The natural product brartemicin is a high affinity ligand for the carbohydrate-recognition domain of the macrophage receptor mincle

Article abstract of DOI:10.1039/c4md00512k

We demonstrate that the natural product brartemicin, a newly discovered inhibitor of cancer cell invasion, is a high-affinity ligand of the carbohydrate-recognition domain (CRD) of the C-type lectin mincle. Recent studies have revealed that mincle is a ke

Full text of DOI:10.1039/c4md00512k

MedChemComm
View Article Online
View Journal
CONCISE ARTICLE
The natural product brartemicin is a high affinity
ligand for the carbohydrate-recognition domain
of the macrophage receptor mincle†
Cite this: DOI: 10.1039/c4md00512k
Kristian M. Jacobsen,a Ulrik B. Keiding,ac Lise L. Clement,a Eva S. Schaffert,a
b
c
b
*
Neela D. S. Rambaruth, Mogens Johannsen, Kurt Drickamer‡
a
*
and Thomas B. Poulsen‡
We demonstrate that the natural product brartemicin, a newly discovered inhibitor of cancer cell invasion, is
a high-affinity ligand of the carbohydrate-recognition domain (CRD) of the C-type lectin mincle. Recent
studies have revealed that mincle is a key macrophage receptor for the mycobacterial virulence factor treha-
lose dimycolate (TDM), which is a glycolipid component of the mycobacterial cell wall. Major uncertainties,
however, remain concerning the mechanism of TDM-binding and subsequent signal transduction as well as
interplay of potential co-receptors. Due to the lipid nature of TDM, functional studies are difficult and soluble
mincle-ligands are therefore of significant interest. Brartemicin, together with designed analogs also
presented in this paper, may thus serve as useful molecular probes for future studies of mincle. Through
computational studies, we further provide an insight into the probable mode of binding of brartemicin.
Received 10th November 2014,
Accepted 19th December 2014
DOI: 10.1039/c4md00512k
www.rsc.org/medchemcomm
functional receptor complex.4 As TDM in vivo is constrained
in the mycobacterial membrane, it is not clear how much
Introduction
Trehalose-6,6-dimycolate (TDM, cord factor, Fig. 1) is an
abundant component of the mycobacterial cell wall. This
complex glycolipid is known to play pivotal roles in the patho-
genesis of mycobacteria such as Mycobacterium tuberculosis,
and it can stimulate granuloma formation in vivo.1 The
biomolecules involved in immune cell activation by TDM has
only recently become clear, with the discovery that mincle,
macrophage-inducible C-type lectin (CLEC4E), is a key media-
tor of the recognition of TDM by immune cells.2 This cell
surface recognition event initiates a signaling cascade through
FcRγ-Syk-Card9 running in parallel to signaling pathways
downstream of other important pattern-recognition receptors,
such as the Toll-Like Receptors (TLRs).2,3 Details about the
precise molecular mechanism of signal transduction, however,
are lacking, and in addition to mincle, a constitutively
expressed C-type lectin receptor MCL (CLEC4D) also appears
to be intimately involved in the cellular response to TDM and
it has been proposed that a mincle-MCL dimer constitutes the
of the structure is in fact exposed for interactions with the
receptorIJs). As a consequence of their immunostimulatory
activities, TDM, as well as synthetic analogs, are under investi-
gation as potential vaccine adjuvants3 and have further been
shown to block tumor formation and metastasis in mice
through an adjuvant mechanism.5
Brartemicin (1, Fig. 1) is a natural product isolated from
Nonomuraea sp that was recently reported to block matrigel-
invasion of cancer cells and which comprises a doubly esteri-
fied α,α-trehalose core structure similar to TDM.6 A number
of ester analogs of brartemicin have recently been reported
and their effects upon cancer cell invasion have been studied.
However, the direct binding targets of these compounds and the
molecular mechanisms by which they work remain obscure.7
a Chemical Biology Laboratory, Department of Chemistry, Aarhus University,
DK-8000, Aarhus C, Denmark. E-mail: thpou@chem.au.dk
b Department of Life Sciences, Imperial College, London SW7 2AZ, UK.
E-mail: k.drickamer@imperial.ac.uk
c Department of Forensic Medicine, Bioanalytical Unit, Aarhus University,
Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark
† Electronic supplementary information (ESI) available: Detailed experimental
procedures and compound characterization data. See DOI: 10.1039/c4md00512k
‡ These authors contributed equally to this work.
