3-fluoro- and 3,3-difluoro-3,4-dideoxy-KRN7000 analogues as new potent immunostimulator agents: Total synthesis and biological evaluation in human invariant natural killer t cells and mice

Article abstract of DOI:10.1021/jm201368m

We propose here the synthesis and biological evaluation of 3,4-dideoxy-GalCer derivatives. The absence of the 3- and 4-hydroxyls on the sphingoid base is combined with the introduction of mono or difluoro substituent at C3 (analogues 8 and 9, respectively) to evaluate their effect on the stability of the ternary CD1d/GalCer/TCR complex which strongly modulate the immune responses. Biological evaluations were performed in vitro on human cells and in vivo in mice and results discussed with support of modeling studies. The fluoro 3,4-dideoxy-GalCer analogues appears as partial agonists compared to KRN7000 for iNKT cell activation, inducing T_H1 or T_H2 biases that strongly depend of the mode of antigen presentation, including human vs mouse differences. We evidenced that if a sole fluorine atom is not able to balance the loss of the 3-OH, the presence of a difluorine group at C3 of the sphingosine can significantly restore human iNKT activation.

Full text of DOI:10.1021/jm201368m

Article
pubs.acs.org/jmc
3-Fluoro- and 3,3-Difluoro-3,4-dideoxy-KRN7000 Analogues as New
Potent Immunostimulator Agents: Total Synthesis and Biological
Evaluation in Human Invariant Natural Killer T Cells and Mice
Julie Hunault,† Mette Diswall,‡ Jean-Cedric Frison,† Virginie Blot,† Jezabel Rocher,‡
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Severine Marionneau-Lambot,§ Thibauld Oullier,§ Jean-Yves Douillard,∥ Stephane Guillarme,⊥
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Christine Saluzzo,⊥ Gilles Dujardin,⊥ Denis Jacquemin,† Jerome Graton,† Jean-Yves Le Questel,†
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Michel Evain,# Jacques Lebreton,† Didier Dubreuil,† Jacques Le Pendu,*,‡ and Muriel Pipelier*,†
†Universite
Faculte des Sciences et des Techniques, 2 rue de la Houssinier
‡INSERM, U892, Universite
de Nantes, IRT UN, 8 Quai Moncousu, 44007 Nantes Cedex 1, France
§Plateforme in vivo du Cancer
opole Grand Ouest, 8 Quai Moncousu, 44093 Nantes Cedex, France
∥Centre Rene
Gauducheau, Centre Regional de Lutte Contre le Cancer Nantes-Atlantique, 44805 Saint-Herblain Cedex, France
⊥Universite
du Maine, CNRS, Unite de Chimie Organique Moleculaire et Macromoleculaire, (UCO2M), UMR CNRS 6011,
Faculte des Sciences et Techniques, Universite du Maine, Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, France
#Universite
de Nantes, Nantes Atlantique Universite, CNRS, Faculte des Sciences et des Techniques, Institut des Mater
Rouxel, UMR CNRS 6502, 2 rue de la Houssiniere, BP 92208, 44322 Nantes Cedex 3, France
́
de Nantes, CNRS, Chimie et Interdisciplinarite:
́
Synthes
̀ ́
e, Analyse, Modelisation (CEISAM), UMR CNRS 6230,
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e, BP 92208, 44322 Nantes Cedex 3, France
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́
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́
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iaux Jean
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S
* Supporting Information
ABSTRACT: We propose here the synthesis and biological evaluation of
3,4-dideoxy-GalCer derivatives. The absence of the 3- and 4-hydroxyls on
the sphingoid base is combined with the introduction of mono or difluoro
substituent at C3 (analogues 8 and 9, respectively) to evaluate their effect
on the stability of the ternary CD1d/GalCer/TCR complex which strongly
modulate the immune responses. Biological evaluations were performed in
vitro on human cells and in vivo in mice and results discussed with support of modeling studies. The fluoro 3,4-dideoxy-GalCer
analogues appears as partial agonists compared to KRN7000 for iNKT cell activation, inducing TH1 or TH2 biases that strongly
depend of the mode of antigen presentation, including human vs mouse differences. We evidenced that if a sole fluorine atom is
not able to balance the loss of the 3-OH, the presence of a difluorine group at C3 of the sphingosine can significantly restore
human iNKT activation.
rationalization of the structure−activity relationship (SAR)2 has
INTRODUCTION
■
been attempted for several synthetic analogues, facilitated by
the availability of the CD1d/KRN7000/TCR trimolecular
complex crystal structure.10,11 Although the stability of the
CD1d/GalCer/TCR complex is believed to contribute greatly
to the shifting of the immunostimulation profile, numerous
factors play a role in the cytokine polarization.
α-Linked galactosylceramides (α-GalCers) are a special class of
exogenous glycolipid antigens with a potent immunoregulatory
effect recognized by the semi-invariant T cell receptors (TCR)
of invariant natural killer T cells (iNKT) when presented by the
CD1d molecule of antigen presenting cells.1−3 The trimolecular
complex formation initiates a rapid release of pro-inflammatory
T helper (TH1) (e.g., TNF-α, IFN-γ, and IL-2) and anti-
inflammatory T helper (TH2) cytokines (e.g., IL-4, IL-10, and
IL-13) by the iNKT cells.4−7 Disruption of the TH1/TH2
balance may lead to disease induction as TH1 and TH2 type
cytokines can antagonize each other’s biological functions.8
The opposing activities displayed by TH1 and TH2 cytokines
induced by α-GalCers limit their value as potential therapeutic
agents. Production of analogues able to favor either of the two biases
without severely altering the immune response is currently the focus
of intense research.
α-GalCers are loaded into the CD1d cleft with only the sugar
headgroup exposed for the interaction with the iNKT TCR.
The nature of the sugar unit, D-galactose, is strongly preferred,12
while modifications of the C3 and C6 positions were found to be
tolerated.13−15 Recently, Van Calenbergh and others16 described
the potent antigenic activity of 6″-amido and 6″-ureido derivatives
bearing an intact phytoceramide moiety that present a TH1
orientation in mice. Carbasugar17 and some open chains mimick-
ing sugar architectures18,19 also orient the cytokine response with
Since the discovery of KRN7000, an α-GalCer with a lack of
Received: October 11, 2011
Published: January 13, 2012
2′-OH group inducing iNKT activation at nM level9 (1, Chart 1),
© 2012 American Chemical Society
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Chart 1. Structure of Selected α-GalCers and 4-Deoxy KRN7000 Analogues
a TH1 bias. The α-O-anomeric configuration of the ceramide
aglycone moiety is believed to be critical20 as β-galactosylcer-
amides21,22 display far lower agonist activities, while enhanced
TH1 response in mice is induced by a nonhydrolyzable α-1C-
galactoside analogue (C-KRN7000, 2, Chart 1).23 Complemen-
tarily, SAR studies focused on the ceramide moiety have
concentrated efforts on modifications of the phytosphingosine
and acyl chains. Truncation of the sphingosine alkyl chain
illustrated by the OCH analogue (3, Chart 1), leads to a marked
decrease in the IFN-γ/IL-4 ratio in favor of the TH2 biased
immune response.24,25 On the other hand, aromatic residues on
either the acyl or sphingoid base of KRN7000 seem to restore the
TH1 cytokine profile.26,27
Throughout all these alterations, one of the remaining
features concerns the polarity of the sphingoid base.
Concerning its OH groups’ influence, we and others9,28−32
investigated the importance of the 4-OH group in the
stimulatory potency of 4-deoxy-KRN700033 (4, Chart 1). We
have recently confirmed its lesser importance compared to that
of the 3-OH group as mentioned in some earlier contradictory
studies, either in vitro with human cells or in vivo in
mice.9,12,32,34,35 According to previous investigations on 3,4-
dideoxy-KRN7000 analogue (5, Chart 1),9,12,34 the sphingoid
base 3-OH group is indisputably required for inducing iNKT
cell responses.32 In accordance with these observations,
crystallography data have clearly shown that only the key 3-
OH group is involved in the stabilization of the trimolecular
CD1d/KRN7000/TCR complex by concomitant donor
participation with CD1d Asp80 and a suspected acceptor effect
with Arg95 of the CDR3α-loop of the TCR.10,36 In addition,
examining the stereochemistry of KRN7000, Chung et al.37
showed that the relative spatial orientations of 2-NH and 3-OH
groups are both important for activity, with a greater impact for
the configuration of 2-NH.
hydrogen bond from the TCR. In vivo evaluation of the latter
gem-difluoro analogue resulted in a minimal loss in IFN-γ
secretion compared to KRN7000. The authors concluded that a
tighter bonding of the 3-OH group with CD1d is beneficial for
a TH1 polarization, while the hydrogen bond with the TCR
seems of lesser importance. The same authors also reported
biological evaluation of 2′,2′-difluoro KRN700040 (7, Chart 1)
in which the acidity of N−H amide group of the ceramide
fragment should be increased by introduction of the two
neighboring difluorine atoms. While expecting to favor a TH1
bias by reinforced hydrogen bonding with Thr156 of mCD1d
(Thr154 for hCD1d) to stabilize the CD1d/GalCer/TCR
complex, a surprising TH2 bias was observed. The authors
suggested that the latter unexpected polarization of the immune
response indicates that hydrogen bonding between the amide and
the TCR receptor does not have a significant impact on the
stability of the ternary complex.
In our continuing efforts to highlight the potency of deoxy
analogues of α-GalCers to modulate the iNKT response, we
evaluated the effect of combined alterations of the sphingoid
base devoid of both 4-OH and 3-OH groups by substituting the
3-OH groups by one or two fluorine atoms (3,4-dideoxy-3-
fluoro-KRN7000 8 and 3,4-dideoxy-3,3-difluoro-KRN7000 9,
respectively, Chart 1). Our aim was to contribute to establish
the relative part of the TCR interaction with the 3-OH group of
α-GalCers. We anticipated that, despite suppression of the
hydrogen-bond donating capacity with CD1d, electron
isoelectronic effect of one or two fluorine atoms, vs oxygen,
would modulate the lack of the sphingosine 3-OH on the
destabilization of the CD1d/GalCer/TCR complex by
reinstating favorable interaction with the TCR.
