Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity

Article abstract of DOI:10.1016/j.tet.2017.11.076

We report on the efficient synthesis of linear trehalose diesters (TDEs) and iso-branched TDEs (maradolipids or iso-TDEs) and their ability to activate bone marrow-derived macrophages (BMDMs) as determined by cytokine (IL-1β IL-12, IL-6, IL-10) and chemokine (MIP-2) production. Both classes of TDEs were found to activate BMDMs in a Mincle-dependent manner, with longer-chain (≥C18) lipids leading to a robust inflammatory response. On the whole, the iso-branched TDEs led to greater cytokine production and a faster immune response when compared to their linear counterparts. Moreover, C12-TDE and iso-C12-TDE elicited the production of MIP-2 by BMDMs, thereby providing the first example of TDEs with a chain length of ≤ C12 leading to a Mincle-dependent immune response and one that is less inflammatory in nature.

Full text of DOI:10.1016/j.tet.2017.11.076

Tetrahedron xxx (2017) 1e9
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Lipid length and iso-branching of trehalose diesters influences Mincle
agonist activity
Ayesha Khan b, Kristel Kodar a b, Mattie S.M. Timmer a
a
,
,
,
b
,
**
, Bridget L. Stocker a
, b, *
a
School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
Centre for Biodiscovery, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 September 2017
Received in revised form
We report on the efficient synthesis of linear trehalose diesters (TDEs) and iso-branched TDEs (mar-
adolipids or iso-TDEs) and their ability to activate bone marrow-derived macrophages (BMDMs) as
determined by cytokine (IL-1b, IL-12, IL-6, IL-10) and chemokine (MIP-2) production. Both classes of TDEs
were found to activate BMDMs in a Mincle-dependent manner, with longer-chain (ꢀC18) lipids leading
to a robust inflammatory response. On the whole, the iso-branched TDEs led to greater cytokine pro-
duction and a faster immune response when compared to their linear counterparts. Moreover, C12-TDE
and iso-C12-TDE elicited the production of MIP-2 by BMDMs, thereby providing the first example of TDEs
with a chain length of ꢁ C12 leading to a Mincle-dependent immune response and one that is less in-
flammatory in nature.
30 November 2017
Accepted 30 November 2017
Available online xxx
Keywords:
Glycolipid
Trehalose
Iso-branched fatty acid
Macrophage
Mincle
© 2017 Published by Elsevier Ltd.
1. Introduction
promising adjuvant activity, while
rivatives
b
-GlcCer13 and cholesterol de-
were recently identified as endogenous Mincle li-
14,15
Since the identification of the Macrophage inducible C-type
lectin (Mincle, Clec4e, or Clecf9) and the knowledge that the
gands, thereby highlighting the breadth of compounds found to
activate this receptor.
1,2
mycobacterial glycolipid, trehalose dimycolate (TDM, 1, Fig. 1),
binds and activates this receptor, there has been much interest in
the potential of Mincle agonists as immunostimulators and in un-
To better understand the scope and specificity of the Mincle-
binding site and how ligand binding correlates to a functional im-
mune response, a number of potential Mincle ligands have been
3
derstanding how changes to the ligand structure influences the
ensuing immune response. Notably, trehalose dibehenate (TDB, 2,
synthesised and their immunomodulatory properties asses-
4
11,12,16e23
sed.
Early studies by our group revealed that TDEs with a
n ¼ 20), which is a simplified trehalose diester (TDE), activates
carbon chain of ꢀ C18 (but not ꢁ C12) elicited a pro-inflammatory
16
Mincle in a manner similar to TDM, with induction of the FcR
g-Syk-
response by bone marrow derived macrophages (BMDMs), and
that trehalose monoesters activate macrophages in a Mincle-
Card9-Bcl10-Malt1 signalling axis and a T helper (Th)-1-polarised
immune response.5
e7
When formulated in dimethyldioctadecyl
dependent manner. The immunomodulatory profile of the Min-
17
8
18
ammonium (DDA) liposomes, TDB has found wide application as a
vaccine adjuvant in a number of pre-clinical and clinical vaccina-
cle ligands
b
-gentiobiosyl diacylglyceride,
glucose mono-
19
0
corynomycolate,
and 6 -acylated glucose and mannose
tion studies.9 Other synthetic Mincle agonists, such as 6 -acylated
,10
0
monoesters were also found to be influenced by lipid length. To a
lesser extent, changing the mycolic acid structure altered the
cytokine response of macrophages and dendritic cells (DCs) to
11
0
11
mannose and 6 -acylated glucose sugars, homogeneous TDMs,
trehalose monomycolate and glucose mycolate, also exhibit
12
12,20
12
TDMs
and trehalose monomycolates, with modifications to
stereochemistry also affecting Mincle agonist activity for both
12
mycolic
monocorynomycolates.
acid-containing
glucolipids
and
glycerol
*
Corresponding author. School of Chemical and Physical Sciences, Victoria Uni-
versity of Wellington, PO Box 600, Wellington, New Zealand.
* Corresponding author. School of Chemical and Physical Sciences, Victoria Uni-
versity of Wellington, PO Box 600, Wellington, New Zealand.
E-mail addresses: mattie.timmer@vuw.ac.nz (M.S.M. Timmer), bridget.stocker@
vuw.ac.nz (B.L. Stocker).
21
*
Given the potential of TDEs to bind and activate Mincle, we
became interested in exploring the ability of maradolipids to act as
Mincle agonists. Maradolipids consist of a mixture of symmetrical
https://doi.org/10.1016/j.tet.2017.11.076
0
040-4020/© 2017 Published by Elsevier Ltd.
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2
A. Khan et al. / Tetrahedron xxx (2017) 1e9
Fig. 1. TDM (1) and TDE (2).
and asymmetrical glycolipids containing mainly iso-branched and
straight chain fatty acids (Fig. 2), and are produced by the dauer
2
4,25
larva of the nematode Caenorhabditis elegans.
While mar-
adolipids are structurally very similar to TDEs, structure-activity
studies from previous Mincle-agonist work indicates that the
incorporation of the iso-branch might be sufficient to give com-
pounds with distinct immunomodulatory properties. Accordingly,
we sought to prepare symmetrical iso-branched maradolipids 3a-f
Scheme 1. Synthesis of iso-branched maradolipids (iso-TDEs) and linear TDEs.
(
iCnþ1) and the linear TDEs (2a, n ¼ 9; 2b, n ¼ 11; 2c, n ¼ 15) to
assess their immunomodulatory profiles by measuring cytokine
production by wild-type and Mincle
ꢂ/ꢂ
BMDMs. Herein, we
successful conjugation of the lipid moieties. The protected linear
TDEs (9a, b and d) were prepared in a similar manner via the
conjugation of hexa-TMS protected trehalose 5 with the commer-
cially available carboxylic acids 7a, (n ¼ 9), 7b (n ¼ 11), and 7d
determine that lipid length, as well as the presence or absence of
the iso-branch, has a remarkable effect on the response of macro-
phages to TDEs.
(
n ¼ 15).
Deprotection of protected iso-TDEs 8a-f was then achieved by
2
. Results and discussion
þ
using Dowex-H in DCM and MeOH (1:1), which provided excellent
yields of the desired iso-TDEs 3a-f after purification by silica gel
flash column chromatography. Again, 1D- and 2D-NMR techniques
were used to analyse the products, and the presence of only one
To synthesise the iso-branched and linear TDEs we envisioned
using the strategy of Toubiana and co-workers to prepare
0
0
0
0
a,a
2
a
lipid.
