Efficient Divergent Synthesis of New Immunostimulant 4″-Modified α-Galactosylceramide Analogues

Article abstract of DOI:10.1021/acsmedchemlett.7b00107

A synthesis strategy for the swift generation of 4″-modified α-galactosylceramide (α-GalCer) analogues is described, establishing a chemical platform to comprehensively investigate the structure-activity relationships (SAR) of this understudied glycolipid

Full text of DOI:10.1021/acsmedchemlett.7b00107

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Letter
Efficient divergent synthesis of new immunostimulant
4”-modified #-galactosylceramide analogues
Jonas Janssens, Tine Decruy, Koen Venken, Toshiyuki Seki, Simon Krols, Johan
T.A. Van der Eycken, Moriya Tsuji, Dirk Elewaut, and Serge Van Calenbergh
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 05 May 2017
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Efficient divergent synthesis of new immunostimulant 4”-modified α-
galactosylceramide analogues.
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Jonas Janssens,a,b Tine Decruy,c,d Koen Venken,c,d Toshiyuki Seki,e,f Simon Krols,a Johan Van der
Eycken,b Moriya Tsuji,e Dirk Elewaut,c,d and Serge Van Calenbergh.a,*
aLaboratory for Medicinal Chemistry, Department of Pharmaceutics (FFW), Ghent University, Ottergemsesteenweg
460, B-9000 Ghent (Belgium)
bLaboratory for Organic and Bioorganic Synthesis, Department of Organic and Macromolecular Chemistry, Ghent
University, Krijgslaan 281 (S4), B-9000 Ghent (Belgium)
cDepartment of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, B-
9000 Ghent (Belgium)
dVIB Inflammation Research Center, Ghent University, B-9000 Ghent (Belgium)
eAaron Diamond AIDS Research Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (USA)
fDepartment of Obstetrics and Gynecology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Mina-
to-ku, Tokyo 105-8461 (Japan)
Keywords: α-galactosylceramide, KRN7000, CD1d, Th1/Th2, cytokine secretion, immunomodulators.
ABSTRACT: A synthesis strategy for the swift generation of 4”-modified α-galactosylceramide (α-GalCer) analogues is
described, establishing a chemical platform to comprehensively investigate the structure-activity relationships (SAR) of
this understudied glycolipid part. The strategy relies on a late-stage reductive ring-opening of a p-methoxybenzylidene
(PMP) acetal to regioselectively liberate the 4”-OH position. The expediency of this methodology is demonstrated by the
synthesis of a small yet diverse set of analogues, which were tested for their ability to stimulate invariant natural killer T-
cells (iNKT) in vitro and in vivo. The introduction of a p-chlorobenzyl ether yielded an analogue with promising im-
munostimulating
properties,
paving
the
way
for
further
SAR
studies.
4 (IL-4), counteract the development of autoimmune
diseases. However, the antagonizing effect of both cyto-
kine types severely hampers clinical potential of α-GalCer
as an immunomodulator. This renders research towards
new analogues with an improved immunological profile,
slanting towards secretion of either Th1- or Th2-
cytokines, highly relevant.
For over two decades, α-galactosylceramide (α-GalCer
or KRN7000; 1) has been serving as a lead structure for
the development of new glycosphingolipids targeting the
immune system.1-4 α-GalCer is a synthetic glycolipid re-
sulting from the structural optimization of agelasphins, a
class of amphiphilic natural products isolated from the
marine sponge Agelas mauritianus.5,6 It is composed of a
polar
philic ceramide tail. This ceramide, in turn, is built from
-ribo-phytosphingosine, which is N-acylated at C2’ with
D-galactose unit, α-anomerically linked to a lipo-
D
cerotic acid (hexacosanoic acid).
α-GalCer binds to the major histocompatibility complex
(MHC) class I-like glycoprotein CD1d, associated with the
membrane of antigen-presenting cells (APCs).7 This bina-
ry CD1d-glycolipid complex is presented to the T-cell
receptor (TCR) of invariant natural killer T-cells (iNKT
cells), evoking simultaneous release of T-helper 1 (Th1)
and T-helper 2 (Th2) cytokines.8,9 The pro-inflammatory
Th1-cytokines, such as interferon-γ (IFN-γ), are involved
in antitumor, antiviral and antibacterial effects, whereas
the anti-inflammatory Th2-cytokines, such as interleukin
Figure 1. Structural formula of 1 and some reported 4”-
analogues.