RESULTS
■
Synthesis of 3,4-Dideoxy-3-fluoro-KRN7000 8. We
have recently reported an access to a 4-deoxy-KRN7000
analogue (4, Chart 1) from the key ethylenic galactoside
intermediate 11 (Scheme 1).30 This latter derivative was readily
obtained by coupling of the α-fluoro-perbenzylglycosyl donor
12 with the acceptor sphingosine fragment 13. Cross-coupling
metathesis reaction between the glycosidic primer 11 and a
corresponding ethylenic partner gave the sphingosine intermediate
10 (R1 = n-C12H25). The last step, prior to total deprotection of
More recently, fluorine atom(s),38 that imparts various
properties to certain drugs, affecting binding interactions,
metabolic stability, and selective reactivities, has been intro-
duced into α-GalCer structures, leading to alterations on
cytokine releases. Linclau et al.39 reported the synthesis of a
4-deoxy-4,4-difluoro-KRN7000 analogue (6, Chart 1) in which
the hydrogen-bond donating capacity with CD1d was rein-
forced vs a concomitant decrease in its ability to accept the
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Scheme 1. Retrosynthetic Pathway to 4-Deoxy-KRN7000 4
a
Scheme 2. Synthesis of 3,4-Dideoxy-3-fluoro-KRN7000 4 and RX Structure of 16
a
Reagents and conditions: (a) DAST, CH2Cl2, 10 min, rt, 16 54%, 15 35%; (b) 1-tetradecene, Grubbs II catalyst, CH2Cl2, reflux, 24 h, 83%; (c) HCl
2N solution in Et2O, THF, reflux; (d) nC25H51COCl, 50% aq NaOAc, THF, 40 °C, 1 h, 38% over two steps; (e) H2, Pd/C, CHCl3/MeOH, 48 h, rt,
then Ac2O/pyridine, DMAP, separation by flash chromatography on silica gel 19 45%, 20 29%; (f) MeONa, CH2Cl2/MeOH (1:1), for 5 47% and
for 8 52%.
the target, consisted of an acylation of the free amine group to
complete the ceramide moiety of the 4-deoxy-KRN7000.
from 14 led to the major formation of the undesired
oxazolidinone 15 (4S). The latter side reaction can be minimized
by running the fluorination reaction at higher temperature (rt vs
−78 °C), yielding the desired fluorinated intermediate 16 in an
optimized 54% yield, along with a minor amount of 15 (35%).
At this stage, Grubbs II catalyzed cross-metathesis reaction
between the fluoro allyl 16 and 1-tetradecene provided access to
the 3-fluorinated sphingosine 17 in 83% yield (Scheme 2). After
acidic removal of the N-Boc group, the acylation of sphingosine
fragment, following a well-established method described in
GalCers series (4-nitrophenyl hexacosanoate, in the presence of
Et3N and catalytic DMAP), failed to give the expected ceramide
derivative 18 from 17. A probable electronic deactivation by the
vicinal fluorine atom should be taken into account to explain the
loss in reactivity of the amine partner. Eventually, success was
encountered by using N-acylation conditions described by De
Jonghe43 and Teare44 for the synthesis of fluorinated sphinganine
and sphingosine, respectively. The crude mixture, obtained after
cleavage of the N-Boc protecting group from 17, was treated with
hexacosanoyl chloride in 50% NaOAc aqueous solution, at 40 °C,
to give the N-acylated derivative 18 in a modest 38% yield. Final
In the light of this previous work, we planed to synthesize
the 3,4-dideoxy-3-fluoro-KRN7000 derivative 8 via the ethyl-
enic 3-deoxy-3-fluoro-galactoside 16 as outlined in Scheme 2.
The fluorine substituent was expected to be introduced in the
required configuration (3R) from the allylic alcohol 11 following
a double SN2 inversion of configuration. Thus, fluorination of 11,
by DAST at −78 °C in CH2Cl2,41 promoted the formation of the
expected fluoro derivative 16, in moderate 30% yield, along with
the oxazolidinone byproduct 15 formed predominantly (60%).
The absolute configuration 3R of fluoro derivative 16 was
unambiguously assigned at solid state by X-ray crystallography
(see Supporting Information for selected data). After 3-OH
activation by DAST, the NHBoc neighboring group cyclized,42
giving rise to intramolecular SN2 formation of an oxazolidinium
salt 14. Subsequent nucleophilic addition of the released fluoride
anion to the iminium 14 provided the fluorinated derivative 16
(3R) upon a second inversion of configuration at the C-3
stereogenic center. Nevertheless, a favorable loss of isobutene
residue from 14, via the release of stable tert-butyl carbocation,
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Scheme 3. Retrosynthetic Scheme for the Synthesis of 3,3-gem-Difluoro Analogue 9
a
Scheme 4. Synthesis of 3,4-Dideoxy-3,3-difluoro Analogue 9
a
Reagents and conditions: (a) LiHMDS, THF, −80 °C, 30 min, then CF2Br2, −100 °C, 1 h, 53% after recrystallization; (b) (i) allyltri-n-butyltin,
AIBN cat., benzene, reflux, 3 h, (ii) EtOH/HCl 2N (9:1), 50 °C, 1 h; (c) Boc2O, NaHCO3, THF, rt, overnight, 39% (over 3 steps); (d) NaBH4,
EtOH, 0 °C to rt, 4 h, 22 81% and 27 <5%; (e) 12, SnCl2, AgClO4, THF/Et2O (1:3), 0 °C, 1 h then rt, 1 h; (f) Boc2O, NaHCO3, dioxane, 21 40%
over 2 steps; (g) HCl gas, Et2O, rt, 3 h; (h) nC25H51COCl, pyridine, 80 °C, 24 h, 38%; (i) 1-tetradecene, Grubbs II catalyst, CH2Cl2, reflux, 4 h,
45%; (j) Pd(OH)2/C, H2, CHCl3/EtOH (1.5:1), 40%.
hydrogenolysis of the benzyl protecting groups with Pd/C and
concomitant hydrogenation of the unsaturation, achieved the
synthesis of the targeted 3,4-dideoxy-3-fluoro-KRN7000 8. In the
latter conditions, an equimolar amount of the byproduct, 3,4-
dideoxy derivative 5, was formed when Pd or Pd(OH)2 on
carbon catalysts were used. The sensitivity of the allylic fluorine
atom to hydrogenolysis conditions has been also observed
by Hsin45 and Hudlicky.46 Because compounds 8 and 5 are
inseparable by chromatography on silica gel, their purification was
achieved via acetylation of the crude hydrogenation mixture (Ac2O/
pyridine 1:2, and DMAP cat.), giving the peracetylated derivatives
19 and 20 that have been separated by flash chromatography in
45% and 29% yields, respectively. Deprotection of the peracetylated
intermediates by sodium methanolate in methanol gave the
expected 3,4-dideoxy-3-fluoro-KRN7000 8 and 3,4-dideoxy-
KRN7000 5 in 52% and 47% yields, from 20 and 19, respectively.
Synthesis of 3,4-Dideoxy-3,3-difluoro-KRN7000 9. To
supplement our investigation on the impact of a fluorine atom
in place of 3-OH group, the synthesis of 3,4-dideoxy-3,3-
difluoro KRN7000 9 was undertaken. To be faithful to our synthetic
strategy, the preparation of gem-difluoro analogue 9 relies on the
building of a 3,3-gem-difluorinated ethylenic galactoside fragment
21 to be engaged in a cross-metathesis process (Scheme 3).
Schiff’s base 23 with CF2Br2, radical allylation of bromide 24
followed by hydrolysis of the chiral group, and N-Boc protection
of the resulting amine 25 prior reduction of corresponding ester
26 (Scheme 4). The latter procedure was retained as (2S)-ethyl-
2-tert-butoxycarbonylamino-hex-5-enoate 26 has been already
used for other chemistry in our group. The reduction of ester 26
into alcohol 22, by NaBH4 in EtOH, proceeded without major
difficulties in good 81% yield, although some HF elimination
product 27 (that could not be separated from the alcohol 22)
was formed concomitantly. However, its formation could be kept
minimal (<5%) by controlling carefully the temperature and the
reaction time.
The reaction sequence was then pursued from the alcohol 22
by a glycosylation under Mukaiyama conditions in the presence
of fluorogalactosyl donor 12. The reaction led to a mixture of
protected N-Boc protected and free NH2 α-galactosides 21 and
28, isolated in low 10% and 25% yields, respectively, after
purification. The amine derivative 28 was quite difficult to
purify, and flash chromatography led to a loss of material.
Consequently, the crude mixture of the glycosidic coupling
reaction was treated under Boc protection conditions (Boc2O,
NaHCO3) to give the gem-difluoro-α-galactoside 21 in 40%
yield over the two steps. Then N-Boc group was removed by
HClg in Et2O, and the resulting crude amine 28 was directly
engaged in the next step without purification. Introduction
of the hexacosanoyl acyl chain was first attempted from
The required homoallylic gem-difluoro alcohol 22 could be
obtained via sulfoxide chemistry47 or following the five-step pro-
cedure described by Katagiri et al.:48 alkylation of ethyl glycinate
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Figure 1. (A) Flow cytometric analysis of hCD1d-transduced HeLa cells, CD1d-transduced and nontransduced Namalwa cells. Gray lines represent
CD1d expression detected by anti-CD1d-PE mAb 51.1 and black lines represent controls where only the secondary antihuman IgG Ab was used. A
total of 250000 cells were analyzed in each experiment. The figure is representative of at least 3 separate experiments. (B and C) Relative potencies
of compound 1 (KRN7000, represented by circles) and analogues 9 (3,4-dideoxy-3,3-difluoro KRN, represented by squares), 8 (3,4-dideoxy-3-fluoro
KRN, represented by triangles), and 5 (3,4-dideoxy KRN, represented by diamonds) to induce TNFα production by the Vα24 iNKT cell line
MAD11. The CD1d-transduced antigen-presenting cell lines HeLa (B) and Namalwa (C) were pulsed with increasing concentrations of glycolipids
and iNKT cells were stimulated for 6 h. Background values obtained when no exogenous glycolipid was added to the CD1d-transduced antigen-
presenting cells were subtracted from the presented values. Negative controls with MAD11 cells alone and CD1d nontransduced antigen presenting
cells in the presence and absence of the glycolipid compounds 1, 9, 8, and 5 are shown on the right-hand side. TNFα secretion was measured by a
cellular assay, and results are shown as mean of triplicates from one representative experiment out of at least three with similar results. Error bars
represent mean values SD.
hexacosanoyl chloride (n-C25H51COCl, NaOAc, THF/H2O,
45 °C) or from hexacosanoic acid (EDC, NMM, HOBt,
CH2Cl2, rt), affording only trace of the expected acylated
product 29. Similarly, the reaction of 28 in the presence of
hexacosanoyl chloride in pyridine at 50 °C proceeded
extremely slowly, and the expected product 29 was obtained
in a poor 6% yield. However, heating the reaction mixture at
80 °C for 24 h increased the yield of the acylation to 36%.
Ruthenium-catalyzed cross-metathesis reaction between 1-tetradecene
and homoallylic gem-difluoro-compound 29, under the same
conditions as for preparation of compound 17 (see Scheme 2), led
to the desired unsaturated compound 30 isolated in 45% yield after
purification. Finally, treatment of the intermediate 30 under hydrogen
atmosphere in the presence of Pd(OH)2/C performed the
subsequent hydrogenolysis of the benzyl protective groups and the
reduction of the double bond to give 3,4-dideoxy-3,3-difluoro-
KRN7000 9 in 40% yield.
Biological Evaluations. In Vitro Results. The ability of
the 3,4-dideoxy analogues to activate iNKT cells in vitro was
investigated using two antigen-presenting cell lines transduced
to express hCD1d. FACS analysis verified that the two cell
types expressed comparable levels of hCD1d after transduction
(Figure 1A). HeLa cells of epithelial origin are spontaneously
CD1d negative (result not shown), whereas Namalwa cells of
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leucocytic origin spontaneously express low levels of CD1d.
Parts B and C of Figure 1 show the TNF-α secretion by the
iNKT MAD11 cell line after stimulation with antigen-
presenting cells pulsed with compound 1 (KRN7000) and
the dideoxy α-GalCer analogues 5 (3,4-dideoxy KRN7000), 8
(3,4-dideoxy-3-fluoro KRN7000), and 9 (3,4-dideoxy-3,3′-
difluoro KRN7000). We have previously shown that the 4-
deoxy analogue of KRN7000 stimulates iNKT cells nearly to
the same extent as reference 1.30 However, as shown in Figure 1,
removal of the hydroxyl groups at both C3 and C4 in the 3,4-
dideoxy KRN7000 analogue 5 renders it a poor stimulator of
human iNKT cells and lowers its stimulatory effect by a 1000-
fold when presented by HeLa-CD1d cells (Figure 1B, left) and
>10000-fold when using Namalwa-CD1d cells (Figure 1C, left).