,3,4,2 ,3 ,4 -hexa-O-trimethylsilyl-
-trehalose in one step from
0
,a -trehalose, with subsequent elaboration to the target glyco-
signal for the anomeric centres (at ca.
formation of symmetric products. Formation of the linear TDEs (2a,
b and 2d) was achieved in an analogous manner, with the spectral
d
¼ 5.9 ppm) confirmed the
26
1
6,27
To this end, bis(trimethylsilyl)acetamide (BSA) and cata-
2
lytic tetrabutylammonium fluoride (TBAF) were used to persilylate
16,29
data for 2b and 2d matching those previously reported.
0
a
6
,
a
-D
-trehalose (4), with the most labile TMS groups at the 6- and
CO
With the iso-branched maradolipids in hand, we then assessed
the production of cytokines and chemokines by granulocyte-
monocyte colony-stimulating factor (GM-CSF) BMDMs upon stim-
ulation with the ligands. Here, the linear TDEs 2a (C12), 2b (C14),
and 2d (C18) could be directly compared to 3a (iC12 þ 1), 3b
0
-positions being subsequently removed via the agency of K
2
3
to afford hexa-TMS protected trehalose 5 in a moderate yield (67%
over two-steps, Scheme 1). Esterification with the commercially
available iso-branched fatty acids 6a and 6c-f, or the C14þ1 fatty acid
6
b, which can be synthesised via a Wittig reaction using the tri-
(
iC14 þ 1) and 3d (iC18 þ 1) to ascertain whether the incorporation
phenylphosphonium salt derived from 11-bromoundecanoic acid
of a single methyl group affected the type and magnitude of im-
2
8
and Ph
occurred
dimethylaminopropyl)carbodiimide
3
P, and isobutyraldehyde, followed by hydrogenation, then
mune response, while the C4 linear TDE, which we had previously
under
the
mediation
of
1-ethyl-3-(3-
and 4-
16,17
synthesised and found not to activate BMDMs,
served as a
(EDCI)
negative control. TDB and LPS served as positive controls.
It is known that TDM and TDB lead to a pro-inflammatory Th-1
immune response upon the binding and activation of Mincle.
Accordingly, we first determined the production of the pro-
inflammatory cytokine interleukin (IL)-1b by BMDMs upon stim-
ulation with the glycolipids (Fig. 3A). To this end, the TDEs were
assessed for their ability to activate BMDMs at two different con-
ꢃ
dimethylaminopyridene (DMAP) in toluene at 56 C for 8e10 h to
give the corresponding esters 8a-f in good to excellent yield
6,7
(
71e90%) following purification by silica gel flash column chro-
matography. For each glycolipid, Heteronuclear Multiple Bond
Correlations (HMBCs) between H-6a and H-6b of the trehalose
moiety with the carbonyl carbons of the lipids confirmed the
centrations (20 mM and 40 mM), with cytokine production being
measured at 24 h and 48 h. Here, maradolipids 3a,b (iC12 þ 1 and
iC14 þ 1) and the corresponding linear TDEs (C12 and C14) did not
result in significant IL-1b production at either time point, however
cytokine production was observed by all TDEs with a lipid
length ꢀ C16. The iso-branched maradolipid 3d (iC18 þ 1) led to
greater IL-1b production when compared to the analogous linear
C18, while cytokine production upon BMDM stimulation with the
longest iso-TDE (iC20 þ 1) was similar to, if not better than, TDB. In
all instances, IL-1b production was concentration dependent and
Fig. 2. Maradolipids and target structures.
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A. Khan et al. / Tetrahedron xxx (2017) 1e9
3
ꢂ/ꢂ
Fig. 3. WT and Mincle
BMMs were stimulated with linear or iso-branched TDEs (20
m
M, 40
m
M), or LPS (100 ng/mL) as a positive control. The production of IL-1
b
(A), IL-12p40
(
B), IL-6 (C), MIP-2 (D) and IL-10 (E) were measured at 24 and 48 h using ELISA. Mean ± SEM of triplicate samples from a representative experiment of three performed are shown.
*
P ꢁ 0.05; **P ꢁ 0.01; ***P ꢁ 0.005; ****P ꢁ 0.001. Statistical significance was calculated in comparison to unstimulated cells.
Please cite this article in press as: Khan A, et al., Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity,
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4
A. Khan et al. / Tetrahedron xxx (2017) 1e9
was also dependent on Mincle.
Next, the production of IL-12 by BMDMs in response to the
TDEs was determined by measuring IL-12p40, which is the 40 kDa
3
0
heavy-chain that makes up the IL-12 heterodimer. The cytokine
IL-12 has an important role in the development of the Th-1 im-
mune response, and in particular, in inducing IFN-g production by
3
0
activated natural killer (NK) and T cells. After 24 h, there was
significant IL-12 production by BMDMs in response to the iso-
TDEs with a chain length of ꢀ C18, however, of the linear TDEs,
only TDB led to a statistically significant increase in IL-12 (Fig. 3B).
The amount of IL-12 produced by the BMDMs at 24 h in response
Fig. 4. A comparison of the production of IL-12p40 (A) and IL-10 (B) by WT BMMs,
treated with 40
samples from a representative experiment of three performed.
m
M of TDB and iC20 þ 1 at 24 and 48 h. Mean ± SEM of triplicate
to 40
m
g/mL of maradolipids 3e (iC19 þ 1) and 3f (iC20 þ 1) was
also significantly greater than that produced by TDB, however
after 48 h, IL-12 production by BMDMs in response to the mar-
adolipids had reached a plateau. Notwithstanding, IL-12 produc-
tion at 72 h was greater for BMDMs stimulated with 3f (iC20 þ 1)
than for cells stimulated with TDB at both concentrations of
glycolipid (data not shown). Once again, IL-12 production was
Mincle-dependent. Taken together, this suggests that the iso-TDEs
elicit a greater and more rapid immune response than their non-
branched counterparts.
the regulation of the inflammatory response via the production of
IL-10 occurs at an earlier time point as well. This is illustrated by
the more direct comparison of IL-12 and IL-10 production at 24
and 48 h for two representative TDEs, TDB and iC20 þ 1 (Fig. 4A
and B), whereby both IL-12 and IL-10 production by iC20 þ 1 is
greater at 24 rather than 48 h, which is also reflected in the
amount of IL-10 produced at these time points. Conversely, TDB
appears to have slower kinetics, with slower onsets of both IL-12
and IL-10. There have only been few studies on IL-10 production
by macrophages in response to TDB, with IL-10 being produced by
The ability of GM-CSF BMDMs to produce the cytokine IL-6 and
the macrophage inflammatory protein-2 (MIP-2 or CXCL2) upon
stimulation with the glycolipids was then explored. Although
mostly regarded as a pro-inflammatory cytokine, IL-6 also has
37
the human THP-1 cell line upon treatment with TDB, and with
beads coated with TDB promoting IL-10 production by BMDMs,
thereby down-regulating IL-12 secretion in response to the Toll-
31
regenerative or anti-inflammatory activities, while MIP-2 is a
3
8
chemotactic for polymorphonuclear leukocytes, such as neutro-
3 4
like receptor (TLR)-2 ligand Pam CSK . Thus, our findings sup-
phils, and hematopoietic stem cells, including monocytes and
port these earlier observations and further demonstrate the
reciprocal relationship between the production of IL-12 and IL-10.
