Lipidated Brartemicin Analogues Are Potent Th1-Stimulating Vaccine Adjuvants

Article abstract of DOI:10.1021/acs.jmedchem.7b01468

Effective Th1-stimulating vaccine adjuvants typically activate antigen presenting cells (APCs) through pattern recognition receptors (PRRs). Macrophage inducible C-type lectin (Mincle) is a PRR expressed on APCs and has been identified as a target for Th1-stimulating adjuvants. Herein, we report on the synthesis and adjuvanticity of rationally designed brartemicin analogues containing long-chain lipids and demonstrate that they are potent Mincle agonists that activate APCs to produce inflammatory cytokines in a Mincle-dependent fashion. Mincle binding, however, does not directly correlate to a functional immune response. Mutation studies indicated that the aromatic residue of lead compound 9a has an important interaction with Mincle Arg183. In vivo assessment of 9a highlighted the capability of this analogue to augment the Th1 response to a model vaccine antigen. Taken together, our results show that lipophilic brartemicin analogues are potent Mincle agonists and that 9a has superior in vivo adjuvant activity compared to TDB.

Full text of DOI:10.1021/acs.jmedchem.7b01468

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Article
Lipidated brartemicin analogues are potent Th1-stimulating vaccine adjuvants
Amy J. Foster, Masahiro Nagata, Xiuyuan Lu, Amy T. Lynch, Zakaria Omahdi,
Eri Ishikawa, Sho Yamasaki, Mattie S. M. Timmer, and Bridget L. Stocker
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01468 • Publication Date (Web): 30 Dec 2017
Downloaded from http://pubs.acs.org on December 31, 2017
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Lipidated brartemicin analogues are potent Th1ꢀ
stimulating vaccine adjuvants
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a
b
b,c,d
a
b,c,d
Amy J. Foster, Masahiro Nagata, Xiuyuan Lu,
Amy T. Lynch, Zakaria Omahdi,
Eri
b,d
b,c,d,e,
a,
a,
Ishikawa, Sho Yamasaki,
* Mattie S. M. Timmer, * and Bridget L. Stocker *
a.
School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600,
Wellington 6140, New Zealand. Email: bridget.stocker@vuw.ac.nz; mattie.timmer@vuw.ac;
yamasaki@biken.osakaꢀu.ac.jp.
b.
Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka
University, Suita, 565ꢀ0871, Japan.
c.
Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University,
Fukuoka 812ꢀ8582, Japan
d.
Laboratory of Molecular Immunology, Immunology Frontier Research Center, Osaka
University, Suita, 565ꢀ0871, Japan
e.
Division of Molecular Immunology, Medical
Keywords: Mincle, Vaccine adjuvant, Glycolipid
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Abstract
Effective Th1ꢀstimulating vaccine adjuvants typically activate antigen presenting cells (APCs)
through pattern recognition receptors (PRRs). Macrophage inducible Cꢀtype lectin (Mincle) is a
PRR expressed on APCs and has been identified as a target for Th1ꢀstimulating adjuvants.
Herein, we report on the synthesis and adjuvanticity of rationally designed brartemicin analogues
containing longꢀchain lipids and demonstrate that they are potent Mincle agonists that activate
APCs to produce inflammatory cytokines in a Mincleꢀdependent fashion. Mincle binding,
however, does not directly correlate to a functional immune response. Mutation studies indicated
that the aromatic residue of lead compound 9a has an important interaction with Mincle Arg183.
In vivo assessment of 9a highlighted the capability of this analogue to augment the Th1 response
to a model vaccine antigen. Taken together, our results show that lipophilic brartemicin
analogues are potent Mincle agonists and that 9a has superior in vivo adjuvant activity compared
to TDB.
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Introduction
Adjuvants have traditionally been used to enhance the adaptive immune response to a vaccine,
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with most current vaccines providing protection primarily through humoral (Th2) immunity. A
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strong and enduring antibody response is suitable for protection against many pathogens
however, humoral immunity is insufficient to confer protection against diseases such as malaria,
2
ꢀ4
tuberculosis, and leishmaniasis. In these cases, adjuvants stimulating acquired cellular (Th1)
2
,5
immunity, which is characterised by the release of interferon (IFN)ꢀγ, have proven effective.
Effective Th1ꢀstimulating adjuvants often engage the innate immune system by activating
4
,6
pattern recognition receptors (PRRs) on professional antigen presenting cells (APCs). Pathogen
associated molecular patterns (PAMPs) bind to PRRs, with the specificity of the ensuing immune
response being directed by the type of PRR activated and the structure of each specific PAMP.
3
,4
There has been much interest in the Tollꢀlike receptors (TLRs) as targets for vaccine adjuvants,
however more recently, Macrophage inducible Cꢀtype lectin (Mincle, Clec4e, or Clecf9) has
7
,8
been identified as a PRR on the surface of macrophages and dendritic cells (DCs) and is a
9
10ꢀ11
promising new target for vaccine development. Mincle is activated by a number of PAMPs,
including the Mycobacterium tuberculosis cell wall glycolipid trehalose dimycolate (TDM, 1,
Figure 1), with Mincle activation leading to the induction of the FcRγꢀSykꢀCard9ꢀBcl10ꢀMalt1
12ꢀ15
signalling axis and a Th1ꢀpolarised immune response.
This response involves ligand binding
and activation of Mincle, followed by dual phosphorylation of the immunoreceptor tyrosineꢀ
based activation motif (ITAM) of the Fc receptor γꢀchain (FcRγ) which then becomes a highꢀ
15
affinity docking site for Syk (spleen tyrosine kinase) family proteins. The subsequent formation
of the Card9ꢀBcl10ꢀMalt1 complex then leads to the activation of transcription factors that
mediate the production of Th1 cytokines.
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In addition to TDM, a number of synthetically derived ligands also bind and activate Mincle.
Notably, trehalose dibehenate (TDB, 2), a synthetic analogue of TDM, activates Mincle with a
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comparable efficacy to TDM.
TDB has been formulated into dimethyldioctadecylꢀ
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ammonium bromide (DDA):TDB cationic liposomes leading to a mixed Th1 and Th17 immune
13,22
response, as evidenced by IFNꢀγ and ILꢀ17 production, respectively.
These liposomes have
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25,26
shown efficacy in vaccination studies for HIV
compounds have been shown to bind to and activate Mincle,
mannose esterified at the 6ꢀposition with αꢀbranched fatty acids (GlcC14C18, ManC14C18)
and tuberculosis.
Various additional
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7ꢀ28
and in particular glucose and
19
exhibited agonist activity similar to TDM. There has also been interest in the ability of the
2
9,30
natural product brartemicin (3) to bind and activate Mincle,
however, a functional Mincleꢀ
dependent immune response to this ligand was not reported.
O
n
R =
HO
x
y
z
OR
O
HO
HO
O
OMe
O
HO
O
OH
OH
OH
A
B
C
D
O
RO
TDM (1) e.g., n = 13, x = 16, y = 12, z = 17
O
OH
O
R =
R =
20
OH
TDB (2)
Brartemicin (3)
Figure 1. Representative trehalose glycolipids.
