Simplified deoxypropionate acyl chains for mycobacterium tuberculosis sulfoglycolipid analogues: Chain length is essential for high antigenicity

Article abstract of DOI:10.1002/cbic.201300482

The longer, the better: Increasing the lengths of the 1,3-methyl-branched fatty acyl chain units in mycobacterial diacylated sulfoglycolipid (Acyl ₂SGL) analogues led to dramatic improvements in their antigenic properties and gave products more

Full text of DOI:10.1002/cbic.201300482

CHEMBIOCHEM
COMMUNICATIONS
DOI: 10.1002/cbic.201300482
Simplified Deoxypropionate Acyl Chains for
Mycobacterium tuberculosis Sulfoglycolipid Analogues:
Chain Length is Essential for High Antigenicity
Benjamin Gau,[a] Aurꢀlie Lemꢀtais,[b] Marco Lepore,[d] Luis Fernando Garcia-Alles,[a]
Yann Bourdreux,[b] Lucia Mori,[d, e] Martine Gilleron,[a] Gennaro De Libero,[d, e] Germain Puzo,[a]
Jean-Marie Beau,*[b, c] and Jacques Prandi*[a]
Human tuberculosis remains
a major worldwide
health problem, with about 1.5 million casualties
every year. Despite numerous efforts over decades to
implement better diagnosis and extensive availability
of antibiotic treatments, this number is only slowly
decreasing, making tuberculosis still one of the most
lethal infectious diseases in the world.[1] New anti-
biotic treatments against Mycobacterium tuberculosis,
the etiologic agent of human tuberculosis, are
needed.[2] A new vaccinal approach is also mandatory,
because the bacillus of Calmette and Guꢀrin (BCG),
Scheme 1. Structures of the natural Acyl2SGLs 1 from M. tuberculosis.
the only existing vaccine against tuberculosis, does not afford
protection in adults.[3]
conclusion that lipid-specific T-cells are primed during infection
and persist in patients with latent tuberculosis.[6]
In this context, lipidic antigens presented by the CD1 (clus-
ter of differentiation 1) family of proteins might be worth con-
sideration as potential candidates for a subunit vaccine.[4]
Indeed, experimental demonstration of the vaccinal interest of
lipidic antigens in the case of mycobacterial infections was
provided in 2003, when it was shown that immunization of
guinea pigs with an uncharacterized mixture of mycobacterial
lipids improved the pulmonary pathology of the animals after
M. tuberculosis infection.[5] We found strong responses to diac-
ylsulfoglycolipids (Acyl2SGLs, Scheme 1) only in patients with
clinical histories of mycobacterial infection; this supported the
Most of the mycobacterial lipidic antigens characterized so
far are presented by the CD1b protein. Among these antigens,
Acyl2SGLs 1 (Scheme 1) stimulated human CD1b-restricted ab
T-cells to release interferon-g (IFN-g) and to kill intracellular my-
cobacteria residing in M. tuberculosis-infected antigen-present-
ing cells (APCs).[6] The scarcity of this lipid antigen in mycobac-
terial cells prompted us to synthesize Acyl2SGL analogues in
which the hydroxyphthioceranoic acyl unit (Scheme 1) had
been replaced by chiral multiply methylated saturated and
a,b-unsaturated fatty acyl chains.[7]
Some of these analogues were able to stimulate the
Acyl2SGL-specific T-cell clone, with the T-cell response gov-
erned by the presence of the sulfate group,[6] the location of
the fatty acyl chains on the trehalose core, and the fine struc-
ture of the multiply methylated fatty acyl chain, including chir-
ality and number of methyl groups.[7,8]
[a] B. Gau, Dr. L. F. Garcia-Alles, Dr. M. Gilleron, Dr. G. Puzo, Dr. J. Prandi
Institut de Pharmacologie et de Biologie Structurale (IPBS)
CNRS and Universitꢀ de Toulouse
BP 64182, 205 route de Narbonne, 31077 Toulouse (France)
E-mail: jacques.prandi@ipbs.fr
[b] A. Lemꢀtais, Dr. Y. Bourdreux, Prof. J.-M. Beau
Institut de Chimie Molꢀculaire et des Matꢀriaux d’Orsay
Laboratoire de Synthꢁse de Biomolꢀcules, Universitꢀ Paris-Sud and CNRS
Bꢂtiment 430, 91405 Orsay (France)
The very recent total synthesis of an Acyl2SGL with a specific
hydroxyphthioceranoic acyl side chain (1, n=7, Scheme 1) re-
vealed antigenic properties of the synthetic glycolipid identical
to those of the natural mixture of antigens.[9] We show here
that synthetic structures much simpler in their 1,3-methyl-
branched lipid part are as potent as or more potent than the
natural antigens 1. Their synthesis is based on an efficient
preparation of long-chain chiral 1,3-methyl-branched fatty
acids of very high optical purity and on a new sequence of re-
gioselective steps initiated by a tandem catalytic approach for
the one-pot regioselective protection of trehalose. We further
demonstrate the crucial role of the length of the lipid chain in
the antigenic properties of synthetic SGLs.
