Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity

Article abstract of DOI:10.1039/C8CC07322H

Mincle, expressed in activated phagocytes, recognizes the lipid ligand to activate the innate immune system. We have synthesized glycerol derivatives possessing simple alkyl chains or aromatic rings and elucidated their structure-activity relationships us

Full text of DOI:10.1039/C8CC07322H

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Synthesis of glycerolipids containing simple linear
acyl chains or aromatic rings and evaluation of
their Mincle signaling activity†
Cite this: Chem. Commun., 2019,
55, 711
Received 9th September 2018,
Accepted 8th December 2018
Takanori Matsumaru,*ab Risa Ikeno,a Yusuke Shuchi,a Toshiki Iwamatsu,a
b
a
Takashi Tadokoro,a Sho Yamasaki,c Yukari Fujimoto,
Atsushi Furukawa
and
a
DOI: 10.1039/c8cc07322h
Katsumi Maenaka
*
rsc.li/chemcomm
Mincle, expressed in activated phagocytes, recognizes the lipid glucose monoesters14,17,18 and mannose monoesters18 also
ligand to activate the innate immune system. We have synthesized exhibit Mincle agonist activity. Additionally, glycerolipids can
glycerol derivatives possessing simple alkyl chains or aromatic rings exhibit Mincle-mediated signaling activity. Glycerol monobehenate
and elucidated their structure–activity relationships using a Mincle- (GroMB)19 and glycerol monocorynomycolate (GroMCM)14 are
mediated signaling assay. The activity depends on the length of the glycerolipids of the typical Mincle ligands whose acyl moieties
simple acyl chains of the glycerol derivatives.
consist of behenic acid and mycolic acid, respectively. Of note,
glycerolipids exhibit only human Mincle-mediated signaling
Pattern recognition receptors (PRRs), which are expressed in activity, while other glycolipid types of Mincle ligands including
phagocytes such as macrophages and dendritic cells, play key TDM demonstrate both human and murine Mincle signaling
roles in the innate immune system. PRRs include Toll-like activity. However, in contrast to glycolipids, the molecular
receptors (TLRs),1,2 Nucleotide-binding Oligomerization Domain mechanism of the Mincle-mediated signaling of glycerolipids
(NOD)-like receptors (NLRs),3,4 and C-type lectin receptors (CLRs), is largely unknown. In this study, we focused on glycerolipids as
which possess calcium ions in a carbohydrate recognition domain Mincle ligands and elucidated the structure–activity relation-
(CRD).5,6 Mincle (also called Clec4e and Clecsf9) is one of the CLRs ships between the acyl moiety of glycerolipids and Mincle
expressed in activated phagocytes.7 Mincle has been known to agonist activity. We designed glycerol monoesters whose acyl
recognize dead cells through the binding to SAP1308 and trehalose chain lengths are from C22 to C30. Glycerol monoacyl ester (C22)
dimycolate (TDM) as an agonist.9 Because TDM is one of the is glycerol monobehenate (GroMB), and glycerol monoacyl
glycolipids in the cell wall of Mycobacterium tuberculosis, Mincle ester (C30) is reported as a natural product found in wheat
is being assessed to serve as a sensor of M. tuberculosis. The bran20 and Abaca (Musa textilis) leaf fibers.21 Meanwhile, we
recognition of ligands such as TDM leads to the activation of also designed other glycerolipids associated with aromatic rings
NF-kB via SYK-Card9-Bcl10-MALT1 signaling through ITAM- based on brartemicin, which is a trehalose-containing natural
containing adaptor molecule FcRg.10 NF-kB induces the transcrip- compound that has signaling activity (Fig. 1).
tion of cytokines to shape the development of T cells into various
effector helper T cells such as TH1 and TH17 subtypes.
First, we focused on the length of the acyl chain of glycerolipids.