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Given the ability of Mincle ligands to promote a Th1/Th17 immune response, we were interested
in developing a potent Mincle agonist for use as a vaccine adjuvant. The crystal structure of
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1
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human Mincle (hMincle) and bovine Mincle (bMincle) revealed a common GluꢀProꢀAsn
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(EPN) motif (residues 169ꢀ171), which is often observed in calciumꢀdependent lectin receptors
2+
(
CLRs). Mincle uses the Ca ion in this EPN motif to bind the equatorial 3ꢀ and 4ꢀOH groups of
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one glucose residue in trehalose,
with molecular modelling studies showing similar binding
1
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to monoesters of glucose. On one side of the EPN motif there is a binding site for the second
glucose residue in trehalose, while the other side contains a hydrophobic groove capable of
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binding linear or branched fatty acids.
The precise molecular mechanisms of glycolipidꢀ
Mincle interactions are far from being understood, with subtle changes in lipid structure leading
19,29,30
to different proposed binding modes for similar molecules.
Notwithstanding, it has been
suggested that in hMincle, Arg183 is in a suitable position to interact with the hydroxyl groups
3
1
of trehalose, and that the hydrophobic groove plays an essential role in binding Mincle
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ligands.
This is supported by structureꢀactivity studies that indicate that trehalose diesters
with lipid chains ≥ C are required for bone marrow derived macrophages (BMDMs) to
18
produce the proꢀinflammatory cytokines interleukin (IL)ꢀ6, ILꢀ1β, and the cellular mediator
16
nitric oxide. Computational studies using bMincle have also determined that brartemicin
strongly binds to Mincle with one aromatic ester occupying the hydrophobic groove and the
second ester potentially being involved in πꢀcation interactions with the aforementioned arginine
2
9
residue (Arg182/183, bovine/human). Taken together, we proposed that trehalose glycolipids
containing an aromatic residue and long lipophilic tails would be potent Mincle agonists and
Th1ꢀstimulating vaccine adjuvants.
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Results and Discussion
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Design of brartemicin analogues. While brartemicin can bind Mincle,
the ability of this
natural product to signal through Mincle has not been reported. Notwithstanding, we envisioned
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that incorporation of lipophilic groups onto the brartemicin scaffold would significantly increase
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Mincle affinity, and that such derivatives would have strong immunostimulatory activity.
In
particular, attachment of lipid chains to the 4ꢀhydroxyls of the brartemicin benzoates could allow
for the formation of compounds capable of strong interactions with Mincle’s hydrophobic
grooves; an effect previously observed in binding experiments using linear trehalose monoꢀ and
3
0,31
diꢀesters of increasing lipid lengths.
As changes to the glycolipid structure can affect binding
1
9,29,30
modes of Mincle ligands,
we set out to determine whether lipidated brartemicin analogues
could be accommodated in the Mincle binding site. To this end, molecular docking of
brartemicin containing lipophilic ethers at the 4ꢀhydroxyls of the brartemicin benzoates was
3
3,34
undertaken using UCSF Chimera/DOCK6.
These studies suggested that, like brartemicin
itself, the lipidated structures would bind in the carbohydrate recognition domain (CRD) of
2+
Mincle with the 3ꢀ and 4ꢀhydroxyls of the trehalose moiety anchoring the ligand to the Ca ion
(Figure 2, SI Fig 1). Moreover, the docking study suggested that the lipidꢀportion of one of the
aromatic esters occupies a hydrophobic region comprised of the Leu199, Phe198, Leu176, and
Val145 side chains. The second aromatic ester was calculated to reside 3.37 Å away from
Arg183, which suggested πꢀcation interactions, as previously observed in molecular modelling
2
9
studies of brartemicin itself. While further modelling studies could be conducted, the
relationship between Mincle binding and signalling is not well understood. For example, C and
5
3
0
C diacylated linear trehalose glycolipids bind Mincle, however in vitro assays showed that C ,
6
4
1
6
C , and C derivatives are unable to activate BMDMs. Accordingly, we set out to synthesise a
7
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variety of 4ꢀOꢀalkylated brartemicin analogues with different lipid chain lengths and varying
functionalities on the aromatic rings, so that these could be tested in functional assays.
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Figure 2. Molecular docking pose of lipidated brartemicin interacting with hMincle (Protein
3
3,34
Data Bank ID code 3WH2) using UCSF Chimera/DOCK6.
Synthesis of brartemicin analogues. To determine whether brartemicin derivatives can bind
and activate Mincle, we synthesised a series of 4ꢀOꢀalkyl, desmethyl, and dehydroxy brartemicin
analogues. In addition, in order to investigate the effect of lipid length on macrophage activation,
we prepared substituted 4ꢀalkoxyꢀbenzoic acid derivatives that incorporated long (C ), medium
1
8
(
C ), or short (C or C ) alkyl chains (Scheme 1). To this end, methyl 2,4ꢀdihydroxybenzoate (4)
7 4 1
was selectively alkylated at the 4ꢀposition, before benzyl protection and ester hydrolysis, to
afford benzoic acid derivatives 4aꢀe in good to excellent yield over the three steps (50ꢀ87%). The
effect of the substitution pattern on the aromatic ring was then considered via the alkylation of 4ꢀ
hydroxybenzoic acid 5 with long and medium length alkyl groups, followed by hydrolysis to
provide 2ꢀdeoxyꢀderivatives 5a and 5b, again in excellent yield (62ꢀ79%). Finally, alkylation of
ethyl 2,4ꢀdihydroxyꢀ6ꢀmethylbenzoate (6), the core aromatic residue found in brartemicin,
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followed by benzylation of the 2ꢀposition and hydrolysis of the ester group gave the benzyl
protected alkoxybenzoate 6a. In addition, 6 was dibenzylated and hydrolysed (→ 6b) en route to
the total synthesis of brartemicin.
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Scheme 1. Reagents and conditions a) CH (CH ) Br/TBAI, CH (CH ) I, CH (CH ) I, or MeI,
3
2 17
3
2 6
3
2 3
K CO , acetone, reflux; b) BnBr, TBAI, K CO , acetone, reflux; c) NaOH (5M), MeOH, reflux.
2
3
2
3
The overall yield for each benzoate is reported in parentheses.
The final glycolipids were assembled via esterification of partially protected trehalose 7
1
6
(prepared in three steps according to literature procedures) with the aforementioned benzoic
acids. The esterification reactions were performed in the presence of 1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide (EDCI) and 4ꢀ(dimethylamino)pyridine (DMAP) and gave
the desired benzylꢀprotected trehalose diesters 8aꢀi in good yields (Table 1). Global
debenzylation using Pearlman’s catalyst and H then gave brartemicin (9i) and analogues 9aꢀh in
2
good to excellent yields.
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Table 1. Synthesis of Brartemicin analogues
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a
Yield (%)
Entry
Benzoate
7 → 8
8a, 66
8 → 9
9a, 68
1
2
8b, 70
9b, 83
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4
5
8c, 61
8d, 65
8e, 58
9c, 86
9d, 62
9e, 92
6
7
8f, 80
8g, 83
9f, 51
9g, 73
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a
All synthesised compounds were determined to be endotoxin free (≤ 0.1 EU/mL) by using the
limulus amebocyte lysate (LAL) chromogenic assay.