Fig.
1 Chemical structures of brartemicin and trehalose-6,6′-
dimycolate = TDM. The side chains of TDM displayed are those of the
α-mycolic acid subclass.
This journal is © The Royal Society of Chemistry 2015
Med. Chem. Commun.

View Article Online
Concise Article
MedChemComm
Realizing the potential importance of discovering high-
affinity, non-lipid ligands for mincle, we took note of the
significant structural similarity between brartemicin and TDM
(Fig. 1). The mycolic acid side chains in TDM are branched at
the α-carbon and hydroxylated on the β-carbon, so the part of
the structure in proximity to the trehalose core maps closely on
to the aromatic groups present in brartemicin. Here we report
our initial results demonstrating that brartemicin as well as
synthetic analogs are competent binders of mincle.
protection group strategy allowed quick access to the 6,6′-diol
that could undergo efficient double ester coupling with
carboxylic acid 6 using DCC and DMAP followed by global
debenzylation. Carboxylic acid 6 was prepared from orcinol
in 3 steps.
Through the same general route, we prepared two analogs
2 and 3. As a side note, both these compounds were reported
to have anti-invasive activity on cancer cells in the same
range as brartemicin.7 Structurally, these two analogs provide
preliminary information about the importance of the substi-
tution pattern on the aromatic rings for mediating binding
to the mincle CRD. In order to investigate the importance of
the dimeric structure, we prepared monoester derivative 4
starting from the monoacetate of protected α,α-trehalose
Results and discussion
Mincle binds ligands through an extracellular carbohydrate-
recognition domain (CRD). A recently published crystal struc-
ture of the CRD from bovine mincle in complex with the simple
disaccharide α,α-trehalose reveals that one glucose residue is
coordinated to a Ca2+-ion through the C4 and C5 hydroxyls that
anchors the ligand in the primary binding site. The second
glucose residue interacts with an adjacent, secondary binding
site with e.g. Glu135 and Arg182 forming key hydrogen bonds
to the C2′-hydroxyl group (Fig. 2).8 Moreover, in close vicinity to
the Ca2+-ion there is a hydrophobic groove that may be involved
in recognition of the mycolic acid portions of TDM. Due to
the presence of phenylalanine (Phe197 and Phe198) residues
using
a mitsunobu esterification protocol. Finally, we
designed a close structural analog, epi-brartemicin (5), having
altered stereochemistry at the glycosidic junction, by starting
the synthesis from α,β-trehalose (see experimental section
and ESI† for details). This compound would be expected to
have a drastically different conformation in the disaccharide
moiety, with potential deleterious effects on interaction with
the receptor. All compounds were rigorously purified by
HPLC before biochemical testing.
we hypothesized that this part of the CRD could potentially inter- Binding studies of putative ligands to
act strongly with ligands containing aromatic groups, such as
those found in brartemicin (1). In order to investigate the pro-
the CRD of mincle
posed interaction of brartemicin with mincle and to probe key
aspects of the structural requirements for binding, the natural
product and four additional analogs based on the brartemicin
structure were synthesized.
The collection of compounds was tested in competition
binding experiments with immobilized bovine mincle CRD8
(Fig. 3a). Strikingly, we found that brartemicin displayed
strong affinity for mincle (KI = 5.5 0.9 μM), which is approx-
imately 300-fold higher than the affinity of α,α-trehalose
itself. Analog 2 was equipotent (KI = 5.4 0.3 μM) and analog
3 had a two-fold reduced affinity (KI = 11.3 0.9 μM). Inter-
estingly, removal of one of the ester groups (monoester 4)
resulted in a significant reduction in binding affinity of roughly
30-fold (KI = 164 13 μM) compared to brartemicin. This loss
in affinity indicates that both ester groups may be directly involved
in interactions with the receptor or that one may be required
for locking the other ester in a binding-competent conformation.
Finally, epi-brartemicin shows an almost 100-fold loss
Synthesis of brartemicin and analogs
We prepared brartemicin (1) through a short synthesis
sequence from α,α-trehalose (Scheme 1). An orthogonal
in affinity (KI
=
440
70 μM) compared to natural
brartemicin and thus has only marginally higher affinity than
α,α-trehalose. This observation indicates strong specificity of
the binding site in mincle for the α,α-trehalose-core struc-
ture. Importantly, we found that brartemicin also binds with
high affinity to the CRD of human mincle9 (Fig. S1†).