Taken together, our findings further support the observation
that minor changes to Mincle-ligands can affect the activation
status of macrophages. In particular, we demonstrate that lipid
length and the iso-branching of TDEs can result in immuno stim-
ulatory compounds with quite distinct biological profiles. As was
first described in our earlier studies exploring the ability of linear
macrophages.3
2e34
At 24 h and 48 h, the iso- and linear-TDEs with
chain lengths ꢀ C14 led to the production of IL-6 in a Mincle-
dependent manner (Fig. 3C). At 24 h, the production of IL-6 by
BMDMs in response to the iso-TDEs 3a (iC14 þ 1), 4b (iC16 þ 1)
and 4d (iC18 þ 1) was greater than that elicited by the corre-
sponding TDE analogues 2a (C14), 3b (C16) and 3d (C18), while
comparable amounts of IL-6 were produced at 48 h for both the
branched and non-branched TDEs. Absolute levels of IL-6 also
appeared to decline for both the iso-TDEs and the linear-TDEs at
16
TDEs to activate BMDMs, the pro-inflammatory immune response
is most notable for both the linear- and iso-branched TDEs of me-
dium to long lipid length. Herein, we also demonstrate that this
immune response is Mincle-dependent for both classes of TDE.
While there are subtle differences between the type and amount of
cytokines produced for each respective glycolipid, on the whole,
the most robust pro-inflammatory immune responses are observed
for the linear TDEs with a lipid length of ꢀC18, while iso-TDEs of a
slightly shorter chain length (i.e. ꢀ C14) are able to induce a similar
response. Moreover, on the whole, a more rapid immune response
is observed in response to the iso-TDEs, as compared to their linear
counterparts, with cytokine production at 24 h typically being
higher for the iso-derivatives. When directly comparing cytokine
and chemokine production elicited by BMDMs in response to
iC18 þ 1 and the linear C18 TDE, iC18 þ 1 generally leads to a
stronger immune response at both 24 and 48 h. The reason for this
is unclear, and while one could speculate that the lipophilic iso-
branch is better accommodated in the major hydrophobic grove
adjacent to the carbohydrate-binding domain of the Mincle-
4
8 h. The production of MIP-2 by BMDMs upon stimulation with
the glycolipids was also greater at 24 rather than 48 h, with both
the iso- and linear-TDEs leading to comparable levels of this
chemokine in a Mincle-dependent manner (Fig. 3D). In contrast to
the production of cytokines IL-1b, IL-12 and IL-6, MIP-2 is pro-
duced by BMDMs in response to TDEs with a shorter chain length.
Notably, the ability of both C12 TDE (2a) and iC12 þ 1 (3a) to
induce MIP-2 production by BMDMs with no (i.e. C12) or minor
(
i.e. iC12 þ 1) production of any of the aforementioned cytokines is
unprecedented.
Finally, the ability of the linear- and iso-TDEs to skew the im-
mune response to more anti-inflammatory phenotype was
explored, as indicated by the production of the potent anti-
3
5
inflammatory cytokine IL-10.
Here, further differences be-
tween the responses elicited by BMDMs following stimulation
with the TDEs and the iso-TDEs was observed (Fig. 3E). The iso-
TDEs with chain length ꢀ C18 led to the production of IL-10 in a
Mincle-dependent manner at both the 24 h and 48 h time points,
while the C18 linear TDE was unable to induce IL-10 production.
Moreover, IL-10 production by BMDMs in response to iC18 þ 1,
iC19 þ 1 and iC20 þ 1 was greater than that produced by the
BMDMs upon stimulation with TDB (2), particularly at lower
concentrations of the glycolipids. It is well established that IL-10
plays an essential role in down-regulating the immune response
to cytokines such as IL-12, so as to limit chronic and acute
3
9e41
binding site,
this major hydrophobic groove is best suited for
binding the first few methylenes of the lipid, not so much their
termini. Thus, it is possible that the difference in immune response
between the linear and iso-branched TDEs is physiochemical in
nature, and is due to the increased fluidity that iso-branching
4
2
typically confers on the membrane. Notwithstanding, our data
indicates that while lipid length remains an important consider-
ation in the development of Th-1-promoting Mincle-agonist, the
addition of an iso-branch further supports agonist activity in vitro.
More advanced in vivo studies would be required to determine
3
5,36
inflammation that could be detrimental to the host.
Given
that the iso-TDEs appear to result in a more rapid immune
response than their linear counterparts, it is not surprising that
Please cite this article in press as: Khan A, et al., Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity,
Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.076

A. Khan et al. / Tetrahedron xxx (2017) 1e9
5
whether the ability of the longer chain iso-TDEs to more rapidly
engage the immune response is beneficial in terms of vaccine
development, however, it would seem intuitive that this is the case
given that the innate immune response enhances the activity of
methylnonadecanoic acid (Larodan AB), 19-methyleicosanoic acid
(Larodan AB), PPh (Acros Organics), BuLi (2 M in cyclohexane,
Alridch), isobutyraldehyde (Aldrich), pyridine (ROMIL), C
(Apollo), CD OD (Cambridge Isotopes Laboratories Inc.), CDCl
(Aldrich) were used as received. Reactions were monitored by TLC
analysis on Macherey-Nagel silica gel coated plastic sheets
(0.20 mm with fluorescent indicator UV254) via detection by UV
3
5 5
D N
3
3
4
3
antigen-presenting cells and the ensuing T cell response.
Another key observation from our studies is the ability of
medium length TDEs, such as C12 and iC12 þ 1, to lead to the
induction of the chemokine MIP-2, and to a lesser extent IL-6, by
BMDMs in the absence of other inflammatory cytokines. In the
case of C12-TDE, only MIP-2 was produced by the BMDMs. MIP-2
elicits chemotaxis, and thus the ability of C12- and iC12 þ 1 to
recruit immune cells such as neutrophils and macrophages in the
absence of inflammatory cytokines may have unique applications,
including wound healing. Inflammatory signals are required to
recruit immune cells for tissue repair and the removal of debris,
however, sustained inflammation can lead to maladaptive pro-
cesses like loss of functional parenchyma, fibrosis and carcino-
absorbtion (254 nm) where relevant and dipping in 10% H
EtOH followed by charring or dipping in a solution of KMnO
(0.05 M), K CO (0.4 M), and NaOH (0.06%) in water. Column
chromatography was performed using Pure Science silica gel
(40e63 m). All solvents were removed by evaporation under
2 4
SO in
4
2
3
m
reduced pressure. High resolution mass spectra were recorded on
an Agilent 6530 Q-TOF mass spectrometer utilising a JetStream™
electro-spray ionisation (ESI) source in positive or negative mode.
Optical rotations were recorded on a Autopol II (Rudolph Research
Analytical) at 589 nm (sodium Dline). Infrared (IR) spectra were
recorded as thin films using either a Bruker Platinum-ATR spec-
4
4
genesis in a variety of organs. In the same way that Toll-like-
receptor (TLR) ligands can impair or contribute to the healing
process depending on the exact type of TLR and its associated
ligand,45 it is possible that C12-TDE or iC12 þ 1 could enhance
wound healing in situations where immune cell recruitment but a
limited inflammatory response are required. Moreover, the pro-
duction of cytokines and/or chemokines by BMDMs in response to
C12 and iC12 þ 1 illustrates, for the first time, that medium-length
TDEs can activate macrophages in a Mincle-dependent manner.
Thus, to fully explore the potential of TDEs as adjuvants or other
immunostimulating agents, it is prudent to assay for a variety of
cytokines and chemokines.
ꢂ1
trometer and are reported in wave numbers (cm ). Nuclear
ꢃ
magnetic resonance spectra were obtained at 20 C in CDCl
3
,
C
5
D
5
N, D
2
O, or CD
3
OD using a Varian INOVA operating at 500 MHz.