Lipidꢀcontaining brartemicin analogues bind and activate mMincle and hMincle. To test
whether human and murine Mincle can recognise the synthesised brartemicin analogues, the
compounds were tested in an enzymeꢀlinked immunosorbent assay (ELISA) using soluble
MincleꢀIg fusionꢀproteins, in which the extracellular domain of mouse Mincle was fused to
8
,10
human IgG Fc for detection (Figure 3a). Here, compounds lacking a lipophilic group, i.e. 4′ꢀ
Oꢀmethylꢀdesmethylꢀbrartemicin (9d), desmethylꢀbrartemicin (9e), and brartemicin (9i), along
with 4′ꢀOꢀbutylꢀdesmethylꢀbrartemicin (9c), which contains a short lipid, did not bind hMincleꢀ
or mMincleꢀIg to any great extent. By comparison, the C containing analogues, 9a, 9f, and 9h,
18
showed good binding affinity. Strongest binding, however, was observed for the analogues
containing C lipid tails (9b and 9g) for both the human and murine fusionꢀproteins. The
7
enhanced binding of the C lipidated brartemicin derivatives compared to those with longer (C )
7
18
lipid lengths was unexpected, particularly as competition assays involving the displacement of
mannoseꢀconjugated serum albumin from bMincle by monoꢀ and diꢀacylated trehalose diesters
bearing 5–12ꢀcarbon linear fatty acids revealed increasing binding affinity as lipid length
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Figure 3. Lipidꢀcontaining brartemicin analogues bind and signal through mMincle and
hMincle (a) Plates coated with Brartemicin analogues (0.1 nmol/well) were incubated with Igꢀ
mMincle, IgꢀhMincle, or Igꢀonly and ligand bound protein was detected via ELISA. Data is
representative of three independent experiments performed in triplicate (mean ± SD). (b) NFATꢀ
GFP 2B4 reporter cells expressing mMincle + FcRγ or (c) hMincle + FcRγ were stimulated
using ligandꢀcoated plates (0.01, 0.1, or 1 nmol/well) for 18 hours. The reporter cells were
harvested and examined for NFATꢀGFP expression. Data represents the mean of three
independent experiments performed in duplicate (mean ± SEM).
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increased. Earlier studies investigating the binding affinities of monoacylated trehalose
derivatives with different carbon lengths (C , C and C ) by surface plasmon resonance (SPR)
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also revealed that the C derivative showed much lower binding affinity to hMincle than the C
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and C analogues. As we are the first to investigate the binding affinity of longer chain
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trehalose diesters, it would seem that there is an optimum lipid length for brartemicinꢀderived
trehalose diesters binding to Mincle that is centered around lipids of intermediate length.
However, it should also be noted that previous competitive binding assays demonstrated that
29
brartemicin (9i) bound bMincle with strong affinity (K = 5.5 ± 0.9) while we did not observe
i
binding of brartemicin to hMincle or mMincle. This difference in binding affinity could be
attributed to differences in the hMincle and bMincle binding sites, but could also point to the
general differences that arise when comparing competition assays and more direct measures,
such as SPR or ELISAꢀbased Igꢀfusion assays, to determine relative binding affinities.
Next, we were curious to determine whether the observed Mincle binding correlated with cellular
activation. To this end, we stimulated nuclear factor of activated T cells (NFAT)ꢀgreen
8
fluorescent protein (GFP) reporter cells expressing mMincle or hMincle coupled to FcRγ using
plates coated with TDM, TDB, brartemicin (9i), or analogues 9aꢀ9h. Activation of the reporter
cells was measured through the production of GFP, which was monitored by flow cytometry,
with cells only expressing FcRγ being used as negative controls (Figure 3b and 3c). In contrast to
our binding studies, ligands incorporating C lipids (9a, 9f, and 9h) strongly activated both
1
8
murine and human Mincle NFATꢀGFP reporter cells in a doseꢀdependent manner, while the C7ꢀ
containing analogues (9b and 9g) also activated mMincle and hMincle NFATꢀGFP reporter cells
but to a lesser extent than the C analogues. The C analogue (9c) was able to activate mMincle
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and hMincle only at high ligand concentrations, while the nonꢀlipidated analogues 9e and
brartemicin (9i) did not significantly activate mMincle, with 9d showing only modest activation
of hMincle at a high ligand concentration. These findings support previous structureꢀactivity
studies with acylated trehalose derivatives whereby trehalose diesters with lipid chains ≥ C18
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were required for the production of inflammatory cytokines by BMDMs and moreover
illustrate that ligand binding does not exactly correlate to Mincle activation. The reason for the
absence of a direct correlation between ligand binding and Mincleꢀactivation is unclear, though it
is possible that the functional receptor undergoes a conformational change upon ligand binding,
which might alter the receptor activation state and receptorꢀligand affinity. Our findings also
2
0,21
demonstrate the subtle speciesꢀspecific differences between hMincle and mMincle,
despite
7
the sequence of mMincle and hMincle being highly conserved. Notwithstanding, at all
concentrations tested, 9a was best able to activate hMincle.
Lipidꢀcontaining brartemicin analogues induce BMDMs to produce inflammatory
cytokines in a Mincleꢀdependent manner. Although the NFATꢀGFP reporter assay is useful
for the identification of Mincle ligands, we sought to assess the interaction of the receptor with
candidate ligands in a more physiological setting. Accordingly, we examined the capability of
the brartemicin analogues to induce an inflammatory response by APCs. First, the ability of the
glycolipids to activate GMꢀCSF BMDMs was determined by monitoring the secretion of the
inflammatory cytokines TNF, ILꢀ6, and ILꢀ1β and the chemokine, MIPꢀ2, in a ligandꢀcoated
plate assay (Figure 4). As illustrated, stimulation of BMDMs with the C brartemicin derivatives
1
8
(9a, 9f and 9h) led to the significant production of all cytokines and MIPꢀ2. This response was
similar to that induced by TDM and TDB, whereby the latter has deemed safe and tolerable as
35
part of the CAF01 liposome formulation in clinical trials. On the other hand, C ꢀderivatives (9b
7
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Figure 4. Lipidated brartemicin analogues induce Mincle dependent inflammatory
ꢀ
cytokine production. TNF, MIPꢀ2, ILꢀ6 and ILꢀ1β production by harvested wildꢀtype or Mincle
/ꢀ
GMꢀCSF BMDMs treated with brartemicin derivatives. Harvested GMꢀCSF BMDMs were
stimulated using TDB, TDM, or brartemicin derivativeꢀcoated plates (0.1 or 1 nmol/well) or
solubilised LPS (100 ng/mL). Cytokine production was measured by ELISA from the
supernatant collected after 24 hours. Data is representative of three independent experiments
performed in triplicate (± SD).
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and 9g) resulted in only modest production of MIPꢀ2, ILꢀ1β, TNF, and ILꢀ6, while the C4
brartemicin derivative (9c) induced MIPꢀ2 but not TNF, ILꢀ6, and ILꢀ1β. The nonꢀlipidated
derivatives (9d and 9e) and brartemicin itself (9i) did not induce the production of any of the
cytokines measured. This data supports the observation that BMDM stimulation with trehalose
diesters with longer lipid tails leads to an enhanced inflammatory response. In the absence of
Mincle, the production of MIPꢀ2, ILꢀ1β, TNF, and ILꢀ6 in response to the synthetic ligands was
abolished, suggesting that Mincle is the major receptor involved in mediating BMDM activation
by lipophilic brartemicin derivatives. At this stage, the C ꢀalkylated desmethyl brartemicin
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analogue (C18dMeBrar, 9a) was chosen as the lead candidate for subsequent assays. Of the three
C brartemicin derivatives best able to activate mMincle and hMincle (9a, 9f and 9h), 9a
18
exhibited the greater hMincle activation at low glycolipid concentrations (0.01 ng/mL) in the
NFATꢀGFP reporter assay (Figure 3). Moreover, 9a, which contains an additional hydroxyl at
the 2ꢀposition of the aromatic ring, was easier to synthesise than the other potential lead agonist,
9
f, and was more soluble in the in vitro assays.