Computational studies of brartemicin
and epi-brartemicin
To deepen the understanding of the binding mode of
these new, non-lipid mincle ligands we inspected the crystal
structure of bovine mincle co-crystallized with α,α-trehalose
(PDB entry 4KZV), in which both rings of α,α-trehalose make
contacts with the receptor (Fig. 2). The crystal structure was
Fig. 2 Structure of bovine mincle co-crystallized with α,α-trehalose
(PDB entry 4KZV). The Ca2+-ion in the primary binding site is coloured
magenta. A hydrophobic groove comprised of Leu172, Val173, Val194,
Phe197, and Phe198 is shown in the front.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2015

View Article Online
MedChemComm
Concise Article
Scheme 1 Syntheses of brartemicin (1), analogs 2–4, and epi-brartemicin (5). For detailed information about reagents and conditions, please
see the ESI.† DMF = N,N-dimethylformamide, Trt-Cl = trityl chloride, Pyr = pyridine, BnCl = benzyl chloride, BnBr = benzyl bromide, DCC =
dicyclohexylcarbodiimide, DMAP = 4-dimethylaminopyridine, DIAD = diisopropylazodicarboxylate, PPh3 = triphenylphosphine.
Fig. 3 a) Data for inhibition of mannose-conjugated serum albumin binding to the CRD from bovine mincle by brartemicin and analogs. KI-values
(average s.d. for three independent experiments) and affinities relative to α,α-trehalose. b) Top view and c) side view of highest scoring docking
pose of brartemicin with bovine mincle. The Ca2+ is coloured magenta.
prepared for modeling by Protein Preparation Wizard in
Maestro. We then performed molecular docking of brartemicin
in bovine mincle using the Glide algorithm,10 and identified
a population of binding capable conformations (Fig. 3b, c
and Fig. S2a†). In these conformations the disaccharide core
of brartemicin was found to overlap closely with the crystal
structure of α,α-trehalose in complex with mincle (Fig. S3†).
Furthermore, as initially predicted, the calculations provide
This journal is © The Royal Society of Chemistry 2015
Med. Chem. Commun.

View Article Online
Concise Article
MedChemComm
strong support for hydrophobic interactions between one
of the aromatic esters of brartemicin and the side chains of
residues Leu172, Val173, Val194, Phe197, and Phe198 that
together comprise the hydrophobic groove adjacent to the
Ca2+-ion. The size of the aromatic ring is closely matched with
the size of the groove. The second aromatic ester is oriented in
the opposite direction within the binding site in the vicinity of
Arg182 indicating possible π-cation interactions and in the
vicinity of Asp165/Asp183 where polar contacts are possible
(Fig. 3b, c). Overall the calculations suggest a possible position
of the second ester substituent and an overall elongated con-
formation of brartemicin in the binding site. The above men-
tioned interactions can explain the 290-fold increase in bind-
ing affinity of brartemicin compared to α,α-trehalose.
the benzyl-protected α,α-trehalose (50 mg, 0.06 mmol, 1.0
equiv.) and 2,4-bis-IJbenzyloxy)-6-methylbenzoic acid (43 mg,
0.12 mmol, 2.2 equiv.) in dry benzene and this was concen-
trated to dryness. This was repeated three times. The residue
was then dissolved in dry DCM (0.24 mL) in a flame-dried
schlenk flask containing a stirring bar and flushed with Ar.
