Corynomycolic acid-containing glycolipids signal through the pattern recognition receptor Mincle

Article abstract of DOI:10.1039/c5cc00085h

An enantioselective synthesis of (+)-corynomycolic acid, and its elaboration to esters of trehalose, glucose and glycerol, is described. Trehalose dicorynomycolate and trehalose monocorynomycolate activate human and mouse Mincle as effectively as trehalose dicorynomycolate (cord factor). Glucose monomycolate is revealed to be a potent activator of both mouse and human Mincle. Glycerol monocorynomycolate signals through human Mincle, with the activity predominantly residing in the 2′S-isomer.

Full text of DOI:10.1039/c5cc00085h

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Corynomycolic acid-containing glycolipids signal
through the pattern recognition receptor Mincle†
Cite this: Chem. Commun., 2015,
1, 5100
5
a
a
b
b
Phillip L. van der Peet, Christian Gunawan, Shota Torigoe, Sho Yamasaki* and
a
Received 6th January 2015,
Accepted 14th February 2015
Spencer J. Williams*
DOI: 10.1039/c5cc00085h
www.rsc.org/chemcomm
An enantioselective synthesis of (+)-corynomycolic acid, and its
elaboration to esters of trehalose, glucose and glycerol, is described.
Trehalose dicorynomycolate and trehalose monocorynomycolate
activate human and mouse Mincle as effectively as trehalose
dicorynomycolate (cord factor). Glucose monomycolate is revealed
to be a potent activator of both mouse and human Mincle. Glycerol
monocorynomycolate signals through human Mincle, with the activity
0
predominantly residing in the 2 S-isomer.
Macrophage inducible C-type lectin (Mincle) is a germline-
encoded pattern recognition receptor that provides immune
1
sensing of bacterial and fungal pathogens. The discovery that
the Mycobacterium tuberculosis glycolipid trehalose dimycolate
(TDM, cord factor) signals through Mincle has led to a surge of
activity to understand how PRR-costimulation through this
2
–4
receptor can alter immune responses (Fig. 1). Ligand binding
to Mincle leads to phosphorylation of the immunoreceptor
tyrosine-based activation motif of the Fc receptor g (FcR g)-chain
molecule, and activation of nuclear factor kappa-B (NF-kB) via
5,6
Card9–Bcl10–MALT1 signalosomes. NF-kB drives transcription
of cytokines that shape the development of na ¨ı ve T cells into
various effector helper T cell (Th) subtypes. In particular, there is
growing interest in the development of Mincle ligands as vaccine
adjuvants to provide Th1/Th17 biased immune responses.
Emerging structure-activity relationships of trehalose-based for Mincle. Treatment of TDM with trehalase, which ostensibly
glycolipids as ligands that signal through Mincle have revealed cleaves the glycolipid into two molecules of glucose monomycolate,
Fig. 1 Structures of
a representative mycobacterial mycolic acid,
(
+)-corynomycolic acid and various esters thereof.
7
1
0
2
that trehalose dibehenate (TDB) possesses activity similar to resulted in loss of Mincle activation. Glycerol monomycolate
2
,3
TDM, and that monoacyl trehalose derivatives also possess the (GroMM) is also a Mincle ligand, but is selective for binding to
8
11
capacity to activate through Mincle, with longer chain lengths the human, and not the mouse form of the receptor.