Arginine 183 is essential for hMincle activation by 9a. We then examined the contribution of
Arg183 to hMincleꢀligand binding, as our molecular modelling studies of lipidated brartemicin
analogues indicated a potentially important interaction between the benzoate group of 9a and
Arg183 in hMincle via πꢀcation interactions. To this end, we established an NFATꢀGFP reporter
cell line expressing hMincle mutants, where Arg183 was replaced with an alanine residue
R183A
(hMincle
). The mutant Mincle was expressed normally on the cell surface of the reporter
cells (Figure 5a) and NFATꢀGFP production was induced when stimulated with TDM (Figure
R183A
5b). Conversely, the hMincle
mutant failed to recognise 9a, indicating that this residue is
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essential for hMincle binding of 9a. Previously it was suggested that Arg183 might enhance the
binding affinity of linear acylated trehalose derivatives by binding to additional hydroxyl
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residues on the trehalose moiety. Indeed, the slightly lower binding affinity of TDM to
R183A
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hMincle
compared to hMincle WT further supports the general importance of Arg183 in
binding Mincle ligands.
a
b
c
Figure 5. Arginine 183 is essential for the activation of hMincle expressing NFATꢀGFP
R183A
2B4ꢀcells by 9a. a) hMincle
NFATꢀGFP reporter cells were stained with antiꢀhMincle
R183A
(bold) or isotype control (dashed). b) hMincle WT or c) hMincle
reporter cells were
stimulated using plates coated with increasing amounts of TDM or 9a and cultured for 18 hours.
The cells were harvested and analysed for NFATꢀGFP expression. Data represents the mean of
two independent experiments performed in duplicate (mean ± SEM).
C18dMeBrar 9a is a Th1/Th17 inducing adjuvant in vitro. The ability of an adjuvant to
qualitatively affect the outcome of the immune response is an important consideration as most
licensed subunit vaccines contain the adjuvant alum, which elevates serum antibody titres but
36
elicits a Th2 antibody response that can be weak and often requires repeated immunisations. In
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addition, alumꢀbased adjuvants are not sufficient for eliminating intracellular pathogens. To this
end, the vaccine adjuvant monophosphoryl lipid A (MPL), which binds TLRꢀ4 to elicit a Th1
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immune response has been developed and used in the licensed AS04 vaccine adjuvant system.
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Notwithstanding, the need for vaccines against chronic infections [e.g., HIV, hepatitis C virus
(HCV), tuberculosis and herpes simplex virus (HSV)] remains, and in particular, the
development of adjuvants that generate Th1/Th17 cellular immune responses is a pressing
3
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goal.
To evaluate the potential of 9a as a Th1/Th17 adjuvant, we coꢀcultured GMꢀCSF BMDMs and
Tꢀcells from OVAꢀspecific OTꢀII TCR Tg mice on plates coated with TDM (0.1 nmol/well),
TDB (0.1 nmol/well), or 9a (0.1 nmol/well), stimulated the cells with OVA (0, 0.1, and 1 µM),
and after 48 hours, collected the supernatant. Levels of the key Th1 cytokine IFNꢀγ, which is
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indicative of an enhanced host defense response, and the Th17 cytokine ILꢀ17 were measured
via ELISA (Figure 6). The OVAꢀspecific production of ILꢀ17 was significantly augmented by
9
a, albeit to a lesser extent than TDM and TDB. Antigenꢀspecific secretion of IFNꢀγ however,
was not enhanced by treatment with 9a.
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Figure 6. Brartemicin 9a displays adjuvant activity in vitro. OTꢀII CD4 Tꢀcells were coꢀ
cultured with GMꢀCSF BMDMs in the presence of TDM (0.1 nmol/well), TDB (0.1 nmol/well),
9a (0.1 nmol/well) and OVA323ꢀ339 peptide (0, 0.1, and 1 ꢁM). After 48 hours, the supernatant
was collected and levels of IFNꢀγ and ILꢀ17 were measured using ELISA. Data is representative
of two experiments performed in triplicate (mean ± SD). *P≤ 0.05; **P≤ 0.01; ***P≤ 0.005;
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C18dMeBrar 9a is a potent Th1ꢀstimulating adjuvant in vivo. Adjuvant 9a was then
evaluated in vivo to confirm its capacity to induce a Th1 antigenꢀspecific response. While Mincle
agonists are often formulated into cationic (CAF01) liposomes for in vivo testing, these
liposomes are thought to directly activate antigenꢀpresenting cells and can effect both CD4 and
4
1
CD8 T cell responses. Thus we sought to explore the adjuvanticity of 9a in the absence of other
immunostimulatory agents, such as DDA, so as to obtain an immune profile that is associated
with 9a alone. Accordingly, we employed a delayedꢀtype hypersensitivity immunisation
protocol, as it is a convenient, sensitive, and well established model for the assessment of in vivo
immune responses. Four groups of C57BL/6 mice were immunised by subcutaneous injection
with oilꢀinꢀwater emulsions containing either OVA only, OVA + 9a, OVA + TDB, or no OVA.
After seven days the mice were challenged with OVA (100 µg per footpad) and after a further
seven days, the splenocytes were isolated and restimulated with OVA at three concentrations
(
10, 30, or 100 µg/mL). The immune response was measured by determining footpad swelling
SI Fig 2), T cell proliferation (Figure 7a), production of IFNꢀγ and ILꢀ17 (Figure 7b), and
(
antibody titres (Figure 7c). While neither 9a nor TDB induced footpad swelling (SI Fig 2) or ILꢀ
17 production, immunisation of mice using 9a gave rise to a polarised immune response with a
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distinct Th1 profile. A trend towards increased antigenꢀspecific IFNꢀγ production was observed
for all concentrations of OVA when 9a was used as the adjuvant, with significantly enhanced
cytokine production when reꢀstimulating with 100 µg/mL of antigen. In contrast, the Mincle
agonist TDB did not lead to a significant increase in IFNꢀγ at any of the concentrations of OVA
tested. A significantly larger number of splenocytes was also observed from mice that received
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a, as compared to those that received OVA alone (Figure 7a). Moreover, at all concentrations of
OVA reꢀstimulation, 9a led to a significant increase in cell count compared to TDB. The limited
efficacy of TDB in these in vivo assays was initially surprising, however studies addressing the
adjuvanticity of TDB have largely focussed on the TDB:DDA (CAF01) liposome
1
3,22,25,26,42,43
system.
Accordingly, the ability of 9a to lead to an enhanced Th1 immune response
was even more striking.
This overall immune profile was also reflected in the antibody production by immunised mice.
While 9a and TDB elevated the production of IgG antibodies, the increase in antibody
production was only significant for C18dMeBrar (9a). When looking at the subclasses of
antibodies produced, both Th1 associated subclasses IgG2b and IgG2c and Th2 associated
4
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IgG1
showed increases for 9a and TDB, with the largest increases observed for 9a, for which
IgG1 doubled and IgG2c tripled. In conclusion, C18dMeBrar (9a) is qualitatively similar to TDB
with regard to its ability to enhance antibody production, however 9a leads to a stronger immune
response.