The mixture was cooled to 0 °C and DCC (0.03 g, 0.14 mmol,
2.4 equiv.) and a catalytic amount of DMAP was added. The
reaction was allowed to reach rt and was stirred overnight
under an argon atmosphere. After 26 h the reaction mixture
was filtered to remove the urea product of DCC. The filtrate
was concentrated in vacuo. Purification of the crude prod-
uct by FC (SiO2, 15 × 2 cm, EtOAc/pentane 1 : 5 → EtOAc/
pentane 1 : 4 → EtOAc/pentane 1 : 2) yielded the diester (62 mg,
0.04 mmol, 71%) as a colorless oil. Rf 0.50 (EtOAc/pentane 1 : 5
Docking of epi-brartemicin (5) and the monoester (4) under
the same conditions as 1 suggests subtle changes in the bound
conformations, which could explain the reduced affinity of
these compounds. Whereas the ensemble of poses found
for brartemicin (Fig. S2a†) show a very well defined binding
mode, the ensembles of poses for both monoester (4) and
epi-brartemicin (Fig. S2b–c†) are much more diffuse suggesting
that key stabilizing interactions are diminished. In both these
structures, the glucose ring in the secondary binding site is
rotated significantly relative to the brartemicin structure. Most
notably, the altered glycosidic configuration in epi-brartemicin
is incompatible with an elongated conformation in the binding
site and key hydrogen bonds are absent. Surprisingly, the
computational studies of the monoester (4) indicate that in this
molecule the aromatic ester occupies an alternative cavity
in close vicinity to the hydrophobic groove (Fig. S2c†). The
reduced interactions in these structures reflect in lower docking
scores in accord with the relative affinities (Table S1, Fig. S4†).
Overall, our experiments and computational studies indicate
that brartemicin and epi-brartemicin constitute a pair of mole-
cules that will allow affinity correlations in future functional
assays. The structural requirements for immune cell activation
by TDM-mimetics are not well understood and the situation is
further complicated by the presence of additional receptors
for TDM, such as MCL, which directly impact the expression
of mincle.11 At the cytokine level, TDM-analogs with simple
saturated ester groups display disparate behaviour and seem to
show maximum activation with intermediate length chains.12
Due to the highly amphiphilic nature of these compounds, solu-
bility issues are unavoidable and may complicate interpretation
of some experiments. Our discovery that phenol-containing
esters, like brartemicin, are strong binders of mincle CRD that
are soluble at millimolar concentrations in aqueous media, will
enable the preparation of new classes of soluble TDM analogs
that may help shed light on the mechanism involved in
immune cell activation through the mincle pathway.
1
(CAM-stain)). H NMR (400 MHz, CDCl3) δ 7.39–7.14 (m, 50H),
6.36 (d, J = 1.8 Hz, 2H), 6.31 (d, J = 1.8 Hz, 2H), 5.10 (d, J =
3.5 Hz, 2H), 5.02–4.92 (m, 12H), 4.83–4.74 (m, 4H), 4.57–4.48
(m, 8H), 4.26 (d, J = 10.2 Hz, 4H), 4.00 (t, J = 9.6 Hz, 2H), 3.59
(t, J = 9.6 Hz, 2H), 3.43 (dd, J = 3.5, 9.6 Hz, 2H), 2.24 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 168.1, 160.4, 157.2, 138.9, 138.4,
138.2, 138.0, 136.7, 136.6, 128.7, 128.6, 128.5, 128.4, 128.4,
128.2, 128.2, 127.9, 127.8, 127.7, 127.6, 127.5, 126.8, 117.2,
108.2, 98.5, 93.7, 81.5, 79.2, 77.9, 75.5, 75.3, 72.6, 70.1, 69.4,
63.2, 20.1. IR (neat) νmax/cm−1 3031, 2926, 2864, 1725, 1602,
1586, 1497, 1453, 1263, 1157, 1089, 1070, 995, 733, 698. [α]D26.8
=
+55.2 (c 0.50, CHCl3).
6,6′-Bis-IJ2,4-dihydroxy-6-methylbenzoate)-α,α-trehalose (1).
The benzyl-protected brartemicin (33.1 mg, 21.4 μmol, 1.0
equiv.) was dissolved in MeOH/CHCl3 1 : 1 (5 mL) in a 50 mL
round-bottom flask and the flask was then purged with
argon. To the solution was added PdIJOH)2/C (23 mg, 20%,
32 μmol, 1.5 equiv.) and the mixture was stirred while
flushed with argon. The atmosphere in the flask was subse-
quently saturated with H2-gas from a balloon. After 6 h the
reaction had run to completion as judged by TLC and the
mixture was filtered to remove residues of the catalyst. The
solvents were removed in vacuo to yield brartemicin (1) as an
analytically pure off-white solid (13.5 mg, 21.0 μmol, 98%).