E-mail: jean-marie.beau@u-psud.fr
[c] Prof. J.-M. Beau
Centre de Recherche de Gif
Institut de Chimie des Substances Naturelles du CNRS
Avenue de la Terrasse, 91198 Gif-sur-Yvette (France)
[d] Dr. M. Lepore, Dr. L. Mori, Prof. Dr. G. De Libero
Experimental Immunology, Department of Biomedicine
University Hospital Basel
Hebelstrasse 20, 4031 Basel (Switzerland)
[e] Dr. L. Mori, Prof. Dr. G. De Libero
Singapore Immunology Network (SIgN)
Agency for Science, Technology and Research
8A Biomedical Grove, 138648 Singapore (Singapore)
We devised a new route to the deoxypropionate fatty acids
that overcome the drawbacks (purification and solubility prob-
lems) of our previous synthesis[7] (Scheme 2).
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300482.
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 2413 – 2417 2413

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
The other major challenge in the synthesis of the
SGLs is the rapid construction of a trehalose-based
core structure suitably protected to differentiate the
2-, 2’-, 3-, and 3’-positions. Solutions to the problem
have usually resulted in a lengthy generation of the
appropriately protected disaccharide building
block.[16] To simplify synthetic sequential methods, we
recently developed one-pot regioselective protection
approaches to mono- and disaccharides through
Lewis-acid-catalyzed transformations on per-O-tri-
methylsilylated glycosides.[17,18] This approach was
applied to the regioselective protection of trehalose
with use of FeCl3·6H2O as an inexpensive and envi-
ronmentally friendly catalyst.[18,19] In this procedure,
two catalyzed acetalations and two regioselective re-
ductive etherifications yielded C2-symmetrical 14 in
a good 61% yield[18] (Scheme 4). Desymmetrization[20]
of diol 14 by monosilylation at a low temperature
(!15) was followed by regioselective mono-3’-de-O-
benzylation under hydrogen transfer conditions on
the least hindered unit to afford diol 16, leaving the
Scheme 2. Synthesis of chiral 1,3-methyl-branched alcohols 5a–c. a) LDA (3.0 equiv), LiCl
(7.0 equiv), THF, ꢀ788C, then iodide (1.0 equiv), 08C, 1 h for 3, RT, 20 h for 6a and 6b,
85–99%; b) LiNH2BH3 (4.0 equiv), 08C to RT, 2 h, 90%; c) I2 (1.2 equiv), PPh3 (1.3 equiv),
toluene, RT, 6 h, 90%.
It started with the Myers alkylation of the lithium enolate of
(1R,2R)-(ꢀ)-pseudoephedrine derivative 2[10] with iodide 3[11] in
the presence of lithium chloride, which provided derivative 4a
in high yield. Evaluation of the diastereoselectivity of the reac-
tion was difficult at this step because of the presence of ro-
tamers around the amide bond in the NMR spectrum. Amide
4a was thus reduced with an excess of lithium amidotrihydri-
doborate to give alcohol 5a in 90% yield, and its optical purity
(>98% ee) was established by derivatization of a sample of
the alcohol to the (1S)-camphanate ester.[12] Conversion of 5a
into iodide 6a was carried out in a 90% yield under standard
Garreg conditions.[13]
benzylidene acetals unaffected.[21] Finally, regioselective esterifi-
cation at the C-2 position of diol 16 with palmitic acid led to
the key common intermediate 17 in good yield.