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Figure 7. Brartemicin analogue 9a displays potent adjuvant activity in vivo. C57BL/6 mice
(n = 5 per group) were immunised subcutaneously with oilꢀinꢀwater emulsions containing OVA
only (200 µg), OVA + TDB (OVA = 200 µg, TDB = 0.3 µmol) OVA + 9a (OVA = 200 µg, 9a =
0.3 µmol), or emulsion only. After seven days, the mice were challenged with OVA (100
µg/footpad). After a further seven days, the mice were sacrificed, blood samples taken, and their
spleens were harvested. Total splenocyte number (a) and cytokine production (b) was measured
following restimulation with OVA. Blood serum was analysed for OVAꢀspecific antibody
production (c). *P≤ 0.05; **P≤ 0.01; ***P≤ 0.005; ****P≤ 0.001.
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To date, TDB is the leading Mincle agonist and has found application as a key constituent of the
2
5,26
23,24
highly promising CAF01 liposomal vaccine adjuvant for the treatment of TB
and HIV.
This, in turn, has led to much interest in the development of other Mincle ligands as adjuvants,
particularly as combinations of adjuvants may be required to confer protection against pathogens
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46
that have thus far evaded vaccination efforts. Indeed, the recently identified GlcC14C18 Mincle
agonist has shown promise as a Th1/Th17 adjuvant when administered in a liposomal
1
9
formulation containing immunostimulatory DDA. The synthesis of GlcC14C18, however,
19
appears challenging (5.7% overall yield as a mixture of diastereoisomers). In contrast, our lead
Mincleꢀagonist, 9a, can be synthesised using a practical and scalable synthesis involving fiveꢀ
linear steps to give the glycolipid as a single compound and in 22% overall yield. Moreover, 9a
exhibited excellent Th1 adjuvant activity, which was greater than TDB. Accordingly,
C18dMeBrar (9a) is one of the most promising Mincle agonists to date and is an ideal candidate
for further vaccination studies.
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Conclusion
In conclusion, we have developed a highly potent Th1 stimulating vaccine adjuvant,
C18dMeBrar (9a), which was based on the structure of the natural product brartemicin. This
glycolipid was synthesised from readily available starting materials in five linear steps and in
excellent overall yield. Computational studies provided insight into the potential binding of the
brartemicin derivatives to hMincle and suggested that the benzoate of brartemicin may interact
with Arg183 by πꢀcation interactions. This was confirmed by the absence of activity of
C18dMeBrar (9a) in an NFATꢀGFP reporter cell line containing Mincle with an alanine, rather
than arginine, residue at position 183. During the course of our studies we also demonstrated that
longer lipids led to a better functional immune response. This was an important observation for
while trehalose glycolipids incorporating shorter lipid chains have previously been shown to bind
Mincle, binding does not always correlate to cellular activation. In our assays, brartemicin did
not activate mMincle or hMincle NFATꢀGFP reporter cells or BMDMs. Notwithstanding, all
three C lipidated brartemicin analogues 9a, 9f and 9h activated both mMincle and hMincle,
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8
with high levels of TNF, ILꢀ6, ILꢀ1β and MIPꢀ2 being produced by the BMDMs upon
stimulation with the ligands. When 9a was combined with an antigen in vivo, a strong Th1 recall
response was observed, and the adjuvanticity of 9a was greater than TDB. Accordingly, our
Mincle agonist C18dMeBrar (9a) is not only a powerful adjuvant in its own right, but it could be
used in tandem with other adjuvants, such as TLR agonists, to develop an optimal adjuvant
combination for vaccination against pathogens that have thus far resisted our efforts in this
respect.
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Experimental
General experimental. Unless otherwise stated, all reactions were performed under an
atmosphere of argon. Acetone, methyl iodide, 1ꢀiodoheptane and 1ꢀiodobutane were distilled and
stored over molecular sieves (4Å). Methyl 2,4ꢀdihydroxy benzoate (BDH), methyl 4ꢀ
hydroxybenzoate (BDH), 1ꢀbromooctadecane (Aldrich), TBAI (Riedelꢀde Haen), benzyl
1
1
1
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5
6
0
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2
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6
7
8
9
0
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7
8
9
0
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8
9
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0
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0
bromide (Aldrich), methanol (Fischer Scientific), sodium hydroxide (Vickers), K CO (Panreac)
2
3
ethanol (Fischer Scientific), EDCI (Chem Impex), DMAP (Lab Supply), toluene (ROMIL),
Pd(OH) (Aldrich), CH Cl (Fischer Scientific), pyridine (ROMIL), C D N (Apollo), CDCl
3
2
2
2
5
5
(
Aldrich), CD OD (Cambridge Isotopes Laboratories Inc.), H (BOC), EtOAc (Pure Science),
3
2
and petroleum ether (Pure Science) were used as received. 2,2',3,3',4,4'ꢀhexaꢀOꢀbenzylꢀα,α'ꢀDꢀ
16
47
trehalose was prepared according to a literature procedure. Methyl 2,4ꢀdihydroxy benzoate
4
8
and ethyl 2,4ꢀdihydroxyꢀ6ꢀmethylbenzoate were prepared according to adapted literature
procedures. All solvents were removed by evaporation under reduced pressure. Reactions were
monitored by TLCꢀanalysis on MachereyꢀNagel silica gel coated plastic sheets (0.20 mm with
fluorescent indicator UV ) via detection by UVꢀabsorption (254 nm), dipping in 10% H SO in
2
54
2
4
EtOH followed by charring, dipping in KMnO solution (2% in H O), or dipping in ceric
4
2
ammonium molybdate solution. Column chromatography was performed using Pure Science
silica gel (40ꢀ63 µm), and size exclusion chromatography was performed using lipophilic
Sephadex (25ꢀ100 µm, Sigma). All compounds were confirmed to be ≥95% pure by NMR
analysis. High resolution mass spectra were recorded on an Agilent 6530 QꢀTOF mass
spectrometer utilising a JetStream electrospray ionisation source in positive and negative mode.
Optical rotation were recorded on an Autopol II or IV (Rudolph Research Analytical) at 589 nm
(sodium
D
ꢀline). Infrared spectra were recorded as thin films using a Bruker Platinum ATR and
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ꢀ1
are reported in wave numbers (cm ). Nuclear magnetic resonance spectra were recorded at 20 °C
in C D N, CD OD, or CDCl using a varian INOVA operating at 500 or 600 MHz. Chemical
5
5
3
3
shifts are given in ppm (δ) relative to residual solvent peaks. NMR peak assignments were made
using COSY, HSQC, and HMBC 2D experiments. Melting points were obtained using a
Gallenkamp Melting Point Apparatus. Quantitative NMR was used to confirm the purity of 9a
was ≥95%.
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General procedure for the synthesis of 4ꢀOꢀalkylꢀbenzoates. To a solution of benzoate
(
1 equiv.) in acetone (20 mL) was added K CO (1.4ꢀ2 equiv.), alkyl halide (1.2–1.5 equiv.) and
2 3
TBAI (for 4ꢀOꢀoctadecyloxyꢀbenzoates only, 0.05ꢀ0.1 equiv.) The mixture was heated at reflux
until the reaction was completed (as gauged by TLC analysis, 18ꢀ48 h). The reaction mixture
was cooled to rt and concentrated under reduced pressure. The resulting residue was purified
using gradient silica gel flash column chromatography (petroleum ether to petroleum
ether:EtOAc; 9:1; v/v).