The product was purified further by semi-preparative C-18 RP
HPLC prior to binding assays (5% → 70% MeOH in H2O over
17 min, hold 3 min, then 70% → 100% MeOH, hold 1 min,
10 mL min−1, RT = 16.7 min, Phenomenex Luna 5u C18(2)
100 A, New Column, 250 × 10 mm). Rf 0.58 (EtOAc/MeOH/
1
H2O 4 : 1 : 1 (CAM-stain)). H NMR (400 MHz, CD3OD) δ 6.21
(d, J = 2.3 Hz, 2H), 6.15 (d, J = 2.3 Hz, 2H), 5.13 (d, J = 3.7 Hz,
2H), 4.58 (dd, J = 2.0, 12.0 Hz, 2H), 4.46 (dd, J = 4.9, 12.0 Hz,
2H), 4.21–4.17 (m, 2H), 3.83 (t, J = 9.6 Hz, 2H), 3.49 (dd, J =
3.7, 9.6 Hz, 2H), 3.43 (t, J = 9.6 Hz, 2H) 2.51 (s, 6H). 13C NMR
(100 MHz, CD3OD) δ 172.8, 166.3, 163.9, 144.9, 112.5, 105.6,
101.7, 95.6, 74.5, 73.2, 72.2, 71.3, 65.4, 24.9. HRMS IJm/z):
[M–H]− calcd for C28H33O17, 641.1723; found, 641.1723. IR
(neat) νmax/cm−1 3292, 2928, 2856, 1644, 1617, 1447, 1312,
1256. [α]2D6.5 = +78.8 (c 0.50, MeOH).
Experimental
Synthesis
2,3,4,2′,3′,4′-Hexa-IJbenzyloxy)-6,6′-bis-IJ2,4-bis-IJbenzyloxy)-6-
methylbenzoate)-α,α-trehalose. An azeotrope was formed with
2,3,4,2′,3′,4′-Hexa-IJbenzyloxy)-6,6′-bis-IJ2,4-bis-IJbenzyloxy)-6-
methylbenzoate)-α,β-trehalose. The hexabenzylated α,β-trehalose
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2015

View Article Online
MedChemComm
Concise Article
(0.050 g, 56.7 μmol, 1.0 equiv.) was added to a flame-dried flask
and an azeotrope was formed with dry benzene, concentrated
to dryness and subsequently placed under high vacuum. This
step was repeated once. The flask was purged with argon and
the compound was redissolved in dry THF/toluene (2 mL, 1 : 1)
and to this was added 2,4-bis-IJbenzyloxy)-6-methylbenzoic acid
(0.059 g, 170 μmol, 3.0 equiv.) and PPh3 (0.045 g, 170 μmol, 3.0
equiv.). The mixture was cooled to 0 °C in an ice bath. To the
cooled solution was added DIAD (33 μL, 170 μmol, 3.0 equiv.)
the reaction was stirred at 0 °C for 2.5 h until TLC showed full
conversion of starting material. The reaction mixture was
diluted with ice-water and extracted with EtOAc (3×), after
which the organic phases were washed with brine, dried over
Na2SO4 and concentrated in vacuo. The crude product was puri-
fied by FC (SiO2, 2 × 11 cm, EtOAc/pentane 10% → 20% →
25% → 30%). The product was isolated as a colorless oil
(0.043 g, 27 μmol, 48%). Rf 0.71 (EtOAc/heptane 1 : 1 (CAM-stain)).
1H NMR (400 MHz, CDCl3) δ 7.41–7.08 (m, 50H), 6.36 (d, J =
2.1 Hz, 1H), 6.34 (d, J = 2.2 Hz, 1H), 6.29 (d, J = 2.1 Hz, 2H),
5.11–5.02 (m, 4H), 5.02–4.98 (m, 1H), 4.98–4.91 (m, 5H), 4.87
(t, J = 10.8 Hz, 2H), 4.79–4.67 (m, 6H), 4.59–4.44 (m, 6H),
4.39 (dd, J = 12.0, 3.9 Hz, 1H), 4.29–4.18 (m, 2H), 3.96 (t, J =
9.3 Hz, 1H), 3.66 (t, J = 9.5 Hz, 1H), 3.62–3.56 (m, 2H),
3.53–3.47 (m, 1H), 3.46–3.35 (m, 2H), 2.23 (s, 3H), 2.20 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 167.9, 167.8, 160.3, 160.3,
157.0, 157.0, 138.7, 138.5, 138.5, 138.4, 138.3, 137.9, 137.8,
136.8, 136.7, 136.6, 136.5, 128.6, 128.6, 128.5, 128.5, 128.4,
128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.8, 127.7, 127.7,
127.6, 127.6, 127.5, 127.5, 127.5, 127.4, 127.3, 126.7, 126.6,
117.1, 116.8, 108.0, 107.9, 103.7, 99.1, 98.4, 98.3, 84.3, 81.6,
81.5, 79.6, 77.5, 75.6, 75.3, 75.1, 75.0, 74.4, 73.3, 73.0, 70.1,
70.0, 70.0, 69.9, 20.1, 20.0. HRMS IJm/z): [M + H]+ calcd for
C98H95O17, 1543.6564; found, 1543.6603. IR (neat) νmax/cm−1
3031, 2924, 1725, 1602, 1497, 1453, 1325, 1263, 1158, 1072,
1043, 1027, 909, 732, 695.