The synthesis of the diacylated SLGs was completed by a
standard acylation of alcohol 17 (1–1.5 equiv) with the saturat-
ed and unsaturated chiral fatty acids to provide diacyl deriva-
tives 18. Quantitative desilylation to afford 19 and sulfation
The same sequence was repeated starting from 6a for fur-
ther introduction of methyl groups on the chain. When using
more sterically hindered iodides 6a and 6b, the alkylation
reaction had to be warmed to room temperature and left for
20 h to ensure high yields of alkylation products 4b and 4c
(90 to 98%). The overall yield for each iteration from 6a to 6b
was reproducibly over 70%, and the whole cycle could be run
on multigram quantities in three days.
Once the required number of methyl groups had been in-
corporated, alcohol 5c was protected as a tert-butyldiphenyl
silyl ether (95%), and the benzyl ether was removed by hydro-
genolysis to give alcohol 7 in high yield (Scheme 3). Tosylate 8
(90%) was obtained from 7 and subjected to copper-catalyzed
nucleophilic displacement with suitable Grignard reagents[14]
(CnH2n+1MgBr, THF, n=4, 12, 20, 22) to give the corresponding
silyl ethers 9a–d in moderate to good yields (45–60%). Remov-
al of the silyl ether in 9 (90%) followed by PCC oxidation gave
aldehydes 10a–d. Carboxylic acid 11 was obtained from 10d
by using a slight excess of Jones’s reagent in acetone, whereas
unsaturated acids 12a–c were prepared by coupling of 10a–
c with the Wittig reagent (carbethoxyethylidene)triphenylphos-
phorane, followed by saponification as previously de-
scribed.[7,15]
Scheme 3. Synthesis of the chiral 1,3-methyl-branched fatty acids.
a) tBuPh2SiCl, NEt3, CH2Cl2, RT, 10 h; b) H2, cat. Pd(OH)2/C, RT, 20 h, 81% over
two steps; c) TsCl, NEt3, CH2Cl2, RT, 4 h, 90%; d) CnH2n+1MgBr, CuBr·SMe2, THF,
RT, 20 h, 45–60%; e) NBu4F, THF, RT, 10 h; f) PCC, AcONa, CH2Cl2, RT, 2 h;
g) Jones reagent, acetone/water, RT, 30 min; h) Ph3P=C(Me)ꢀCOOEt, CH2Cl2,
RT, 4 d; i) KOH (2m), ethanol/water, 808C.
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 2413 – 2417 2414

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
Scheme 5. Structures of the natural Acyl2SGLs (1) and of the synthetic ana-
logues 21a–d. The lengths of the R1 acyl chains are given in parentheses.
Scheme 4. Preparation of the sulfoglycolipids. a) PhCHO, Et3SiH, cat.
FeCl3·6H2O, 61% (see ref. [17]); b) TBSOTf (1.1 equiv), 2,6-lutidine (2 equiv),
CH2Cl2, ꢀ788C, 3.5 h, 74%; c) HCO2NH4 (20 equiv), Pd (10%)/C, MeOH, 658C,
45 min, 65%; d) C15H31CO2H (1.1 equiv), DCC (1.5 equiv), DMAP (1.7 equiv),
CH2Cl2, RT, 4 h, 85%; e) RCO2H (0.7 to 1 equiv), DCC (0.7 to 1 equiv), DMAP (1
to 2 equiv), CH2Cl2, RT, 6 to 16 h, 32 to 64%; f) Bu4NF (2 to 2.6 equiv), THF,
RT, 2.0 h; 76 to 97%; g) i: SO3·pyr (5 equiv), pyridine, 908C, 3 h; ii: Amberlite
IR120 Na, 81 to 91%; h) for saturated acyl chains: H2 (1 atm), Pd (5%)/C,
MeOH/CH2Cl2, RT, 2 h, then Amberlite IR120 Na, 70 to 72%; i) for unsaturated
acyl chains: FeCl3, 15 equiv, CH2Cl2, 08C, 2.5 h, then Amberlite IR120 Na, 47
to 51%. TBS=tert-butyldimethylsilyl; TMS=trimethylsilyl.
methylated unsaturated fatty esters on position 3 of the treha-
lose (see Scheme 5). They are substituted by fatty esters with
chain lengths of 16, 24, and 32 carbon atoms, respectively.
