatograms of final compounds 4, 12, 13 and 14. See
DOI: 10.1039/c1ob06235b
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capacity to stimulate NKT cells.17 The Gal(a1→6)GalCer was
able to stimulate NKT cells without processing to the parent
monoglycosylceramide, in contrast to the corresponding diglyco-
sylceramides at the other OH-groups (i.e., Gal (a1→2)GalCer, Gal
(b1→3)GalCer and Gal(a1→4)GalCer), which lost their activity
if the additional sugar cap was not enzymatically processed.18
Guzma´n and coworkers used a 6¢¢-amide group to construct a
water soluble pegylated a-GalCer analogue with specificity for
CD1d and stimulatory properties on immune cells (e.g., DC and
NK cells).19 Mori and coworkers reported a series of 6¢¢-modified
analogues of a-GalCer. Among others, the a-D-fucopyranosyl
analogue (RCAI-58, 4, Fig. 2) was found superior to a-GalCer
in inducing IFN-g in mice.20 Finally, we recently reported a series
of 6¢¢-amide and 6¢¢-urea derivatives (e.g., 5) that retained or
even surpassed the antigenic potency of KRN7000, and as an
additional benefit, proved capable of producing a significant Th1-
skewed cytokine response leading to superior tumor protection
in vivo.21,22
CD1d bound to a-GalCer, which indicates that the Gal 6¢¢-OH is
the only sugar hydroxyl group not involved in H-bond formation.
Recently isolated immunoresponsive glycolipids from Sphin-
gomonas species, which were capable of specifically stimulating
human and mouse NKT cells in a CD1d-dependent manner, con-
tained another 6¢¢-OH modification.24 Structurally these mainly
differ from a-GalCer in that they contain an a-linked glucuronic
(GSL-1) or galacturonic acid glycone (GSL-1¢, 6).
In view of the interesting NKT-cell mediated responses induced
by the 6¢¢-derivatised a-GalCer analogues, synthetic methodology
that allows late-stage introduction of a sugar 6¢¢-substituent and
sphingosine amine functionalisation is desired for subsequent
S.A.R. studies. Apart from limiting sugar protecting group manip-
ulations, the situation for galacturonic acid and fucosyl analogues
is compounded by the anomeric glycosylation selectivity (see
below).
Recently, Cox/Besra and co-worker reported a practical proce-
dure to synthesize 6¢¢-N-derivatized a-GalCer analogues, relying
on the orthogonal protection of the 2- and 6¢¢-amino groups
(Scheme 1, top).25 Selective 6¢¢-TMS-ether cleavage nicely allowed
The tolerance for 6¢¢-OH modifications could be explained by
the crystal structure23 of the human NKT TCR in complex with
Fig. 2 Structures of key 6¢¢-modified glycosylceramides.
Scheme 1 The synthesis of KRN7000 analogues with late-stage functionalisation of the sphingosine amino group and sugar 6¢¢-position. Top: the
Cox/Besra method illustrated for a 6¢¢-amino analogue PBS-57; and bottom: our proposed method, illustrated for fucosyl and galacturonic acid-modified
analogues.
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introduction of the 6¢¢-azide group. Prompted by this report, we
want to disclose an alternative divergent approach towards 6¢¢-
modified analogues (Scheme 1, bottom), in which the difficult
galacturonic acid glycosylation was circumvented by the use of
a protected galactose donor which 4¢¢,6¢¢-benzylidene protecting
group not only contributed to selective glycosylation,26,27,28 but also
provided a handle for selective 6¢¢-OH deprotection. In contrast to
the Cox/Besra method, the nature of the chemistry involved did
not allow global deprotection before functionalisation. To demon-
strate the versatility of our method, it was first used to gain access
to the known a-D-fucopyranosyl analogue 4, the galacturonic
analogue 13,29 and two derivatives in which the conventional acyl
moiety has been replaced by a 6-phenylhexanoyl (C6Ph) moiety
known to considerably enhance the overall production of both Th1
and Th2 type cytokines and to skew the balance toward a Th1 type
response.30 Compound 13 (Scheme 1) represents an analogue of
GSL-1¢ (6) in which the ceramide part was replaced by that found
in a-GalCer.29 GSL-1¢ was shown to induce a more Th1-based
immunity and to suppress tumor growth and prolong survival of
mice bearing lung cancer more effectively than a-GalCer at equal
doses. In contrast to GSL-1¢, i.v. administration of compound 13
results in a drastically lower secretion of INF-g than that caused
by a-GalCer administration. Despite this fact, it is more effective
towards lung and breast cancer in mice compared to a-GalCer.
highly a-stereoselective due to the fact that the cis-decalin ring
system with the equatorial phenyl group prevents attack from the
b-face.27,32 We decided to exploit the selectivity of both reactions
for the synthesis of KRN7000 derivatives functionalised at the
galactose 6¢¢-position, in particular galacturonic acid, ester, amide
and amino derivatives, as well as 6¢¢-deoxy (fucosyl) KRN7000.
An added beneficial aspect of this strategy concerns the
glycosylation of galacturonic acid donors, which surprisingly is
not well-precedented.12,33,34 This may be due to the deactivating
effect of the electron-withdrawing C-5 carboxylic group, making
the galacturonidation particularly challenging.35 In addition, in
their efforts to assess the influence of a pyranosyl C-5 carboxylate
ester on the stereochemical outcome of glycosylation reactions,
van der Marel and coworkers demonstrated that a C-5 ester is 1,5-
cis or b-directing as opposed to C-5 methylene oxybenzyl, which
induces little selectivity.36 This is reflected in the synthesis of the
Sphingomonas glycolipid 6 by Seeberger et al., who found that
even after extensive optimization (including galactose protecting
groups), glycosylation was achieved with a maximum 4.2 : 1 ratio
of the a- and b-anomers.34
The synthesis of the a-D-fucosyl analogue RCAI-58 by
Mori and coworkers involved glycosylation under Mukaiyama
conditions.20 Unfortunately, neither yield nor anomeric ratio
were mentioned. In contrast to the scarce reports about D-
fucosylation, L-fucosylation is more prevalent, with reasonable
a-selectivities.37,38,39 Neglecting the potential influence on the a/b
ratio due to the asymmetry of the sphingosine, glycosylation of
this acceptor with D- and L-fucose is expected to proceed with the
same stereoselectivity.