The list of new α-GalCer analogues continues to grow,
with much attention being devoted to modifications of
the hydroxyl groups of the galactose unit, thereby gradu-
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ally revealing more of the SAR of this key glycolipid part. This divergent strategy uses PMB ethers as hydroxyl pro-
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3
4
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6
7
8
Modifications at the 2”-position lead to a complete disap-
pearance of antigenicity, due to abrupt disturbance of a
vital hydrogen bonding interaction with Gly96α of the
TCR.10,11 A sulfate group can be substituted for the 3”-OH-
group.12 The SAR of the 6”-position has been extensively
studied, mainly for two reasons. First, the 6”-OH is the
only primary hydroxyl group of the sugar part, which
from a chemoselective point of view implies that straight-
forward modification of this position is possible. Indeed,
our laboratory has already explored the mild yet powerful
regioselective benzylidene ring-opening route to access
the 6”-position for the synthesis of fucosyl, galacturonic
acid, triazole, carbamate and urea analogues.13-15 Second,
and more importantly, this hydroxyl group does allow for
modifications due to the absence of any major interac-
tions with either CD1d or the TCR,16 and this has given
rise to an array of new immunoactive analogues.17-22
tecting groups, in contrast to most other syntheses, which
use benzyl groups. Although they are widely used because
of their easy introduction and relative inertness towards a
plethora of conditions, benzyl groups suffer from the
major drawback that by deprotection via catalytic hydro-
genolysis some medicinally interesting functionalities,
such as alkenes, alkynes, thioethers, naphthalenes,
(iso)quinolines, furans, thiophenes, cyclopropanes and
chloroarenes, might be (partly) reduced. This unavoidably
restricts the structural diversity of the analogues. PMB
ethers, on the other hand, can be cleaved under mild and
widely tolerated conditions, therefore permitting a broad-
er substrate scope.26
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The previously reported trichloroacetimidate donor
(13)27 was synthesized via an improved scalable route from
cheap α-D-galactose pentaacetate (9) (Scheme 1). Intro-
duction of the p-thiotolyl moiety as an anomeric protect-
ing group and subsequent deacetylation under Zemplén
conditions was followed by installation of the p-
methoxybenzylidene acetal and protection of the remain-
ing hydroxyl groups as PMB ethers to yield the fully pro-
tected intermediate 11. Attempts to remove the p-thiotolyl
group with N-iodosuccinimide (NIS) in acetone/H2O 10:1
In contrast to this, only few 4”-analogues of α-GalCer
are known (Figure 1) and, as a consequence, the SAR of
this position has been poorly investigated. Crystallo-
graphic studies have shown that the 4”-OH forms a hy-
drogen bond to the main chain carbonyl group of Phe29α
of the TCR, indicating that it acts as a hydrogen bond
donor.16 4”-Deoxygenation leads to a less active analogue
(2) as compared to α-GalCer, yet recognition by the TCR
is not severely disturbed.23 Derivatization of the 4”-OH as
O-methyl ether (3) or O-ethanol ether (4) gives slightly
less potent analogues, whereas the N-acetyl analogue (5)
is a significantly weaker antigen.24 Additionally, a couple
of active analogues bearing an aromatic ring on the 4”-
position have been synthesized (6-8).25 Inversion of the
4”-OH gives α-glucosylceramide (α-GlcCer), which is
slightly less active as compared to α-GalCer.7
resulted
in
partial
conversion
of
the
p-
methoxybenzylidene acetal to an isopropylidene acetal.
This inconvenience was overcome by performing the
reaction in acetonitrile/H2O 10:1, giving hemiacetal 12 as a
mixture of anomers (α:β ~ 6:4 at 25 °C in CDCl3) in a near-
ly quantitative yield. Finally, 12 was converted to the cor-
responding α-trichloroacetimidate under thermodynamic
control, furnishing donor 13 in an overall yield of 75%
over 6 steps. All intermediates, except for 13, can be puri-
fied by simple crystallization from a suitable solvent sys-
tem.
Scheme 1. Synthesis of trichloroacetimidate donor
13.a
Efforts were undertaken to perform the glycosylation of
the known acceptor 1428 with the thioglycoside donor 11,
as this would shorten the synthesis route. However, the
application of neither benzenesulfinyl morpholine
(BSM),29 nor NIS or copper(II) triflate30 as thiophilic pro-
moters proved to be successful. These highly electrophilic
conditions, typically used in thioglycosylations,31 were
found to be incompatible with the p-methoxybenzylidene
acetal. Indeed, p-methoxybenzylidene and p-thiotolyl
cleavage were observed as the main side reactions.