Addition of a fluorine atom at C3 in monofluoro derivative 8
further lowers the activity >10000 times compared to 1, using
both types of antigen-presenting cell lines (Figure 1B and C,
left). Intriguingly, addition of a second fluorine atom at C3 in
3,3-difluoro KRN7000 analogue 9 partly restores the activity
with a stimulatory effect only 20 times lower than that of
compound 1 when presented by HeLa-CD1d cells (Figure 1B,
left) and 100 times lower when presented by Namalwa-CD1d
cells (Figure 1C, left). These results indicate that the loss of the
hydroxyl group at C3 in the sphingoid base of analogues 5 and
8 destabilizes the CD1d/glycolipid/TCR complex, as expected.
In contrast, the presence of two geminal fluorine atoms in the
3,3-difluoro analogue 9 restabilizes the complex. As a control,
all glycolipids were also loaded onto nontransduced HeLa and
Namalwa cells in order to confirm that they were presented by
CD1d. Glycolipids 1, 5, 8, and 9 loaded onto HeLa cells
induced only background TNF-α secretion (Figure 1B, right).
When loaded onto Namalwa cells (Figure 1C, right), TNF-α
release was about 20−90% of the secretion obtained when
loaded to CD1d-transduced cells, in agreement with the
FACS results showing significant native expression of CD1d
in Namalwa cells (Figure 1A).
To determine if stimulation by the 3,4-dideoxy analogues led
to a bias in the TH1/TH2 balance of the response, we measured
the secretion of a TH1 type (IFN-γ) and a TH2 type (IL-13)
cytokines. IL-13 was chosen because IL-4 secretion was always
extremely low in our in vitro assay conditions. HeLa and
Namalwa cell lines were pulsed with increasing concentrations
of glycolipids, and iNKT cells were stimulated for 6 and 48 h
for the IFN-γ and IL-13 analysis, respectively. Figure 2 shows
the ratio between the releases of the TH1 type cytokine IFN-γ
(left y axis, solid line) and the TH2 type cytokine IL-13 (right y
axis, dotted line) induced by the glycosylceramides 1, 5, 8, and 9.
Because compound 1, KRN7000, is taken as the reference for
the TH1/TH2 balance (ratio IFN-γ/IL-13 = 1), the curves
illustrating the release of IFN-γ and IL-13 induced by this
glycolipid were superimposed and compared to the super-
imposed curves generated by the dideoxy analogues in order to
establish their TH1/TH2 profiles. When presented by HeLa-
CD1d cells (Figure 2A−C, left), analogues 5, 8, and 9 all
generated IFN-γ/IL-13 relative secretion similar to that
obtained by compound 1, and intensities of the responses for
each of these cytokines paralleled those obtained for TNF-α. In
contrast, when Namalwa-CD1d cells were used as antigen-
presenting cells, 3,3-difluoro analogue 9 gave an IFN-γ/IL-13
profile identical to that of 1, whereas analogues 8 and 5
appeared to induce slightly elevated levels of IL-13 relative to
IFN-γ indicative of a weak TH2 orientation by comparison with
1 (Figure 2A−C, right). However, regardless of the glycolipid,
IL-13 secretion was always weaker upon presentation by
Namalwa-CD1d cells than by HeLa-CD1d cells (note that the
scales for IL-13 are different between the left and right panels of
Figure 2). In addition, no difference in IL-13 secretion was
noted between compounds 9, 8, and 5 when presented by
Namalwa-CD1d, suggesting a distinct behavior depending on
the antigen presenting cells. The factors that determine
whether an immunologic challenge will trigger a TH1 or a
TH2 response are not fully understood, but a correlation has
been established between the stability and interaction time of
the CD1d/glycolipid/TCR complex and the type of cytokines
that are being secreted. A more stable complex allows for a
longer interaction and secretion of TH1 type cytokines, whereas
a destabilized complex leads to a shorter interaction time and
release of TH2 cytokines. Our results show a slight TH2
orientation compared to reference compound 1 for analogues
3,4-dideoxy-3-fluoro KRN7000 (8) and the 3,4-dideoxy
KRN7000 (5), visible upon presentation by Namalwa-CD1d
cells. This is in agreement with the results obtained in the TNF-
α analysis (above), i.e., that the loss of the hydroxyl H-donating
group at C3 in the long chain base destabilizes the CD1d
complex, while addition of a second fluorine atom in gem-
difluoro analogue 9 increases the complex stability. However,
the differences observed in terms of IL-13 secretion between
presentation by either the HeLa-CD1d or Namalwa-CD1d cells
indicate that other factors related to the presenting cells
contribute to dictate the response orientation. The negative
controls in Figure 2D (right charts) confirm that, when loaded
to non-CD1d transduced HeLa cells, the glycolipids induced
only background quantities of IFN-γ and IL-13 release. When
loaded to Namalwa cells, the cytokine releases were about
0−30% (IFN-γ) and 10−40% (IL-13) of the secretion obtained
when loaded to CD1d-transduced cells, in agreement with the
FACS analysis shown in Figure 1A.
In Vivo Results. To investigate whether 3,4-dideoxy-
KRN7000 analogues 5, 8, and 9 could elicit CD1d-dependent
NKT cell activation in vivo, mice received intraperitoneally (ip)
100 μg/kg of glycolipids in 0.1 mL of vehicle after which TNF-
α, IFN-γ, and IL-4 kinetic releases were measured and
compared to the secretion induced by KRN7000. Injection
of 3,4-dideoxy-3-fluoro 8 and 3,4-dideoxy-3,3-difluoro 9
KRN7000 analogues resulted in notably lower serum cytokine
levels relative to KRN7000. The area under curve (AUC) for
TNF-α release induced by both fluorine derivatives 8 and 9
appeared quite similar, arising at 23% and 32% of KRN7000
potency. Intriguingly, the 3,4-dideoxy-derivative 5 promoted
TNF-α release with a 40% relative efficiency (Figure 3a). This
behavior was highlighted by the INF-γ cytokine levels induced
by compound 5, providing an AUC over 70% close to that of
KRN7000 (Figure 3b). The difluorinated analogue 9 induced a
noticeable in vivo INF-γ stimulation only twice lower than that
of KRN7000, 8 h after injection, while that of monofluorinated
compound 8 leveled off at a maximum of 30%. IL-4 amounts
induced by all dideoxy-analogues 5, 8, and 9 appeared quite
similar (Figure 3c), providing AUC dideoxy analogues/
KRN7000 ratio ranging between 0.35 and 0.39. Consequently,
a TH1 orientation was observed with the 3,4-dideoxy-3,
3-difluoro-KRN7000 9 and more surprisingly also with the
3,4-dideoxy-KRN7000 5, providing AUC IFN-γ/IL-4 ratio of
1.31 and 1.86, respectively (Figure 4). In contrast, mono-
fluorinated derivative 8, that stimulated a 3-fold lower release
of IFN-γ than KRN7000, favored a slight TH2 profile with an
AUC IFN-γ/IL-4 ratio of 0.78. These results clearly indicate
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Figure 2. Relative potencies of compound 1 (KRN7000, represented by circles) and analogues 9 (3,4-dideoxy-3,3-difluoro KRN, represented by
squares), 8 (3,4-dideoxy-3-fluoro KRN, represented by triangles), and 5 (3,4-dideoxy KRN, represented by diamonds) to induce secretion of IFNγ
(left y-axis, represented by solid lines and filled symbols) and IL-13 (right y-axis, represented by dotted lines and open symbols) by the Vα24 iNKT
cell line MAD11. The CD1d-transduced antigen-presenting cell lines HeLa (left-hand side) and Namalwa (right-hand side) were pulsed with
increasing concentrations of glycolipids and iNKT cells were stimulated for 6 and 48 h for the IFNγ and IL-13 analysis, respectively. Compound 1 is
shown in all graphs A−C as a TH1/TH2 neutral reference glycolipid. Background values obtained when no exogenous glycolipid was added to the
CD1d-transduced antigen-presenting cells were subtracted from the presented values. Negative controls with MAD11 cells alone and CD1d
nontransduced antigen presenting cells in the presence and absence of the glycolipid compounds 1, 9, 8, and 5 are shown in (D). Cytokine secretion
was measured by ELISA, and results are shown as mean of triplicates. Error bars represent mean values SD.
that the behavior of the 3,4-dideoxy compounds differ
between the in vivo assay in mice and the in vitro assay with
human cells, the most notable difference being observed for
compound 5.
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Figure 3. AUC TNF-α (a), IFN-γ (b), and IL-4 (c) levels in C57BL/6J Rj mice serum after ip injection of Galcers 1, 8, 9, and 5. Compound 1
always stimulates significantly higher levels of cytokines than the other compounds (p < 0.001 for all, except for IFN level of compound 5
(p < 0.01)). p < 0.01 = **, p < 0.001= ***.
with the CD1d, was thought to be offset by reinforcing the
donating NH-bond of the group potentially involved in a TCR
interaction.13 The existence of such a TCR−HNCO bond has
emerged from X-ray crystallographic studies36 through a critical
interaction with adjacent Thr residue (Thr156 for mCD1d and
Thr154 for hCD1d), without involvement of the amide oxygen
in CD1d. Consequently, on the basis of the hydrogen bond
arguments, the increased acidity of the NH-amide induced by
the withdrawing effect of fluorine activation would bias the
iNKT cells response toward a TH1 orientation by restabilizing
the GalCer/TCR complex or, at least, would favor selected
Figure 4. AUC IFN-γ/AUC IL-4 ratio in C57BL/6J mice serum after
ip injection of GalCers 1, 8, 9, and 5.
cytokine secretion. In contrast with this prediction, Linclau et al.
have found that introduction of a gem 2′,2′-difluorine group on
the acyl chain of KRN7000 (compound 7, Chart 1) elicits a
TH2 polarization in mice.40 Considering a small conformational
impact of a gem difluorine group, relative to an OH group, the
authors have correlated this outcome to a negative impact of
the modified acyl chain on the polar group stabilization,
consistent with a lower affinity with the TCR, rather than an
effect of the amide NH...mice Thr156 bond. Obviously, the
cytokine secretion restored by difluoro analogue 9 gives a better
insight into the amide network by delivering more effective
inductive electronic effects from the 3,3-gem difluoro group
through the F2C3−C2−NHC1 linkage.