However, these SGLs show strong differences in their abilities
to activate T-cells: the longer the chain, the more stimulatory
the analogue (Figure 1A).
This was clearly indicated by comparison of the potencies
(doses of antigen giving 50% of maximum activation) and of
the efficacies (maximum quantities of released cytokines) of
the different sulfoglycolipids. Interestingly, 21a, with a short
C16 alkyl chain, showed lower potency and efficacy than the
other analogues (Figure 1B). Compounds 21b and 21c
showed similar potencies, but efficacies that were clearly differ-
ent from one another, with 21c being more active than 21b
(Figure 1B). The potency differences suggest that whereas 21a
forms smaller numbers of active CD1b-antigen complexes than
the other analogues, 21b and 21c form similar numbers of
complexes with CD1b. This issue was addressed through
CD1b-plate-bound experiments in which recombinant soluble
CD1b molecules were immobilized on the plastic, loaded with
different doses of 21b, 21c, or natural 1, and then incubated
with T-cells.
provided the 2’-O-sulfated derivatives 20 in high yields. The
final complete deprotection was achieved by hydrogenolysis
for SGL analogues 21d and 21e, each substituted at O-3 with
a saturated chiral lipid, or by treatment with an excess of anhy-
drous FeCl3 in dichloromethane, a procedure that preserves
the conjugated unsaturation of chiral lipids in SGL analogues
21a–c.[22] Some of the new SGLs are presented in Scheme 5.
The antigenicities of these new Acyl2SGLs were then as-
sessed with the aid of the Acyl2SGL-specific T-cells.[6–8] GM-CSF
(granulocyte-macrophage colony-stimulating factor) released
by the T-cells was quantified, and the results are summarized
in Figure 1. We had demonstrated earlier[7,8] that the number
of methyl groups on the 1,3-methyl-branched fatty acyl chain
of the SGL was an important structural element, with increas-
ing antigenicity of the analogues as the number of methyl
groups increased. Furthermore, with acyl chains no longer that
26 carbon atoms, when the same number of methyl groups
was present on the fatty acyl chain, analogues containing a,b-
unsaturated acyl groups—structures not found in the natural
SGLs—were more active than analogues with saturated acyl
groups of the same chain length.[7,8] We show here that the
chain length of the unsaturated fatty acyl chain is also decisive
for the efficient activation of the T-cells (Figure 1). Synthetic
SGLs 21a–c differ only in the lengths of the chains of the tetra-
Also in the absence of APCs, 21b and 21c stimulated T-cells
with comparable potencies, which were dramatically higher
than that of 1 (Figure 1C). In another set of experiments the
stabilities of the stimulatory complexes formed by these two
analogues with CD1b were evaluated in vivo by pulse-chase
experiments. Dendritic cells (DCs) were separately pulsed with
the sulfoglycolipids, washed, and then used to stimulate
CD1b-restricted T-cells after different chase times. Because the
extent of T-cell activation is directly correlated with the
amounts of stimulatory Ag-CD1b complexes present on the
surfaces of the APCs at each time point, these experiments are
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 2413 – 2417 2415

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
In contrast, 21d is highly anti-
genic, and it is as active as the
natural product. Moreover, 21d,
substituted with a C32 saturated
fatty acyl chain with only three
methyl groups, and 21c, which
carries a C32 unsaturated fatty
acyl chain with four methyl
groups, were found to be equal-
ly antigenic (Figure 1E and 1F).