generally providing greater potency of activation in Mincle The reported structure-activity relationships for Mincle
reporter assays, which correlates with direct binding affinities activation have in some cases used heterogeneous glycolipids
9
2
,3,11
isolated from bacterial sources,
structure(s) of the glycolipid is unclear. On the other hand
studies performed using authentic synthetic materials,
which represent the ‘gold standard’ for immunological studies,
have utilized much simpler structures bearing straight chain acyl
groups, and relating the measured activities to homogeneous
full length natural materials has not been possible. Mycolic acids
for which the precise
a
b
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Victoria 3010, Australia. E-mail: sjwill@unimelb.edu.au
Division of Molecular Immunology, Medical Institute of Bioregulation,
8
–10,12
Kyushu University, Fukuoka 812-8582, Japan. E-mail: yamasaki@bioreg.kyushu-u.ac.jp
Electronic supplementary information (ESI) available: Full synthetic proce-
†
dures, characterisation of novel compounds and immunological methods and
data. See DOI: 10.1039/c5cc00085h
5100 | Chem. Commun., 2015, 51, 5100--5103
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from corynebacteria (termed corynomycolic acids) are shorter
1
3
in length and simpler in structure than mycobacterial mycolic
acids and constitute synthetically accessible targets whose
relationships to the authentic mycobacterial materials is more
readily appreciated. In this work we have developed a total
synthesis of a range of corynebacterial glycolipids, using homo-
geneous synthetic (+)-corynomycolic acid. These glycolipids
were used to study activation of a reporter cell line by human
and mouse Mincle, revealing a comprehensive structure-activity
relationship that provides new leads for the development of
simpler ligands for Mincle activation.
A range of approaches have previously been reported for the
1
4
15–17
synthesis of racemic and enantiopure
corynomycolic acids.
Our strategy for the synthesis of (+)-2R,3R-corynomycolic acid
aimed to set the crucial anti stereochemistry using the boron-
mediated aldol reaction of chiral ketones developed by the
1
8
Paterson group (Scheme 1). Paterson reported that aldol
reactions of sterically-demanding boron enolates of ketones
derived from 2-O-benzoyl lactate undergo aldol reactions to
1
8
afford anti-configured products. While this approach has been
0
Scheme 2 Synthesis of TDCM (3), TMCM (4), GMCM (6), 2 R-GroMCM
0
0
0
widely applied using lactate-derived ketones bearing small (2 R-8), and 2 S-GroMCM (2 S-8).
it has seen only limited application to larger
groups. The ketone 12 was prepared by treatment of (S)-ethyl
1
8,19
a-substituents,
1
8
lactate derived Weinreib amide 10 with pentadecylmagnesium over the two steps. The relative anti-stereochemistry was defined
bromide, followed by benzoylation (Scheme 2). Boron-mediated by conversion to the acetonide, which displayed a characteristic
1
8
anti-aldol reaction of 12 with palmitaldehyde afforded the large coupling constant ( J = 10.5 Hz) between the a- and
2
1
aldol adduct 13 in 76% yield. The relative and absolute stereo- b-protons. The absolute stereochemistry was then defined
chemistry was assumed as 2R,3R, and subsequently confirmed by comparison of the optical rotation with literature data for
2
4
16
as described below. Completion of the synthesis of (+)-2R,3R- (+)-corynomycolic acid [a]
D 3 D
8.51 (c 1.02, CHCl ), lit. [a] 7.81
1
4
corynomycolic acid required manipulation of 13 to a carboxylic (c 1.0, CHCl
acid. In practice this was best achieved through sequential While various dehydrative coupling approaches have been
reduction with NaBH and then debenzoylation (NaOMe/MeOH) utilized for the formation of sugar esters with various mycolic
to give triol 14. In this way epimerization at the ketone a-position acids,
3
).
4
1
7,22–24
an alternative approach utilizes substitution of a
of 14 followed by leaving group by an alkali salt, preferably cesium, of a mycolic
2
0
was avoided. Glycol cleavage (NaIO
Lindgren oxidation, afforded (+)-corynomycolic acid (9) in 91% acid.
tion of 15 or 17 with 9 in the presence of CsHCO in THF/DMF
4 2
/SiO )
2
5–27
TDCM (3) and TMCM (4) were prepared by substitu-
2
8
3
followed by deprotection (Scheme 2). Reaction of 9 with the known
25
tosylate 19 under the same conditions, followed by deprotection,
afforded GMCM (6). To our knowledge this comprises the first
complete report of the synthesis of enantiopure GMCM, a com-
pound that appears to have only been synthesized as a mixture of
2
3,29
diastereoisomers from racemic corynomycolic acid.