A hybrid QM/QM′ model (see Supporting Information for
computational details) has been applied for the fluoro
KRN7000 analogues to quantify the strength of the hydrogen-
bond interaction with Thr154. Because we are only interested in
relative variations, a rather small model was defined, including the
KRN7000 analogue and seven AA residues (Figure 5) but
allowing full ab initio calculations. The results are summarized in
DISCUSSION
■
Elimination of the CD1d Asp80 hydrogen bond by replacing
the 3-OH by a fluorine atom in α-GalCer structures drastically
reduced the magnitude of cytokine releases, both in mice and
human iNKT cells. The weak stimulation induced by the 3,4-
dideoxy-3-fluoro KRN7000 analogue 8 was consistent with the
established crucial role of the donating hydrogen bond that
stabilizes the CD1d/GalCer complex.24,49−52 Consequently, the
slight TH2 profile of 3,4-dideoxy-3-fluoro analogue 8, observed
either in vitro on human iNKT cells (IL-13 vs IFN-γ when
presented by Namalwa-CD1d cells) and in mice (AUC IFN-γ/
IL-4 ratio of 0.78), was anticipated due to a predictable
destabilization of the long chain base in the (C′)F′-binding
groove. Nevertheless, intriguing effects were observed when
two geminal fluorine atoms were introduced at the C3 position
of the sphingosine chain. The agonist activity on the TNF-α
release of 3,4-dideoxy-3,3-difluoro-KRN7000 9 appeared only
20- and 100-fold lower than that of KRN7000 1 on human
iNKT cells when presented by either HeLa-CD1d and
Namalwa-CD1d cells, respectively. Furthermore, difluoro
analogue 9 expressed an IFN-γ/IL-13 profile identical to that
of KRN7000 when loaded on Namalwa-CD1d cells. This
outcome was pronounced in vivo, inducing a TNF-α secretion
in mice only twice lower than that of KRN7000, with a slight
TH1 polarization (AUC IFN-γ/IL-4 ratio = 1.31).
Several interpretations can be made from these observations,
taking into account a combination between the close isosteric
relationship of fluorine atom to hydroxyl and the strong
withdrawing electronic induction. It is well-known that addition
of fluorine atom(s) on carbon induces a drastic increase of
acidity of a vicinal acidic group (pK = 4.76, 2.59, and 1.24 for
CH3COOH, CH2FCOOH, and CHF2COOH, respectively).
Obviously, such an electronic effect can be transposed to the
ceramide NH−CO bound to contribute in the CD1d/GalCer/
TCR interaction. Thus, the destabilization of the ternary
complex, due to the lack of the donating 3-OH hydrogen bond
Figure 5. (A) Partially optimized structure of a model of 9 in
interaction with seven AA residues of the protein at the PBE0/
6-311G(d,p):PBE0/6-31G level of theory. Red atom = O, dark-blue
atom = N, purple atom = F, light-blue atom = C of the ligand, orange
atom = C of protein residue. (B) Model system investigated: in ball-
and-stick the QM part [level of theory: PBE0/6-311G(d,p)], in
wireframe the QM′ part [level of theory: PBE0/6-31G]. Red atom =
O, blue atom = N, gray atom = C.
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Table 1 for the above-mentioned amide NH-bond, the fluorine-
free structure 1 being used as reference. First, it should be noted
Thus, modeling studies confirm that positioning the
modified C3 difluoro analogue 9 in the A′ pocket of hCD1d
exposes the 3(S)-fluorine atom in a syn-periplanar conformation
to the NH amide group (3(S)F−C3−N−H: 0.6°, Figure 5),
allowing an optimum electron withdrawing effect.
a
Table 1. Theoretical Results
model dH (NH-Thr154)
of
An additional aspect should be also discussed concerning a
possible C3−F···H−Arg95 hydrogen bond participation in the
complex stabilization, as hydrogen bonds involving fluorine
atom have emerged from several protein structures cocrystal-
lized with fluorine-containing ligands.53 Even if C−F···H−O
bond energy was measured at less than half the strength of a
C−O···H−O bond (between 2 and 3.38 kcal mol−1), consistent
interaction can occur when the fluorine atom possessed a short
C−F···H− contact of ≤2.35 Å.54,55 Thus interaction of the
fluorine atom pointing toward the CDR3α loop of the TCR
from the sphingoid base can be envisaged with respect to
CD1d/9/TCR modeling data, giving the C3(S)-F···H−Arg95
and C3(R)−F···H−Arg95 distances at 2.286 and 2.292 Å,
respectively. Both are closer than the 3-HO···H distance in
reference 1 measured at 2.471 Å. However, as confirmed in
vitro by the cytokine release levels using 3,4-dideoxy-3(R)-
fluoro KRN7000 analogue 8, this interaction does not balance
the lack of the donating hydrogen bond from C3-OH group.
The weak stimulatory potency of 3(R)-fluoro compound 8
allowed also to eliminate the occurrence of a double acceptor
binding by the same fluorine atom in the ternary complex
network that could involve two H-donor groups.53
Along this discussion, it is intriguing that 3,4-dideoxy-KRN7000
analogue 5, used as negative control, promotes TNF-α and INF-γ
releases in mice with 40% and 70% respective efficiencies relative
to KRN7000 in spite of the absence of both hydroxyl groups on
the sphingoid base. These data are consistent with previous
works,9,25 that regarded the 3,4-dideoxy-KRN7000 5 as a partial
agonist in mice in spite of a nonstimulatory effect on human cells
in vitro. Thus, despite a pronounced TH1 polarization by 3,4-
dideoxy-KRN7000 5 in mice (AUC IFN-γ/IL-4 ratio of 1.86), this
behavior was definitively not observed with human cells.
Additionally, Sidobre et al.32 have noted the poor potency of
3,4-dideoxy-KRN7000 5 in stimulating IL-2 from human NKT
cells derived from hybridoma, while Chung et al.37 observed a
potent effect toward the secretion of IL-13 in other human iNKT
cell clones (TH2 bias). Altogether, these observations increase our
knowledge on the sensitive and versatile role of CD1d and TCR
receptors in the stabilization of GalCer substrates and highlight
some sensitive differences between mice and human that remain
difficult to rationalize.
(2)
groups
qH
qO
qN
E
n→σ*
R1 = OH;
1
1.819
1.813
1.818
1.842
0.07
−0.46
−0.66
8.23
8.49
8.69
7.75
R2 = R4 = H
R1 = F;
R2 = R4 = H
8
9
7
0.08
0.19
0.10
−0.46
−0.52
−0.44
−0.62
−0.69
−0.44
R1 = R2= F;
R4 = H
R1 = OH;
R2 = H;
R4 = F, F
a
dH is the amide N-hydrogen-bond distance (in Å), q are the partial
atomic MK charges (in |e|) computed for the three atoms implied in
(2)
the α-GalCer analogues−Thr154 hydrogen bond, and E
are the
n→σ*
averaged n → σ* interaction energies (in kcal·mol−1) that characterize
the H-bond strength. See Chart 1 for substitution positions (in all
these models X = O, R3 = OH, and the sphingoid base contains 18
carbon atoms).
that in models 1, 8, and 9, neither the hydrogen-bond distances
nor the atomic charges are strongly affected by the substitution
with fluorine atoms on the C3 carbon atom. Therefore, we
predict no drastic modifications of the nature of the interaction.
Nevertheless, it appears that a 2′-CH2 substitution by CF2 on the
acyl chain of 7 implies a less negative nitrogen atom (−0.44e
instead of −0.66e) accompanied by a lengthening of the
hydrogen bond (ca. +0.02 Å). Considering the nearly constant
charge of the shared hydrogen atom, this elongation suggests a
less favorable interaction between the two moieties, as confirmed
by the smaller En(→2)σ* value between the oxygen lone pair n(O) and
the unoccupied antibonding orbital σ*(N−H) of the amide moiety.
Despite the presence of two electron-withdrawing fluorine atoms,
the hydrogen-bond interaction with Thr154 is therefore slightly
decreased in the analogue 7, a finding consistent with its TH2
profile. This behavior actually originates in the weak intra-
molecular interaction taking place between an occupied non-
bonding orbital of one fluorine atom, n(F) and the unoccupied
antibonding orbital σ*(N−H) of the amide moiety. Our model
(2)
n→σ*
yields a E
value of 0.40 kcal mol−1 for this intramolecular
interaction (data not shown) that does not occur in the
nonfluorinated analogue 1 nor in the monofluorinated 8 where
the fluorine and the NH moieties are spatially distant. For the
3,3-difluoro analogue 9, the second fluorine atom is favorably
oriented toward the NH bond but the En(→2)σ* value is significantly
smaller (0.13 kcal mol−1), hinting that the intermolecular
interaction with Thr154 is not weakened for this difluorinated
derivative. Theory therefore foresees for 9 similar geometrical
parameters as in KRN7000 1 but a twice more positive amide
CONCLUSION
■
In summary, after having shown that 4-deoxy-KRN7000 can be
regarded as a full agonist of KRN7000 in the stimulation of
human iNKT cells,30 we have performed the synthesis of 3,4-
dideoxy-3-fluoro 8 and 3,4-dideoxy-3,3-difluoro 9 KRN7000
analogues. Along with the aim to pursue our investigations on
the ability of deoxy sphingosine derivatives to release selected
cytokines from human and murine iNKT cells, substitutions of
3-OH group by one or two fluorine atoms on the ceramide
substrate were sought to evaluate the importance of the TCR
hydrogen-bond interactions through either a restored acceptor
effect with Asp95 of the CDR3α-loop and/or an increasing
donating NH-amide linkage with hTrp154 of the TCR.
hydrogen, implying a stronger donating interaction with Thr154.
(2)
n→σ*
This assumption is consistent with the larger E
energy
reported in Table 1. Eventually, the features of 8 are very similar
to those of the reference molecule 1, and one can assume a
relatively trifling impact of this substitution on the NHCO/TCR
participation. Although the results of such simulations should
certainly not be considered blindly, they seem to indicate a posi-
tive relationship between the strength of the difluoro KRN7000
analogue 9−Thr154 hydrogen bond of the TCR and the
experimental findings, i.e., the TH1 bias of 9 in mice or the
clear recovery of cytokine secretion of human iNKT cells.
In agreement with the literature, the loss of the hydroxyl
H-donating group at C3 in the sphingoid base induced a
destabilization of the GalCer/CD1d complex that could not be
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4.57 (AB syst, J = 11.7, 2H, CH2Ph), 4.79 (d, J = 3.6, 1H, H1″), 4.71
and 4.69 (AB syst, J = 12.0, 2H, CH2Ph), 4.40 and 4.30 (syst AB,
J = 12.0, 2H, CH2Ph), 3.98 (dd, J = 9.9, 3.6, 1H, H2″), 3.87−3.80 (m,
4H, H4, H3″, H4″, H5″), 3.71 (m, 1H, H6″), 3.65 (m, 1H, H6″), 3.44 (m,
2H, H5), 1.34 (s, 9H, O−C(CH3)3). 13C NMR (75 MHz, CDCl3) δ
154.3 (CO), 137.7−136.8 (Cqar), 132.4 (d, J = 18.8, C2), 127.4−126.5
(CHar), 118.2 (C1), 98.1 (C1″), 90.6 (d, J = 172.5, C3), 78.8 (O-
C(CH3)3), 78.6 (C3″ or/and C4″ or/and C5″), 76.7 (C2″), 74.8, 73.6,
73.4, 73.0 (CH2Ph), 69.8 (C3″ or C4″ or C5″), 69.1 (C5), 67.8 (C6″),
53.2 (d, J = 26.4, C4), 28.4 (O−C(CH3)3). 19F NMR (282 MHz,
CDCl3) δ −185.8 (dt, J = 52.5, 11.3). HRMS (ESI+) calcd for
C44H52FNNaO8 [M + Na]+ 764.3575; found 764.3539. Anal. Calcd for
C44H52FNO8: C, 71.23; H, 7.06; F, 2.56; N, 1.89. Found: C, 71.21; H,
7.18; F, 2.79; N, 1.82.
balanced by its substitution with one isoelectronic fluorine
atom. This result indicates a weak participation of donating
Asp95-H···3-OH bond in the stabilization of the CD1d/
Galcer/TCR ternary complex. Nevertheless, we found that
addition of a second fluorine atom in 3,4-dideoxy-3,3-difluoro
KRN7000 analogue 9 restores the stability of the ternary
CD1d/GalCer/TCR complex both in vivo in mice and in vitro
in human iNKT cells. Modeling studies showed that the syn-
periplanar orientation of the 3(R)-F with NH amide group on
the acyl chain enhances its donating effect with hTCR Trp154
stabilizing the ternary complex through the NH-bond.