This shows that the unsaturation
of the acyl chain has secondary
functional effects. These find-
ings, together with our previous
studies, confirm that the major
features controlling SGL antige-
nicity were indeed the length of
the deoxypropionate fatty acyl
chain and the presence of
methyl groups on this lipid
chain.[8]
The relevance of the methyl
groups was also highlighted by
the crystal structure of the solu-
ble CD1b·21b complex,[23] which
showed that the first three
methyl groups of the 1,3-methyl-
Figure 1. Immunological activities of synthetic sulfoglycolipid analogues. Antigenicities of Acyl2SGLs 1 and com-
pounds 21a–d. A) GM-CSF release by T-cells activated by Acyl2SGLs 1 and compounds 21a–c. B) Lineweaver–Burk
plot representing efficacies and potencies of Acyl2SGLs (–·–) and compounds 21a (–··–), 21b (—) and 21c (––).
C) Plate-bound assay of sulfoglycolipid complexes 21b and 21c with the recombinant soluble CD1b, GM-CSF re-
lease by T-cells;. D) Persistence of the stimulatory activity of DCs pulsed with sulfoglycolipids Acyl2SGLs, 21b and
21c over time. E) GM-CSF release by T-cells activated by compounds 21c and 21d. F) IFN-g release by T-cells acti-
branched fatty acyl chain of 21b
are located outside the hydro-
phobic A’ channel of CD1b. This
unique localization suggested
that they might interact with the
~
&
&
*
*
vated by compounds 21c and 21d. : Acyl2SGLs, : 21a, : 21b, : 21c, : 21d.
TCR; this would thus explain
their absolute requirement for
antigenicity. Finally, an in-depth
informative about the stabilities of active complexes over time
in living cells. DCs pulsed with 21b and with natural Acyl2SGLs
1 induced 70% of the maximum T-cell GM-CSF release after
24 h, dropping to about 60% after 72 h (Figure 1D). In con-
trast, DCs pulsed with 21c were significantly more efficient,
their stimulatory capacity being almost unaffected in the first
48 h of chasing and still 85% after 72 h (Figure 1D). These find-
ings indicate that the presence of the long acyl chain stabilizes
the immune-active CD1b-sulfoglycolipid complexes, prolong-
ing their availability within living cells.
reevaluation of the structures of natural Acyl2SGLs showed that
some phthioceranoic-acid-substituted SGLs were also present
in the natural mixture.[24] Compound 21d can thus be consid-
ered a true analogue of these phthioceranoic-acid-substituted
SGLs that have high antigenic capacities. The presence of a hy-
droxy group on the fatty acyl chain is thus not critical for the
antigenicity of a SGL towards CD1b-restricted T-cells. These re-
sults seem to contradict modeling studies predicting that the
hydroxy groups in the hydroxyphthioceranoic side chains of
the natural Acyl2SGLs contribute to the binding mode and play
a key role in the chain positioning.[9]
Compound 21c also showed higher efficacy than 21b; this
suggests that it induces a more productive triggering of T-cells
than 21b, ultimately resulting in higher cytokine release. Ana-
logue 21c differs from 21b in the presence of a longer (C32 vs.
C24) alkyl chain, so this structural difference might be important
for more efficient interaction with the T-cell receptors (TCRs) of
responding cells.
In conclusion, we have developed an efficient synthetic
route to a collection of Acyl2SGL analogues; it includes regiose-
lective steps for the desymmetrization of the trehalose core,
combined with highly diastereoselective access to chiral poly-
methylated fatty acids of any chain length. This new synthetic
route is compatible with the incorporation of very-long-chain
fatty acyl chains (length >C30) in the SGLs. These long chains
have been shown to be of utmost importance for high antige-
nicities of the SGL analogues. Products 21c and 21d showed
the same potencies as natural Acyl2SGLs, together—surprising-
ly—with higher efficacies. These unique features suggest they
Another striking example of the importance of the chain
length for the antigenicity of a SGL is emphasized by compari-
son of 21d, which carries a saturated trimethylated C32 chain,
with 21e, with a saturated trimethylated C24 chain (Scheme 5).