Methyl 2ꢀhydroxyꢀ4ꢀmethoxybenzoate. By subjecting methyl 2,4ꢀdihydroxybenzoate (400
mg, 2.39 mmol), methylꢀiodide (0.19 mL, 3.12 mmol), and K CO (528 mg, 3.82 mmol) to the
2
3
general procedure for the synthesis of 4ꢀOꢀalkylꢀbenzoates, the title compound was obtained as a
1
clear oil (390 mg, 2.14 mmol, 90%). R = 0.58 (CH Cl ); H NMR (500 MHz, CDCl ) δ 10.98 (s,
f
2
2
3
1
H, OH), 7.73 (d, J6,5 = 9.0 Hz, 1H, Hꢀ6), 6.43 (m, 2H, Hꢀ3 & Hꢀ5), 3.91 (s, 3H, CO Me), 3.82
2
1
3
(
s, 3H, OMe); C NMR (125 MHz, CDCl ) δ 170.4 (Cꢀ7), 165.6 (Cꢀ4), 163.8 (Cꢀ2), 131.3 (Cꢀ6),
3
107.6 (Cꢀ5), 105.5 (Cꢀ1), 100.7 (Cꢀ3), 55.5 (OMe), 52.0 (CO Me); IR (film): 3080, 3013, 2957,
2
ꢀ1
1
665, 1620, 1583, 1504, 1434, 1349, 1255, 1225, 1191, 1139, 773 cm ; HRMS (ESI) m/z calcd.
+
for [C H O +H] : 183.0652; obsd.: 183.0650.
9
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Methyl 4ꢀbutoxyꢀ2ꢀhydroxybenzoate. By subjecting methyl 2,4ꢀdihydroxybenzoate (205 mg,
1
.22 mmol), 1ꢀiodobutane (0.20 mL, 1.55 mmol), and K CO (291 mg, 2.11 mmol) to the
2
3
general procedure for the synthesis of 4ꢀOꢀalkylꢀbenzoates, the title compound was obtained as a
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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4
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0
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9
0
clear oil (248 mg, 1.10 mmol, 90%). R = 0.58 (CH Cl ); H NMR (500 MHz, CDCl ) δ 10.96 (s,
f
2
2
3
1
H, OH), 7.72 (d, J = 7.2 Hz, Hꢀ6), 6.41ꢀ6.44 (m, 2H, Hꢀ3 & Hꢀ5), 3.98 (t, J = 6.5 Hz, 2H,
6,5 8,9
CH ꢀ8), 3.91 (s, 3H, CO Me), 1.77 (p, J = J = 6.8 Hz, 2H, CH ꢀ9), 1.48 (sext, J
10,9
= J10,11
=
2
2
9,8
9,10
2
1
3
7
.5 Hz, 2H, CH ꢀ10), 0.97 (t, J
= 7.5 Hz, 3H, CH ꢀ11). C NMR (125 MHz, CDCl ) δ 170.4
2
11,10 3 3
(
Cꢀ7), 165.2 (Cꢀ4), 163.7 (Cꢀ2), 131.2 (Cꢀ6), 108.0 (Cꢀ5), 105.2 (Cꢀ1), 101.1 (Cꢀ3), 68.0 (Cꢀ8),
5
2.0 (CO Me), 31.1 (Cꢀ9), 19.2 (Cꢀ10), 13.9 (Cꢀ11). IR (film): 3111, 2957, 2874, 1666, 1621,
2
ꢀ1
+
1505, 1439, 1395, 1347, 1251, 1137, 730 cm ; HRMS (ESI) m/z calcd. for [C H O +H] :
1
2
16
4
2
25.1121; obsd.: 225.1118.
Methyl 4ꢀ(heptyloxy)ꢀ2ꢀhydroxybenzoate. By subjecting methyl 2,4ꢀdihydroxybezoate (310
mg, 1.84 mmol), K CO (395 mg, 2.86 mmol), and 1ꢀiodoheptane (0.38 mL, 2.31 mmol) to the
2
3
general procedure for the synthesis of 4ꢀOꢀalkylꢀbenzoates, the title compound was obtained as a
1
colourless oil (479 mg, 1.80 mmol, 98%). R = 0.85 (CH Cl ); H NMR (500 MHz, CDCl ) δ
f
2
2
3
1
6
0.96 (s, 1H, OH), 7.72 (d, J = 9.3 Hz, 1H, Hꢀ6), 6.41ꢀ6.44 (m, 2H, Hꢀ3 & Hꢀ5), 3.96 (t, J =
6,5 8,9
.5 Hz, 2H, CH ꢀ8), 3.91 (s, 3H, CO Me), 1.77 (p, J = J = 7.5 Hz, 2H, CH ꢀ9), 1.47ꢀ1.40
2
2
9,8
9,10
2
13
(
m, 2H, CH ꢀ10), 1.38ꢀ1.26 (m, 6H, CH ꢀ11ꢀCH ꢀ13), 0.89 (t, J
= 6.7 Hz, 3H, CH ꢀ14); C
2
2
2
14,13 3
NMR (500 MHz, CDCl ) 170.6 (Cꢀ7), 166.3 (Cꢀ4), 163.9 (Cꢀ2), 131.3 (Cꢀ6), 108.1 (Cꢀ3), 105.3
3
(Cꢀ1), 101.2 (Cꢀ5), 68.4 (Cꢀ8), 52.1 (CO Me), 31.9 , 29.2, 29.1, 26.1, 22.7 (Cꢀ9–Cꢀ13), 14.2 (Cꢀ
2
ꢀ1
1
4); IR (film): 3143, 2928, 2857, 1669, 1623, 1582, 1505, 1468, 1440, 1254, 1140, 780 cm ;
+
HRMS (ESI) m/z calcd. for [C H O +H] : 267.1591; obsd.: 267.1593.
15
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Journal of Medicinal Chemistry
Page 26 of 66
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Methyl 2ꢀhydroxyꢀ4ꢀ(octadecyloxy)benzoate. By subjecting methyl 2,4ꢀdihydroxybenzoate
(
357 mg, 2.12 mmol), K CO (429 mg, 3.10 mmol), 1ꢀbromooctadecane (836 mg, 2.50 mmol),
2
3
and TBAI (71 mg, 0.19 mmol) to the general procedure for the synthesis of 4ꢀOꢀalkylꢀbenzoates,
0
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0
1
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0
the title compound was obtained as an amorphous offꢀwhite solid (695mg, 1.65 mmol, 78%). Rf
1
=
0.83 (CH Cl ); H NMR (500 MHz, CDCl ) δ 10.95 (s, 1H, OH), 7.72 (d, J = 9.5 Hz, 1H, Hꢀ
2
2
3
6,5
6
), 6.41ꢀ6.43 (m, 2H, Hꢀ3 & Hꢀ5), 3.96 (t, J8,9 = 6.6 Hz, 2H, CH ꢀ8), 3.91 (s, 3H, CO Me), 1.78
2 2
(
p, J9,8 = J9,10 = 7.1 Hz, 2H, CH ꢀ9), 1.40ꢀ1.47 (m, 2H, CH ꢀ10), 1.24ꢀ1.35 (m, 28H, CH ꢀ11ꢀ
2 2 2
13
CH ꢀ24), 0.88 (t, J
= 6.6 Hz, 3H, CH ꢀ25); C NMR (125 MHz, CDCl ) δ 170.6 (Cꢀ7), 165.4
3 3
2
25,24
(
Cꢀ4), 163.9 (Cꢀ2), 131.3 (Cꢀ6), 108.1 (Cꢀ5), 105.3 (Cꢀ1), 101.2 (Cꢀ3), 68.4 (Cꢀ8), 52.1
(CO Me), 32.1, 29.9, 29.84, 29.83, 29.81, 29.80, 29.73, 29.70, 29.52, 29.49, 22.9 (Cꢀ11ꢀCꢀ24),
2
2
1
9.14 (Cꢀ9), 26.1 (Cꢀ10), 14.3 (Cꢀ25); IR (film): 2916, 2850, 1674, 1583, 1473, 1441, 1350,
ꢀ1
+
258, 781 cm ; HRMS (ESI): calcd. for [C H O +H] : 421.3312; obsd.: 421.3303.