Mice iNKT responses show strong divergences with human
cells (already noted by others) and cannot be considered
reliable to study partial agonists. In addition, the fact that no
difference in IL-13 secretion was noted between all 3,4-dideoxy
KRN7000 compounds 9, 8, and 5 when presented by Namalwa-
CD1d, in contrast to HeLa-CD1d cells, suggests a distinct
behavior depending on the antigen presenting cells.
(4S,5S)-(2,3,4,6-Tetra-O-benzyl-α-D-galactopyranosylmethyl)-5-
1
vinyl-1,3-oxazolidin-(3H)-2-one 15. [α]20 +4.2 (c 1.3, CHCl3). H
D
NMR (300 MHz, CDCl3) δ 7.32−7.18 (m, 20H, Har), 6.20 (d, J = 9.6,
1H, NH), 5.76 (ddd, J = 17.1, 10.5, 6.6, 1H, HCCH2), 5.28 (d, J =
16.8, 1H, HCCH2), 5.18 (d, J = 10.5, 1H, HCCH2), 4.86 and
4.57 (AB syst, J = 12.0, 2H, CH2Ph), 4.78 and 4.67 (AB syst, J = 12.0,
2H, CH2Ph), 4.71−4.69 (m, 2H, H1″, CH2Ph), 4.53−4.47 (m, 2H, H5,
CH2Ph), 4.42 and 4.34 (AB syst, J = 11.7, 2H, CH2Ph), 3.99 (dd,
J = 10.5, 3.6, 1H, H2″), 3.88−3.80 (m, 3H, H3″, H4″, H5″), 3.65−3.59
(m, 2H, sugar−O-CH2, H4) 3.48−3.35 (m, 3H, sugar−O-CH2, H6″).
13C NMR (75 MHz, CDCl3) δ 158.3 (CO), 138.4−137.6 (Cqar),
134.0 (HCCH2), 128.4−127.5 (CHar), 118.7 (HCCH2), 98.6
(C1″), 78.7 (C5, C3″ or C4″ or C5″), 76.4 (C2″), 74.8 (C3″ or C4″ or C5″),
74.6, 73.8, 73.5, 73.0 (CH2Ph), 70.1 (sugar-O-CH2, C3″ or C4″ or C5″),
69.5 (C6″), 57.5 (C4). HRMS (ESI+) calcd for C40H43NaNO8 [M + Na]+
688.2886; found 688.2868
(2S,3R)-2-(tert-Butyloxycarbonylamino)-3-fluoro-1-(2,3,4,6-tetra-
O-benzyl-α-D-galactopyranosyl)heptadec-4-ene 17. To fluorine
compound 16 (96 mg, 0.13 mmol, 1.0 equiv) dissolved in dry
CH2Cl2 (2 mL) at room temperature under argon were added
1-tetradecene (330 μL, 1.30 mmol, 10.0 equiv) and Grubbs II catalyst
(5 mg, 6 μmol, 0.05 equiv). The mixture was heated to reflux for 24 h.
1-Tetradecene (330 μL, 1.3 mmol, 10 equiv) and Grubbs II catalyst
(5 mg, 6 μmol, 0.05 equiv) were added (after cooling) and the
solution continued to stir for 4 h under reflux. Without treatment,
CH2Cl2 was evaporated. Purification by flash chromatography on silica
Considering recent studies56,57 showing that α-GalCer
derivatives induce iNKT cell anergy, the discovery of novel
immunostimulators able to shorten the unresponsiveness of
human iNKT cells is of interest for therapeutic strategies. Thus,
3,4-dideoxy-α-GalCer analogues, which deliver a suitable cytokine
secretion while maintaining TH1 polarization, may pave the way
toward candidates for anticancer immunotherapy.
EXPERIMENTAL SECTION
■
General. Solvents were purified and dried by standard methods
prior to use.58 1H and 13C NMR spectra were recorded on Bruker
Avance 300 (spectrometer fitted with a 5 mm i.d. BBO probe), Avance
III 400 (spectrometer fitted with a 5 mm i.d. BBFO+ probe,
temperature of the probe was set at 303K), and Avance III 500
(spectrometer fitted with a 5 mm i.d. 13C/1H dual cryoprobe,
temperature of the probe was set at 303K). Spectra were recorded at
room temperature with TMS as internal standard for 1H spectra.
Chemical shifts (δ) are given in ppm and coupling constants (J) in Hz.
1
All assignments were confirmed with the aid of two-dimensional H,
1
1H (COSYDFTP) or H, 13C (INVBTP) experiments using standard
gel (petroleum ether/EtOAc 90:10) afforded 17 as a colorless oil
1
(98 mg, 83%); [α]20 +36.6 (c 0.9, CHCl3). H NMR (400 MHz,
D
Bruker pulse programs. All reactions were monitored by TLC on
commercially available precoated plates (Kieselgel 60 F254), and the
products were visualized with mostaine solution (250 mL of H2O,
10.5 g of (NH4)6Mo7O24·4H2O, 0.5 g of Ce(SO4)2, and 15 mL of
H2SO4). Kieselgel 60, 230−400 mesh (Merck), was used for column
chromatography. Optical rotations were measured at 20 1 °C on
Perkin-Elmer 341 in the indicated solutions whose concentrations are
expressed in g/100 mL. Mass spectra were measured by CI with NH3
on a quad. Hewlett-Packard 5989A. HRMS were measured on an LCT
spectrometer from Micromass (Lokspray, channel 2795 from Waters,
flow, MeOH/H2O 50/50, 0.2 mL/min). FTIR spectra were obtained
in the 500−4000 cm−1 range on a Bruker Vector 22 FT-IR spectro-
meter. The tested compounds gave >95% purity as determined by
elemental analysis. Elemental analyses were performed at the Service
Central d’AnalyzeCNRS in Solaize (France).
CDCl3) δ 7.41−7.26 (m, 20H, Har), 5.75−5.68 (m, 1H, H5), 5.59−
5.50 (m, 1H, H4), 5.01 (d, J = 9.0, 1H, NH), 4.98 and 4.58 (AB syst,
J = 11.2, 2H, CH2Ph), 4.91 (d, J = 3.6, 1H, H1″), 4.88 (m, 1H, H3),
4.85 and 4.76 (AB syst, J = 12.0, 2H, CH2Ph), 4.82 and 4.66 (AB syst,
J = 11.6, 2H, CH2Ph), 4.50 and 4.41 (AB syst, J = 12.0, 2H, CH2Ph),
4.06 (dd, J = 10.0, 3.6, 1H, H2″), 3.97−3.90 (m, 4H, H2, H3″, H4″, H5″),
3.83−3.73 (m, 2H, H6″), 3.53 (d, J = 6.3, 2H, H1), 2.02−1.99 (m, 2H,
H6), 1.43 (s, 9H, O−C(CH3)3), 1.35−1.27 (m, 20H, H7−H16), 0.90
(t, J = 6.6, 3H, H17). 13C NMR (100 MHz, CDCl3) δ 155.2 (CO),
138.7−137.9 (Cqar, C5), 128.4−127.4 (CHar), 125.1 (d, J = 18.2, C4),
99.1 (C1″), 92.0 (d, J = 170.7, C3), 79.4 (O-C(CH3)3), 78.8 (C3″ or C4″
or C5″), 76.7 (C2″), 74.9 (C3″ or C4″ or C5″), 74.4, 73.5, 73.1, 73.0
(CH2Ph), 69.7 (C3″ or C4″ or C5″), 69.0 (C1), 68.1 (C6″), 53.3 (d, J =
27.6, C2), 32.3 (C6), 31.9 (C15), 29.6−28.8 (C7−C14), 28.3 (O−
C(CH3)3), 22.6 (C16), 14.1 (C17). 19F NMR (376 MHz, CDCl3)
δ −177.3 (d, J = 47.4). HRMS (ESI+) calcd for C56H76FNNaO8 [M + Na]+
932.5453; found 932.5492.
(2S,3R)-3-Fluoro-2-(hexacosanoylamino)-1-(2,3,4,6-tetra-O-ben-
zyl-α-D-galactopyranosyl)heptadec-4-ene 18. The Boc protected
amino compound 17 (108 mg, 119 μmol) in THF (1 mL) was treated
with HCl solution (2 mol·L−1 in Et2O, 580 μL, 10 equiv), and the
mixture was heated to reflux until complete consumption of starting
material. To the solution of the resulting amine dissolved in THF (2 mL)
were added a 50% aqueous NaOAc solution (1 mL) and hexacosanoyl
chloride (98 mg, 236 μmol, 1.9 equiv). The mixture was heated to 40 °C
for 1 h 30 min and then diluted with THF and brine. The combined
organic layer was washed with water combined, dried over MgSO4. and
concentrated. Purification of the residue by flash chromatography on
(3R,4S)-4-(tert-Butyloxycarbonylamino)-3-fluoro-5-(2,3,4,6-tetra-
O-benzyl-α-D-galactopyranosyl)pent-1-ene 16. Under argon, DAST
(262 μL, 1.99 mmol, 3.0 equiv) was added to a solution of alcohol 11
(490 mg, 0.66 mmol, 1.0 equiv) in CH2Cl2 (6.6 mL). The mixture was
stirred for 10 min at rt and then quenched with saturated aqueous
NaHCO3 solution. The aqueous layer was extracted with CH2Cl2. The
organic layers were combined, dried over MgSO4, and concentrated.
Purification of the crude by flash chromatography on silica gel
(EP/EtOAc 90:10 to 60:40) afforded fluoride 16 as a white solid (268
mg, 54%) and oxazolidinone 15 as a colorless oil (155 mg, 35%);
[α]20D +39.0 (c 1.0, CHCl3); mp 98−99 °C (crystallization: pentane/
Et2O 1:2). 1H NMR (300 MHz, CDCl3) δ 7.31−7.16 (m, 20H,
Harom), 5.84−5.70 (m, 1H, H2), 5.19 (m, 3H, H1, NH), 4.88 and 4.49
(AB syst, J = 11.4, 2H, CH2Ph), 4.87 (md, J = 52.5, 1H, H3), 4.80 and
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silica gel (EP/EtOAc 90:10) afforded N-acyl compound 18 as a white
solid after purification by flash chromatography on silica gel (CHCl3/
wax (54 mg, 38% over two steps); [α]20 +22.5 (c 1.0, CHCl3).
MeOH 97:3); [α]20 + 26.4 (c 0.6, pyridine); mp 100−101 °C.