Results and discussion
Chemistry
So, 10 was synthesized by Schmidt glycosidation,27 forming
only a negligible amount of the b-anomer which could be easily
removed by flash chromatography. Starting from the glycosidation
product 10, Scheme 2 summarizes the divergent route followed
The selective ring opening of 4,6-O-benzylidene protected car-
bohydrates is a well-described method for obtaining protected
saccharides having a free 6-OH group.31 In addition, glycosylation
of 4,6-O-benzylidene protected galactosyl donors is known to be
Scheme 2 Reagents and conditions: (a) BH3·THF, Cu(OTf)2, CH2Cl2, 78%; (b) PMe3, THF, NaOH, EDC, RCOOH, CH2Cl2, 71–81%; (c) PPh3, I2,
imidazole, toluene, 85%; (d) TEMPO, BAIB, CH2Cl2, H2O, 81–97%; (e) Pd black, H2, 49–86%.
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Table 1 Equilibrium binding affinity measurements by surface plasmon
for the synthesis of both types of target analogues. In order to
selectively address the C-6¢¢ position for further modifications,
a regioselective opening of the benzylidene ring was required.
This key step could be realized by treating 10 with BH3·THF
and Cu(OTf)2.31 The regioselective opening was confirmed by an
HSQC experiment, which unambiguously proved that the carbon
attached to the hydroxyl group possesses two protons, indicating
that the free hydroxy-group was connected to C-6¢¢. Staudinger
reduction of the azide followed by acylation of the resulting amino
group with the appropriate acids gave the versatile intermediates
16 and 17.
resonance
Glycolipid
K
ass/M-1 s-1
K
diss/s-1
KD (Kdiss/Kass)
KRN7000 (1) 1.3E+05 1E+03 1.45E-03 4.5E-05 11.2 0.2 nM
4
0.8E+05 3E+03 5.26E-03 2.9E-04 64.8 6.0 nM
1.0E+05 2E+04 6.48E-03 1.8E-03 74.4 25.0 nM
1.0E+05 9E+03 16.6E-03 5.5E-03 165 38.3 nM
1.1E+05 6E+03 8.11E-03 1.2E-03 75.3 15.2 nM
12
13
14
The low cytokine release of the C6Ph analogues 12 and 14 is
Towards the deoxygenation of the 6¢¢-OH, 16 and 17 were
first converted to the corresponding iodo analogues 18 and 19
upon treatment with triphenylphosphine, imidazole and iodine.
Our plan was to carry out the reductive dehalogenation and
debenzylation in a single step. However, upon treatment with
palladium black under hydrogen atmosphere, the reaction stopped
at the stage of the deiodinated products still containing all
benzyl groups, probably due to poisoning of the catalyst. After
flash chromatography of the reaction mixture, the deiodinated
intermediates were again subjected to catalytic hydrogenation
(same conditions) to afford the target compounds 4 and 12.
The galacturonic acid derivatives were prepared upon oxidation
of the 6¢¢-OH groups of 16 and 17 into the corresponding
carboxylic acids 20 and 21 via a TEMPO/BAIB oxidation.40 Final
deprotection was accomplished by Pd-catalyzed hydrogenation to
afford the desired compounds 13 and 14.
surprising. Wong et al. demonstrated that this and related acyl
moieties enhanced the stability for mCD1d, a finding that was
further substantiated by cocrystal structures with that protein.41
Furthermore, introduction of this acyl group in a-GalCer afforded
a compound that produced more INF-g and less IL-4 from human
NKT cells compared to a-GalCer. It remains to be investigated if
the very low antigenicity found for 12 and 14 is due to the fact that
this acyl moiety negatively influences cytokine secretion in the
mouse system (despite good affinity for mCD1d). Alternatively,
our results may also indicate that modification of the carbohydrate
moiety can significantly alter the influence of the lipid moiety
of the ligand on CD1d presentation and iNKT cell responses.
This is in accordance with observations made by Besra and
coworkers.42
We also determined the equilibrium binding affinities of the
mouse Va14Vb8.2 NKT TCR toward the four CD1d-glycolipid
complexes using surface plasmon resonance (Table 1). Purified
and biotinylated mouse CD1d was loaded with the four individual
glycolipid ligands and coated on a Biacore sensor chip. Increasing
concentrations of TCR were simultaneously passed over each
flow channel of the chip to measure real time binding kinetics.
Interestingly, the binding phase (kass) of the TCR to each of the
CD1d-presented glycolipids is similar, ranging from 0.8 ¥ 105–
1.3 ¥ 105 (M-1 s-1) while the dissociation of the TCR (kdiss) can
differ up to 10-fold between KRN7000 1 and 13 (compare 1.45
¥ 10-3 s-1 with 16.6 ¥ 10-3 s-1), resulting in over 10-fold different
binding affinities (KD = 11.2 nM–165 nM).
Biological evaluation
To assess the biological activity of the galacturonic acid and
a-D-fucopyranosyl analogues, serum levels of INF-g and IL-
4 were measured after intraperitoneal injection of 5 mg of the
corresponding glycolipids in mice. The cytokine secretion induced
by these compounds is presented in Fig. 3. Consistent with the
results of Mori,20 6¢¢-deoxy-analogue 4 is a superior INF-g inducer.
However, where Mori and coworkers only reported the INF-g
secretion, we measured the IL-4 levels as well, revealing a strong
Th2 response, hence resulting in no polarization. In combination
with a C6Ph modified acyl chain, fucosyl analogue 12 induces
only a weak cytokine secretion. So, combination of the two
modifications known to enhance antigenic activity results in a
remarkably decreased activity. In contrast to thein vitro response in
human NKT cells,29 galacturonic acid 13 shows a minor cytokine
secretion in vivo in mice. Combination with the C6Ph modified
acyl chain in compound 14 abolishes all activity.