) relative to the solvent residual
Chemical shifts are given in ppm (
d
peak. NMR peak assignments were made using COSY, HSQC, and
HMBC 2D experiments.
3.2. 13-Methyltetradecanosanoic acid (6b)
11-Bromoundecanoic acid (500 mg, 2 mmol) and triphenyl
ꢃ
phosphine (530 mg, 2 mmol) were combined and heated to 100 C
In conclusion we have synthesised a variety of linear- and iso-
branched-TDEs in good yields and determined that both classes
of TDEs activate macrophages in a Mincle-dependent manner.
Notably, the iso-TDEs lead to greater cytokine production by
BMDMs when compared to their analogous linear counterparts,
and moreover, the iso-TDEs had more rapid kinetics. A long lipid
for 10 min under an Ar atmosphere. The resulting yellow oil was
dissolved in 10 mL of dry DMSO and heated for 10 min. The solution
was cooled to room temperature and butyl lithium (2.0 M in
cyclohexane, 2.5 mL, 5 mmol) was added drop wise over the course
of 1 h, with each addition leading to an orange colouration
appearing in the reaction mixture. The reaction was stirred for half
an hour and then isobutyraldehyde (0.50 mL, 5.0 mmol) was added
slowly. The reaction remained yellow after the addition of iso-
butyraldehyde was completed and the mixture was stirred for 2 h.
The suspension was diluted with EtOAc, and washed with 1 M HCl
and brine solution. The combined aqueous layers were extracted
with EtOAc (2 ꢄ 20 mL), and the combined organic phases were
dried with anhydrous magnesium sulfate, filtered, and concen-
trated in vacuo to give a yellow oil. The crude product was purified
by silica gel flash chromatography (PE to PE/Acetic acid, 99:1, v/v) to
yield E/Z-13-methyltetradec-11-enoic acid as a white solid. E/Z-13-
methyltetaredec-11-enoic acid was dissolved in DCM (5 mL),
(ꢀC18) is required for a robust inflammatory immune response by
both the linear and iso-TDEs, however it was observed that the C12
and iC12 þ 1 TDEs elicited the production of MIP-2 by BMDMs. This
is the first time that TDEs with a chain length of ꢁC12 have been
shown to lead to a Mincle-dependent functional immune response
and one that is less inflammatory in nature. While the translation of
this work to more advanced in vivo assays remains, our findings
nonetheless suggest that long-chain iso-TDEs may have superior
adjuvant activity, while the C12 linear and iso-branched TDEs may
have application in situations such as wound healing, where im-
mune cell recruitment along with a minimal inflammatory
response is required.
Pd(OH)
2
/C (57.6 mg, 22 wt% Pd) was added, and the black sus-
and stirred for 21 h. The
pension put under an atmosphere of H
2
suspension was filtered through Celite and concentrated to give a
white solid. The crude product was recrystallised from acetone to
give 13-methyltetradecanoic acid as a crystalline white solid
3
. Experimental
3
.1. General chemical
(223 mg, 0.92 mmol, 46%). R ¼ 0.24 (PE/EtOAc, 9:1, v/v);
f
ꢃ
28
ꢃ
Mp ¼ 51.0e51.9 C (lit. 51.5e51.7 C); IR (film) ¼ 2917, 2850, 1698,
ꢂ1
1
All reactions were performed under an atmosphere of argon.
1472 cm ; H NMR (500 MHz, CDCl )
d
2.3 (t, J ¼ 7.7 Hz, 2H, H-2),
3
2,3
Prior to use, methanol (Fisher Scientific) was distilled. Toluene
ROMIL) was dried and stored over Na wire, and the following
1.63 (p, J3,2 ¼ J ¼ 7.3 Hz, 2H, H-3), 1.51 (m, 1H, H-13), 1.37e1.21 (m,
3,4
(
16H, H-4eH-11), 1.18e1.12 (m, 2H, H-12), 0.86 (d, J
¼ 6.7 Hz, 6H,
13,14
1
3
solvents were distilled: acetone, ethyl acetate (EtOAc) and petro-
H-14); C NMR (125 MHz, CDCl3) d 179.0 (C-1), 39.0 (C-12), 33.89
0
leum ether (PE).
a,
a
-
D
-Trehalose dihydrate (Sigma), behenic acid
(Pure Science), NaCl (Chem Solute), Et
LabServ), BSA (Aldrich), Pd(OH) /C (Acros Organics), DMAP (Lab
(Pure Sci-
ence), HCl (Chem Solute), isopropanol (Fischer Scientific), KMnO
AnalR), 11-bromoundecanoic acid (Aldrich), 11-
(C-2), 29.9, 29.69, 29.65, 29.64, 29.59, 29.4, 29.2, 29.1 (C-4eC10), 28
(
(
BDH Biochem), MgSO
4
2
O
(C-13), 27.4 (C-11), 24.7 (C-3), 22.7 (C-14); HRMS (ESI) m/z calcu-
ꢂ
2
lated for [C15
H
29O] : 241.2173 found 241.2168. The data corre-
2
8
Supply), EDCI (Chem Impex), AcOH (Pancreac), NaHCO
3
sponded to those published.
4
(
3.3. General esterification procedure
methyldodecanosanoic acid (Larodan AB), 15-methylhexadecanoic
acid (Larodan AB), 17-methyloctadecanoic acid (Larodan AB), 18-
Maradolipids of different chain lengths were synthesised
Please cite this article in press as: Khan A, et al., Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity,
Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.076

6
A. Khan et al. / Tetrahedron xxx (2017) 1e9
1
6
0
0
0
0
0
0
0
according to a procedure by Khan et al. 2,2 ,3,3 ,4,4 -hexa-O-tri-
3.6. 6,6 -Di-O-15-methylhexadecanoyl-2,2 ,3,3 ,4,4 -hexa-O-
0
0
methylsilyl-
a,
a
-D
-trehalose (1 mmol, 1 equiv.) and carboxylic acid
trimethylsilyl-
a
,a
-D
-trehalose (8c)
(
(
4.4 mmol, 4 equiv.) were co-evaporated together with dry toluene
1 mL), then suspended in dry toluene (5 mL). To the reaction
By subjecting diol
5
(65 mg, 0.084 mmol), 15-
mixture, EDCI (6.6 mmol, 6.6 equiv.) and DMAP (1 mmol, 1 equiv.)
were added and the resulting suspension was heated to 56 C until
TLC analysis (PE/EtOAc, 9:1, v/v) showed complete conversion of
the starting material to a higher running product. The reaction was
methylhexadecanoic acid 6c (98 mg, 0.4 mmol), EDCI (106 mg,
0.55 mmol) and DMAP (10 mg, 0.084 mmol) to the general pro-
cedure for esterification (8 h), the title compound 8c was obtained
ꢃ
as a colourless oil (79.6 mg, 0.062 mmol, 74%). R
f
¼ 0.8 (PE/EtOAc,
2
3
cooled to r.t. and diluted with of Et
then washed with water (5 mL), sat. aq. NaHCO
(
2
O (5 mL). The organic layer was
(5 mL), and brine
5 mL). The combined aqueous phases were re-extracted with of
9
:1, v/v); ½
a
ꢅ
¼ þ140 (c ¼ 0.1, CHCl
3
); IR (film) ¼ 2953, 2924, 2841,
D
ꢂ1
1
3
1
(
741, 1249, 1109, 1099, 1075, 1008, 897, 870, 837, 746 cm ; H NMR
500 MHz, CDCl
3
)
d
4.92 (d, J1,2 ¼ 3.1 Hz, 2H, H-1), 4.27 (dd,
Et
2
O (5 mL) and the combined organic phases were dried with
, filtered, and concentrated in vacuo. The product
J6a,6b ¼ 12.0 Hz, J5,6a ¼ 2.0 Hz, 2H, H-6a), 4.05 (dd, J6a,6b ¼ 12.0 Hz,
J5,6b ¼ 4.4 Hz, 2H, H-6b), 4.02e3.97 (m, 2H, H-5), 3.90 (t,
J2,3 ¼ J3,4 ¼ 9.0 Hz, 2H, H-3), 3.48 (t, J3,4 ¼ J4,5 ¼ 9.0 Hz, 2H, H-4), 3.44
anhydrous MgSO
4
was purified using gradient silica-gel column chromatography (PE
to PE/EtOAc, 9:1, v/v).