We had shown earlier that 21e was very weakly antigenic.[7,8]
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 2413 – 2417 2416

CHEMBIOCHEM
COMMUNICATIONS
www.chembiochem.org
of the camphanoyl CH2O group by 1H NMR. Only one isomer was de-
tected; this showed that the purity of the starting alcohol was >98%
ee.
should be good candidates as components of anti-tuberculosis
subunit vaccines.
[13] P. J. Garegg, B. Samuelsson, J. Chem. Soc. Chem. Commun. 1979, 978–
980.
[14] B. ter Horst, B. L. Feringa, A. J. Minnaard, Chem. Commun. 2007, 489–
491.
Acknowledgements
We thank the Ministꢁre de la Recherche for a grant to A.L., and
the CNRS and the Institut Universitaire de France (IUF) for finan-
cial support. This work has received funding from the European
Community’s Seventh Framework Program under grant agree-
ment no. 241745 (New TB-VAC) and from the Mꢀrieux foundation
through a Mꢀrieux Research Grant.
[15] No epimerization at the C-4 stereocenter occurred during the saponifi-
cation of esters to acids 12a–d, as was evidenced by the 1H NMR spec-
tra in the 1.8–0.5 ppm region. The signal of the methyl group at C-4 in
the 4,6-syn esters and acids 12a–d (Scheme 3) appears as a doublet
(J=6.5 Hz) at 0.98–0.99 ppm for all compounds, whereas a 4,6-anti
isomer would exhibit a distinct doublet (same J value) at a slightly
higher field (0.96–0.97 ppm), as previously observed.[7,8]
[16] a) H. H. Baer, X. Wu, Carbohydr. Res. 1993, 238, 215–230; b) P. A. Wallace,
D. E. Minnikin, J. Chem. Soc. Chem. Commun. 1993, 1292–1293; c) C. D.
Leigh, C. R. Bertozzi, J. Org. Chem. 2008, 73, 1008–1017.
[17] A. FranÅais, D. Urban, J.-M. Beau, Angew. Chem. 2007, 119, 8816–8819;
Angew. Chem. Int. Ed. 2007, 46, 8662–8665.
Keywords: antigens · glycolipids · trehalose · tuberculosis ·
vaccines
[18] a) Y. Bourdreux, A. Lemꢀtais, D. Urban, J.-M. Beau, Chem. Commun.
2011, 47, 2146–2148; b) A. Lemꢀtais, Y. Bourdreux, P. Lesot, J. Farjon, J.-
M. Beau, J. Org. Chem. 2013, 78, 7648–7657.
[19] For selected reviews on iron salts as agents in selective transformations
see: a) C. Bolm, J. Legros, J. Le Paih, L. Zani, Chem. Rev. 2004, 104,
6217–6254; b) D. D. Diaz, P. O. Miranda, J. I. Padrꢂn, V. S. Martin, Curr.
Org. Chem. 2006, 10, 457–476; c) B. D. Sherry, A. Fꢃrstner, Acc. Chem.
Res. 2008, 41, 1500–1511; d) E. B. Bauer, Curr. Org. Chem. 2008, 12,
1341–1369.
[20] For very recently reported C2-desymmetrization studies on trehalose
see: a) M. K. Patel, B. G. Davis, Org. Biomol. Chem. 2010, 8, 4232–4235;
b) V. A. Sarpe, S. S. Kulkarni, J. Org. Chem. 2011, 76, 6866–6870; c) U.
Pꢄssler, M. Gruner, S. Penkov, T. V. Kurzchalia, H.-J. Knçlker, Synlett 2011,
2482–2486.
[21] For the selective removal of benzyl groups in the presence of benzyli-
dene acetals by using ammonium formate as hydrogen donor, see: a) T.
Bieg, W. Szeja, Synthesis 1985, 76–77; b) D. Beaupere, I. Boutbaiba, G.
Demailly, R. Uzan, Carbohydr. Res. 1988, 180, 152–155.
[22] Selected references for the removal of O-benzyl groups with iron(III)
chloride: a) M. H. Park, R. Takeda, K. Nakanishi, Tetrahedron Lett. 1987,
28, 3823–3824; b) J. I. Padrꢂn, J. T. Vꢅsquez, Tetrahedron: Asymmetry
1995, 6, 857–858.