These data indicate that modifications of the galactose moiety
and the acyl chain do affect NKT TCR affinity but not to a
degree that has been seen for weak microbial antigens, such as
borrelial a-galactosyl diacylglycerolipids (KD = 6.2 mM). As a
result, the apparent disconnect between relatively high affinity
TCR interaction in vitro and weak NKT cell activation in vivo,
likely is a result of different pharmacokinetics of the lipids inside
cells, rather than attributed to altered TCR binding kinetics.
Fig. 3 IL-4 and INF-g secretion, measured at respectively 4 h and 16 h, after intraperitoneal injection of 5 mg of the glycolipids in mice.
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was purified by column chromatography (hexanes/EtOAc: 9/1 +
1 V% Et3N) to afford compound 10 (486 mg, 67%) as a white solid.
1H NMR (300 MHz, CDCl3): d 7.53–7.51 (m, 2H, arom. H),
7.41–7.21 (m, 23H, arom. H), 5.45 (s, 1H, O-CHPh-O), 4.97 (d,
J = 3.2 Hz, 1H, H-1¢), 4.86 (d, J = 12.0 Hz, 1H, CH2-Ph), 4.81 (d,
J = 13.2 Hz, CH2-Ph), 4.77 (d, J = 10.4 Hz, 1H, CH2-Ph), 4.74 (d,
J = 12.3 Hz, 1H, CH2-Ph), 4.67 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.60
(d, J = 11.4 Hz, 1H, CH2-Ph), 4.58 (d, J = 11.7 Hz, 1H, CH2-Ph),
4.50 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.16 (app. d, J = 3.5 Hz, 1H,
H-4¢), 4.14–4.07 (m, 2H, H-2¢, H-6¢), 4.04–3.99 (m, 2H, H-3¢, H-
1), 3.88 (dd, J = 12.6 Hz and 1.3 Hz, 1H, H-6¢), 3.73–3.69 (m, 3H,
H-1, H-2, H-3), 3.65–3.60 (m, 1H, H-4), 3.57 (app. s, 1H, H-5¢),
1.65–1.26 (m, 26H, CH2), 0.88 (t, J = 6.6 Hz, 3H, CH3).
13C NMR (75 MHz, CDCl3): d 139.00, 138.60, 138.26, 138.07,
129.90, 129.10, 128.62, 128.59, 128.51, 128.47, 128.34, 128.15,
128.04, 128.00, 127.94, 127.91, 127.85, 127.75, 127.70, 126.58,
121.12, 101.30, 99.38, 79.65, 79.20, 76.04, 75.69, 74.91, 74.02,
73.74, 72.44, 72.31, 72.28, 69.56, 68.69, 63.21, 62.04, 54.71, 53.64,
32.16, 30.26, 30.01, 29.94, 29.92, 29.90, 29.88, 29.85, 29.72, 29.60,
29.47, 25.70, 22.92, 21.58, 14.35.
Conclusions
Modification of the 6¢¢-position of the prototypical NKT-cell
agonist KRN7000, has resulted in a number of analogues with
interesting biological properties. In this report we describe a
practical synthetic route to modify the 6¢¢-position after the
glycosidation, featuring the regioselective opening of a 4¢¢,6¢¢-O-
benzylidene ring as the key step. As a proof-of-concept this method
was employed to prepare the 6¢¢-deoxy-analogue of KRN7000, as
well as the otherwise not so accessible galacturonic acid analogue.
The carboxylate group of the latter may be suitable for flexible
substitution through an amide linkage. An additional advantage
of the reported procedure is that it allows to introduce alternative
acyl moieties in the phytosphingosine moiety in the final stages of
the synthesis.
Experimental section
Synthesis
Exact mass (ESI-MS) for C59H75N3O8 [M + Na]+ found,
976.5492; calcd, 976.5507.
General. Precoated Macherey-Nagel SIL G/UV254 plates were
used for TLC, and spots were examined under UV light at
254 nm and further visualized by sulfuric acid–anisaldehyde
spray. Column chromatography was performed on Biosolve silica
2-Azido-3,4-di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-hydroxy-a-
D-galactopyranosyl)octadecane-1,3,4-triol (15). To a solution of
10 (50 mg, 0.05 mmol) in anhydrous CH2Cl2 (1.6 mL) under
argon atmosphere were added copper(II) triflate (3 mg, 0.008
mmol) and BH3·THF (0.26 mL, 0.26 mmol). After stirring for
1.5 h at room temperature, the brown reaction mixture was
quenched with methanol. Subsequently the mixture was diluted
with EtOAc and washed with sat. NaHCO3, water and brine.
The organic layer was dried over Na2SO4 and concentrated
under reduced pressure. Purification by column chromatography
(hexanes/EtOAc: 8.5/1.5) yielded 15 (37 mg, 73%) as a colorless
oil.
1H NMR (300 MHz, DMSO-d6): d 7.38–7.7.22 (m, 25H, arom.
H), 4.97 (d, J = 2.25 Hz, 1H, H-1¢), 4.82 (d, J = 11.4 Hz, 1H, CH2-
Ph), 4.79–4.69 (m, 5H, CH2-Ph, OH), 4.60–5.54 (m, 4H, CH2-Ph),
4.44 (d, J = 11.6 Hz, 1H, CH2-Ph), 4.08 (br.s, 1H, H-3¢), 3.97–
3.88 (m, 3H, H-1, H-3, H-2¢), 3.79–3.73 (m, 3H, H-2, H-4¢, H-5¢),
3.67–3.62 (m, 2H, H-1, H-4), 3.52–3.46 (m, 2H, H-6¢), 2.49–1.20
(m, 26H, CH2) 0.85 (t, J = 6.6 Hz, 3H, CH3).