Glycosylation was successful at the trichloroacetimidate
stage through application of the “inverse protocol”
(Scheme 2).32 By slow addition of donor 13 to a solution of
acceptor 14 and BF3⋅OEt2 as the promoter at -20 °C, the
desired glycoside 15 was obtained as α-anomer only in
85% yield. The use of trimethylsilyl trifluoromethanesul-
fonate (TMSOTf) as glycosylation promoter led to silyla-
tion of the acceptor, while the common protocol with
BF3⋅OEt2, wherein the promoter is added to a solution of
donor and acceptor, gave the glycoside in lower yields
(30-50%) due to more extensive decomposition of the
donor before glycosylation. The high stereoselectivity
observed in this reaction is caused by a well-known con-
aReagents and conditions: (a) p-thiocresol, BF3⋅Et2O,
CH2Cl2, 0 °C (94%); (b) NaOMe, MeOH, rt (quant.); (c) p-
anisaldehyde dimethylacetal, CSA, CH2Cl2, 4Å MS, rt (87%);
(d) NaH, PMBCl, TBAI, DMF, 0 °C to rt (98%); (e) NIS,
MeCN, H2O, 0 °C (99%); (f) Cl3CCN, DBU, CH2Cl2, 0 °C
(94%).
To explore the SAR more thoroughly, a reliable and
scalable synthesis route is highly needed. Here, we pre-
sent the synthesis of two powerful precursors (16 and 19)
with a free 4”-OH, permitting the fast generation of new
4”-α-GalCer-analogues in a late stage of the synthesis.
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acylated with N-succinimidyl hexacosanoate (NSHC, 18)
formational effect exhibited by the 4”,6”-O-acetal, form-
ing a cis-decalin-like system with the galactose ring.33
under basic conditions to deliver amide 19 in 86% yield
over 2 steps (Scheme 3). To drive the reaction to comple-
tion within a reasonable amount of time, the amide for-
mation was carried out at 70 °C.
1
2
3
4
Scheme 2. Glycosylation of 13 with 14 and subse-
quent regioselective ring-opening of glycoside 15.a
5
6
7
8
Both 16 and 19 were now suited for late-stage diversifi-
cation. Alkylation of the 4”-OH had to be performed at
the azide stage, since methylation of 19 with methyl io-
dide and BaO/Ba(OH)2 in DMF yielded an unseparable
mixture of O- and N-alkylated products in a 4:1 ratio (as
determined via 1H NMR spectroscopy). Thus, alkylation of
16 with p-chlorobenzyl bromide gave derivative 22
(Scheme 4). Both 17 and 22 were subjected to H2S-
mediated azide reduction and subsequent amide for-
mation. The final PMB ether cleavage was performed
using HCl in 1,4-dioxane, swiftly delivering analogues 21
and 24. Anisole was added in large excess (10 equiv.) as a
scavenger for the highly reactive p-methoxybenzyl carbo-
cation.
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12
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aReagents and conditions: (a) 14, BF3⋅Et2O, THF, Et2O, 4Å
MS, -20 °C (85%); (b) DIBALH, toluene, -80 °C (89%); (c)
BaO, Ba(OH)2, MeI, DMF, rt (93%).
Scheme 4. Synthesis of O-alkylated analogues 21
and 24.a
OR
MeO
O
(CH2)24CH3
NH OR
(CH2)13CH3
Next, attempts were made to regioselectively open the
p-methoxybenzylidene acetal in order to liberate the 4”-
OH function. Experiments involving BH3⋅THF/Cu(OTf)2,34
BH3⋅THF/nBu2BOTf35 or PhBCl2/TESH36 as reducing
agents mainly gave rise to degradation of the p-
methoxybenzylidene acetal and the glycosidic bond. A
more successful approach was the use of diisobutylalu-
minium hydride (DIBALH) in toluene,37 while keeping the
temperature at -80 °C to avoid azide reduction (Scheme
2). This afforded azido alcohol 16 as a single regioisomer
in 89% yield. The structure of 16 was unambiguously
proven by derivatization as the corresponding methyl
ether (17) and analysis of the relevant cross peaks in the
HSQC and HMBC spectra (see Supporting Information).
