Total synthesis, stereochemical elucidation and biological evaluation of Ac2SGL; A 1,3-methyl branched sulfoglycolipid from Mycobacterium tuberculosis

Article abstract of DOI:10.1039/c2sc21620e

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), continues to represent a challenging pathogen causing many deaths. A reason for the persistence of this pathogen is the cell-envelope composition, which consists of long-tailed (glyco)lipids, involved in the modulation of the host immune response. Diacylated sulfoglycolipid Ac₂SGL (1), found in the cell envelope, is a potent antigen that stimulates the immune response towards TB. This observation suggests the application of 1 as part of a vaccine. Here, we report the first asymmetric total synthesis of 1. Two approaches were developed for the synthesis of hydroxyphthioceranic acid (4), its polypropionate part, thereby establishing the absolute stereochemistry of the C17 hydroxyl function to be of (R)-configuration. Subsequently, 4 was regioselectively connected to the trehalose core and after selective sulfation and a final fourfold deprotection step, pure 1 was obtained. The identity of synthetic and natural 1 was confirmed by NMR and mass analysis, Furthermore, synthetic 1 shows identical biological function to 1 and activates CD1b-restricted and Ac ₂SGL-specific T cells that are highly sensitive to minimal structural modifications of 1 with the same potency. A modeling study is presented to point out the structural features of 1 that are important for binding to the antigen-presenting molecule CD1b and to the T-cell receptor. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2sc21620e

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ARTICLE TYPE
Total Synthesis, Stereochemical Elucidation and Biological Evaluation
of Ac2SGL; a 1,3-Methyl Branched Sulfoglycolipid from Mycobacterium
tuberculosis
Danny Geerdink,a Bjorn ter Horst,a Marco Leporeb, Lucia Morib,e, Germain Puzoc,d, Anna K. H. Hirsch,a
5
Martine Gilleron,c,d Gennaro de Liberob,e and Adriaan J. Minnaarda*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), continues to represent a challenging pathogen causing many
deaths. A reason for the persistence of this pathogen is the cell envelope composition, which consists of longꢀtailed (glyco)lipids,
10 involved in the modulation of the host immune response. Diacylated sulfoglycolipid Ac2SGL (1), found in the cell envelope, is a potent
antigen that stimulates the immune response towards TB. This observation suggests the application of 1 as part of a vaccine. Here, we
report the first asymmetric total synthesis of 1. Two approaches were developed for the synthesis of hydroxyphthioceranic acid 4, its
polypropionate part, thereby establishing the absolute stereochemistry of the Cꢀ17 hydroxy function to be of (R)ꢀconfiguration.
Subsequently, 4 was regioselectively connected to the trehalose core and after selective sulfation and a final fourfold deprotection step,
15 pure 1 was obtained. The identity of synthetic and natural 1 was confirmed by NMR and mass analysis, Furthermore, synthetic 1 shows
identical biological function to 1 and activates CD1bꢀrestricted and Ac2SGLꢀspecific T cells that are highly sensitive to minimal
structural modifications of 1 with the same potency. A modeling study is presented to point out the structural features of 1 that are
important for binding to the antigenꢀpresenting molecule CD1b and to the Tꢀcell receptor.
(glyco)lipids, creating a hydrophobic barrier against antibiotics.5ꢀ7
45 Two of these glycolipids, which are found exclusively in
20 Introduction
M. tuberculosis, are acyl2sulfoglycolipid (Ac2SGL, 1) and
sulfolipidꢀ1 (SLꢀ1 / Ac4SGL, 2) (Figure 1). SLꢀ1, a constituent of
the cell envelope, was discovered by Goren in the 1970s and was
indicated as a virulence factor.8ꢀ10 The role played by SLꢀ1 in the
50 mycobacterial cell wall remains unclear, and different studies
have provided contradicting results as to whether SLꢀ1 has an
influence on the in vivo virulence of mycobacteria.11ꢀ16
Ac2SGL, and possibly other SLꢀ1 intermediates, might also
modulate M. tuberculosis pathogenesis. A ꢁmmpL8 mutant,
55 which accumulates Ac2SGL but lacks SLꢀ1, shows diminished
virulence in mice. By contrast, a ꢁpks2 mutant, which lacks both
Ac2SGL and SLꢀ1, is indistinguishable from wildtype
M. tuberculosis in mice and guinea pigs.17ꢀ19 Recent studies have
also described that lack of SLꢀ1 facilitates intracellular survival
60 of M. tuberculosis.20 These findings were observed when human
macrophages and not mouse macrophages were infected,
indicating a speciesꢀspecific effect probably associated with
differences in antimycobacterial effector mechanisms of
macrophages.