˚
gel (63–200 mm, 60 A). NMR spectra were obtained with a
Varian Mercury 300 Spectrometer or a Bruker Avance II 500
spectrometer. Chemical shifts are given in ppm (d) relative to the
residual solvent signals, in the case of CDCl3: d = 7.26 ppm for 1H
and d = 77.4 ppm for 13C, in the case of DMSO-d6: d = 2.54 ppm
for 1H and d = 40.5 ppm for 13C, and in the case of pyridine-d5: d =
8.71, 7.56 and 7.18 ppm for 1H and d = 149.9, 135.5 and 123.5 ppm
for 13C. Exact mass measurements were performed on a Waters
LCT Premier XE TOF equipped with an electrospray ionization
interface and coupled to a Waters Alliance HPLC system. Samples
were infused in a CH3CN/HCOOH (1000 : 1) mixture at 10 mL
min-1. The purity of the target compounds was assessed by
HPLC. An Agilent Technologies 1100 HPLC system (Agilent
Technolgies, Waldbronn, Germany) was coupled to a Varian
385-LC Evaporative Light Scattering Detector (ELSD) (Agilent
Technologies). The compounds were subjected to an Xbridge
Shield C18 column (Waters, Millford, MA, USA) operated at
◦
65 C. Mobile phases, delivered at a flow rate of 2 mL min-1,
13C NMR (75 MHz, CDCl3): d 138.96, 138.88, 138.63, 138.43,
138.30, 128.82, 128.72, 128.66, 128.63, 128.61, 128.54, 128.22,
128.13, 128.09, 128.00, 127.98, 127.86, 127.84, 127.78, 98.84,
79.56, 79.46, 79.06, 77.72, 77.30, 76.87, 76.69, 75.43, 74.73, 73.96,
73.69, 73.48, 72.30, 71.12, 68.68, 62.70, 62.25, 32.19, 30.27, 30.02,
29.97, 29.93, 29.89, 29.87, 29.63, 25.61, 22.96, 14.39.
consisted of 25 mM ammonium formate pH 5 (solvent A) and
methanol (solvent B). A linear gradient from 70% B to 100% B in
10 min. was applied, followed by elution with 100% B during
3 min. The target compounds were dissolved in pyridine and
further diluted a hundred fold in acetonitrile prior to injecting
2 ml. Final concentrations were 92 ng ml-1, 97 ng ml-1, 75 ng ml-1
and 20 ng ml-1 for 4, 12, 13 and 14, respectively.
Exact mass (ESI-MS) for C59H77N3O8 [M + Na]+ found,
978.5695; calcd, 978.5603.
2-Azido-3,4-di-O-benzyl-1-O-(2,3-di-O-benzyl-4,6-O-benzyl-
idene-a-D-galactopyranosyl)octadecane-1,3,4-triol (10). A solu-
tion of 2-azido-3,4-di-O-benzyl-1-hydroxy-octadecane-1,2,4-triol
(400 mg, 0.76 mmol) in THF (10 mL) was added to a solu-
tion of 2,3-di-O-benzyl-4,6-O-benzylidene-a-D-galactopyranosyl
trichloroacetimidate (680 mg, 1.15 mmol) in THF (5 mL) followed
by dropwise addition of TMSOTf (0.02 mL, 0.11 mmol) at
-20 ◦C. After stirring for 1 h at -20 ◦C, the reaction mixture
was neutralized with Et3N and evaporated to dryness. The residue
3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-hydroxy-a-D-gala-
ctopyranosyl)-2-hexacosylamino octadecane-1,3,4-triol (16). To a
solution of 15 (157 mg, 0.16 mmol) in THF (1.6 mL) was added
dropwise trimethylphosphine (0.8 mL, 0.82 mmol). After stirring
for 2.5 h, a NaOH solution (3 mL, 1 M) was added and the
mixture was allowed to stir for an additional 2.5 h. The reaction
mixture was extracted with EtOAc and the organic layer was
washed with brine, dried over Na2SO4 and concentrated under
reduced pressure. The crude amine was dissolved in CH2Cl2
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(2 mL) and added to a solution of EDC (63 mg, 0.33 mmol) and
hexacosanoic acid (97 mg, 0.25 mmol) in CH2Cl2 (0.5 mL). This
reaction mixture was stirred overnight at room temperature after
which it was extracted with CH2Cl2. The organic layer was washed
with brine and dried over Na2SO4. After evaporation of the organic
solvent, the residue was purified by column chromatography
(hexanes/EtOAc: 6/4) affording compound 16 (167 mg, 78%) as
a yellow oil.
1H NMR (300 MHz, CDCl3): d 7.39–7.25 (m, 25H, arom. H),
5.83 (d, J = 9.0 Hz, 1H, NH), 4.94 (d, J = 11.5 Hz, 1H, CH2-Ph),
4.88 (d, J = 3.8 Hz, 1H, H-1¢), 4.82 (d, J = 12.7 Hz, 1H, CH2-Ph),
4.79 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.73 (d, J = 11.8 Hz, 1H, CH2-
Ph), 4.71 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.64 (d, J = 12.1 Hz, 1H,
CH2-Ph), 4.62 (d, J = 11.8 Hz, 1H, CH2-Ph), 4.58 (d, J = 11.8 Hz,
1H, CH2-Ph), 4.50 (d, J = 11.5 Hz, 1H, CH2-Ph), 4.47 (d, J = 11.8
Hz, 1H, CH2-Ph), 4.41–4.33 (m, 1H, H-2), 4.03 (dd, J = 3.7 Hz
and 9.6 Hz, 1H, H-2¢), 3.95 (dd, J = 7.6 Hz and 11.6 Hz, 1H, H-1),
3.85-3.82 (m, 3H, H-1, H-3¢, H-4¢), 3.70–3.62 (m, 3H, H-3, H-5¢,
H-6¢), 3.55–3.50 (m, 1H, H-4), 3.48–3.42 (m, 1H, H-6¢), 2.39 (t,
1H, OH), 1.92–1.87 (m, 2H, COCH2), 1.69–1.14 (m, 72H, CH2),
0.88 (t, J = 6.8 Hz, 6H, CH3).
2.57 (t, J = 7.1 Hz, 2H, CH2-Ph), 1.97–1.80 (m, 2H, COCH2),
1.69–1.26 (m, 34H, CH2), 0.88 (t, J = 6.6 Hz, 3H, CH3).