O
20 (R = PMB)
21 (R = H)
RO
a
17
b
RO
O
OR
OPMB
O
O
Cl
PMBO
N3 OPMB
(CH2)13CH3
c
PMBO
16
O
OPMB
22
OR
O
O
(CH2)24CH3
O
Cl
23 (R = PMB)
24 (R = H)
RO
d
NH OR
e
RO
O
(CH2)13CH3
OR
Scheme 3. Amide formation of 16.a
aReagents and conditions: (a) (i)H2S, pyridine, H2O, rt; (ii)18,
Et3N, THF, 70 °C (78% over 2 steps); (b) HCl, 1,4-dioxane,
anisole, rt (32%); (c) p-chlorobenzyl bromide, BaO, Ba(OH)2,
DMF, rt (92%); (d) (i)H2S, pyridine, H2O, rt; (ii)18, Et3N, THF,
70 °C (66% over 2 steps); (e) HCl, 1,4-dioxane, anisole, rt
(30%).
O
O
N
O
(CH2)24CH3
O
NSHC (18)
OPMB
HO
OPMB
HO
O
(CH2)24CH3
NH OPMB
O
O
PMBO
PMBO
N3 OPMB
a
Besides alkylation, some other chemistries were ex-
plored to create a diverse set of analogues (Scheme 5).
Carbamoylation of 19 with 1-naphthyl isocyanate deliv-
ered, after deprotection, naphthyl carbamate analogue 26.
This analogue structurally resembles the 6”-naphthyl
urea, which exerts a strong Th1-bias as a result of the
naphthyl moiety occupying an additional binding pocket
in CD1d.18 Oxidation of the alcohol in 16 using a Swern
oxidation delivered the corresponding ketone, which,
without intermediate purification, smoothly underwent a
mild Julia-Kocienski olefination with 1-methyl-2-
(methylsulfonyl)-1H-benzo[d]imidazole (MSBI, 27)38 to
give the exocyclic methylene derivative. Following azide
reduction, amide formation and overall deprotection,
analogue 29 was obtained. Finally, the OH-group was
converted into the corresponding azide 30 via a DPPA-
PMBO
PMBO
O
O
(CH2)13CH3
OPMB
(CH2)13CH3
OPMB
16
19
aReagents and conditions: (a) (i)H2S, pyridine, H2O, rt; (ii)18,
Et3N, THF, 70 °C (86% over 2 steps).
Azido alcohol 16 was subjected to azide reduction under
the classical Staudinger conditions with PMe3, followed
by EDC-mediated amide formation with hexacosanoic
acid (cerotic acid). However, it was observed that the
intermediate iminophosphorane was highly stable and
even with concentrated sodium hydroxide at elevated
temperatures its hydrolysis proceeded sluggishly, provid-
ing amide 19 in a rather low yield (43% over 2 steps). To
reduce the azide in a more efficient way, we turned our
attention to hydrogen sulfide in a pyridine/H2O mixture.
This readily furnished the amine, which was immediately
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mediated Mitsunobu reaction. The azide was reduced and ly delivering
Page 4 of 7
32.
analogue
1
2
derivatized as the corresponding naphthyl urea, eventual-
Scheme 5. Synthesis of naphthyl analogues 26 and 32, and alkenyl compound 29.a
3
4
5
6
7
O
OR
N
O
O
(CH2)24CH3
NH OR
(CH2)13CH3
H
O
S
N
N
O
O
a
RO
RO
O
8
9
OR
MSBI (27)
OR
O
(CH2)24CH3
NH OR
(CH2)13CH3
25 (R = PMB)
26 (R = H)
O
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
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26
27
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b
RO
amide
c
16
19
RO
O
formation
OR
O
OPMB
OR
HN
28 (R = PMB)
29 (R = H)
O
(CH2)24CH3
NH OPMB
O
(CH2)24CH3
NH OR
(CH2)13CH3
d
O
O
N3
PMBO
HN
RO
e
f
PMBO
RO
O
O
(CH2)13CH3
OPMB
30
OR
31 (R = PMB)
32 (R = H)
g
aReagents and conditions: (a) 1-naphthyl isocyanate, DMF, rt (89%); (b) HCl, 1,4-dioxane, anisole, rt (44%); (c) (i)(COCl)2,
DMSO, Et3N, CH2Cl2, -78 °C; (ii)27, KOtBu, DMF, rt; (iii)H2S, pyridine, H2O, rt; (iv)18, Et3N, THF, 70 °C (61% over 4 steps); (d) HCl,
1,4-dioxane, anisole, rt (31%); (e) DPPA, DEAD, PPh3, THF, -20 °C to rt (74%); (f) (i)H2S, pyridine, H2O, rt; (ii)1-naphthyl isocya-
nate, DMF, rt (22% over 2 steps); (g) HCl, 1,4-dioxane, anisole, rt (48%).