Previously, we have demonstrated that the length of the acyl chain
To date, several Mincle ligands have been reported.11,12 in trehalose monoesters (C8, C10, C12) influences the binding activity
Typical Mincle ligands are trehalose esters such as TDM and of Mincle using surface plasmon resonance (SPR) and demon-
trehalose dibehenate (TDB).9,10,13 Trehalose dicorynomycolates strated that a minimum lipid length of C10 was required for the
(TDCMs), whose acyl moieties consist of corynomycolic acid binding.22 The lipid structure–activity relationships of various
from Corynebacteria14 and brartemicin and its derivatives,15,16 Mincle ligands such as trehalose diesters,23 trehalose mono-
are also known as Mincle agonists. Other glycolipids such as esters,24 iso-branched trehalose diesters,25 b-gentiobiosyl diacyl-
glycerides26 and glucose mono-corynomycolate27 have been
a Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo, Hokkaido 060-0812, Japan. E-mail: maenaka@pharm.hokudai.ac.jp
b Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,
Yokohama, Kanagawa 223-8522, Japan. E-mail: matsumaru@keio.chem.ac.jp
c Department of Molecular Immunology Research Institute for Microbial Diseases,
and Laboratory for Molecular Immunology, Osaka University, 3-1 Yamadaoka,
Suita, Osaka 565-0871, Japan
reported, but the lipid length–signaling activity relationship of
glycerolipids is still unclear. In order to investigate the lipid
length of glycerolipids–signaling activity relationship, we prepared
glycerolipids possessing C22 (GroMB), C24, C26, C28 and C30 acyl
chains. The appropriate carboxylic acid was converted to acid
chloride and subjected to isopropylidene glycerol to obtain
acylated isopropylidene glycerol. The glycerolipids were obtained
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc07322h
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expected to interact with the hydrophobic groove were prepared.
Moreover, we synthesized glycerolipids possessing aromatic
rings with additional alkyl chains. These compounds were
prepared by the same method as in Fig. 2A and C.
Mincle-mediated signaling activity was evaluated using the
nuclear factor of activated T cells (NFAT)-green fluorescent
protein (GFP) reporter assay, which has been widely used for
the functional evaluation of Mincle ligands.9,14,16,19,27,30–33 The
reporter cells expressing human or murine Mincle with FcRg
were stimulated with plate-coated derivatives. The signaling
activity was determined by measuring GFP production. The
positive control, TDM, induced the activation of both human
and murine Mincles in a dose-dependent manner. Regarding
the series of glycerol derivatives possessing different lengths of
Fig. 1 The structures of glycerolipids containing linear acyl chains or
aromatic rings.
by the deprotection of the acetal group (Fig. 2A, method A). acyl chains, the signaling intensity increased with the length of
Another method is condensation with the appropriate carboxylic acyl chains. Thus, the lengths of the acyl chains of the glycero-
acid and isopropylidene glycerol, following the deprotection of lipids have a vast impact on the signaling activity among acyl
isopropylidene to provide glycerolipids (Fig. 2A, method B). chains from C22 to C30 (Fig. 3A). Compared to 20S-GroMCM,
Unfortunately, these synthetic methods gave a mixture of the which is known as the best Mincle agonist of glycerol derivatives
desired compound with the migrated 2-acyl compound in each with chirality at the 20-position and corynomycolate as the acyl
glycerolipid. The proportions of the 2-acyl compound in each moiety, GroC30 also demonstrated potent Mincle-mediated
inseparable mixture are approximately 4.1–13.8% detected by signaling activity.
1H NMR.
On the other hand, glycerolipids containing an aromatic
Next, we designed novel glycerolipids with aromatic rings ring did not show any detectable signaling activity (Fig. 3B).