13C NMR (75 MHz, CDCl3): d 173.26, 142.68, 138.84, 138,
76, 138.66, 138.47, 128.68, 128.65, 128.62, 128.60, 128.51, 128.17,
128.14, 128.09, 127.98, 127, 95, 127.86, 127.84, 127.66, 125.93,
100.23, 80.57, 79.50, 79.43, 77.67, 77.45, 77.25, 76.82, 75.04, 74.76,
73.79, 73.40, 73.33, 72.06, 71.51, 69.98, 62.69, 51.08, 50.89, 36.87,
35.95, 32.16, 31.36, 31.16, 30.44, 30.02, 29.96, 29.92, 29.60, 29.12,
26.01, 25.73, 22.93, 14.36.
Exact mass (ESI-MS) for C71H93NO9 [M + H]+ found,
1104.7063; calcd, 1104.6923.
3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-iodo-a-D-galactopy-
ranosyl)-2-hexacosylamino octadecane-1,3,4-triol (18). PPh3
(18 mg, 0.07 mmol) was added to a solution of 16 (74 mg,
0.06 mmol) in toluene (0.4 mL) under argon followed by refluxing
during 10 min. The mixture was cooled down to 80 ◦C and
imidazole (11 mg, 0.17 mmol) and I2 (19 mg, 0.07 mmol) were
added. After refluxing for 20 min, the solution was concentrated
under reduced pressure. The resulting residue was diluted with
EtOAc and washed with a saturated Na2S2O3 solution and water.
The organic layer was dried on Na2SO4 and evaporated to dryness.
Purification by column chromatography (hexanes/EtOAc: 9/1)
yielded 18 (68 mg, 85%) as a white solid.
13C NMR (75 MHz, CDCl3): d 173.37, 138.86, 138.79, 138.67,
138.65, 138.48, 128.66, 128.64, 128.60, 128.17, 128.13, 128.06,
127.96, 127.94, 127.83, 127.67, 100.31, 80.68, 79.46, 77.66, 77.44,
77.24, 76.91, 76.81, 75.09, 74.77, 73.77, 73.43, 73.32, 72.08, 71.50,
70.04, 62.70, 50.90, 37.05, 32.16, 30.43, 29.95, 29.93, 29.89, 29.83,
29.66, 29.59, 26.01, 26.93, 22.92, 14.43, 14.35.
1H NMR (300 MHz, CDCl3): d 7.40–7.21 (m, 25H, arom. H),
5.84 (d, J = 8.6 Hz, 1H, NH), 5.03 (d, J = 11.2 Hz, 1H, CH2-Ph),
4.84 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.83 (d, J = 3.4 Hz, 1H, H-1¢),
4.79–4.72 (m, 3H, CH2-Ph), 4.65–4.58 (m, 3H, CH2-Ph), 4.52 (d,
J = 11.7 Hz, 1H, CH2-Ph), 4.49 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.30
(m, 1H, H-2), 4.08 (m, 1H, H-4¢), 4.02 (dd, J = 10.0 Hz and 3.5
Hz, 1H, H-2¢), 3.91–3.87 (m, 2H, H-1, H-3¢), 3.84–3.77 (m, 3H,
H-1, H-3, H-5¢), 3.53 (ddd, J = 7.2 Hz, and 3.6 Hz, 1H, H-4), 3.18
(dd, J = 9.9 Hz and 7.1 Hz, 1H, H-6¢), 3.09 (dd, J = 9.9 Hz and
7.0 Hz, 1H, H-6¢), 2.02–1.86 (m, 2H, COCH2), 1.72–1.06 (m, 72H,
CH2), 0.88 (t, J = 6.7 Hz, 6H, CH3).
Exact mass (ESI-MS) for C85H129NO9 [M + H]+ found,
1308.9780; calcd, 1308.9746.
3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-hydroxy-a-D-gala-
ctopyranosyl)-2-(6-phenylhexanoyl)amino octadecane-1,3,4-triol
(17). To a solution of 15 (120 mg, 0.13 mmol) in THF (1.3 mL)
was added dropwise trimethylphosphine (0.6 mL, 0.63 mmol).
After stirring for 2.5 h, a NaOH solution (2.3 mL, 1 M) was
added and the mixture was allowed to stir for an additional 2.5 h.
The reaction mixture was extracted with EtOAc and the organic
layer was washed with brine, dried over Na2SO4 and concentrated
under reduced pressure. The crude amine was dissolved in CH2Cl2
(1.4 mL) and added to a solution of EDC (48 mg, 0.25 mmol) and
6-phenylhexanoic acid (36 mg, 0.19 mmol) in CH2Cl2(0.5 mL).
This reaction mixture was stirred overnight at room temperature
after which it was extracted with CH2Cl2. The organic layer was
washed with brine and dried over Na2SO4. After evaporation
of the organic solvent, the residue was purified by column
chromatography (hexanes/EtOAc: 7/3) affording compound 17
(107 mg, 77%) as a colorless oil.
1H NMR (300 MHz, CDCl3): d 7.38–7.15 (m, 30H, arom. H),
5.84 (d, J = 8.6 Hz, 1H, NH), 4.94 (d, J = 11.4 Hz, 1H, CH2-Ph),
4.86 (d, J = 3.7 Hz, 1H, H-1¢), 4.83 (d, J = 11.7 Hz, 1H, CH2-Ph),
4.80 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.74 (d, J = 11.7 Hz, 1H, CH2-
Ph), 4.69 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.66 (d, J = 11.7 Hz, 1H,
CH2-Ph), 4.60 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.55 (d, J = 11.4 Hz,
1H, CH2-Ph), 4.52 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.48 (d, J = 11.4
Hz, 1H, CH2-Ph), 4.44 (d, J = 11.4 Hz, 1H, CH2-Ph), 4.37 (m,
1H, H-2), 4.02 (dd, J = 9.9 and 3.7 Hz, 1H, H-2¢), 3.94 (dd, J =
11.4 and 7.7 Hz, 1H, H-1), 3.85–3.82 (m, 3H, H-1, H-3¢and H-4¢),
3.70–3.62 (3H, H-3, H-5¢ and H-6¢), 3.55–3.41 (m, 2H, H-4, H-6¢),
13C NMR (75 MHz, CDCl3): d 169.41, 135.12, 135.05, 135.00,
134.87, 134.74, 124.95, 124.90, 124.87, 124.39, 124.37, 124.30,
124.23, 124.16, 124.12, 124.08, 123.93, 101.28, 95.36, 76.43, 75.71,
75.61, 73.94, 73.94, 73.72, 73.52, 73.10, 72.70, 71.88, 71.63, 69.97,
69.80, 69.70, 68.44, 64.81, 46.66, 33.29, 28.44, 26.63, 26.37, 26.24,
26.21, 26.17, 26.12, 25.97, 25.91, 25.89, 25.87, 22.46, 22.26, 19.21,
10.63.