(dd, J2,3 ¼ 9.0 Hz, J1,2 ¼ 3.0 Hz, 2H, H-2), 2.39e2.28 (m, 4H, H-8),
1.69e1.58 (m, 4H, H-9), 1.56e1.47 (m, 2H, H-21), 1.38e1.20 (m, 40H,
H-10eH-19), 1.19e1.11 (m, 4H, H-20), 0.86 (d, J21,22 ¼ 6.7, 12H, H-
0
0
0
0
3
.4. 6,6 -Di-O-11-methyldodecanoyl-2,2 ,3,3 ,4,4 -hexa-O-
13
2
2), 0.15 (s, 18H, TMS), 0.135 (s, 18H, TMS), 0.130 (s, 18H, TMS);
C
0
trimethylsilyl-
a,
a
-
D-trehalose (8a)
NMR (125 MHz, CDCl 173.8 (C-7), 94.5 (C-1), 73.5 (C-3), 72.6 (C-
3
) d
2
2
2
), 71.9 (C-4), 70.7 (C-5), 63.2 (C-6), 39.05 (C-20), 34.1 (C-8), 29.9,
9.68, 29.6, 29.4, 29.3, 29.1 (C-10eC-18), 27.9 (C-21), 27.4 (C-19),
By subjecting trehalose diol 5 (80 mg, 0.10 mmol), 11-
methyldodecanoic acid 6a (97 mg, 0.44 mmol), EDCI (130 mg,
4.7 (C-9), 22.6 (C-22),1.0, 0.8, 0.1 (TMS); HRMS (ESI) m/z calculated
0
.66 mmol) and DMAP (12.6 mg, 0.103 mmol) to the general pro-
cedure for esterification (8 h), the title compound 8a was obtained
as a yellow oil (52.9 mg, 0.072 mmol, 71%). R
¼ 0.8 (PE/EtOAc, 9:1,
); IR (film) ¼ 2954, 2924, 2854,
738, 1459, 1250, 1163, 1110, 1075, 1044, 1009, 963, 897, 871, 841,
þ
for [C64
H134NaO13Si
6
] : 1301.8332, found 1301.8363.
f
0
0
0
0
3.7. 6,6 -Di-O-17-methyloctadecanoyl-2,2 ,3,3 ,4,4 -hexa-O-
2
3:1
v/v); ½
aꢅ
¼ þ50 (c ¼ 0.1 CHCl
3
0
a,a -D
D
trimethylsilyl-
-trehalose (8d)
1
ꢂ1
1
7
47, 683 cm ; H NMR (500 MHz, CDCl
3
)
d
4.92 (d, J1,2 ¼ 3.1 Hz, 2H,
By subjecting diol
methyloctadecosanoic acid 6d (101 mg, 0.34 mmol), EDCI (98 mg,
.5 mmol) and DMAP (9.4 mg, 0.077 mmol) to the general pro-
cedure for esterification (10 h), the title compound 8d was obtained
as a colourless oil (86 mg, 0.064 mmol, 76%). R
¼ 0.8 (PE/EtOAc, 9:1,
); IR (film) ¼ 2923, 1742, 1249,
5
(60 mg, 0.077 mmol), 17-
H-1), 4.27 (dd, J6a,6b ¼ 12.0 Hz, J5,6a ¼ 2.0 Hz, 2H, H-6a), 4.06 (dd,
J
3
4
6a,6b ¼ 12.0 Hz, J5,6b ¼ 4.5 Hz, 2H, H-6b), 4.02e3.97 (m, 2H, H-5),
0
.90 (t, J2,3 ¼ J3,4 ¼ 9.0 Hz, 2H, H-3), 3.48 (t, J3,4 ¼ J4,5 ¼ 9.0 Hz, 2H, H-
), 3.44 (dd, J2,3 ¼ 9.0 Hz, J1,2 ¼ 3.0 Hz, 2H, H-2), 2.40e2.28 (m, 4H,
f
H-8), 1.66e1.58 (m, 4H, H-9), 1.54e1.47 (m, 2H, H-17), 1.35e1.21 (m,
2
3:9
v/v); ½
aꢅ
¼ þ96 (c ¼ 0.1, CHCl
3
D
2
4H, H-10eH-15), 1.18e1.11 (m, 4H, H-16), 0.86 (d, J17,18 ¼ 6.7, 12H,
13
ꢂ1
1
1163, 1075, 1008, 870, 837, 747 cm ; H NMR (500 MHz, CDCl
4.92 (d, J1,2 ¼ 3.1 Hz, 2H, H-1), 4.27 (dd, J6a,6b ¼ 12.0 Hz,
5,6a ¼ 2.0 Hz, 2H, H-6a), 4.06 (dd, J6a,6b ¼ 12.0 Hz, J5,6b ¼ 4.4 Hz, 2H,
H-6b), 4.02e3.98 (m, 2H, H-5), 3.90 (t, J2,3 ¼ J3,4 ¼ 9.0 Hz, 2H, H-3),
.48 (t, J3,4 ¼ J4,5 ¼ 9.0 Hz, 2H, H-4), 3.44 (dd, J2,3 ¼ 9.0 Hz,
1,2 ¼ 3.0 Hz, 2H, H-2), 2.40e2.28 (m, 4H, H-8), 1.66e1.58 (m, 4H, H-
), 1.56e1.47 (m, 2H, H-23), 1.38e1.19 (m, 48H, H-10eH-21),
.18e1.11 (m, 4H, H-22), 0.86 (d, J23,24 ¼ 6.7, 12H, H-24), 0.15 (s, 18H,
3
)
H-18), 0.15 (s,18H, TMS), 0.135 (s,18H, TMS), 0.130 (s,18H, TMS);
C
d
3
NMR (125 MHz, CDCl ) d 173.8 (C-7), 94.3 (C-1), 73.4 (C-3), 72.6 (C-
J
2
2
9
), 71.8 (C-4), 70.7 (C-5), 63.2 (C-6), 39.0 (C-16), 34.1 (C-8), 29.8,
9.6, 29.4, 29.3, 29.1 (C-10eC-14), 27.9 (C-17), 27.4 (C-15), 24.7 (C-
3
J
9
1
), 22.6 (C-18), 1.0, 0.8, 0.1 (TMS); HRMS (ESI) m/z calculated for
þ
[C
56
H118NaO13Si
6
] : 1189.7080, found 1189.7083.