[23] L. F. Garcia-Alles, A. Collmann, C. Versluis, B. Lindner, J. Guiard, L. Ma-
veyraud, E. Huc, J. S. Im, S. Sansano, T. Brando, S. Julien, J. Prandi, M. Gil-
leron, S. A. Porcelli, H. de La Salle, A. J. R. Heck, L. Mori, G. Puzo, L.
Mourey, G. De Libero, Proc. Natl. Acad. Sci. USA 2011, 108, 17755–
17760.
[1] http://www.who.int/tb/country/en/index.html.
[2] V. Makarov, G. Minina, K. Mikusova, U. Mçllmann, O. Ryabova, B. Saint-
Joanis, N. Dhar, M. R. Pasca, S. Buroni, A. P. Licarelli, A. Milano, E. De Ros-
si, M. Belanova, A. Bobovska, P. Dianiskova, J. Kordulakova, C. Sala, E.
Fullam, P. Schneider, J. D. McKinney, P. Brodin, T. Christophe, S. Waddell,
P. Butcher, J. Albrethsen, I. Rosenkrands, R. Brosch, V. Nandi, S. Bharath,
S. Gaonkar, R. K. Shandil, V. Balasubramanian, T. Balganesh, S. Tyagi, J.
Grosset, G. Riccardi, S. T. Cole, Science 2009, 324, 801–804.
[3] P.-H. Lambert, T. Hawkridge, W. A. Hanekom, Clin. Chest Med. 2009, 30,
811–826.
[4] D. B. Moody, G. S. Besra, I. A. Wilson, S. A. Porcelli, Immunol. Rev. 1999,
172, 285–296.
[5] C. C. Dascher, K. Hiromatsu, X. Xiong, C. Morehouse, G. Watts, G. Liu,
D. N. McMurray, K. P. LeClair, S. A. Porcelli, M. B. Brenner, Int. Immunol.
2003, 15, 915–925.
[6] M. Gilleron, S. Stenger, Z. Mazorra, F. Wittke, S. Mariotti, G. Bçhmer, J.
Prandi, L. Mori, G. Puzo, G. De Libero, J. Exp. Med. 2004, 199, 649–659.
[7] J. Guiard, A. Collmann, M. Gilleron, L. Mori, G. De Libero, J. Prandi, G.
Puzo, Angew. Chem. 2008, 120, 9880–9884; Angew. Chem. Int. Ed. 2008,
47, 9734–9738.
[8] J. Guiard, A. Collmann, L. F. Garcia-Alles, L. Mourey, T. Brando, L. Mori, M.
Gilleron, J. Prandi, G. De Libero, G. Puzo, J. Immunol. 2009, 182, 7030–
7037.
[9] D. Geerdink, B. ter Horst, M. Lepore, L. Mori, G. Puzo, A. K. H. Hirsch, M.
Gilleron, G. De Libero, A. J. Minnaard, Chem. Sci. 2013, 4, 709–716.
[10] a) A. G. Myers, B. H. Yang, D. J. Kopecky, Tetrahedron Lett. 1996, 37,
3623–3626; b) A. G. Myers, B. H. Yang, H. Chen, L. McKinstry, D. J. Ko-
pecky, J. L. Gleason, J. Am. Chem. Soc. 1997, 119, 6496–6511.
[11] Iodide 3 was prepared in two steps from the commercially available 4-
O-benzyl butane-1,4-diol.
[24] E. Layre, D. Cala-De Paepe, G. Larrouy-Maumus, J. Vaubourgeix, S. Mun-
dayoor, B. Lindner, G. Puzo, M. Gilleron, J. Lipid Res. 2011, 52, 1098–
1110.
[12] Derivatization of alcohol 5a was achieved through the (1S)-camphanate
ester [(1S)-(ꢀ)-camphanoyl chloride (98% ee), NEt3, CH2Cl2]. Purity was
established by integration of the signals for the diastereotopic protons
Received: July 25, 2013
Published online on October 31, 2013
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 2413 – 2417 2417