Tuberculosis (TB) is a disease that remains difficult to cure as it
requires the administration of antibiotic drugs for long periods of
time. Active TB has a clinical course with bad prognosis and
untreated patients have a death rate exceeding 50%. The
25 estimated annual death rate of TB patients is approximately 1.7
million, predominantly in developing countries.1ꢀ2
Mycobacterium tuberculosis is the causative agent of TB and
mostly localizes to the lungs. It is a highly infectious pathogen
transmitted with sputum and aerosol droplets from infected
30 individuals. Although only a small part of the population will
develop clinically active TB, it is estimated that oneꢀthird of the
world’s population is currently infected with a dormant or latent
form of M. tuberculosis. With the emergence of multidrugꢀ
resistant and extensively drugꢀresistant mycobacterial bacilli,
35 there is a great need to enhance our understanding of how
mycobacteria achieve a dormant and immunologically neutral
3
state and how the immune system provides efficient protection.
65 In 2004, Gilleron et al. reported the isolation and structure of a
diacylated sulfoglycolipid (Ac2SGL, 1),21 exclusively found in
M. tuberculosis.22 Ac2SGL 1 also features a trehalose 2'ꢀsulfate
core, but is diacylated with either a palmitic or stearic acid
residue at the 2ꢀposition and hydroxyphthioceranic acid at the 3ꢀ
70 position. Additionally, it was proven that 1 functions as a potent
There is broad consensus that the development of novel vaccines
capable of limiting bacilli growth and eradicating intracellular
40 latent forms would represent an important achievement.4
One of the main reasons why M. tuberculosis is so elusive, is
impermeability of the cell envelope to most antibiotics. The outer
membrane consists of
a variety of complex, longꢀtailed
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45
Figure 2 Phthioceranic (3) and hydroxyphthioceranic (4) acid and the
absolute configuration of their stereocenters.
We have been involved in the synthesis of (glyco)lipids from
M. tuberculosis for several years,26 and an efficient catalytic
iterative protocol for the synthesis of deoxypropionates has been
50 developed and applied to the synthesis of 3, among other
polyketides.27ꢀ30 Taking advantage of this knowledge, the
synthesis of 4 has been attempted with the final goal of
Figure 1 Structures of sulfoglycolipids Ac2SGL (1) and SLꢀ1 (2) as found
in the cell wall of M. tuberculosis.
generating
a synthetic sample of 1. To elucidate the
stereochemistry of the C17 stereocenter of 4, an initial synthetic
55 route has been devised. A second route to 4 has been developed
to optimize its synthesis and here we report our results for the
complete asymmetric synthesis of 1 and the elucidation of its
stereochemistry.
antigen of CD1b restricted ab T cells in humans infected by
M. tuberculosis.23 T cells activated by 1 release interferonꢀγ,
recognize M. tuberculosisꢀinfected cells, and kill intracellular
mycobacteria. In light of these properties, 1 has been proposed as
a component of a new vaccine against tuberculosis.
5
Synthesis and stereochemical elucidation of
Comparison of the antigenicity of a panel of synthetic analogues
60 hydroxyphthioceranic acid: A first approach
10 of Ac2SGL 1, sharing the same trehaloseꢀsulfate polar headgroup
but differing in the structure of their acyl tails, showed that the
number of Cꢀmethyl substituents on the chain, the configuration
of these chiral centres, and the respective location of the two
different acyl chains on the trehalose moiety govern Tꢀcell
15 receptor (TCR) recognition and T lymphocyte activation. A direct
correlation was observed between the ability of SGLs to stimulate
Ac2SGLꢀspecific T cells and the number of methylꢀbranched
groups on the aliphatic chain at the 3ꢀposition of the trehalose.