(100 MHz, CD3OD) δ 172.7, 172.3, 166.3, 166.1, 163.9, 163.7,
144.7, 112.6, 112.5, 105.9, 102.8, 101.7, 77.4, 75.5, 75.3, 74.9,
73.8, 71.6, 71.5, 65.2, 64.4, 24.9, 24.8. HRMS IJm/z): [M − H]−
calcd for C28H33O17, 641.1723; found, 641.1725. IR (neat)
νmax/cm−1 3391, 2920, 2851, 1645, 1456, 1315, 1263, 1167,
1080.
Binding studies
A biotin-tagged version of the CRD from bovine mincle was
prepared in a bacterial expression system as previously
described.8 An equivalent fragment of human mincle was
generated using an analogous system.9 Binding competition
assays, based on inhibition of binding of radioiodinated
Man31-bovine serum albumin to the biotin-tagged CRDs
immobilized on streptavidin-coated plates, were performed
as previously described.8 Results are reported as average
standard deviation for at least 3 independent experiments,
each performed in duplicate.
Docking experiments
The computational studies were performed with a crystal
structure of bovine mincle (PDB entry 4KZV). The pdb file
was loaded in the Schrödinger (2014-2) software Maestro 9.813
and prepared using Protein Preparation Wizard14 in accordance
with Schrödinger's guidelines. The structures of brartemicin
and epi-brartemicin were loaded into the software and mini-
mized.15 Using Glide 5.6,16 a ligand binding domain was defined
at the trehalose binding site and the minimized structures
were docked generating up to 25 poses of each structure in
the binding site. The structures were scored using Glide XP
algorithm.
Conclusions
6,6′-Bis-IJ2,4-dihydroxy-6-methylbenzoate)-α,β-trehalose (5).
The globally benzylated epi-brartemicin (0.042 g, 27 μmol,
1.0 equiv.) was dissolved in CHCl3/MeOH (1 : 1, 5 mL) and
the flask was purged with argon before PdIJOH)2/C (0.020 g,
20%, 28 μmol, 1.05 equiv.) was added. The flushing with
argon was continued until the atmosphere in the flask was
exchanged for H2-gas. The reaction mixture was stirred under
the H2-atmosphere for 4 h at which point TLC showed full
conversion of the starting material. The mixture was filtered
over celite with MeOH and solvents were evaporated. This
yielded the product quantitatively as an analytically pure
brownish solid. The product was subsequently purified by
semi-preparative HPLC before binding assays (5% → 65%
MeCN in H2O over 20 min, hold for 1 min, then 65% →
100% MeCN, hold for 2 min, RT = 5.5 min, 10 mL min−1,
Phenomenex Luna 5u CN 100 A, 250 × 10 mm 5 micron).
Rf 0.58 (EtOAc/MeOH/H2O 4 : 1 : 1 (CAM-stain)). 1H NMR
(400 MHz, CD3OD) δ 6.19–6.03 (m, 4H), 5.12 (d, J = 3.7 Hz,
1H), 4.61–4.49 (m, 2H), 4.48–4.35 (m, 2H), 4.28 (dd, J = 11.9,
2.6 Hz, 1H), 4.06 (dt, J = 10.2, 2.9 Hz, 1H), 3.72–3.57 (m, 2H),
3.50–3.35 (m, 5H), 2.48 (s, 3H), 2.43 (s, 3H). 13C NMR
In conclusion, we have discovered that the natural product
brartemicin is a new ligand for the CRD of mincle. The affin-
ity is higher than that of long chain alkyl (C6) diesters8 and
thus brartemicin constitutes a new lead structure for identify-
ing soluble mincle-ligands with even higher affinity for this
receptor. We also report that inverting one stereogenic center
at the core trehalose leads to a dramatic reduction in affinity.