D
D
1H NMR (400 MHz, CDCl3) δ 7.41−7.25 (m, 20H, Har), 6.18 (d, J =
9.0, 1H, NH), 5.77−5.69 (m, 1H, H5), 5.55−5.47 (m, 1H, H4), 4.94 and
4.58 (AB syst, J = 11.7, 2H, CH2Ph), 4.98−4.86 (m, 1H, H3), 4.89 (d,
J = 3.6, 1H, H1″), 4.82 and 4.76 (AB syst, J = 11.7, 2H, CH2Ph), 4.81−
4.66 (m, 2H, CH2Ph), 4.49 and 4.40 (AB syst, J = 12.0, 2H, CH2Ph),
4.22−4.17 (m, 1H, H2), 4.06 (dd, J = 10.4, 3.6, 1H, H2″), 3.98−3.89 (m,
4H, H3″, H4″, H5″, H1), 3.70−3.67 (m, 1H, H1), 3.56−3.45 (m, 2H, H6″),
2.38 (t, J = 7.4, 2H, H2′), 2.04−1.99 (m, 2H, H6), 1.58−1.53 (m, 2H,
H3′), 1.27−1.26 (m, 64H, H7−H16, H4′−H25′), 0.89 (t, J = 6.9, 6H, H17,
H26′). 13C NMR (100 MHz, CDCl3) δ 172.8 (C1′), 138.7−137.7 (Cqar),
138.1 (d, J = 11.1, C5), 128.4−127.2 (CHar), 125.2 (d, J = 18.3, C4),
99.5 (C1″), 91.9 (d, J = 170.3, C3), 78.8 (C3″), 77.2 (C4″ or C5″), 76.8
(C2″), 74.8, 73.5, 72.9 (CH2Ph), 70.0 (C4″ or C5″), 69.2 (C6″), 68.1 (C1),
51.9 (d, J = 27.5, C2), 36.7 (C2′), 32.3 (C6), 31.9 (C15, C24′), 29.6−28.7
(C7−C14, C4′-C23′), 25.6 (C3′), 22.7 (C16, C25′), 14.1 (C17, C26′). 19F NMR
(376 MHz, CDCl3) δ −178.9 (d, J = 47.7). HRMS (ESI+) calcd for
C77H118FNNaO7 [M + Na]+ 1210.8790; found 1210.8789.
1H NMR (500 MHz, pyridine-d5) δ 8.66 (d, J = 9.0, 1H, NH), 5.42 (d,
J = 3.8,1H, H1″), 5.10 (m, JHF = 50.0, 1H, H3), 5.02−4.70 (m, 5H, OH,
H2), 4.66 (dd, J = 10.0, 3.8, 1H, H2″), 4.57 (d, J = 2.9, 1H, H4″), 4.42−
4.44 (m, 3H, H3″, H5″, H6″), 4.39 (dd, J = 10.8, 5.1, 1H, H6″), 4.32 (ddd,
J = 10.9, 3.7, 1.5, 1H, H1), 4.19 (m, 1H, H1), 2.48 (t, J = 7.5, 2H, H2′),
1.95−1.82 (m, 4H, H4, H3′), 1.33−1.25 (m, 68H, H5−H16, H4′−H25′),
0.88 (2t, J = 6.8, 6H, H17, H26′). 13C NMR (125 MHz, pyridine-d5) δ
173.4 (CO), 102.2 (C1″), 93.9 (d, J = 172.0, C3), 73.2, 71.6 (C3″, C5″),
71.4 (C4″), 70.6 (C2″), 68.2 (d, J = 4.1, C1), 62.8 (C6″), 52.8 (d, J =
24.0, C2), 37.0 (C2′), 32.1, 30.0−29.6, 26.3, 25.6 (C4−C16, C3′−C25′),
14.3 (C17, C26′). 19F NMR (376 MHz, pyridine-d5) δ −188.73 (m).
HRMS (ESI+) calcd for C49H96FNNaO7 [M + Na]+ 852.7069; found
852.7071. Anal. Calcd for (C49H96FNO7·0.5H20): C, 70.12; H, 11.65;
F, 2.26; N, 1.67. Found: C, 70.21; H, 11.88; F, 2.47; N, 1.53.
(2S)-2-Hexacosanoylamino-1-(α-D-galactopyranosyl)-
heptadecane 5. Unprotected glycoceramide 5 was synthesized
following the general deacetylation procedure. From 19 (18 mg, 18
μmol) and MeONa (0.4 mM solution in MeOH, 550 μL, 22 μmol) in
CH2Cl2/MeOH (1 mL), 3,4-dideoxy-α-GalCer 5 (7 mg, 47%) was
obtained as a white solid after purification by flash chromatography on
(2S,3R)-3-Fluoro-2-(hexacosanoylamino)-1-(2,3,4,6-tetra-O-ace-
tyl-α-D-galactopyranosyl)heptadecane 20 and (2S)-2-Hexacosa-
noylamino-1-(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-
heptadecane 19. To derivative 18 (85 mg, 72 μmol, 1.0 equiv)
dissolved in a CHCl3/MeOH mixture (1:1, 3.7 mL) were added Pd/C
10% (85 mg) in one portion and AcOH (100 μL). The mixture was
stirred overnight under H2 atmosphere. The reaction mixture was
diluted with pyridine, and the catalyst was removed by filtration over
Celite. The filtrate was concentrated. The residue was diluted in a
pyridine/Ac2O mixture (2:1, 3 mL), and a catalytic amount of DMAP
was added. The mixture was stirred overnight at rt. After evaporation
of the solvent, the crude was purified by chromatography on silica gel
(EP/EtOAc 90:10 to 80:20) to afford the fluorinated compound 20
1
silica gel (CHCl3/MeOH 97:3); mp 91−92 °C. H NMR (400 MHz,
pyridine-d5) δ 8.23 (d, J = 8.8, 1H, NH), 5.78−5.44 (m, 4H, OH),
5.40 (d, J = 3.6,1H, H1″), 4.66 (dd, J = 10.0, 3.6, 1H, H2″), 4.65 (m, 1H,
H2), 4.60 (m, 1H, H4″), 4.54−4.49 (m, 2H, H3″, H5″), 4.49−4.39 (m,
2H, H6″), 4.10 (dd, J = 10.2, 5.4, 1H, H1), 3.91 (dd, J = 10.2, 5.4, 1H,
H1), 2.47 (t, J = 7.8, 2H, H2′), 1.89−1.75 (m, 2H, H3′), 1.42−1.26 (m,
72H, H3−H16, H4′−H25′), 0.89−0.86 (m, 6H, H17, H26′). 13C NMR
(100 MHz, pyridine-d5) δ 173.2 (CO), 101.7 (C1″), 73.1 (C3″ or C4″ or
C5″), 71.8 (C1), 73.1 (C3″ and/or C4″ and/or C5″), 70.6 (C2″), 62.9
(C6″), 49.7 (C2), 36.9 (C2′), 32.3, 32.1, 30.0−29.6, 22.9 (C3−C16, C4′−
C25′), 26.6 (C3′), 14.3 (C17, C26′). HRMS (ESI+) calcd for
C49H97NNaO7 [M + Na]+ 834.7157; found 834.7184. Anal. Calcd
for C49H97NO7: C, 72.45; H, 12.04; N, 1.72. Found: C, 72.33; H,
12.20; N, 1.63.
(34 mg, 45%) and the 3-deoxy compound 19 (20 mg, 29%); 20 [α]20
D
+ 33.9 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3) δ 5.83 (d, J = 9.2,
1H, NH), 5.45 (d, J = 3.2,1H, H4″), 5.33−5.29 (m, 1H, H3″), 5.15−
5.12 (m, 2H, H1″, H2″), 4.53 and 4.41 (2 m, 1H, H3), 4.28−4.18
(m, 2H, H2, H5″), 4.16−4.02 (m, 2H, H6″), 3.79−3.68 (m, 2H, H1),
2.26−2.20 (m, 2H, H2′), 2.14, 2.05, 2.04, 2.00 (4s, 12H, CH3 Ac),
1.66−1.61 (m, 4H, H4, H3′), 1.33−1.25 (m, 68H, H5−H16, H4′-H25′),
0.88 (t, J = 6.8, 6H, H17, H26′). 13C NMR (100 MHz, CDCl3) δ 172.7
(C1′), 170.3−170.1 (CO), 97.3 (C1″), 92.8 (d, J = 173.0, C3), 68.0,
67.9, 67.5 (C2″, C3″, C4″, C5″), 67.6 (C1), 61.8 (C6″), 51.2 (d, J = 23.0,
C2), 42.8 (C2′), 32.1, 31.9, 29.6−29.3, 25.7, 22.7 (C4−C16, C3′−C25′),
20.6 (CH3 Ac), 14.1 (C17, C26′). 19F NMR (376 MHz, CDCl3) δ
Ethyl (2S)-2-(tert-Butoxycarbonylamino)-3,3-difluoro-hex-6-
enoate 26. A mixture of the imine 24 (4.2 g, 9.3 mmol), prepared
following Katagiri procedure,48 and allyltri-n-butyltin (5.8 mL, 2 equiv)
was dissolved in benzene (10 mL), and argon was bubbled through the
solution for 15 min. The reaction mixture was then heated to 80 °C
and stirred at this temperature for 5 min. AIBN (150 mg, 0.91 mmol
0.1equiv, in 5 mL benzene) was subsequently added in portions until
complete consumption of the starting material (monitored by NMR,
typically 4 h). The mixture was cooled to room temperature, diluted
with ethyl acetate (50 mL), and a half saturated aqueous solution of
KF (50 mL) was added. After 1 h of stirring, the biphasic system was
filtered through Celite and the layers separated. The organic phase was
dried over Na2SO4 and the solvent removed under reduced pressure.
The oily residue was taken up in ethanol (20 mL), and 2 M aqueous
HCl solution (20 mL) was added. The mixture was stirred at 50 °C
until consumption of the imine (typically 1 h), and the mixture was
then diluted with 20 mL of water. The aqueous phase was washed
three times with diethyl ether. The solvent was removed under
reduced pressure. The oily residue, corresponding to amine 25, was
taken up in dioxane (20 mL), and this solution was cooled to 0 °C
(precipitation occurs). Boc2O (1.5 eq relative to starting imine 24) was
added, followed by the dropwise addition of 50 mL of aqueous
saturated NaHCO3, and the mixture was stirred overnight. The
reaction mixture was extracted with diethyl ether (3 × 50 mL), the
combined organic layers were dried over MgSO4, and the solvent was
removed under reduced pressure. The oily residue was purified by
column chromatography (PE/AcOEt 4:1), affording the title
compound 26 as an oil in 38% yield (from the imine). Data are
consistent with the literature.48
−191.1 (m). HRMS (ESI+) calcd for C57H104FNNaO11 [M + Na]+
1
1020.7491; found 1020.7492. 19. [α]20 + 45.4 (c 1.0, CHCl3). H
D
NMR (400 MHz, CDCl3) δ 5.51 (d, J = 8.8, 1H, NH), 5.45 (d, J =
3.2,1H, H4″), 5.32 (dd, J = 3.2, 10.4, 1H, H3″), 5.14−5.08 (m, 2H, H1″,
H2″), 4.15 (m, 1H, H5″), 4.10−4.02 (m, 3H, H2, H6″), 3.70 (dd, J =
10.0, 3.4, 1H, H1), 3.49 (dd, J = 10.8, 3.4, 1H, H1), 2.21−2.17 (m, 2H,
H2′), 2.14, 2.06, 2.04, 2.00 (4s, 12H, CH3 Ac), 1.64−1.53 (m, 4H, H3,
H3′), 1.36−1.18 (m, 70H, H4−H16, H4′−H25′), 0.88 (t, J = 6.8, 6H, H17,
H26′). 13C NMR (100 MHz, CDCl3) δ 172.7 (CO), 170.4−170.1
(CO), 97.1 (C1″), 71.0 (C1), 68.2, 68.0, 67.6 (C2″, C3″, C4″), 66.6 (C5″),
61.9 (C6″), 48.8 (C2), 36.9 (C2′), 31.9, 31.5, 29.7, 29.5, 29.4, 26.1, 25.8,
22.7 (C3−C16, C3′-C25′), 20.7−20.6 (CH3 Ac) 14.1 (C17, C26′). HRMS
(ESI+) calcd for C57H105NNaO11 [M + Na]+ 1020.7491; found
1020.7491.