13
0
0
0
0
TMS), 0.135 (s, 18H, TMS), 0.130 (s, 18H, TMS); C NMR (125 MHz,
CDCl3) 173.8 (C-7), 94.3 (C-1), 73.5 (C-3), 72.6 (C-2), 71.9 (C-4),
0.7 (C-5), 63.3 (C-6), 39.0 (C-22), 34.1 (C-8), 29.9, 29.73, 29.7, 29.6,
3
.5. 6,6 -Di-O-13-methyltetradecanoyl-2,2 ,3,3 ,4,4 -hexa-O-
0
d
trimethylsilyl-
a,
a
-
D-trehalose (8b)
7
2
2
9.4, 29.3, 29.1 (C-10eC-20), 27.9 (C-23), 27.4 (C-21), 24.7 (C-9),
2.6 (C-24), 1.0, 0.8, 0.1 (TMS); HRMS (ESI) m/z calculated for
By subjecting trehalose diol 5 (80 mg, 0.103 mmol), 13-
methyltetradecanoic acid 6b (112.5 mg, 0.46 mmol), EDCI
þ
[C
68
H142NaO13Si
6
] : 1357.8953, found 1357.8918.
(
130 mg, 0.66 mmol) and DMAP (12.6 mg, 0.10 mmol) to the general
procedure for esterification (8 h), the title compound 8b was ob-
tained as a colourless oil (111.7 mg, 0.091 mmol, 88%). R
0
0
0
0
f
¼ 0.6 (PE/
3.8. 6,6 -Di-O-18-methylnonadecanoyl-2,2 ,3,3 ,4,4 -hexa-O-
trimethylsilyl-
2
1:8
0
a
,a
-D
-trehalose (8e)
EtOAc, 9:1, v/v); ½
aꢅ
¼ þ60 (c ¼ 0.1 CHCl
3
); IR (film) ¼ 2923,
D
ꢂ1
1
2
853, 1741, 1250, 1164,1076, 871, 843, 747 cm ; H NMR (500 MHz,
By subjecting diol
methylnonadecanoic acid 6e (106 mg, 0.34 mmol), EDCI (98 mg,
.5 mmol) and DMAP (9.4 mg, 0.077 mmol) to the general pro-
cedure for esterification (10 h), the title compound 8e was obtained
as a colourless oil (95.5 mg, 0.069 mmol, 90%). R
¼ 0.6 (PE/EtOAc,
); IR (film) ¼ 2923, 2853,
5
(60 mg, 0.077 mmol), 18-
CDCl
5,6a ¼ 2.0 Hz, 2H, H-6a), 4.07 (dd, J6a,6b ¼ 12.0 Hz, J5,6b ¼ 4.5 Hz, 2H,
H-6b), 4.03e3.99 (m, 2H, H-5), 3.92 (t, J2,3 ¼ J3,4 ¼ 9.0 Hz, 2H, H-3),
.49 (t, J3,4 ¼ J4,5 ¼ 9.0 Hz, 2H, H-4), 3.45 (dd, J2,3 ¼ 9.0 Hz,
1,2 ¼ 3.0 Hz, 2H, H-2), 2.41e2.29 (m, 4H, H-8), 1.66e1.58 (m, 4H, H-
), 1.57e1.48 (m, 2H, H-19), 1.37e1.23 (m, 32H, H-10eH-17),
.19e1.13 (m, 4H, H-18), 0.87 (d, J19,20 ¼ 6.7, 12H, H-20), 0.16 (s, 18H,
3
)
d
4.93 (d, J1,2 ¼ 3.1 Hz, 2H, H-1), 4.29 (dd, J6a,6b ¼ 12.0 Hz,
J
0
3
J
9
f
2
4:7
9:1, v/v); ½
aꢅ
¼ þ150 (c ¼ 0.1, CHCl
1742, 1250, 1164, 1076, 1010, 842, 841, 748 cm ; H NMR (500 MHz,
CDCl
4.92 (d, J1,2 ¼ 3.1 Hz, 2H, H-1), 4.28 (dd, J6a,6b ¼ 12.0 Hz,
3
D
ꢂ1
1
1
13
TMS), 0.15 (s, 18H, TMS), 0.14 (s, 18H, TMS); C NMR (125 MHz,
CDCl 173.8 (C-7), 94.3 (C-1), 73.5 (C-3), 72.6 (C-2), 71.9 (C-4),
3
) d
3
)
d
J
5,6a ¼ 2.0 Hz, 2H, H-6a), 4.06 (dd, J6a,6b ¼ 12.0 Hz, J5,6b ¼ 4.4 Hz, 2H,
7
2
2
0.7 (C-5), 63.2 (C-6), 39.0 (C-18), 34.6 (C-8), 29.9, 29.7, 29.6, 29.4,
9.3, 29.1 (C-10eC-16), 27.9 (C-19), 27.4 (C-17), 24.8 (C-9), 22.6 (C-
H-6b), 4.02e3.97 (m, 2H, H-5), 3.90 (t, J2,3 ¼ J3,4 ¼ 9.0 Hz, 2H, H-3),
3.48 (t, J3,4 ¼ J4,5 ¼ 9.0 Hz, 2H, H-4), 3.44 (dd, J2,3 ¼ 9.0 Hz,
0), 1.0, 0.8, 0.1 (TMS); HRMS (ESI) m/z calculated for
J
1,2 ¼ 3.0 Hz, 2H, H-2), 2.40e2.28 (m, 4H, H-8), 1.66e1.58 (m, 4H, H-
þ
[C
60
H126NaO13Si
6
] : 1245.7706, found 1245.7700.
9), 1.56e1.47 (m, 2H, H-24), 1.36e1.20 (m, 52H, H-10eH-22),
Please cite this article in press as: Khan A, et al., Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity,
Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.076

A. Khan et al. / Tetrahedron xxx (2017) 1e9
7
1
.18e1.10 (m, 4H, H-23), 0.86 (d, J24,25 ¼ 6.7, 12H, H-25), 0.15 (s, 18H,
solid (52 mg, 0.066 mmol, 80%). R
f
¼ 0.63 (EtOAc/MeOH, 9:1, v/v);
13
2
4:8
TMS), 0.135 (s, 18H, TMS), 0.130 (s, 18H, TMS); C NMR (125 MHz,
CDCl 173.8 (C-7), 94.3 (C-1), 73.5 (C-3), 72.6 (C-2), 71.9 (C-4),
7
2
2
½
a
ꢅ
¼ þ7 (c ¼ 0.1, C
5
H
5
N); IR (film) ¼ 3334, 2917, 2849, 1727,
D
3
) d
1
7
1
467, 1383, 13,366, 1214, 1170, 1154, 1101, 1022, 984, 938, 912, 806,
54 cm ; H NMR (500 MHz, C
), 5.14e5.09 (m, 2H, H-5), 5.04e5.00 (m, 2H, H-6a), 4.85 (dd,
0.7 (C-5), 63.3 (C-6), 39.0 (C-23), 34.1 (C-8), 29.9, 29.73, 29.70,
9.69, 29.66, 29.6, 29.4, 29.3, 29.1 (C-10eC-21), 27.9 (C-24), 27.4 (C-
ꢂ1 1
5
H
5
N)
d
5.91 (d, J1,2 ¼ 3.7 Hz, 2H, H-
2), 24.7 (C-9), 22.6 (C-25), 1.0, 0.87, 0.18 (TMS); HRMS (ESI) m/z
J
6a,6b ¼ 11.7 Hz, J5,6b ¼ 5.4 Hz, 2H, H-6b), 4.77 (t, J2,3 ¼ J3,4 ¼ 9.5 Hz,
H, H-3), 4.33 (dd, J2,3 ¼ 9.5 Hz, J1,2 ¼ 3.7 Hz, 2H, H-2), 4.19 (t,
3,4 ¼ J4,5 ¼ 9.5 Hz, 2H, H-4), 2.39e2.28 (m, 4H, H-8), 1.63 (p,
J8,9 ¼ J9,10 ¼ 7.3 Hz, 4H, H-9), 1.54e1.46 (m, 2H, H-19), 1.36e1.13 (m,
þ
calculated for [C70
H
146NaO13Si
6
] : 1185.9271, found 1185.9294.