We speculate that the molecules reported here will constitute
important molecular probes for future studies of mincle and
its role in innate immunity.
Acknowledgements
TBP is grateful for financial support from the Lundbeck
Foundation, the Danish Cancer Society and the Carlsberg
Foundation. KD acknowledges support from Biotechnology
and Biological Sciences Research Council grant BB/K007718/1.
Notes and references
1 D. G. Russell, Nat. Rev. Microbiol., 2007, 5, 39.
This journal is © The Royal Society of Chemistry 2015
Med. Chem. Commun.

View Article Online
Concise Article
MedChemComm
2 E. Ishikawa, T. Ishikawa, Y. S. Morita, K. Toyonaga,
H. Yamada, O. Takeuchi, T. Kinoshita, S. Akira, Y. Yoshikai
and S. Yamasaki, J. Exp. Med., 2009, 206, 2879.
9 S. A. F. Jégouzo, E. C. Harding, O. Acton, M. J. Rex,
A. J. Fadden, M. E. Taylor and K. Drickamer, Glycobiology,
2014, 24, 1291.
3 H. Schoenen, B. Bodendorfer, K. Hitchens, S. Manzanero,
K. Werninghaus, F. Nimmerjahn, E. M. Agger, S. Stenger,
P. Andersen, J. Ruland, G. D. Brown, C. Wells and R. Lang,
J. Immunol., 2010, 184, 2756.
4 A. Lobato-Pascual, P. C. Saether, S. Fossum, E. Dissen and
M. R. Daws, Eur. J. Immunol., 2013, 43, 3167.
5 H. Yamamoto, M. Oda, M. Nakano, N. Watanabe, K. Yabiku,
M. Shibutani, M. Inoue, H. Imagawa, M. Nagahama, S. Himeno,
K. Setsu, J. Sakurai and M. Nishizawa, J. Med. Chem., 2013, 56, 381.
6 Y. Igarashi, T. Mogi, S. Yanase, S. Miyanaga, T. Fujita,
H. Sakurai, I. Saiki and A. Ohsaki, J. Nat. Prod., 2009, 72, 980.
7 Y.-L. Jiang, S. Miyanaga, X.-Z. Han, L.-Q. Tang, Y. Igarashi,
I. Saiki and Z.-P. Liu, J. Antibiot., 2013, 66, 531.
8 H. Feinberg, S. A. F. Jégouzo, T. J. W. Rowntree, Y. Guan,
M. A. Brash, M. E. Taylor, W. I. Weis and K. Drickamer,
J. Biol. Chem., 2013, 288, 28457.
10 R. A. Friesner, R. B. Murphy, M. P. Repasky, L. L. Frye,
J. R. Greenwood, T. A. Halgren, P. C. Sanschagrin and
D. T. Mainz, J. Med. Chem., 2006, 49, 6177.
11 Y. Miyake, K. Toyonaga, D. Mori, S. Kakuta, Y. Hoshino,
A. Oyamada, H. Yamada, K.-I. Ono, M. Suyama, Y. Iwakura,
Y. Yoshikai and S. Yamasaki, Immunity, 2013, 38, 1050.
12 B. L. Stocker, A. A. Khan, S. H. Chee, F. Kamena and
M. S. M. Timmer, ChemBioChem, 2014, 15, 382.
13 Maestro, version 9.8, Schrödinger, LLC, New York, NY, 2014.
14 Schrödinger Suite 2014-2 Protein Preparation Wizard;
Epik version 2.8, Schrödinger, LLC, New York, NY, 2014;
Impact version 6.3, Schrödinger, LLC, New York, NY, 2014;
Prime version 3.6, Schrödinger, LLC, New York, NY, 2014.
15 MacroModel, version 10.4, Schrödinger, LLC, New York, NY,
2014.
16 Glide, version 6.3, Schrödinger, LLC, New York, NY, 2014.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2015