General Procedure for Deacetylation Step. To a stirred solution of
the acetyl-protected glycolipid in a CH2Cl2/MeOH mixture (1:1) were
added MeONa (1.2 equiv, solution in MeOH). After stirring at rt,
resin H+ Amberlyst 15 was added and the mixture was left to stir for
additional 15 min. After filtration, the crude was subjected to column
chromatography using silica gel (using the indicated eluent).
( 2 S , 3 R ) - 3 - F l u o r o - 2 - h e x a c o s a n o y l a m i n o - 1 - ( α - D -
galactopyranosyl)heptadecane 8. Unprotected glycoceramide 8 was
synthesized following the general deacetylation procedure. From
compound 20 (23 mg, 23 μmol) and MeONa (0.1 mM solution in
MeOH, 400 μL, 46 μmol) in a CH2Cl2/MeOH mixture (2 mL), 3,4-
dideoxy-3-fluoro-α-GalCer 8 (10 mg, 52%) was obtained as a white
(5S)-5-(tert-Butoxycarbonylamino)-4,4-difluoro-hex-1-en-6-ol 22
and (5S)-5-(tert-butoxycarbonylamino)-4-fluoro-hex-1,3-dien-6-ol
27. To the ester 26 (586 mg, 2 mmol), in 20 mL of absolute ethanol
at 0 °C, was added NaBH4 (380 mg, 10 mmol, 5.0 equiv) in one
portion. After 10 min stirring, the ice bath was replaced by a water
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bath and the solution was stirred for an additional 2 h, after which time
TLC indicated that the reaction was complete. Saturated aqueous
NH4Cl was cautiously added (gas evolution) and the mixture stirred
for 30 min. Then 100 mL of water and 50 mL of Et2O were added, the
layers were separated, and the aqueous phase was extracted with a
further 50 mL of Et2O. After drying over Na2SO4, the solvent was
removed under reduced pressure and the residue purified by silica gel
chromatography (ethyl acetate) to afford the alcohol 22 in 80% yield
and contaminated by 2−5% of the elimination product 27. 22:
colorless oil. 1H NMR (300 MHz, CDCl3) δ 5.75 (m, 1H, H5), 5.25−
5.05 (m, 3H, H6, NH), 4.05−3.98 (m, 1H, H2), 3.87−3.62 (m, 2H, H1),
2.64 (td, J = 16.4, 7.0, 2H, H4), 1.39 (s, 9H, t-Bu). 13C NMR (75 MHz,
CDCl3) δ 155.8 (CO), 128.4 (C5), 123.3 (t, J = 147.0, C3), 80.4
(C(CH3)3), 60.2 (C1), 54.6 (t, J = 23.4, C2), 39.0 (t, J = 24.6, C4), 28.2
(C9). MS (EI): m/z = 252.1 (M + 1, 40%), 196.0 (M-tBu, 100%). 27:
Data for the elimination product 27 were extracted from a 1:1 mixture
of the two compounds (obtained from another experiment where
temperature was not strictly controlled). 1H NMR (300 MHz CDCl3)
δ 6.51 (dt, J = 10.6, 17.2, 1H, H5), 5.48 (dd, J = 10.8, 35.7, 1H, H4),
5.15−5.00 (m, 3H, 2H6, NH), 4.28 (m, H2), 3.80−3.60 (m, 2H, H1),
2.00 (br, 1H, OH), 1.38 (s, 9H, t-Bu). MS (EI) m/z = 232.1 (M + 1,
40%), 176.0 (M-tBu, 100%).
(5S)-5-(tert-Butyloxycarbonylamino)-4,4-difluoro-6-O-(2,3,4,6-
tetra-O-benzyl-α-galactopyranosyl)-hex-1-ene 21. A Schlenk tube
was charged with 4 g of activated 4 Å molecular sieves in powder, 1.2 g
of AgClO4·H2O (5.7 mmol, 3 equiv) and 1.1 g (5.7 mmol, 3 equiv) of
SnCl2 and 15 mL of THF and the slurry mixture stirred at 0 °C under
argon for 1 h. In a separate flask, the alcohol 22 (480 mg, 1.9 mmol)
and galactosyl fluoride 12 (2 g, 2 eq, 3.8 mmol) were dissolved in diethyl
ether (45 mL), and the solution was cooled to 0 °C. The second solution
was added over 20 min to the Lewis acid, and the resulting mixture was
stirred for 30 min at 0 °C and then 1 h at room temperature. The
solution was quenched with aqueous saturated NaHCO3, filtered over
Celite, and the salts thoroughly washed with diethyl ether. The organic
layer was dried over Na2SO4 and the solvent removed under reduced
pressure. The crude oily residue was taken up in THF, Boc2O (1.5 equiv)
and aqueous saturated NaHCO3 were subsequently added, and the
reaction mixture iwas stirred at rt overnight. The solution was diluted
with 50 mL of water and extracted with 3 × 25 mL of diethyl ether. The
organic layer was dried over Na2SO4 and the solvent removed under
reduced pressure. Purification by column chromatography (PE/Et2O 4:1)
affords the title compound as an oil in 40% yield. Colorless oil. 1H NMR
(400 MHz, CDCl3) δ 7.40−7.30 (m, 20H, Har), 5.71 (m, 1H, H2),
5.17−4.98 (m, 3H, H1 + NH), 4.86 and 4.48 (AB syst, J = 11.5, 2H,
CH2Ph), 4.77 (d, J = 3.6, 1H, H1″), 4.73 and 4.57 (AB syst, J = 11.9, 2H,
CH2Ph), 4.71 and 4.63 (AB syst, J = 11.8, 2H, CH2Ph), 4.42 and 4.32
(AB syst, J = 11.9, 2H, CH2Ph), 4.07 (bs, 1H, H5), 3.97 (dd, J = 10.0, 3.6,
1H, H2″), 3.92−3.84 (m, 2H, H4″, H5″), 3.82 (dd, J = 10.8, 2.8, 1H, H3″),
3.74 (dd, J = 10.8, 3.7, 1H, H6), 3.65 (m, 1H, H6), 3.44 (m, 2H, H6″),
2.58 (td, J = 17.0, 7.2, 2H, H3), 1.35 (s, 9H, t-Bu). 13C NMR (100 MHz,
CDCl3) δ 155.4 (CO), 138.8−138.0 (Cqar), 128.7−127.5 (CHar, C2),
122.6 (t, J = 247.0, C4), 120.1 (C1), 99.0 (C1″), 80.0 (C(CH3)3), 78.8
(C3″), 76.7 (C2″), 75.1 (C4″ or C5″), 74.8 (CH2Ph), 73.4 (CH2Ph), 73.3
(CH2Ph), 69.8 (C4″ or C5″), 69.0 (C4″ or C5″), 69.0 (C6″), 66.3 (C6), 53.6
(t, J = 26.0, C5), 39.0 (t, J = 25.0, C3), 28.3 (C(CH3)3). 19F NMR (376
MHz, CDCl3) δ −105.4 (dd, J = 7.3, 232.0), − 106 (dq, J = 17.7, 232.0).
HRMS (ESI+) Calc. for C45H53F2NNaO8 [M + Na]+ 796.3631; found
796.3652.
ether. After drying the organic layer over Na2SO4 and evaporating
the solvent, the crude product was directly engaged in the next step. The
crude amine 28 (260 mg) was dissolved in 10 mL of dry pyridine,
p-nitrophenyl hexacosanoate (302 mg, 1.5 equiv/21, 0.504 mmol) was
added in one portion, and the mixture was heated to 80 °C for 24 h.
Pyridine was removed under vacuum, and the yellow solid residue was
dissolved in diethyl ether. The solution was washed with 2 portions of a
0.5 M NaHCO3 solution until the aqueous layer was colorless. The
organic layer was dried over Na2SO4 and purified by column
chromatography on silica gel using a 1:1 CH2Cl2/petroleum ether
eluent to afford 160 mg of the title compound (approximately 38%
yield) still contaminated with 5−10% of p-nitrophenol that could not be
1
removed. H NMR (400 MHz, CDCl3) δ 7.40−7.15 (m, 20H, Har),
6.33 (d, 1H, J = 9.1, NH), 5.75 (m, 1H, H2), 5.87−5.13 (m, 2H, H1),
4.85 and 449 (AB syst, J = 11.5, 2H, CH2Ph), 4.77 (d, J = 3.9, 1H, H1″),
4.75 and 4.57 (AB syst, J = 11.5, 2H, CH2Ph), 4.72−4.65 (AB syst, J =
11.7, 2H, CH2Ph), 4.43−4.31 (m + AB syst, 3H, CH2Ph, H5), 3.98 (dd,
J = 10.0, 3.9, 1H, H2″), 3.97−3.90 (m, 2H, H6″, H5″), 3.84−3.79 (m, 2H,
H4″, H3″), 3.62 (dd, J = 11.7, 4.9, 1H, H6″), 3.49 (dd, J = 10.0, 6.9, 1H,
H6), 3.36 (dd, J = 10.0, 5.9, 1H, H6), 2.55 (td, J = 16.8, 7.0, 1H, H4),
1.99−1.88 (m, 2H, H2′), 1.45 (m, 2H, H3′), 1.30−1.17 (m, 40H, H4′−
H23′), 0.80 (t, 3H, J = 7.0, H24′). 13C NMR (100 MHz, CDCl3) δ 173.4
(C1′), 138.6−137.8 (Cqar), 128.6−127.4 (CHar, C2, C3), 120.7 (C1),
99.7 (C1″), 78.8 (C3″), 76.7 (C2″), 74.9 (C4″), 74.7 (CH2Ph), 73.7
(CH2Ph), 73.5 (CH2Ph), 73.0 (CH2Ph), 70.1 (C5″), 69.5 (C6″), 67.0
(C1), 51.4 (m, C5), 39.1 (m, C3), 36.3 (C2′), 31.9 (C3′), 29.7−29.3, 25.4,
22.7 (CH4′-C23′), 14.1 (C24′). 19F NMR (376 MHz, CDCl3) δ −104.8 (d,
J = 248.0), −105.8 (d, J = 248.0).
It should be noted that for compounds 29 and 30, trace of a
byproduct was observed on 19F NMR spectra. This impurity could be
assigned to a fluorinated ceramide residue resulting from hydrolysis of
the glycosidic bound during HCl treatment for the Boc deprotection
step. Trace of this side-product can be removed after the hydro-
genation step.