Exact mass (ESI-MS) for C85H128INO8 [M + H]+ found,
1418.8851; calcd, 1418.8757.
3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-6-iodo-a-D-galactopy-
ranosyl)-2-(6-phenylhexanoyl)amino octadecane-1,3,4-triol (19).
PPh3 (20 mg, 0.08 mmol) was added to a solution of 17 (70 mg,
0.06 mmol) in toluene (0.4 mL) under argon followed by refluxing
during 10 min. The mixture was cooled down to 80 ◦C and
imidazole (13 mg, 0.19 mmol) and I2 (21 mg, 0.08 mmol) were
added. After refluxing for 20 min, the solution was concentrated
under reduced pressure. The resulting residue was diluted with
EtOAc and washed with a saturated Na2S2O3 solution and water.
The organic layer was dried on Na2SO4 and evaporated to dryness.
Purification by column chromatography (hexanes/EtOAc: 8/2)
yielded 19 (67 mg, 85%) as a yellow oil.
1H NMR (300 MHz, CDCl3): d 7.42–7.16 (m, 30H, arom. H),
5.86 (d, J = 8.6 Hz, 1H, NH), 5.05 (d, J = 11.2 Hz, 1H, CH2-Ph),
4.86 (d, J = 3.6 Hz, 1H, H-1¢), 4.85 (d, J = 11.7 Hz, 1H, CH2-Ph),
8418 | Org. Biomol. Chem., 2011, 9, 8413–8421
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4.79 (d, J = 11.7 Hz, 1H, CH2-Ph), 4.78 (d, J = 11.8 Hz, 1H,
CH2-Ph), 4.76 (d, J = 11.5 Hz, 1H, CH2-Ph), 4.66 (d, J = 11.1
Hz, 1H, CH2-Ph), 4.65 (d, J = 10.0 Hz, 1H, CH2-Ph), 4.62 (d, J =
12.0 Hz, 1H, CH2-Ph), 4.54 (d, J = 11.5 Hz, 1H, CH2-Ph), 4.52 (d,
J = 11.7 Hz, 1H, CH2-Ph), 4.34 (m, 1H, H-2), 4.11–4.10 (m, 1H,
H-4¢), 4.05 (dd, J = 10.0 Hz and 3.5 Hz, 1H, H-2¢), 3.95–3.89 (m,
2H, H-1, H-3¢), 3.87–3.85 (m, 1H, H-5¢), 3.83–3.79 (m, 2H, H-1,
H-3), 3.59–3.54 (m, 1H, H-4), 3.21 (dd, J = 10.0 Hz and 7.2 Hz,
1H, H-6¢), 3.12 (dd, J = 10.0 Hz and 6.9 Hz, 1H, H-6¢), 2.60 (t, J =
7.6 Hz, 2H, CH2-Ph), 2.03–1.86 (m, 2H, COCH2), 1.73–1.26 (m,
32H, CH2), 0.91 (t, J = 6.7 Hz, 3H, CH3).
1H NMR (300 MHz, CDCl3): d 7.34–7.14 (m, 30H, arom. H),
5.80 (d, J = 8.2 Hz, 1H, NH), 4.95 (d, J = 3.4 Hz, 1H, H-1¢), 4.91–
4.42 (m, 12H, CH2-Ph, H-4¢, H-5¢), 4.42–4.31 (m, 1H, H-2), 4.06
(dd, J = 10.0 and 3.5 Hz, 1H, H-2¢), 3.93 (dd, J = 10.1 and 2.6 Hz,
1H, H-3¢), 3.89 (dd, J = 10.5 and 4.9 Hz, 1H, H-1), 3.80 (m, 2H,
H-1, H-3), 3.54 (m, 1H, H-4), 2.67 (t, J = 7.7 Hz, 2H, CH2-Ph),
1.92–1.77 (m, 2H, COCH2), 1.67–1.19 (m, 34H, CH2), 0.90 (t, J =
6.7 Hz, CH3).
13C NMR (75 MHz, CDCl3): d 173.33, 170.50, 142.73, 138.57,
138.55, 138.44, 138.42, 138.25, 128.71, 128.70, 128.68, 128.66,
128.62, 128.52, 128.46, 128.32, 128.25, 128.18, 128.16, 128.10,
128.02, 128.00, 127.96, 127.92, 127.64, 125.93, 99.93, 79.64, 79.45,
78.23, 77.72, 77.29, 76.87, 76.18, 75.95, 75.43, 74.07, 73.22, 73.07,
72.12, 71.17, 69.03, 50.23, 36.74, 35.98, 32.18, 31.38, 30.47, 30.06,
29.98, 29.93, 29.63, 29.12, 25.98, 25.72, 22.95, 14.39.
13C NMR (75 MHz, CDCl3): d 169.14, 138,98, 135.09, 135.02,
134.97, 134.84, 134.72, 124.93, 124.88, 124.85, 124.76, 124.38,
124.36, 124.33, 124.29, 124.21, 124.14, 124.11, 124.07, 123.90,
122.17, 95.31, 76.35, 75.71, 75.57, 73.96, 73.54, 73.12, 72.69, 71.83,
71.60, 69.95, 69.75, 69.63, 68.39, 64.75, 56.87, 45.58, 33.11, 32.24,
28.42, 27.67, 26.62, 26.35, 26.21, 26.16, 25.86, 25.42, 22.42, 22.00,
19.19, 17.53, 10.69, 10.63.