SGLs esterified with saturated methylꢀbranched acids required at
20 least two 1,3ꢀmethyl substituents to stimulate Ac2SGLꢀspecific T
cells at all. Although most synthetic analogues showed activation
Our initial approach toward 4 was based on the copperꢀcatalyzed
asymmetric allylic alkylation of 5, which in turn is prepared from
cinnamoyl bromide and acrolein (Scheme 1).31,32 Addition of
pentadecylmagnesium bromide, using CuBr•SMe2 and L1,
65 provided 6 in 76% yield and 98% ee. As the stereochemistry of
of Ac2SGL specific T cells, at low concentrations (<100 nM ~ 0.1
μg/mL) their antigenicity was significantly decreased compared
to 1.24,25
25 Because of their important role in the human immune response
and
their
painstaking
purification
from
pathogenic
M. tuberculosis,18 it might appear remarkable that 1 (as well as 2)
has escaped total synthesis to date. All the more so because the
application of 1 as part of a vaccineꢀdevelopment program would
30 heavily depend on the access to synthetic material. The molecular
complexity of 1 is, however, impressive. The 1,3ꢀarray of eight
methyl substituents in hydroxyphthioceranic acid (4) is
unprecedented in synthesis and the presence of a hydroxy
function at C17 of 4, vicinal to a methyl group and with unknown
35 stereochemistry, thwarts a straightforward synthesis even further.
At the outset, it was not fully obvious whether this
stereochemistry could be unambiguously established by
synthesis, also because of the limited amount of natural reference
material, available only as component of a complex cell wall
40 extract. Additional challenges in the synthesis of 1 are the
regioselective functionalization, including sulfation, of C2ꢀ
symmetric trehalose25 and the necessity to limit the number of
synthetic steps after attachment of precious 4 to trehalose.
Scheme 1 First approach to the synthesis of
hydroxyphthioceranic acid (4).
2
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the hydroxyl group in natural 4 was unknown, we deliberately
chose the Sꢀconfiguration, assuming that this could be adapted at
a later stage via a Mitsunobu reaction should this be necessary.
Ringꢀclosing metathesis of 6 using 1 mol% of secondꢀgeneration
HoveydaꢀGrubbs catalyst (HG II) afforded α,βꢀunsaturated
lactone 7 in 87% yield. Using substrate control, 1,4ꢀaddition with
the Gilman reagent Me2CuLi gave antiꢀ8 as a single diastereomer
in 94% yield.
5
Initially, we attempted to reduce the lactone to the corresponding
10 lactol, followed by Wittig or HornerꢀWadsworthꢀEmmons
(HWE) olefination to prepare compound 10, after silylation.
Although reduction of 7 with DIBALH afforded the lactol
quantitatively, this compound resisted olefination. Initially, ring
opening and esterification of the resulting acid were hampered by
15 the strong tendency to cyclize again. Treatment with one
equivalent of KOH in THF/water, followed by addition of an
excess of isoꢀpropyl bromide in DMF, however, allowed the
isolation of ester 9 in nearꢀquantitative yield. The secondary
hydroxyl group of 9 was protected as its silyl ether, after which
20 the ester moiety was reduced to the corresponding aldehyde with
DIBALH. Subsequent HWE olefination afforded α,βꢀunsaturated
thioester 10 in 73% yield over three steps. With 10 in hand, we
were curious what the influence of the substrate would be on the
diastereoselectivity of the copperꢀcatalyzed 1,4ꢀaddition of
25 MeMgBr using (+)ꢀ(S,RFe)ꢀ or (–)ꢀ(R,SFe)ꢀJosiphos (L2) as the
ligand. Analysis of both reactions by 1HꢀNMR spectroscopy
showed in the case of (+)ꢀ(S,RFe)ꢀJosiphos a clear preference for
the desired syn product 11, which was isolated in high yield and a
de higher than 98%. The antiꢀproduct 12 was obtained with (–)ꢀ
30 (R,SFe)ꢀJosiphos in an acceptable but markedly lower de of 70%
(Figure 1SI).