2
J
0
0
0
0
3.9. 6,6 -Di-O-19-methyleicosanoyl-2,2 ,3,3 ,4,4 -hexa-O-
0
13
trimethylsilyl-
a,
a
-D-trehalose (8f)
36H, H-10eH-18), 0.88 (d, J
¼ 6.5, 12H, H-20); C NMR
19,20
5 5
(125 MHz, C H N) d174.3 (C-7), 96.4 (C-1), 75.5 (C-3), 74.0 (C-2),
By subjecting diol 5 (60 mg, 0.077 mmol), 19-methyleicosanoic
acid 6f (100 mg, 0.36 mmol), EDCI (98 mg, 0.5 mmol) and DMAP
72.6 (C-4), 72.1 (C-5), 65.0 (C-6), 39.9 (C-18), 35.0 (C-8), 30.8, 30.6,
30.61, 30.5, 30.4, 30.2, 30.0 (C-10eC-16), 28.8 (C-19), 28.3 (C-17),
25.9 (C-9), 23.4 (C-20); HRMS (ESI) m/z calculated for
(
(
(
9.4 mg, 0.077 mmol) to the general procedure for esterification
10 h), the title compound 8f was obtained as a colourless oil
þ
[C42H82NO ] : 808.5781, found 808.5776. The data corresponded
13
4
6
82.5 mg, 0.059 mmol, 77%). R
f
¼ 0.6 (PE/EtOAc, 9:1, v/v);
to those published.
2
5:5
½
a
ꢅ
¼ þ121 (c ¼ 0.1, CHCl
3
ꢂ1
); IR (film) ¼ 2923, 2854, 1736, 1250,
D
1
0
0
a,a -D
1
162, 1075, 870, 840, 737 cm ; H NMR (500 MHz, CDCl
1,2 ¼ 3.1 Hz, 2H, H-1), 4.28 (dd, J6a,6b ¼ 12.0 Hz, J5,6a ¼ 2.0 Hz, 2H, H-
a), 4.06 (dd, J6a,6b ¼ 12.0 Hz, J5,6b ¼ 4.4 Hz, 2H, H-6b), 4.02e3.97
m, 2H, H-5), 3.90 (t, J2,3 ¼ J3,4 ¼ 9.0 Hz, 2H, H-3), 3.48 (t,
3,4 ¼ J4,5 ¼ 9.0 Hz, 2H, H-4), 3.44 (dd, J2,3 ¼ 9.0 Hz, J1,2 ¼ 3.0 Hz, 2H,
H-2), 2.40e2.27 (m, 4H, H-8), 1.67e1.57 (m, 4H, H-9), 1.55e1.47 (m,
3
)
d
4.92 (d,
3.13. 6,6 -Di-O-15-methylhexadecanosanoyl-
-trehalose (3c)
J
6
(
J
By subjecting 8c (70 mg, 0.055 mmol) to the general procedure
for desilylation, the title compound 3c was obtained as a white solid
(36.5 mg, 0.043 mmol, 78%). R
f
¼ 0.68 (EtOAc/MeOH, 4:1, v/v);
2
6:6
½ ꢅ
a
¼ þ6 (c ¼ 1.0, C H N); IR (film) ¼ 3321, 2921, 2852, 1728,
5 5
D
2
0
H, H-25), 1.36e1.20 (m, 56H, H-10eH-25), 1.18e1.11 (m, 4H, H-24),
.86 (d, J25,26 ¼ 6.7, 12H, H-26), 0.15 (s, 18H, TMS), 0.135 (s, 18H,
1464, 1365, 1351, 1277, 1243, 1152, 1080, 1018, 983, 937, 806, 756,
ꢂ1
1
721 cm ; H NMR (500 MHz, C H N)
5
5
d
5.90 (d, J1,2 ¼ 3.7 Hz, 2H, H-
13
TMS), 0.130 (s, 18H, TMS); C NMR (125 MHz, CDCl
3
)
d
173.8 (C-7),
1), 5.13e5.07 (m, 2H, H-5), 5.01 (d, J6a,6b ¼ 11.7 Hz, 2H, H-6a), 4.84
(dd, J6a,6b ¼ 11.7 Hz, J5,6b ¼ 5.4 Hz, 2H, H-6b), 4.76 (t,
J2,3 ¼ J ¼ 9.5 Hz, 2H, H-3), 4.32 (dd, J ¼ 9.5 Hz, J ¼ 3.7 Hz, 2H,
9
3
2
4.3 (C-1), 73.5 (C-3), 72.6 (C-2), 71.9 (C-4), 70.7 (C-5), 63.3 (C-6),
9.0 (C-24), 34.1 (C-8), 29.9, 29.73, 29.70, 29.66, 29.63, 29.4, 29.3,
9.1 (C-10eC-22), 27.9 (C-25), 27.4 (C-23), 24.7 (C-9), 22.6 (C-26),
3,4
2,3
1,2
H-2), 4.18 (t, J ¼ J ¼ 9.5 Hz, 2H, H-4), 2.39e2.28 (m, 4H, H-8),
3
,4
4,5
1
.0, 0.8, 0.1 (TMS); HRMS (ESI) m/z calculated for
1.64 (p, J8,9 ¼ J
¼ 7.5 Hz, 4H, H-9), 1.55e1.46 (m, 2H, H-21),
9,10
þ
13
[C
72
H150NaO13Si
6
] : 1413.9584, found 1413.9587.
1.36e1.14 (m, 44H, H-10eH-20), 0.89 (d, J
¼ 6.6, 12H, H-22);
C
21,22
NMR (125 MHz, C
2), 72.4 (C-4), 72.0 (C-5), 64.8 (C-6), 39.7 (C-20), 34.8 (C-8), 30.6,
0.48, 30.46, 30.42, 30.37, 30.2, 30.0, 29.8 (C-10eC-17), 28.6 (C-21),
5 5
H N) d 174.0 (C-7), 96.2 (C-1), 75.3 (C-3), 73.8 (C-
3.10. General desilylation procedure
3
To a solution of TMS protected maradolipids in CH
5 mL, 1/1, v/v) Dowex-H (10% by weight) was added and the re-
2
Cl
2
:MeOH
28.1 (C-19), 25.7 (C-9), 23.2 (C-22); HRMS (ESI) m/z calculated for
þ
þ
(
[C46
H90NO13] : 864.6407, found 864.6407.
action was stirred at room temperature. After 30 min, the reaction
mixture was filtered and concentrated in vacuo. The residue was
subjected to gradient silica-gel column chromatography (EtOAc to
EtOAc/MeOH, 9:1, v/v).