Exact mass (ESI-MS) for C71H97NO10 [M + H]+ found,
1118.6841; calcd, 1118.6716.
1-O-(6-Deoxy-a-D-galactopyranosyl)-2-hexacosylamino octa-
decane-1,3,4-triol (4). A solution of compound 18 (65 mg, 0.05
mmol) in MeOH (2.5 mL) was hydrogenated under atmospheric
pressure in the presence of palladium black (20 mg). After
consumption of the starting material, 1 spot was visible on
TLC, corresponding with the deiodinated product. After a quick
purification by column chromatography (hexanes/EtOAc: 7/3),
the product was dissolved in MeOH (3 mL) and hydrogenated
under atmospheric pressure in the presence of palladium black
(15 mg). Upon completion of the reaction, the mixture was
diluted with MeOH and filtered through celite. The filter cake
was rinsed with MeOH and the filtrate was evaporated to dryness.
After purification by column chromatography (CH2Cl2/MeOH:
8/2), compound 4 (18 mg, 47%) was afforded as a white powder.
1H NMR (300 MHz, pyridine-d5): d 8.43 (d, J = 8.7 Hz, 1H,
NH), 6.94 (d, J = 4.8 Hz, 1H, OH), 6.53 (d, J = 5.8 Hz, 1H, OH),
6.42 (d, J = 6.5 Hz, 1H, OH), 6.16 (d, J = 4.5 Hz, 1H, OH), 6.11
(d, J = 5.8 Hz, 1H, OH), 5.48 (d, J = 3.7 Hz, 1H, H-1¢), 5.31 (m,
1H, H-2), 4.67 (dd, J = 10.5 Hz and 5.3 Hz, 1H, H-1), 4.59–4.56
(m, 1H, H-2¢), 4.42–4.29 (m, 5H, H-1, H-3, H-4, H-3¢, H-5¢), 4.09–
4.04 (m, 1H, H-4¢), 2.47 (t, J = 7.43, COCH2), 2.31–1.27 (m, 72H,
CH2), 1.11 (t, J = 7.1 Hz, 3H, CH3), 0.89 (t, J = 6.7 Hz, CH3).
13C NMR (75 MHz, pyridine-d5): d 171.88, 100.25, 75.62, 72.08,
71.39, 70.44, 68.72, 67.34, 66.32, 59.09, 50.02, 35.63, 33.30, 30.94,
29.20, 28.96, 28.85, 28.83, 28.75, 28.74, 28.69, 28.64, 28.57, 28.44,
28.43, 25.31, 25.23, 21.76, 19.63, 16.03, 13.10.
Exact mass (ESI-MS) for C71H92INO8 [M + K]+ found,
1252.5511; calcd, 1252,5499.
3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-a-D-galactopyranosyl-
uronate)-2-hexacosylamino octadecane-1,3,4-triol (20). TEMPO
(22 mg, 0.14 mmol) and BAIB (578 mg, 1.80 mmol) were added
to a mixture of 16 (940 mg, 0.72 mmol) in CH2Cl2 (4.7 mL) and
H2O (2.4 mL). The emulsion was vigorously stirred overnight at
room temperature and the reaction was quenched with Na2S2O3.
After extraction of the aqueous layer with EtOAc, the organic
layer was washed with sat. NaHCO3 and brine, dried over
Na2SO4 and evaporated. The residue was submitted to column
chromatography (CH2Cl2/MeOH: 24/1 with 1% formic acid),
affording compound 20 (823 mg, 87%) as a yellow oil.
1H NMR (300 MHz, CDCl3): d 7.32–7.19 (m, 25H, arom. H),
4.97 (app. s, 1H, H-1¢), 4.86–4.28 (m, 13H, CH2-Ph, H-2, H-4¢,
H-5¢), 4.02 (dd, J = 3.4 Hz and 9.9 Hz, 1H, H-2¢), 3.90 (dd, J =
2.6 Hz and 10.0 Hz, 1H, H-3¢), 3.88–3.83 (m, 1H, H-1), 3.76–3.69
(m, 2H, H-1 and H-3), 3.53–3.50 (m, 1H, H-4), 1.93–1.74 (m, 2H,
COCH2), 1.59–1.08 (m, 72H, CH2), 0.86 (t, J = 6.8 Hz, 6H, CH3).
13C NMR (75 MHz, CDCl3): d 173.30, 169.69, 138.59, 138.55,
138.38, 138.16, 128.70, 128.66, 128.47, 128.38, 128.21, 128.14,
128.13, 128.09, 128.01, 127.98, 127.66, 99.32, 79.65, 79.57, 78.14,
77.67, 77.45, 77.25, 76.82, 76.47, 75.84, 75.55, 74.07, 73.23, 73.10,
72.16, 71.21, 68.97, 50.15, 36.90, 32.17, 30.47, 30.04, 29.97, 29.94,
29.91, 29.89, 29.87, 29.84, 29.81, 29.67, 29.61, 29.60, 29.54, 25.99,
25.90, 22.93, 14.36.
Exact mass (ESI-MS) for C50H99NO8 [M + H]+ found, 842.7474;
calcd, 842.7444.
Exact mass (ESI-MS) for C85H127NO10 [M + Na]+ found,
1344.9396; calcd, 1344.9352.
1-O-(6-Deoxy-a-D-galactopyranosyl)-2-hexacosylamino octa-
decane-1,3,4-triol (12). A solution of compound 19 (66 mg,
0.05 mmol) in MeOH (2.5 mL) was hydrogenated under
atmospheric pressure in the presence of palladium black (20 mg).
After consumption of the starting material, 1 spot was visible on
TLC, corresponding to the deiodinated product. After a quick
purification by column chromatography (hexanes/EtOAc: 7/3),
the product was dissolved in MeOH (2.5 mL) and hydrogenated
under atmospheric pressure in the presence of palladium black
(15 mg). Upon completion of the reaction, the mixture was
diluted with MeOH and filtered through celite. The filter cake
was rinsed with MeOH and the filtrate was evaporated to dryness.