The preparation of 11 set the stage for the introduction of all
subsequent methyl substituents applying an iterative protocol
following the sequence of DIBALH reduction/HWE
35 olefination/asymmetric conjugate addition.28,29 Repetition of steps
e, f and g (Schemes 1 and 2) led to thioester 13 with all eight
methyl substituents in a 1,3ꢀarray as present in natural 4. The 21
steps, starting from 10, were performed in an excellent 14%
overall yield, affording 13 as a single stereoisomer without
40 formation of antiꢀproduct (for the 1HꢀNMR spectrum, see SI).
Scheme 3 Synthesis of C17 synꢀ and antiꢀhydroxyphthioceranic acid
methyl esters.
convenient to have 4 available as its methyl ester. For this reason,
60 alcohol 15 was oxidized using RuCl3ꢂ(H2O)x and NaIO4 and
immediately treated with trimethylsilyl diazomethane to give
methyl ester 16 in 75% yield over two steps. Deprotection with
TBAF afforded the methyl ester of 4 (17) with the secondary
alcohol at C17 in an anti configuration with respect to the methyl
65 group at C16. To unambiguously assign the stereochemistry of
C17 in natural 4, 17 was also converted into its C17ꢀOH epimer
(18) by Mitsunobu reaction with pꢀnitrobenzoic acid. Transꢀ
esterification with NaCN in MeOH finally afforded 18 in 85%
yield over two steps. With the current synthetic route, 4 was
70 obtained in 1.4% overall yield over 32 steps starting from 5.
The optical rotation of 17 and 18 was virtually identical ([α] =
+16.0 for 17 and [α] = +16.4 for 18, both in CHCl3), and in turn
close to that reported in the literature for a mixture of
homologues ([α] = +23).34 An extract of cellꢀwall lipids
75 containing sulfolipidꢀ1 (2), prepared following the procedure of
Goren, was transꢀesterified with NaCN in MeOH to give a
complex mixture of polypropionate methyl esters.8 The presence
of 17 or 18 in the natural sample was proven by mass
spectrometry. Analysis with HPLCꢀELSD revealed a significant
80 difference in retention time between 17 and 18 and comparison
with the natural sample confirmed that antiꢀ17 was not present in
the natural sample, whereas synꢀ18 was (Figure 2SI). The
esterified natural sample was compared to synthetic 17 and
Scheme 2 Synthesis of the allꢀsyn 1,3ꢀmethyl deoxypropionate array.
To install the carboxylic acid function of 4 at the correct position
starting from 13, one carbon had to be removed (Scheme 3). To
45 do so, the βꢀsubstituted thioester was converted into the
corresponding methyl ketone using Me2CuLi, affording 14 in
84% yield.33 This ketone was subsequently submitted to a
regioselective Baeyer–Villiger oxidation employing mꢀ
chloroperbenzoic acid. To overcome partial hydrolysis of the
50 acetate formed in the Baeyer–Villiger oxidation, the mixture
obtained was hydrolyzed to the primary alcohol 15 in 63% yield
over two steps. Oxidation of the primary alcohol and deprotection
of the secondary hydroxyl group would directly afford the target
compound. However, we realized that in order to compare the
55 synthetic material with that of natural origin, it would be more
85
Scheme 4 Proposed synthetic pathway for the synthesis of
hydroxyphthioceranic acid (4).
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Scheme 5 Synthesis of phthioceranic and hydroxyphthioceranic acid.
18 by 1HꢀNMR spectroscopy. Although the natural sample
consists of a mixture of homologues, the chemical shift for the
methineꢀproton next to the OHꢀgroup is not obscured. Inspection
resulting alkene in order to introduce the hydroxyl group and to
append the required alkyl chain to the terminus. Even though the
asymmetric allylic alkylation is a reasonably well explored
5
of the 1HꢀNMR spectrum of synthetic antiꢀ17 and synꢀ18 35 reaction, we were not certain of its viability in the presence of
revealed a small but distinct difference (3.47 and 3.50 ppm,
respectively). A perfect match with synꢀ18 was found for the
such an extended array of chiral centres nearby. Moreover,
enantioꢀ or diastereoselective difunctionalization of
10 natural sample which provided strong evidence for a syn
monosubstituted aliphatic terminal olefins is notoriously difficult
and only few successful examples have been reported in the
relationship in the natural product (Figure 3SI). Additional and
conclusive evidence was obtained by comparing the 13CꢀNMR 40 literature.36,37 Finally, as 4 will have to be esterified to the
spectra of all samples. Both 17 and 18 displayed distinct
differences in chemical shift for the carbon atoms in the
15 proximity of the secondary hydroxyl group. The natural product
was shown to be identical with synꢀ18, whereas antiꢀ17 was not
trehalose core, its hydroxyl function needed protection in such a
way that deprotection would not be detrimental to the rest of the
molecule.