(2S)-2-(Hexacosanoylamino)-3,3-difluoro-1-O-(2,3,4,6-tetra-O-
benzyl-α-galactopyranosyl)-octadec-5-ene 30. Under an argon
atmosphere, the amide 29 (54 mg) was dissolved in CH2Cl2 to
obtain a 0.05 M solution, 5 equiv of 1-tetradecene (0.72 mmol, 180 μL)
were added, and the mixture was heated to 40 °C. Once the temperature
was stable, 5 mol % of Grubbs II catalyst (0.007 mmol, 6 mg) was added
and the reaction stirred at that temperature until complete disappearance
of the starting material (TLC monitoring, typically 3 h). After the
reaction was complete, the mixture was cooled to room temperature and
the product purified by column chromatography using a 1:1 CH2Cl2/PE
eluent, affording the title compound 30 in 45% yield (28 mg). 1H NMR
(400 MHz) δ 7.40−7.15 (m, 20H, Har), 6.32 (d, J = 9.6, 1H, NH), 5.51
(dt, J = 6.5, 15.5, 1H, H6), 5.34 (dt, J = 6.5, 15.5, 1H, H5), 4.86 and 4.49
(AB syst, J = 11.4, 2H, CH2Ph), 4.77, (d, J = 3.8, 1H, H1″), 4.74 and 4.65
(AB syst, J = 11.8, 2H, CH2Ph), 4.71 and 4.57 (AB syst, J = 11.8, 1H,
CH2Ph), 4.45−4.30 (m and AB syst, J = 11.8, 3H, CH2Ph, H2), 3.97
(dd, J = 10.1, 3.8, 1H, H2″), 3.95−3.90 (m, 2H, H5″, H1), 3.85 (m, 1H,
H4″), 3.62 (dd, J = 11.8, 4.3, 1H, H1), 3.49 (dd, J = 9.4, 6.7, 1H, H6″),
3.36 (dd, J = 9.4, 6.0, 1H, H6″), 2.48 (dt, J = 17.5, 6.7, 2H, H4), 2.02−
1.84 (m, 4H, H2″, H7), 1.60−1.45 (m, 2H, H3′), 1.33−1.07 (m, 62H,
H4′−H23′ and H8−H17), 0.81 (t, J = 6.7, 6H, H24′, H18).). 19F NMR (376
MHz, CDCl3) δ = −104.9 (d, J = 247.0), δ = −105.7 (d, J = 247.0).
(2S)-1-O-(α-D-Galactopyranosyl)-2-hexacosanoylamino-3,3-di-
fluoro-heptadecane 9. Pd(OH)2/C (20% w/w, 27 mg) was
suspended in 1 mL of methanol, and H2 was bubbled through the
solution for 15 min. Then, the protected α-GalCer 32 (27 mg, 0.022
mmol), dissolved in 4 mL of chloroform, was added and the slurry was
stirred under H2 atmosphere. After 16 h, the solution was filtered
through Celite and the cake washed 3 times with 10 mL of a warm
CHCl3/MeOH mixture (4:1). The solvent was removed and the crude
purified by column chromatography on silica gel. Elution with CHCl3/
MeOH 98:2 then CHCl3/MeOH 95:5 afforded 8 mg of the title
compound 9 (40% yield) as a white solid. 1H NMR (500 MHz,
pyridine-d5) δ 9.41 (d, J = 9.3, 1H, NH), 5.44 (d, J = 3.8, 1H, H1″),
4.73 (dd, J = 9.9, 3.8, H2″), 4.64 (bd, J = 3.0, 1H, H4″), 4.59−4.51
(m, 3H, H1, H3″, H5″), 4.51−4.42 (m, 2H, H6″), 4.20 (dd, J = 10.9, 7.6,
(5S)-5-(Hexacosanoylamino)-4,4-difluoro-6-O-(2,3,4,6-tetra-O-
benzyl-α-galactopyranosyl)-hex-1-ene 29. First, 300 mg (0.39
mmol) of (5S)-5-(tert-butyloxycarbonylamino)-4,4-difluoro-6-O-
(2,3,4,6-tetra-O-benzyl-α-galactopyranosyl)-hex-1-ene 21 were dis-
solved in 25 mL of diethyl ether, and the solution was cooled to
0 °C. Subsequently, gaseous HCl was bubbled through the solution until
complete disappearance of the starting material as judged by TLC.
Excess HCl was removed by bubbling nitrogen through the solution, and
additional Et2O (10 mL) was added. Then, 2 equiv of NaHCO3 (1.6 mL
of a 0.5 M aqueous solution) were added at that temperature, and the
mixture was stirred for a further hour at 0 °C. The organic layer was
separated, and the aqueous layer extracted with another 10 mL of diethyl
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1H, H1), 2.63 (t, J = 6.9, H2′), 2.37−2.17 (m, 2H, H4), 1.99−1.82 (m,
2H, H3′), 1.81−1.59 (m, 2H, H5), 1.51−1.09 (m, 66H, H4′‑25′ and
H6−17), 0.91 (t, J = 7.2, 3H, H26′ or H18), 0.91 (t, J = 6.9, 3H, H26′ or
H18). 13C NMR (125 MHz, pyridine-d5) δ 174.9 (C1′), 125.3 (t, J =
246.0, C3), 102.2 (C1″), 73.4 (C5″), 71.7 (C3″), 71.1 (C4″), 70.6 (C2″),
66.8 (C1), 62.8 (C6″), 53.4 (t, J = 28.0, C2), 37.2 (C2′), 35.0 (t, J = 24.0,
C4), 32.3, 32.3, 30.2, 30.1, 30.0, 29.9, 29.9, 29.8, 29.8, 26.5 (C3′), 23.1,
22.3 (C5), 14.5. 19F NMR (470 MHz, pyridine-d5) δ −105.7 (d, J =
247.0), −106.4 (d, J = 247.0). HRMS (ESI+) calcd for C50H97F2-
NNaO7 [M + Na]+ 884.7125; found 884.7144. Anal. Calcd for
(C50H97F2NO7·H20): C, 68.22; H, 11.34; F, 4.32; N, 1.59. Found: C,
68.49; H, 11.03; F, 4.16; N, 1.78.
In Vitro Assays for Human iNKT Cell Dtimulation. The ability
of the 3,4-dideoxy-KRN7000 analogues 5, 8, and 9 to stimulate a
human iNKT cell line was tested using human CD1d-expressing HeLa
and Namalwa cells as presenting cells. iNKT cells were prepared from
bulk human peripheral lymphocytes by two successive rounds of
selection, using first an anti-Vα24 and, second, an anti-Vβ11 monoclonal
antibody. At each round, cells were sorted using antimouse IgG-coated
magnetic beads (Dynal) and cultivated in RPMI 1640 supplemented
with 10% heat-unactivated fetal calf serum (FCS), 2 mM glutamine,
50 U/mL penicillin, 50 μg/mL streptomycin (Gibco), and 300 U/mL
IL-2 (Chiron). Human CD1d-transfected HeLa cells were obtained from
M. Kronenberg (La Jolla, CA). Namalwa cells were transduced with a
lentiviral vector containing a human CD1d insert in order to generate
high expression of cell surface CD1d. These antigen presenting cells
were cultivated in DMEM or RPMI 1640, respectively, containing 1 g/L
glucose, supplemented as described above. Antigen presenting cells were
plated at 30000 per well on 96-well flat bottom plates in complete RPMI
for the TNF-α and IFN-γ secretion analyses and at 7500 per well for the
IL-13 secretion analysis and incubated overnight at 37 °C with varying
concentrations of glycolipids solubilized in DMSO. The cells were then
washed twice with RPMI. Then 15000 iNKT cells in 150 μL of complete
RPMI without IL-2 were then added for 6 h or 48 h at 37 °C for the
TNF-α and IFN-γ or IL-13 analyses, respectively. Cell-free supernatants
were collected and tested for the presence of either TNF-α by an MTT
assay using WEHI 164 cells or IFN-γ and IL-13 by ELISA (BD
Pharmingen). Experiments were repeated from 3 to 5 times for each
glycolipid compound. Untransfected HeLa cells devoid of CD1d were
used as negative control presenting cells. Synthetic KRN7000 1 was used
as reference in all experiments.
In Vivo Assay. Female mice C57BL/6J, 6−8 weeks old, purchased
from Janvier laboratories, were bred and housed at INSERM, U892,
University of Nantes, under the animal care license no. 44278. The
stock solution of KRN7000 (1 mg/mL in DMSO) was dissolved with
10% DMSO in 0.9% NaCl solution (vehicle). Mice received
intraperitoneally (ip) 100 μg/kg KRN7000 or GalCer analogues 5,
8, and 9 in 0.1 mL of vehicle. Groups of six mice were used for each
time point.
ELISA. Sera were collected at different times points after each ip
injection of glycolipid or vehicle. Serum IFN-γ, IL-4, and TNF-α levels
were evaluated using specific ELISA kits (Mouse ELISA Ready-Set-
GO, eBioscience) according to the manufacturer’s instructions.
Statistical Analysis. To analyze the amounts of cytokines secreted
at different time points following injection of glycolipids, areas under
the curves (AUC) were compared. They were computed using the
Prism 5.0 software (Graphpad, La Jolla, CA). Groups were then
compared either by the Mann−Whitney 2-tailed test or by the Kruskal−
Wallis test, and p < 0.05 values were considered as indicative of signi-
ficant differences between groups.
AUTHOR INFORMATION
Corresponding Author
*E-mail: muriel.pipelier@univ-nantes.fr, Phone: 00 33 (0)2 76
64 51 51 (M.P.); jlependu@nantes.inserm.fr, Phone: 00 33 (0)2
28 08 03 17 (J.L.P.).
■
ACKNOWLEDGMENTS
This work was supported by the CNRS, the “Cancer
■
́
̂
opole
Grand Ouest” and “Reg
́
ion des Pays de la Loire” through the
“CIMATH” program for a Ph.D. fellowship (J.H.). We also
thank ANR PCV Galcerdeo 2008 (ANR-08-PCVI-0024-02) for
grant and post doctoral fellowships (J.-C.F.) and also
L’Association pour la Recherche sur le Cancer (ARC) for
grant “Equipements Classiques 2009”. D.J. is indebted to the
“Reg
́
ion des Pays de la Loire” for financial support in the
ique”. This research
framework of a “recrutement sur poste strateg
́
used CPU resources of (1) the GENCI-CINES/IDRIS (grant
c2011085117); (2) the CCIPL (Centre de Calcul Intensif des
Pays de Loire).
ABBREVIATIONS USED
■
IL, interleukin; IFN-γ, interferon γ; TNF-α, tumor necrosis
factor α; iNKT, invariant natural killer T; TH, T helper; TCR, T
cell receptor; CD, cluster of differentiation; hCD1d, human
CD1d; mCD1d, mouse CD1d; SAR, structure−activity
relationship; α-GalCer, α-galactosylceramide; rt, room temper-
ature; DMAP, N,N-dimethylaminopyridine; Boc, tert-butyloxycar-
bonyl; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
NMM, N-methylmorpholine; HOBt, 1-hydroxybenzotriazole;
DMF, N,N-dimethylformamide; AA, amino acids; Asp, aspartic
acid; Arg, arginine; Thr, threonine; DMEM, Dulbecco’s Modified
Eagle’s Medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrasodium bromide; RPMI, Roswell Park Memorial Institute
medium; FCS, fetal calf serum; ELISA, enzyme-linked immuno-
sorbent assay; PE, petroleum ether
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ASSOCIATED CONTENT
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