After purification by column chromatography (CH2Cl2/MeOH:
3,4-Di-O-benzyl-1-O-(2,3,4-tri-O-benzyl-a-D-galactopyranosyl-
uronate)-2-(6-phenylhexanoyl)amino octadecane-1,3,4-triol (21).
TEMPO (3 mg, 0.02 mmol) and BAIB (77 mg, 0.2 mmol)
were added to a mixture of 17 (106 mg, 0.10 mmol) in CH2Cl2
(0.6 mL) and H2O (0.3 mL). The emulsion was vigorously stirred
overnight at room temperature and the reaction was quenched
with Na2S2O3. After extraction of the aqueous layer with EtOAc,
the organic layer was washed with sat. NaHCO3 and brine, dried
over Na2SO4 and evaporated. The residue was submitted to
column chromatography (CH2Cl2/MeOH: 29/1 with 1% formic
acid), affording compound 21 (104 mg, 97%) as a yellow oil.
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9.2/0.8), compound12 (22 mg, 70%) was afforded as a white
powder.
Exact mass (ESI-MS) for C36H61NO10 [M - H]- found, 666.4213;
calcd, 666.4223.
1H NMR (300 MHz, CD3OD): d 7.88 (d, J = 8.9 Hz, 1H, NH),
7.26–7.10 (m, 5H, arom. H), 4.79 (app. s, 1H, H-1¢), 4.22–4.17 (m,
1H, H-2), 3.95 (dd, J = 13.3 Hz and 6.7 Hz, 1H, H-5¢), 3.84 (dd,
J = 10.5 Hz and 4.4 Hz, 1H, H-1), 3.77–3.70 (m, 2H, H-2¢, H-3¢),
3.65–3.52 (m, 4H, H-1, H-3, H-4, H-4¢), 2.61 (t, J = 7.7 Hz, 2H,
CH2-Ph), 2.21 (t, J = 7.5 Hz, 2H, COCH2), 1.69–1.18 (m, 32H,
CH2), 0.89 (t, J = 6.7 Hz, 3H, CH3).
Surface plasmon resonance studies
Recombinant mouse CD1d and TCR preparations, glycolipid
loading and SPR studies were performed as reported previously.43
Briefly, glycolipids were dissolved in DMSO at 1 mg ml-1 and
loaded onto biotinylated CD1d by incubating o/n at RT in
100 mM Tris/HCl, pH 7.0, 0.08% Tween 20 buffer. Approximately
300 response units (RU) of 3 different CD1d-glycolipid complexes
were immobilized onto flow channels 2–4 of a SA capture chip
(GE), while biotinylated CD1d incubated in buffer without lipid
was bound to flow channel 1 for background substraction. After
increasing concentrations of TCR were passed over the individual
flow channels simultaneously, kinetic parameters were calculated
using a simple Langmuir 1:1model in the BIA evaluation software
version 4.1. Experiments were performed 2–3 times for each
glycolipid, using 2 different TCR preparations.
13C NMR (75 MHz, CD3OD): d 174.47, 142.55, 128.21, 128.11,
125.52, 99.86, 74.20, 72.42, 71.77, 70.50, 68.78, 66.81, 66.60, 50.59,
35.98, 35.57, 31.90, 31.85, 31.31, 29.64, 29.59, 29.31, 28.77, 25.82,
25.77, 22.56, 15.52, 13.28.
Exact mass (ESI-MS) for C36H63NO8 [M + H]+ found, 638.4659;
calcd, 638.4626.
1-O-(a-D-Galactopyranosyluronate)-2-hexacosylamino
octa-
decane-1,3,4-triol (13). A solution of compound 20 (105 mg, 0.08
mmol) in CHCl3 (1.3 mL) and EtOH (3.8 mL) was hydrogenated
under atmospheric pressure in the presence of palladium black
(15 mg). Upon reaction completion, the mixture was diluted with
pyridine and filtered through celite. The filter cake was rinsed
with CHCl3 and EtOH and the filtrate was evaporated to dryness.
After purification by column chromatography (CH2Cl2/MeOH:
8/2), compound 13 (54 mg, 78%) was afforded as a white powder.
1H NMR (500 MHz, pyridine-d5): d 8.46 (d, J = 8.7 Hz, 1H,
NH), 5.64 (d, J = 3.4 Hz, 1H, H-1¢), 5.27–5.26 (m, 1H, H-2), 5.15
(app. s, 1H, H-5¢), 5.02 (app. s, 1H, H-4¢), 4.69-4.66 (m, 2H, H-2¢,
H-1), 4.50 (dd, J = 9.9 Hz and 3.1 Hz, 1H, H-3¢), 4.37 (dd, J = 10.6
Hz and 4.5 Hz, 1H, H-3), 4.30 (app. s, 2H, H-1, H-4), 2.46–2.41
(m, 2H, COCH2), 2.26–1.23 (m, 72H, CH2), 0.85 (t, J = 6.7 Hz,
CH3).
13C NMR (75 MHz, pyridine-d5): d 171.93, 171.07, 152.04,
149.40, 105.00, 100.53, 75.48, 71.51, 71.34, 70.99, 70.05, 68.64,
68.08, 49.98, 35.61, 33.18, 30.94, 29.48, 29.20, 28.97, 28.86, 28.83,
28.76, 28.74, 28.69, 28.64, 28.60, 28.45, 28.43, 25.31, 25.22, 21.76,
13.10.
Exact mass (ESI-MS) for C50H97NO10 [M - H]- found, 870.7089;
calcd, 870.7039.
Abbreviations
a-GalCer
i.v.
a-galactosylceramide
intravenous
MHC
NK
Major Histocompatibility Complex
Natural Killer
NKT
S.A.R.
TCR
Th
Natural Killer T
Structure–Activity Relationship
T Cell Receptor
T helper
Acknowledgements
S.V.C. and D.E. received support of the Belgian Stichting tegen
Kanker and the FWO Flanders. D.M.Z is recipient of an Investi-
gator award from the Cancer Research Institute and supported by
NIH grant AI074962.
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