Starting from α,βꢀunsaturated thioester 20, intermediate 19 was
detected (Figure 4SI). The combined data establish that 4/18 have 45 obtained in good yield with seven methyl substituents following
the same stereochemistry as natural hydroxyphthioceranic acid.
With the structure of 4 established, a more straightforward
20 synthetic route could be envisioned without the need to invert the
stereochemistry of the hydroxyl group. In addition, the synthesis
our iterative 1,4ꢀaddition procedure (Scheme 5).38 The aliphatic
tail was introduced by reduction of the thioester, transformation
of the alcohol formed into a leaving group and substitution using
the desired Grignard reagent. Subsequently, the silyl ether was
of 3 and 4 from a common intermediate is appealing, because 50 removed and the resulting primary alcohol was oxidized to afford
both polypropionates are found in sulfolipidꢀ1 (Figure 1).9
Commencing the synthesis with the iterative deoxypropionate
25 synthesis protocol, and subsequent functionalization towards 3
and 4 would streamline their synthesis considerably.
3 in yields corresponding to those reported.
Towards the synthesis of benzyletherꢀprotected 4, intermediate 19
was reduced with DIBALH and the aldehyde was submitted to a
HWE olefination, affording oxoꢀester 22 in 85% yield over two
In Scheme 4, the most advanced common intermediate for both 3 55 steps. Reduction of 22 with DIBALH furnished allylic alcohol
and 4 is 19,29 with seven methyl branches installed.27 In order to
obtain 4, we planned to introduce the eighth methyl branch via
30 copperꢀcatalyzed allylic substitution.35 Unlike conjugate addition,
allylic substitution enables subsequent difunctionalization of the
23, which was, in turn, converted to allylic bromide 24, using
NBS/PPh3 in 88% yield. We were pleased to see that the copperꢀ
catalyzed asymmetric allylic alkylation of 24 with (R,RFe)ꢀL1 and
MeMgBr afforded terminal olefin 25 in a remarkable yield of
4
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Ph
O
O
O
HO
O
O
O
H25C12
O
O
34
O
O
Ph
O
Si
O
Si
i-Pr
i-Pr
i-Pr
i-Pr
31
, 2,4,6-trichloroBzCl,
Et3N, DMAP,
C6H6, rt
76%
Figure 3 Highly diastereoselective allylic alkylation affording syn and
Ph
O
O
anti products.
O
O
C14H29
O
O
O
OBn
O
88%. Preparation of the corresponding anti product with the
enantiomer of L1 confirmed that both syn and anti products can
be obtained in a de >95%. Figure 3 shows that the olefinic
protons B and B’ display a small but distinct difference in
chemical shift in the 1HꢀNMR spectrum. Comparison of both
products with related compounds reported earlier, demonstrated
O
5
O
O
O
Ph
35
O
Si
O
Si
i-Pr
i-Pr
i-Pr
i-Pr
TBAF, pH = 6.5
THF, 40 ºC
85%
10 the correct assignment of the relative and absolute
configurations.39,40
Ph
O
O
With terminal olefin 25 in hand, the Sharpless dihydroxylation
reaction was explored for the introduction of the diol moiety.