The set of new analogues (21, 24, 26, 29 and 32) was
evaluated for its immunostimulating capacity, both in
vitro and in vivo. C57BL/6 mice were injected intraperito-
neally with 5 µg the glycolipids (2.0 x 10-4 µg/kg) and the
blood serum levels of the cytokines were measured by
ELISA (Figure 2). IFN-γ was quantified 16 h after injection,
whereas IL-4 was measured 4 h after injection. All of the
analogues were able to stimulate the immune system,
albeit at levels comparable to or lower than those elicited
by α-GalCer (1). Although the 4”-OH acts as a hydrogen
bond donor to interact with the TCR, derivatization as a
simple ether is allowed. The antigenic effect is most dra-
matic when introducing a methyl group (21). The p-
chlorobenzyl analogue 24 was found to polarize the cyto-
kine response towards Th1, although its antigenicity was
lower than that of α-GalCer. Yet, we believe that this
analogue might be a good lead structure for future opti-
mization towards highly potent Th1-polarizing α-GalCer
analogues.
tural studies are required to gain more insight into this
phenomenon.
IFNꢀγ in mouse serum
7000
6000
5000
4000
3000
2000
1000
0
21
24
26
29
32
DMSO
αGalCer
ILꢀ4 in mouse serum
2500
2000
1500
1000
500
In the same way, introducing a gluco-naphthyl urea on
the 4”-position (32) yields an analogue that is Th1-
polarizing. Future structural studies might shed light on
the mechanism underlying this Th1-polarization, but
enhanced interaction with CD1d may account for this
observation. The galacto-naphthyl carbamate (26), in
which the naphthyl group is unlikely to interact with
CD1d due to its axial configuration, is significantly less
potent.
0
21
24
26
29
32
DMSO
αGalCer
Figure 2. IFN-γ and IL-4 secretion after intraperitoneal
injection of 2.0 x 10-4 µg/kg of each analogues in C57BL/6
mice (5 µg/animal).
Remarkably, even when no heteroatom is present at
C4”, as exemplified by the exocyclic alkene (29), im-
munostimulatory capacity is partly preserved. Apparently,
the exocyclic alkene, which will partly flatten the galac-
tose ring, induces a pyranose conformation that is still
able to bridge between CD1d and the TCR. Further struc-
To assess if these analogues also stimulate human iNKT
cells, the latter were co-cultured with HeLa CD1d cells
with varying concentrations of the glycolipids. After 24 h
of incubation the IFN-γ levels were determined by ELISA
(Figure 3). These results reflect the observations in mice,
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edged for purity analysis of the final compounds. Joren Guil-
namely that all of the compounds are able to stimulate
1
2
laume and dr. Martijn D. P. Risseeuw are acknowledged for
valuable discussions.
iNKT cells, with the methyl ether being most antigenic.
3
4
IFNꢀγ in in vitro coꢀculture
ABBREVIATIONS
30000
5
6
7
8
APC,
antigen-presenting
cell;
CSA,
(1S)-(+)-10-
25000
20000
15000
10000
5000
0
camphorsulfonic acid; DBU, 1,8-diazabicyclo(5.4.0)undec-7-
ene; DEAD, diethyl azodicarboxylate; DIBALH, diisobutylal-
uminium hydride; DPPA, diphenylphosphoryl azide; EDC,
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide;
ELISA,
9
enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; IL,
interleukin; MHC, major histocompatibility complex; MS,
molecular sieves; MSBI, 1-methyl-2-(methylsulfonyl)-1H-
benzo[d]imidazole; NIS, N-iodosuccinimide; NKT, natural
killer T-cell; NSHC, N-succinimidyl hexacosanoate; PMB, p-
methoxybenzyl; PMP, p-methoxyphenyl; SAR, structure-
activity relationship; TBAI, tetra-n-butylammonium iodide;
TCR, T-cell receptor; TESH, triethylsilane; Th, T-helper.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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48
49
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51
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53
54
55
56
57
58
59
60
21
24
1 nM
26
29
0.01 nM
32
control
αGalCer
0.1 nM
Figure 3. IFN-γ levels measured upon incubation of dif-
ferent concentrations of the analogues with human iNKT
and HeLa CD1d cells.
REFERENCES
1.