The two
0
C2 epimeric forms of GroMCM were prepared through a similar
strategy. Thus, known (R)-1,2-isopropylideneglycerol mesylate 21
or (S)-1,2-isopropylideneglycerol mesylate 23 were coupled with 9
30
31
in the presence of CsHCO
3
to afford 22 and 24, respectively. TFA
0
treatment of 22 or 24 afforded the GroMCM epimers 2 R-8 and
0
2
S-8 in 98% and 94% yields, respectively. It appears that this
work represents the first synthesis of the enantiopure GroMCM
epimers, with previous syntheses having utilized racemic
corynomycolic acid affording these epimers as mixtures of
2
4,32
27
anti-diastereoisomers,
or providing no synthetic details.
Activation through Mincle was assessed using an nuclear
factor of activated T cells (NFAT)-green fluorescent protein (GFP)
reporter assay that has been previously used to identify Mincle
Scheme 1 Enantioselective total synthesis of (+)-2R,3R-corynomycolic
acid (9).
6,11,33
ligands.
NFAT-GFP reporter cells transfected with FcRg alone
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its ability to signal through Mincle. Evidence for direct binding
of activating ligands was obtained by an enzyme-linked immuno-
sorbent assay (ELISA) using mouse and human Mincle Ig fusions
(see ESI†).
Previous studies have identified that GroMM selectively
1
1
activates through human, but not mouse Mincle. Here we
0
confirm and extend this result to identify the 2 S-stereoisomer
as that which predominantly signals through human Mincle
(Fig. 2B). This result is the first to demonstrate that Mincle has
the ability to recognize differences in stereochemistry of the
0
mycolic acid head-group. The specific recognition of the 2 S
isomer is opposite to the selectivity displayed by these glycolipids
0
for CD1b-restricted activation of T cells, where 2 R-GroMCM was
27
0
0
identified as a more potent antigen. As both 2 R- and 2 S-GroMM
27
were detected in lipid extracts of M. bovis BCG, these results
show that CD1b-restricted presentation of GroMM to T cells, and
Mincle C-type lectin receptor provide complementary recognition
of the two natural diastereoisomers of this bacterial glycolipid.
In summary, we have completed the total synthesis of single
stereoisomers of various corynomycolate esters and established
their ability to signal through Mincle. These results extend early
work by Nishizawa et al. who demonstrated that TDCM and
TMCM exhibit pro-inflammatory activity through release of
3
6
IL-6, although signalling through Mincle was not considered.
Surprisingly, GMCM (6) also signals through Mincle, albeit less
potently than TDCM, a result that provides a new lead for the
development of simplified vaccine adjuvants. However, simpli-
Fig. 2 NFAT-GFP reporter cells expressing either human Mincle/FcRg or
mouse Mincle/FcRg as well as those expressing FcRg alone were tested for
their reactivity to (A) TDM (1), (+)-corynomycolic acid (CMA, 9), TDCM (3),
0
0
TMCM (4), GMCM (6) and, or (B) TDM, 2 R- and 2 S-GroMCM (8). Assays fying the lipid chain to form glucose 6-monobehenate yielded
were performed in triplicate; the mean values and standard deviations are an inactive compound. Further, we reveal that the ability of
shown. Replots of the data using quantities expressed in nmol are available
GroMCM to signal through human but not mouse Mincle
in the ESI.†
0
resides predominantly in the 2 S-isomer.
This work was supported by the Australian Research Council
or together with human or mouse Mincle were stimulated with and the JSPS Grant-in-Aid for Scientific Research. We thank
plate-coated mycobacterial TDM, and the NFAT-dependent Profs Mark Rizzacasa and Ian Paterson for helpful advice.
activation of cells was monitored by the detection of GFP-
Notes and references
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