Although the yield of the dihydroxylation was satisfactory, the
15 5:1 syn:anti ratio obtained on model substrate 32 having two
methyl substituents was somewhat disappointing (Scheme 6).41 In
2009, an intriguing enantioselective diboration reaction has been
O
O
C14H29
O
O
O
OBn
O
O
O
36
HO
O
Ph
OH
-OTf
N+
O
37
N
S
O
O
61%
reported, employing bis(pinacolato)diboron (B2pin2) and
a
Cl3C
combination of [Pt2(dba)3] and phosphonite L3 as the catalyst.42
20 Oxidation of the diboronate with H2O2 afforded diols in high
enantioselectivities. As the reaction was reported to be applicable
to unfunctionalized aliphatic terminal olefins as well, this
alternative approach was employed.
1,2-di-methylimidazole,
THF, rt
Ph
O
O
O
O
C14H29
O
O
O
OBn
O
(DHQ)2Phal (2.5 mol%)
O
O
O
O
38
K2OsO2(OH)4 (1 mol%), K2CO3,
O
O
Ph
S
K3FeCN6
,
methanesulfonamide,
OH
O
t-BuOH/H2O, 0 ºC
CCl3
(S)
OTBDPS
OTBDPS
(R)
(S)
HO
79%
32
OH
de = ~70%
33
Pd(OH)2 / C,
NH4HCO2, H2,
DCM/MeOH, rt
49%
a) B2pin2, L3 (5.1 mol%),
[Pt2(dba)3] (2.5 mol%)
b) NaOH, H2O2
(S)
OTBDPS
OTBDPS
(R)
(S)
HO
75%
HO
HO
OH
32
33
de > 95%
O
O
C14H29
O
O
O
25
Scheme 6 Dihydroxylation of terminal olefins using procedures by
OH
O
Sharpless and Morken.
OH
OH
O
O
O
1
O
S
OH
O-Na+
When we subjected model substrate 32 to the conditions
42
Scheme 7 Functionalization towards Ac2SGL (1)
described in the study of Morken’s group we observed the syn
product with complete selectivity by 13CꢀNMR spectroscopy
with tetradecylmagnesium bromide, employing copper catalysis.
Analysis of alcohol 28 by 13CꢀNMR spectroscopy provided
45 strong evidence for the formation of the syn alcohol (Figure 5SI).
Its chemical shifts match those of synꢀ18. Whereas we were
unable to protect alcohol 28 under basic conditions, Lewis acidic
conditions, in particular TMSOTf with benzyl 2,2,2ꢀ
trichloroacetimidate, afforded benzyl ether 29 in 76% yield. The
50 silyl ether of 29 was cleaved with TBAF to afford primary
alcohol 30 in high yield. Finally, oxidation with TEMPO, NaOCl
and NaClO2 gave 31 in an excellent 90% yield.43 Overall 31 was
obtained in 3.0% yield over 32 steps.
30 (Figure 6SI). As the yield was comparable to that obtained in the
Sharpless dihydroxylation reaction, we decided to use the
Morken dihydroxylation procedure in our synthesis. However,
when the same conditions were applied to substrate 25 with eight
methyl groups, only 5–10% of the desired product was obtained.
35 We discovered that with a twofold increase of the catalyst
concentration and three equivalents of B2pin2, diol 26 was
obtained in a gratifying 98% yield and >95% de. This example
represents the first application of this methodology to the
synthesis of a natural product. Epoxidation of 26 under phaseꢀ
40 transfer conditions afforded 27 in 88% yield, which was opened
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Synthesis of diacylsulfoglycolipid: combining
Modeling studies
hydroxyphthioceranic acid and the trehalose core
To investigate how the natural product 1 might be bound by
CD1b, the CD1b–diacylsulfoglycolipid coꢀcrystal structure (PDB
code: 3T8X)46 was used as a template to model the natural
55 product into the deep binding pocket. 1 was manually docked into
the binding groove, and the energy of the system minimized
using the MAB force field as implemented in the computer
program MOLOC,47 while keeping the protein coordinates fixed.
Given that the chain at C3 of 1 is considerably longer than the
60 corresponding substituent of the diacylsulfoglycolipid found in
the coꢀcrystal structure with CD1b, we decided not to include the
model of the endogenous spacer during the energy
minimization.46
To attach 31 regioselectively to the trehalose moiety, 34 was
prepared according to a literature procedure (Scheme 7).25
Several acylation strategies were investigated, but the procedure
developed by Yamaguchi proved to be most effective in our
system, affording 35 in 76% yield. Using a buffered TBAF
solution, the bis(diisopropylsilyl) ether was removed to provide
36 in 85% yield.