26
44
4
Methyl 4ꢀ(heptyloxy)ꢀbenzoate. By subjecting methyl 4ꢀhydroxybenzoate (550 mg, 3.62
mmol), K CO (1.01 g, 7.33 mmol), and 1ꢀiodoheptane (0.77 mL, 4.70 mmol) to the general
2
3
procedure for the synthesis of 4ꢀOꢀalkylꢀbenzoates, the title compound was obtained as an
amorphous white solid (853 mg, 3.41 mmol, 94%). R = 0.66 (Petroleum ether:EtOAc, 5:1, v/v);
f
1
HꢀNMR (500 MHz, CDCl ) δ 7.98 (d, J = 9.1 Hz, 2H, Hꢀ2), 6.90 (d, J3,2 = 8.8 Hz, 2H, Hꢀ3),
3
2,3
4
1
.00 (t, J6,7 = 6.6 Hz, 2H, CH ꢀ6), 3.88 (s, 3H, CO Me), 1.80 (p, J = J = 6.8 Hz, 2H, CH ꢀ7),
2
2
7,6
7,8
2
.42ꢀ1.49 (m, 2H, CH ꢀ8), 1.37ꢀ1.31 (m, 6H, CH ꢀ9ꢀCH ꢀ11), 0.90 (t, J = 6.1 Hz, 3H, CH3ꢀ
12,11
2
2
2
1
3
12); CꢀNMR (125 MHz, CDCl ) 166.9 (Cꢀ5), 163.0 (Cꢀ4), 131.6 (Cꢀ2), 122.3 (Cꢀ1), 114.0 (Cꢀ
3
3
), 68.2 (Cꢀ6), 51.8 (CO Me), 31.9, 29.18, 22.8 (Cꢀ9ꢀCꢀ11), 29.3 (Cꢀ7), 26.1 (Cꢀ8), 14.2 (Cꢀ12);
2
ꢀ1
IR (film): 2919, 2851, 1716, 1606, 1512, 1436, 1251, 1168, 850 cm ; HRMS (ESI) calcd. for
+
[C H O ] : 251.1642; obsd.: 251.1630.
1
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Methyl 4ꢀ(octadecyloxy)benzoate. By subjecting methyl 4ꢀhydroxybenzoate (406 mg, 2.67
mmol), K CO (565, 4.09 mmol), 1ꢀbromooctadecane (1.31 g, 3.92 mmol), and TBAI (101 mg,
2
3
0.27 mmol) to the general procedure for the synthesis of 4ꢀOꢀalkyl benzoates, the title compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was obtained as an amorphous white solid (968 mg, 2.39 mmol, 90%). R = 0.54
f
1
(
petroleum ether:EtOAc, 24:1, v/v); HꢀNMR (500 MHz, CDCl ) δ 7.98 (d, J = 8.3 Hz, 2H, Hꢀ
3
2,3
2
), 6.90 (d, J3,2 = 8.6 Hz, 2H, Hꢀ3), 4.00 (t, J6,7 = 6.6 Hz, 2H, CH ꢀ6), 3.88 (s, 3H, CO Me), 1.79
2 2
(
p, J7,6 = J7,8 = 7.1 Hz, 2H, CH ꢀ7), 1.45 (p, J = J8,9 = 7.3 Hz, 2H, CH ꢀ8), 1.42ꢀ1.26 (m, 28H,
2 8,7 2
13
CH ꢀ9ꢀCH ꢀ22), 0.88 (t, J
= 6.5 Hz, 3H, CH ꢀ23); CꢀNMR (125 MHz, CDCl ) 166.9 (Cꢀ5),
3 3
2
2
23,22
1
63.0 (Cꢀ4), 131.6 (Cꢀ2), 122.3 (Cꢀ1), 114.0 (Cꢀ3), 68.2 (Cꢀ6), 51.8 (CO Me), 31.9, 29.7, 29.6,
2
29.5, 29.4, 26.0, 22.7 (Cꢀ8ꢀCꢀ22), 29.1 (Cꢀ7), 14.1 (Cꢀ23); IR (film): 2916, 2847, 1723, 1608,
ꢀ1
+
1
510, 1473, 1256, 850 cm ; HRMS (ESI) calcd. for [C H O ] 405.3363; obsd. 405.3359.
26 45 3
Methyl 2ꢀhydroxyꢀ6ꢀmethylꢀ4ꢀ(octadecyloxy)benzoate. By subjecting ethyl 2,4ꢀdihydroxyꢀ
6ꢀmethylbenzoate (500 mg, 2.55 mmol), K CO (478 mg, 3.46 mmol), 1ꢀbromooctadecane
2
3
(
1.047, 3.14 mmol), and TBAI (50 mg, 0.14 mmol) to the general procedure for the synthesis of
4
ꢀOꢀalkylꢀbenzoates, the title compound was obtained as an amorphous pale yellow solid (1.13
1
g, 2.52 mmol, 99%). R = 0.58 (CH Cl ); H NMR (500 MHz, CDCl ) δ 11.85 (s, 1H, OH), 6.31
f
2
2
3
(
d, J = 2.5 Hz, 1H, Hꢀ3), 6.28 (d, J = 2.5 Hz, 1H, Hꢀ5), 4.39 (q, J = 7.2 Hz, 2H, CH ꢀOEt),
3,5 5,3 2
3
.94 (t, J = 6.6 Hz, 2H, CH ꢀ8), 2.50 (s, 3H, 6ꢀMe), 1.76 (p, J = J = 7.4 Hz, 2H, CH ꢀ9),
8,9 2 9,8 9,10 2
1
.39ꢀ1.45 (m, 5H, CH ꢀOEt & CH ꢀ10), 1.24ꢀ1.36 (m, 28H, CH ꢀ11ꢀCH ꢀ24), 0.88 (t, J
25,24
3
2
2
2
13
= 7.0 Hz, 3H, CH ꢀHꢀ25); C NMR (125 MHz, CDCl ) δ 171.9 (Cꢀ7), 165.7 (Cꢀ2), 163.6 (Cꢀ4),
3
3
1
43.2 (Cꢀ6), 111.7 (Cꢀ5), 105.3 (Cꢀ1), 99.3 (Cꢀ3), 68.2 (Cꢀ8), 61.3 (CH ꢀOEt), 32.1, 29.9, 29.83,
2
2
9.82, 29.81, 29.74, 29.70, 29.52, 29.49, 22.9 (Cꢀ11ꢀCꢀ24), 29.2 (Cꢀ9), 26.1 (Cꢀ10), 24.6 (6ꢀMe),
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6
1
8
4.4 (CH ꢀOEt), 14.3 (Cꢀ25); IR (film): 2917, 2848, 1648, 1613, 1581, 1365, 1262, 1178, 1041,
3
ꢀ1
+
15 cm ; HRMS (ESI) calcd. For [C H O +H] : 449.3625; obsd.: 449.3631.