In summary, we have developed a concise and scalable
synthetic route towards two valuable precursors (16 and
19) to perform late-stage diversification of the 4”-OH
position of α-GalCer. In this strategy, a triple role has
been provided for the 4”,6”-O-p-methoxybenzylidene
acetal: as a protecting group, as a stereocontrolling ele-
ment during glycosylation and as a structural element to
enable a regioselective ring-opening. We have demon-
strated the utility of this method by synthesizing a diverse
set of analogues, that was tested for the ability to stimu-
late iNKT cells both in vitro an in vivo. In both cases, the
analogues were able to stimulate iNKT cells, some of
them showing a clear pro-inflammatory activity due to
Th1-polarization. Even though the 4”-OH is involved in
binding to the TCR, we have shown that derivatization is
allowed, which should inspire further biological and
structural research to comprehensively explore the SAR of
this glycolipid part.
Murphy, N.; Zhu, X.; Schmidt, R. R. α-
Galactosylceramide and analogues important im-
munomodulators for use as vaccine adjuvants. Carbo-
hydr. Chem. 2010, 36, 64-100.
–
2. Banchet-Cadeddu, A.; Hénon, E.; Dauchez, M.; Renault,
J.-H.; Monneaux, F.; Haudrechy, A. The stimulating ad-
venture of KRN 7000. Org. Biomol. Chem. 2011, 9, 3080-
3104.
3. Birkholz, A. M.; Howell, A. R.; Kronenberg, M. The alpha
and omega of galactosylceramides in T cell immune func-
tion. J. Biol. Chem. 2015, 290, 15365-15370.
4. Carreño, L. J.; Saavedra-Ávila, N. A.; Porcelli, S. A. Syn-
thetic glycolipid activators of natural killer T cells as im-
munotherapeutic agents. Clin. Trans. Immunol. 2016, 5,
e69; DOI: 10.1038/cti.2016.14.
5. Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Agelas-
phins, novel antitumor and immunostimulatory cerebro-
sides from the marine sponge Agelas mauritianus. Tetra-
hedron 1994, 50, 2771-2784.
6. Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.;
Sawa, E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fuku-
shima, H. Structure-activity relationship of α-
galactosylceramides against B16-bearing mice. J. Med.
Chem. 1995, 38, 2176-2187.
7. Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.;
Motoki, K.; Ueno, H.; Nakagawa, R.; Sato, H.; Kondo, E.;
Koseki, H.; Taniguchi, M. CD1d-Restricted and TCR-
mediated activation of Vα14 NKT cells by glycosylcer-
amides. Science 1997, 278, 1626-1629.
8. Coquet, J. M.; Chakravarti, S.; Kyparissoudis, K.; McNab,
F. W.; Pitt, L. A.; McKenzie, B. S.; Berzins, S. P.; Smyth,
M. J.; Godfrey, D. I. Diverse cytokine production by NKT
cell subsets and identification of an IL-17-producing CD4–
NK1.1– NKT cell population. Proc. Natl. Acad. Sci. U. S. A.
2008, 105, 11287-11292.
9. Vartabedian, V. F.; Savage, P. B.; Teyton, L. The pro-
cessing and presentation of lipids and glycolipids to the
immune system. Immunol. Rev. 2016, 272, 109-119.
10. Barbieri, L.; Costantino, V.; Fattorusso, E.; Mangoni, A.;
Aru, E.; Parapini, S.; Taramelli, D. Immunostimulatory α-
galactoglycosphingolipids: synthesis of a 2’-O-methyl-α-
Gal-GSL and evaluation of its immunostimulating capaci-
ty. Eur. J. Org. Chem. 2004, 2004, 468-473.
ASSOCIATED CONTENT
Supporting Information
The supporting information is available free of charge on the
ACS Publications website at DOI: XXX.
Experimental details and characterization data for the re-
ported compounds, NMR spectra, and biological data (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: Serge.VanCalenbergh@UGent.be.
Funding Sources
Ghent University is gratefully acknowledged for financial
support to this research, through a Special Research Fund
grant to JJ. The work in the laboratory of MT is supported by
the NIH (grant AI070258).
ACKNOWLEDGMENTS
Tim Courtin from the NMR & Structural Analysis Unit of
Ghent University is thanked for recording the NMR spectra
of the final compounds. Izet Karalic is gratefully acknowl-
11. Barbieri, L.; Costantino, V.; Fattorusso, E.; Mangoni, A.;
Basilico, N.; Mondani, M.; Taramelli, D. Immunomodula-
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24. Florence, W. C.; Xia, C.; Gordy, L. E.; Chen, W.; Zhang,
tory α-galactoglycosphingolipids: synthesis of 2’-fluoro-
2’-deoxy-α-galactosylceramide and an evaluation of its
immunostimulating properties. Eur. J. Org. Chem. 2005,
2005, 3279-3285.