10 For the introduction of the sulfate group in a protected form, we
relied on a procedure reported recently by Taylor et al.,
employing sulfuryl imidazolium salts to regioselectively
introduce protected sulfates.44,45 The 2,2,2ꢀtrichloroethyl (TCE)
group was considered to be a suitable protecting group as
15 deprotection can be achieved under hydrogenolysis conditions,
identical to those planned for the removal of the benzylidene
acetals. It was shown that the TCE sulfate moiety could be
introduced using the readily prepared imidazolium salt 37 with a
remarkable regioselectivity for 4,6ꢀOꢀbenzylidene acetals of αꢀ
20 and βꢀglucosides, affording the 2ꢀOH and 3ꢀOH substituted
products, respectively. By taking advantage of this observation,
we were pleased to find mainly the desired 2ꢀsulfated product.
Although traces of 2,3ꢀdisulfated product were observed as well,
38 was obtained in 61% yield.
5
25 Given that the 4,6ꢀOꢀbenzylidene acetals, the benzyl ether, and
the TCE group all are labile under hydrogenolysis conditions, we
envisioned complete deprotection in one step, which would avoid
the purification of very polar intermediates. Using ammonium
formate and Pd(OH)2/C under a hydrogen atmosphere, complete
30 deprotection was indeed achieved. Importantly, 1 was obtained as
its sodium salt in 49% yield as confirmed by a comparison of the
1HꢀNMR spectra of natural and synthetic 1. Even though the
natural product has been isolated as a mixture of homologues, the
chemical shifts and multiplicities of the distinctive CH protons on
35 the carbohydrate core are unambiguous.22
65
Figure 5 MOLOCꢀgenerated molecular model of 1 in the hydrophobic
binding pocket of human CD1b (PDB code: 3T8X).45 Color code: protein
skeleton: C: gray; inhibitor skeleton: C: green; O: red; S: yellow.
Hydrogen bonds are represented as dashed lines. Distances between
70
heavy atoms are given in Å. Image generated with Pymol.49
Biological activity
Analysis of the computed binding mode of 1 to CD1b revealed no
repulsive interactions, numerous vanꢀderꢀWaals interactions and
four hydrogen bonds between the terminal trehalose moiety and
the side chains of Asp79 and Gln152. In addition, a weak
75 hydrogen bond between the C17 hydroxyl group and the
backbone carbonyl group of Leu161 was observed inside the
hydrophobic binding pocket. According to the modeled binding
pose, this hydroxyl group – that can now be modeled with the
correct stereochemistry – not only contributes to the binding
80 energy but could also play an important role in positioning the
chain in the tunnel.
The synthetic sulfoglycolipid 1 was compared with natural 1 for
its ability to activate the Ac2SGLꢀspecific T cells as reported.19
40 Human dendritic cells expressing CD1b protein were incubated
with varying amounts of natural or synthetic antigen, before
addition of specific T cells. Tꢀcell response was quantified by
measuring the amounts of GMꢀCSF and IFNꢀγ cytokines released
by activated T cells in an antigen doseꢀdependent manner
45 (Figure 4). In these very sensitive assays, the activity of
sulfoglycolipid 1 was remarkably comparable to that of purified
natural Ac2SGL.
In close analogy to the binding mode of diacylsulfoglycolipid, 1
was modeled to bind with the sugar moieties at the Tꢀcellꢀ
receptor (TCR) recognition surface and the long, aliphatic chains
85 extending into a deep cleft, consisting of a series of three
hydrophobic channels and a tunnel at the core of the α1α2
domain, referred to as A’, C’, and F’ channels and T’ tunnel,
respectively.48 The chain at C2 occupies the C’ channel nearly
fully and extends into the lateral opening. The chain at C3 is
90 accommodated in the A’ and T’ channels, only partially filling
the latter. The F’ channel remains unoccupied (Figure 5 and
Figure 4 Activity profiles comparing the cytokine release of synthetic
50
and natural Ac2SGL.