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8
48
4
General benzylation procedure. To a solution of benzoate (1 equiv.) in acetone (20 mL) was
added K CO (1.2ꢀ3 equiv.), benzyl bromide (1.2–3 equiv), and TBAI (0.03–0.1 equiv.). The
0
1
2
3
4
5
6
7
8
9
0
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0
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0
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0
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resulting mixture was refluxed for 18 hours, cooled to room temperature, and concentrated in
vacuo. The residue was then purified using silicaꢀgel flash column chromatography (petroleum
ether to petroleum ether:EtOAc; 4:1; v/v).
Methyl 2,4ꢀbis(benzyloxy)benzoate. By subjecting methyl 2,4ꢀdihydroxybenzoate (178 mg,
1
.15 mmol), benzyl bromide (0.41 mL, 3.46 mmol), K CO (478 mg, 3.46 mmol), and TBAI (43
2 3
mg, 0.12 mmol) to the general procedure for benzylation, the title compound was obtained as an
1
amorphous yellow solid (378 mg, 1.08 mmol, 94%). R = 0.82 (CH Cl ); H NMR (500 MHz,
f
2
2
CDCl ) δ 7.88 (d, J = 8.6 Hz, 1H, Hꢀ6), 7.50 (d, J = 7.6 Hz, 2H, CHarom), 7.42ꢀ7.30 (m, 8H,
3
6,5
CHarom), 6.61 (d, J = 2.2 Hz, 1H, Hꢀ3), 6.59 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H, Hꢀ5), 5.15 (s,
3,5
5,6
5,3
1
3
2
H, CH Ph), 5.07 (s, 2H, CH Ph), 3.88 (s, 3H, CO Me); C NMR (125 MHz, CDCl ) δ 166.4
2 2 2 3
(Cꢀ7), 163.4, 160.4 (Cꢀ2, Cꢀ4), 136.3 (C , CH Ph), 134.1 (C , CH Ph), 128.8 (Cꢀ6), 128.7, 128.4,
i 2 i 2
1
27.9, 127.7, 126.9 (CHarom), 113.3 (Cꢀ1), 106.2 (Cꢀ5), 101.6 (Cꢀ3), 70.7 (CH Ph), 70.4
2
(CH Ph), 51.9 (CO Me); IR (film): 3026, 2949, 2853, 1723, 1700, 1605, 1250, 1178, 1141,
2 2
ꢀ1
+
1
3
087, 1024, 848, 734 cm ; HRMS (ESI) m/z calcd. for [C H O +H] : 349.1434; obsd.:
22 20 4
49.1434.
Ethyl 2,4ꢀbis(benzyloxy)ꢀ6ꢀmethylbenzoate. By subjecting ethyl 2,4ꢀdihydroxyꢀ6ꢀ
methylbenzoate (300 mg, 1.53 mmol), K CO (517 mg, 3.74 mmol), benzyl bromide (0.47 mL,
2
3
3
.82 mmol) to the general procedure for benzylation, the title compound was obtained as an
amorphous white solid (566 mg, 1.50 mmol, 98%). R = 0.42 (Petroleum ether:EtOAc, 4:1, v/v);
f
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H NMR (600 MHz, CDCl ) δ 7.40ꢀ7.28 (m, 10H, CHarom), 6.43 (d, J = 2.2 Hz, 1H, Hꢀ3), 6.42
3
3,5
(d, J = 2.2 Hz, 1H, Hꢀ5), 5.04 (s, 2H, CH Ph), 5.02 (s, 2H, CH Ph), 4.34 (q, J = 7.2 Hz, 2H,
5
,3
2
2
1
3
CH ꢀOEt), 2.30 (s, 3H, 6ꢀMe), 1.30 (t, J = 7.2 Hz, 3H, CH ꢀOEt); C NMR (150 MHz, CDCl ) δ
2
3
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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2
3
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6
7
8
9
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8
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6
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8
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2
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7
8
9
0
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2
3
4
5
6
7
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9
0
1
1
7
1
68.3 (Cꢀ7), 160.4 (Cꢀ4), 157.3 (Cꢀ2), 138.4 (Cꢀ6), 136.8 (C , CH Ph) , 136.7 (C , CH Ph), 128.8,
i 2 i 2
28.6, 128.2, 127.9, 127.6, 127.2 (CHarom), 117.6 (Cꢀ1), 108.3 (Cꢀ5), 98.6 (Cꢀ3), 70.5 (CH Ph),
2
0.2 (CH Ph), 61.1 (CH ꢀOEt), 20.0 (6ꢀMe), 14.4 (CH ꢀOEt); IR (film): 2930, 2901, 2873, 1716,
2
2
3
ꢀ1
590, 1432, 1365, 1334, 1286, 1264, 1160, 1096, 1038, 775, 696 cm ; HRMS (ESI) calcd. for
+
[
C H O +H] : 377.1747; obsd.: 377.1760.
2
4
24
4
Methyl 2ꢀ(benzyloxy)ꢀ4ꢀmethoxybenzoate. By subjecting methyl 2ꢀhydroxyꢀ4ꢀ
methoxybenzoate (290 mg, 1.59 mmol), benzyl bromide (0.26 mL, 2.07 mmol), K CO (297 mg,
2
3
2
.15 mmol), and TBAI (55 mg, 0.15 mmol) to the general procedure for benzylation, the title
1
compound was obtained as a pale yellow oil (422 mg, 1.55 mmol, 97%). R = 0.56 (CH Cl ); H
f
2
2
NMR (500 MHz, CDCl ) δ 7.89 (d, J = 9.3 Hz, 1H, Hꢀ6), 7.52 (d, J2',3' = 7.2 Hz, 2H, Hꢀ2'),
3
6,5
7
.40 (t, J3',2' = J3',4' = 7.2 Hz, 2H, Hꢀ3'), 7.32 (d, J4',3' = 7.2 Hz, 1H, Hꢀ4'), 6.50ꢀ6.53 (m, 2H, Hꢀ3 &
13
Hꢀ5), 5.17 (s, 2H, CH Ph), 3.87 (s, 3H, CO Me), 3.82 (s, 3H, OMe); C NMR (125 MHz,
2
2
CDCl ) δ 166.4 (Cꢀ7), 164.2 (Cꢀ4), 160.4 (Cꢀ2), 136.8 (Cꢀ1'), 134.1 (Cꢀ6), 128.7 (Cꢀ3'), 127.9
3
(Cꢀ4'), 126.9 (Cꢀ2'), 113.1 (Cꢀ1), 105.3 (Cꢀ5), 100.8 (Cꢀ3), 70.7 (CH Ph), 55.6 (OMe), 51.9
2
ꢀ1
(CO Me); IR (film): 2948, 2839, 1719, 1698, 1606, 1249, 1167, 1086, 1026, 734, 695 cm ;
2
+
HRMS (ESI) calcd. for [C H O +H] : 273.1121; obsd.: 273.1122.
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Methyl 2ꢀ(benzyloxy)ꢀ4ꢀbutoxybenzoate. By subjecting methyl 4ꢀbutoxyꢀ2ꢀhydroxybenzoate
(243 mg, 1.08 mmol), K CO (240 mg, 1.73 mmol), benzyl bromide (0.21 mmol, 1.73 mmol),
2
3
and TBAI (13 mg, 0.04 mmol) to the general procedure for benzylation, the title compound was
obtained as a white crystalline solid (312 mg, 0.99 mmol, 92%). R = 0.54 (CH Cl ); Mp. 39.8 –
f
2
2
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