1
2
3
4
5
6
7
Y.; Scott-Browne, J. P.; Kinjo, Y.; Yu, K. O. A.; Keshiped-
dy, S.; Pellicci, D. G.; Patel, O.; Kjer-Nielsen, L.;
McCluskey, J.; Godfrey, D. I.; Rossjohn, J.; Richardson, S.
K.; Porcelli, S. A.; Howell, A. R.; Hayakawa, K.; Gapin, L.;
Zajonc, D. M.; Wang, P. G.; Joyce, S. Adaptability of the
semi-invariant natural killer T-cell receptor towards
structurally diverse CD1d-restricted ligands. EMBO J.
2009, 28, 3579-3590.
12. Xing, G.-W.; Wu, D.; Poles, M. A.; Horowitz, A.; Tsuji,
M.; Ho, D. D.; Wong, C.-H. Synthesis and human NKT
cell
stimulating
properties
of
3-O-sulfo-α/β-
galactosylceramides. Bioorg. Med. Chem. 2005, 13, 2907-
2916.
8
9
25. Zhang, W.; Xia, C.; Nadas, J.; Chen, W.; Gu, L.; Wang, P.
G. Introduction of aromatic group on 4’-OH of α-GalCer
manipulated NKT cell cytokine production. Bioorg. Med.
Chem. 2011, 19, 2767-2776.
26. Greene, T. W.; Wuts, P. G. M. Protective Groups in Or-
ganic Synthesis, 2nd ed.; John Wiley & Sons: New York,
1991.
13. Pauwels, N.; Aspeslagh, S.; Vanhoenacker, G.; Sandra, K.;
Yu, E. D.; Zajonc, D. M.; Elewaut, D.; Linclau, B.; Van
Calenbergh, S. Divergent synthetic approach to 6”-
modified α-GalCer analogues. Org. Biomol. Chem. 2011, 9,
8413-8421.
14. Pauwels, N.; Aspeslagh, S.; Elewaut, D.; Van Calenbergh,
S. Synthesis of 6”-triazole-substituted α-GalCer ana-
logues as potent iNKT cell stimulating ligands. Bioorg.
Med. Chem. 2012, 20, 7149-7154.
15. Guillaume, J.; Pauwels, N.; Aspeslagh, S.; Zajonc, D. M.;
Elewaut, D.; Van Calenbergh, S. Synthesis of C-5” and C-
6”-modified α-GalCer analogues as iNKT-cell agonists.
Bioorg. Med. Chem. 2015, 23, 3175-3182.
16. Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.;
Pellicci, D. G.; Koh, R.; Besra, G. S.; Bharadwaj, M.; God-
frey, D. I.; McCluskey, J.; Rossjohn, J. CD1d-lipid-antigen
recognition by the semi-invariant NKT T-cell receptor.
Nature 2007, 448, 44-49.
17. Zhou, X.-T.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.;
Bendelac, A.; Savage, P. B. Synthesis and NKT cell stimu-
lating properties of fluorophore- and biotin-appended 6”-
amino-6”-deoxy-galactosylceramides. Org. Lett. 2002, 4,
1267-1270.
18. Trappeniers, M.; Van Beneden, K.; Decruy, T.; Hillaert,
U.; Linclau, B.; Elewaut, D.; Van Calenbergh, S. 6’-
derivatised α-GalCer analogues capable of inducing
strong CD1d-mediated Th1-biased NKT cell responses in
mice. J. Am. Chem. Soc. 2008, 130, 16468-16469.
19. Hsieh, M.-H.; Hung, J.-T.; Liw, Y.-W.; Lu, Y.-J.; Wong, C.-
H.; Yu, A. L.; Liang, P.-H. Synthesis and evaluation of
acyl-chain- and galactose-6”-modified analogues of α-
GalCer for NKT cell activation. ChemBioChem 2012, 13,
1689-1697.
20. Jervis, P. J.; Graham, L. M.; Foster, E. L.; Cox, L. R.; Por-
celli, S. A.; Besra, G. S. New CD1d agonists: synthesis and
biological activity of 6”-triazole-substituted α-galactosyl
ceramides. Bioorg. Med. Chem. Lett. 2012, 22, 4348-4352.