6
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Figure 7SI). The proposed binding mode shows that the first and
55 Notes and references
third methyl group are surfaceꢀexposed, enabling them to be
involved in recognition of the TCR, whereas the remaining
methyl groups are buried in the A’ channel. The additional
methyl branches of 1 could enhance the strength of the interaction
with CD1b and also have an influence on the conformation of the
whole molecule, in particular the surfaceꢀexposed methyl groups
that might interact with the TCR. This is also supported by
structureꢀfunctional studies performed using analogues of 1 with
aStratingh Institute for Chemistry, University of Groningen, Nijenborgh 7,
NL-9747 AG Groningen, The Netherlands. E-mail: a.j.minnaard@rug.nl
bExperimental Immunology, Department of Biomedicine, University
Hospital Basel, Hebelstrasse 20, CH-4031, Basel, Switzerland
60 cCNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205
route de Narbonne BP 64182, F-31077 Toulouse, France
dUniversité de Toulouse, UPS, IPBS, F-31077 Toulouse, France
eSingapore Immunology Network (SIgN), Agency for Science, Technology
and Research (A*STAR), Biopolis, Singapore.
5
10 less methyl groups.24
65
† Electronic Supplementary Information (ESI) available: HPLC traces,
spectroscopic characterization, modeling studies and experimental details.
See DOI: 10.1039/b000000x/
Given the presumed degree of flexibility in the CD1b binding
groove, the modeling was repeated while leaving the side chains
lining the pocket flexible. Only a few aminoꢀacid side chains
moved to a small extent, illustrating how CD1b is capable of
15 accommodating lipids with different numbers, patterns and types
of substituents (aminoꢀacid residues in question shown as sticks
in Figure 5). This observation is in agreement with the results
reported in the literature and suggests that Tꢀcell activation by
CD1b is controlled by its threeꢀdimensional structure as well as
20 the way in which the polar head group and some of the methyl
groups of 1 are presented to the TCR.50ꢀ52
70
75
1
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Conclusions
10 The synthesis of a sulfolipidꢀI analogue was reported earlier: C.D.
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11 J.P. Brozna, M. Horan, J.M. Rademacher, K.M. Pabst, M.J. Pabst,
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Hydroxyphthioceranic acid 4 has been prepared, for the first time,
via two different routes. The most efficient route afforded the
25 product in 3.0% yield over 32 steps and its stereochemistry was
established by comparison with that of the natural product. The
highestꢀyielding synthesis is based on a very efficient catalytic
iterative protocol for the stereoselective introduction of methyl
substituents, a highly stereoselective catalytic allylic substitution
30 and a extremely stereoselective platinumꢀcatalyzed diboration
reaction. This result was used for the first synthesis of Ac2SGL, a
complex glycolipid isolated from M. tuberculosis. The synthesis
is based on a regioselective functionalization of trehalose, and a
carefully devised protectingꢀgroup strategy. Ac2SGL was
35 prepared in seven steps from trehalose with an overall yield of
4.1% based on 4, and its structure was identical to that of the
natural product. Biological evaluation revealed that the antigenic
potency of synthetic 1 is identical to that of the natural product.
Together with earlier studies on model compounds, this implies
40 that the precise structure and stereochemistry of the
hydroxyphthioceranic acid part in 1 are important for its antigenic
activity. As Ac2SGL (1) is an extremely potent antigen, studies
on its application as a vaccine against tuberculosis are of utmost
importance and can now be performed with the pure synthetic
45 material available. Modeling studies of natural Ac2SGL give an
indication on the influence of altering the complex side chain of
Ac2SGL on binding. This approach could be valuable for the
development of future analogues.
85
12 M.B. Goren, O. Brokl, W.B. Schaeffe, Infect. Immun., 1974, 9, 142.
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15 M. Kato, M.B. Goren, Inf. Immun., 1974, 10, 733.
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19 C. Rousseau, O.C. Turner, E. Rush, Y. Bordat, T.D. Sirakova, P.E.
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100
105
22 M. Gilleron, S. Stenger, Z. Mazorra, F. Wittke, S. Mariotti, G.
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Financial support from The Netherlands Organization for
Scientific Research (NWOꢀCW) is acknowledged.
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