instead of an acyl chain to increase the affinity with human Recently, lipidated brartemicins possessing trehalose as a head
Mincle. Actually, brartemicin, a natural product isolated from group and various lipid moieties containing aromatic rings were
Nonomuraea sp., is a trehalose conjugated with two aromatic synthesized and exhibited Mincle-mediated signaling activity.16
rings (Fig. 2B),28 which bound to Mincle with high affinity.15 The lipid moiety of compound 5b is similar (less than one
A crystallographic study of the brartemicin complex of bovine carbon in the alkyl chain) to the lipid moiety of brartemicin
Mincle29 revealed that one of the aromatic rings interacts with derivatives but did not exhibit signaling activity. A previous
the hydrophobic groove composed of Leu172, Val173, Val194, study showed that one of the two aromatic rings of brartemicin
Phe197 and Phe198 (corresponding to residues Ile173, Ala174, is located in a hydrophobic pocket and the other interacts with
Val195, Phe198 and Leu199 in human Mincle), while the calcium ion neighboring Arg182 (corresponding to Arg183 in
trehalose moiety is located at the calcium ion binding site. human Mincle) p–cation interactions.15 Our derivatives have
Thus, the benzoyl and phenyl acetic acid types of lipid moieties only one aromatic ring and thus possibly could not create
Fig. 2 (A) Synthesis of 2a–2e, (B) the structure of brartemicin, and (C) synthesis of 5a and b and 8a and b.
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Fig. 4 Model of the glycerolipid C30–Mincle complex.
To examine the lipid recognition site, we evaluated the Mincle-
mediated signaling activity for glycerolipid C30 using reporter
cells expressing either the hMincle R183V mutant or the hMincle
MD chimera whose hydrophobic region (residues 195–202)
is replaced by the corresponding region of Dectin-2 (residues
192–199).22 2e exhibited reduced NFAT signaling activity for both
mutants as glycolipids we previously reported (Fig. 3C). Thus, this
result suggests that glycerolipids interact with the hydrophobic
groove similarly to glycolipids, consistent with the model of the
glycerolipid C30–Mincle complex (Fig. 4).
In conclusion, we have synthesized two types of glycerolipids
possessing simple linear acyl chains or aromatic rings and
evaluated their Mincle-mediated signaling activity. The glycero-
lipids having an aromatic ring with an alkoxyl chain do not
exhibit signaling activity, in contrast to lipidated brartemicin
derivatives with similar lipid moieties. On the other hand,
glycerolipids possessing simple acyl chains harbor the relation-
ships of the signaling activity with C22–C30 acyl chain length. To
the best of our knowledge, this is the first report on the
relationships of the length of the lipid moiety of glycerolipids
Fig. 3 The evaluation of (A) monoacyl glycerolipids, and (B) the glycero-
lipids containing aromatic rings using NFAT-GFP reporter cells expressing
human or murine Mincle, or FcRg alone. (C) The evaluation of 2e using
reporter cells expressing mutant Mincles.
p–cation interactions with Arg, resulting in the lack of signaling and the signaling activity. In particular, glycerol C30 showed
activity. potent signaling activity. This agonist can be one of the best
In order to understand the interaction between Mincle and templates that can be easily utilized in and expanded to various
glycerol derivatives, we successfully built complex models with areas of chemical biology research and drug discovery. Cur-
the glycerol derivatives including glycerol C30 using the crystal rently, the structural study of the binding modes of human and
structure of bovine Mincle bound to trehalose (Fig. 4, details in murine Mincles with lipid moieties of conjugated lipids is
the ESI†). The model showed that the ligands could be set up underway.
for their glycerol moiety to interact with calcium ions and for
This research was partially supported by the Platform Project
the part of their acyl chains neighboring glycerol to accommodate for Supporting Drug Discovery and Life Science Research (Basis for
into a hydrophobic groove consisting of Ile173, Ala174, Phe198 Supporting Innovative Drug Discovery and Life Science Research
and Leu199, consistent with previous reports.16,18 However, the (BINDS)) from AMED under Grant Number 18am0101093j and the
hydrophobic groove only has sufficient space to accommodate Middle Molecule Project from AMED under Grant Number
C12 carbons of the acyl chain at most. The distal portion of 18ae0101047h, Hokkaido University, the Global Facility Center
the lipid moiety may be exposed to the outside and/or weakly (GFC), the Pharma Science Open Unit (PSOU), funded by MEXT
fit onto somewhat hydrophobic patches of Mincle. On the under the ‘‘Support Program for Implementation of New Equip-
other hand, Arg183 could interact with aromatic rings next ment Sharing System’’, the Hokkaido University Biosurface
to or around the glycerol moiety in the case of ligands project, the Takeda Science Foundation, the Akiyama Life
possessing them.
Science Foundation and a Grant-in-Aid for Scientific Research
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