21. Hung, J.-T.; Sawant, R. C.; Chen, J.-C.; Yen, Y.-F.; Chen,
W.-S.; Yu, A. L.; Luo, S.-Y. Design and synthesis of galac-
tose-6-OH-modified α-galactosyl ceramide analogues
with Th2-biased immune responses. RSC Adv. 2014, 4,
47341-47356.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27. Yamamoto, K.; Sato, Y.; Ishimori, A.; Miyairi, K.; Okuno,
T.; Nemoto, N.; Shimizu, H.; Kidokoro, S.; Hashimoto, M.
Synthesis of
D-trigalacturonic acid methylglycoside and
conformational comparison with its sulfur analogue. Bi-
osci. Biotechnol. Biochem. 2008, 72, 2039-2048.
28. Du, W.; Gervay-Hague, J. Efficient synthesis of α-
galactosyl ceramide analogues using glycosyl iodide do-
nors. Org. Lett. 2005, 7, 2063-2065.
29. Lu, Y.-S.; Li, Q.; Zhang, L.-H.; Ye, X.-S. Highly direct α-
selective glycosylations of 3,4-O-carbonate-protected 2-
deoxy- and 2,6-dideoxythioglycosides by preactivation
protocol. Org. Lett. 2008, 10, 3445-3448.
30. Hou, D.; Lowary, T. L. 2,3-Anhydrosugars in glycoside
bond synthesis. Application to 2,6-dideoxypyranosides. J.
Org. Chem. 2009, 74, 2278-2289.
31. Lian, G.; Zhang, X.; Yu, B. Thioglycosides in Carbohydrate
Research. Carbohydr. Res. 2015, 403, 13-22.
32. Schmidt, R.; Toepfer, A. Glycosylation with highly reac-
tive glycosyl donors: efficiency of the inverse procedure.
Tetrahedron Lett. 1991, 32, 3353-3356.
33. Yule, J. E.; Wong, T. C.; Gandhi, S. S.; Qiu, D.; Riopel, M.
A.; Koganty, R. R. Steric control of N-acetylgalactosamine
in glycosidic bond formation. Tetrahedron Lett. 1995, 36,
6839-6842.
34. Shie, C.-R.; Tzeng, Z.-H.; Kulkarni, S. S.; Uang, B.-J.; Hsu,
C.-Y.; Hung, S.-C. Cu(OTf)2 as an efficient and dual-
purpose catalyst in the regioselective reductive ring
opening of benzylidene acetals. Angew. Chem. Int. Ed.
2005, 44, 1665-1668.
35. Hernández-Torres, J. M.; Achkar, J.; Wei, A. Tempera-
ture-controlled regioselectivity in the reductive cleavage
of p-methoxybenzylidene acetals. J. Org. Chem. 2004, 69,
7206-7211.
36. Dilhas, A.; Bonnaffé, D. PhBCl2 promoted reductive
opening of 2’,4’-O-p-methoxybenzylidene: new regiose-
lective differentiation of position 2’ and 4’ of α-L-iduronyl
moieties in disaccharide building blocks. Tetrahedron
Lett. 2004, 45, 3643-3645.
22. Compton, B. J.; Tang, C.-w.; Johnston, K. A.; Osmond, T.
L.; Hayman, C. M.; Larsen, D. S.; Hermans, I. F.; Painter,
G. F. Synthesis and activity of 6”-deoxy-6”-thio-α-GalCer
and peptide conjugates. Org. Lett. 2015, 17, 5954-5957.
23. Wun, K. S.; Borg, N. A.; Kjer-Nielsen, L.; Beddoe, T.; Koh,
R.; Richardson, S. K.; Thakur, M.; Howell, A. R.; Scott-
Browne, J. P.; Gapin, L.; Godfrey, D. I.; McCluskey, J.;
Rossjohn, J. A minimal binding footprint on CD1d-
glycolipid is a basis for selection of the unique human
NKT TCR. J. Exp. Med. 2008, 205, 939-949.
37. Sarpe, V. A.; Kulkarni, S. S. Desymmetrization of treha-
lose via regioselective DIBAL reductive ring opening of
benzylidene and substituted benzylidene acetals. Org.
Biomol. Chem. 2013, 11, 6460-6465.
38. Ando, K.; Kobayashi, T.; Uchida, N. Practical methylena-
tion reaction for aldehydes and ketones using new Julia-
type reagents. Org. Lett. 2015, 17, 2554-2557.
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Efficient divergent synthesis of new immunostimulant 4”-modified α-galactosylceramide ana-
logues
Jonas Janssens, Tine Decruy, Koen Venken, Toshiyuki Seki, Simon Krols, Johan Van der Eycken, Moriya Tsuji, Dirk Elewaut and
Serge Van Calenbergh
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