0
0
-D
3.14. 6,6 -Di-O-17-methyloctadecanoyl-
a
,
a
-trehalose (3d)
By subjecting 8d (78 mg, 0.058 mmol) to the general procedure
for desilylation, the title compound 3d was obtained as a white
0
0
-D
3
.11. 6,6 -Di-O-11-methyldodecanoyl-
a,
a
-trehalose (3a)
solid (41.5 mg, 0.046 mmol, 79%). R
f
¼ 0.42 (EtOAc/MeOH, 9:1, v/v);
N); IR (film) ¼ 3330, 2921, 2852, 1728,
1465, 1365, 1214, 1152, 1102, 1080, 1020, 983, 937, 911, 806,
2
6:7
½aꢅ
¼ þ27 (c ¼ 1.0, C
5 5
H
D
By subjecting 8a (50 mg, 0.068 mmol) to the general procedure
for desilylation, the title compound 3a was obtained as a white
solid (40 mg, 0.054 mmol, 79%). R
¼ 0.8 (EtOAc/MeOH, 9:1, v/v);
N); IR (film) ¼ 3325, 2922, 2852, 1727,
ꢂ1
1
755 cm ; H NMR (500 MHz, C H N)
5
5
d
5.91 (d, J1,2 ¼ 3.7 Hz, 2H, H-
1), 5.14e5.09 (m, 2H, H-5), 5.05e5.0 (m, 2H, H-6a), 4.85 (dd,
6a,6b ¼ 11.7 Hz, J5,6b ¼ 5.4 Hz, 2H, H-6b), 4.77 (t, J2,3 ¼ J3,4 ¼ 9.5 Hz,
f
2
1:8
½
a
ꢅ
¼ þ5 (c ¼ 1.0, C
463, 1383, 1365, 1250, 1151, 1109, 988, 756, 721 cm
500 MHz, C N)
5.90 (d, J1,2 ¼ 3.7 Hz, 2H, H-1), 5.14e5.09 (m,
H, H-5), 5.06e4.98 (m, 2H, H-6a), 4.85 (dd, J6a,6b ¼ 11.7 Hz,
5,6b ¼ 5.5 Hz, 2H, H-6b), 4.77 (t, J2,3 ¼ J3,4 ¼ 9.5 Hz, 2H, H-3), 4.33
5
H
5
J
D
ꢂ1
1
1
;
H NMR
2H, H-3), 4.33 (dd, J2,3 ¼ 9.5 Hz, J1,2 ¼ 3.7 Hz, 2H, H-2), 4.19 (t,
(
2
J
(
2
9
J
9
3
5
H
5
d
J
J
3,4 ¼ J4,5 ¼ 9.5 Hz, 2H, H-4), 2.40e2.29 (m, 4H, H-8), 1.64 (p,
8,9 ¼ J9,10 ¼ 7.5 Hz, 4H, H-9), 1.55e1.46 (m, 2H, H-23), 1.37e1.13 (m,
13
52H, H-10eH-22), 0.88 (d, J23,24 ¼ 6.6, 12H, H-24); C NMR
dd, J2,3 ¼ 9.5 Hz, J1,2 ¼ 3.7 Hz, 2H, H-2), 4.19 (t, J3,4 ¼ J4,5 ¼ 9.5 Hz,
5 5
(125 MHz, C H N) d 173.8 (C-7), 96.0 (C-1), 75.0 (C-3), 73.5 (C-2),
H, H-4), 2.39e2.28 (m, 4H, H-8), 1.63 (p, J8,9 ¼ J 9,10 ¼ 7.3 Hz, 4H, H-
), 1.54e1.46 (m, 2H, H-17), 1.36e1.13 (m, 28H, H-10eH-16), 0.88 (d,
72.2 (C-4), 71.7 (C-5), 64.5 (C-6), 39.4 (C-22), 35.6 (C-8), 30.45,
30.24, 30.23, 30.2, 30.1, 29.9, 29.8, 29.5 (C-10eC-20), 28.4 (C-23),
27.9 (C-21), 25.4 (C-9), 22.9 (C-24); HRMS (ESI) m/z calculated for
1
3
17,18 ¼ 6.5, 12H, H-18); C NMR (125 MHz, C N)
6.2 (C-1), 75.3 (C-3), 73.8 (C-2), 72.4 (C-4), 72.0 (C-5), 64.8 (C-6),
9.6 (C-16), 34.8 (C-8), 30.6, 30.3, 30.1, 30.0, 29.8 (C-10eC-14), 28.6
5
H
5
d
174.1 (C-7),
þ
[C50
H
98NaO13] : 920.7033, found 920.7033.
0
0
-D
(
C-17), 28.1 (C-15), 25.7 (C-9), 23.2 (C-18); HRMS (ESI) m/z calcu-
3.15. 6,6 -Di-O-18-methylnonadecanoyl-
a
,
a
-trehalose (3e)
þ
lated for [C38
H74NO13] : 752.5155, found 752.5153.
By subjecting 8e (80 mg, 0.059 mmol) to the general procedure
for desilylation, the title compound 3e was obtained as a white
0
0
3
.12. 6,6 -Di-O-13-methyltetradecanoyl-
a,
a
-D
-trehalose (3b)
solid (43 mg, 0.046 mmol, 78%). R
f
¼ 0.3 (EtOAc/MeOH, 9:1, v/v);
1
6:5
By subjecting 8b (100 mg, 0.082 mmol) to the general procedure
for desilylation, the title compound 3b was obtained as a white
½aꢅ
¼ þ81 (c ¼ 1.0, C
5 5
H
N); IR (film) ¼ 3331, 2918, 2850, 1741,
D
ꢂ1
1
1467, 1365, 1251, 1153, 1103, 1078, 1015, 984, 938, 806, 720 cm ; H
Please cite this article in press as: Khan A, et al., Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity,
Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.076

8
A. Khan et al. / Tetrahedron xxx (2017) 1e9
NMR (500 MHz, C
5
H
5
N)
d
5.91 (d, J1,2 ¼ 3.7 Hz, 2H, H-1), 5.14e5.09
be free of endotoxin at a sensitivity of ꢁ0.125 EU/mL by Limulus
(
m, 2H, H-5), 5.05e5.0 (m, 2H, H-6a), 4.85 (dd, J6a,6b ¼ 11.7 Hz,
amebocyte lysate (LAL) assay using an endotoxin kit (Pyrotell,
Limulus Amebocyte Lysate).
J
(
2
5,6b ¼ 5.4 Hz, 2H, H-6b), 4.77 (t, J2,3 ¼ J3,4 ¼ 9.5 Hz, 2H, H-3), 4.33
dd, J2,3 ¼ 9.5 Hz, J1,2 ¼ 3.7 Hz, 2H, H-2), 4.19 (t, J3,4 ¼ J4,5 ¼ 9.5 Hz,
H, H-4), 2.40e2.29 (m, 4H, H-8), 1.64 (p, J8,9 ¼ J9,10 ¼ 7.5 Hz, 4H, H-
),1.55e1.46 (m, 2H, H-24), 1.37e1.13 (m, 56H, H-10eH-23), 0.88 (d,
3.17.3. Cytokine analysis
9
Levels of IL-10 (R&D systems), MIP-2 (R&D systems), IL-1b (BD
1
3
J
24,25 ¼ 6.6, 12H, H-25); C NMR (125 MHz, C
5
H
5
N)
d
174.2 (C-7),
Biosciences), IL-12p40 (BD Biosciences), and IL-6 (BD Biosciences)
were determined by sandwich ELISA according to the manufac-
turer's instructions.
9
3
3
6.4 (C-1), 75.5 (C-3), 74.0 (C-2), 72.6 (C-4), 72.2 (C-5), 65.0 (C-6),
9.9 (C-23), 35.0 (C-8), 30.8, 30.68, 30.67, 30.63, 30.5, 30.4, 30.2,
0.0 (C-10eC-21), 28.8 (C-24), 28.3 (C-22), 25.9 (C-9), 23.4 (C-25);
þ
HRMS (ESI) m/z calculated for [C52
H
102NaO13] : 948.7346, found
3.17.4. Statistics
9
48.7355.
Statistical significance of differences was assessed with 2-way
ANOVA with Dunnett's multiple comparisons test using Prism v7
software (GraphPad).
0
0
3
.16. 6,6 -Di-O-19-methylleicosanoyl-
a,
a
-D-trehalose (3f)
By subjecting 8f (65 mg, 0.047 mmol) to the general procedure
for desilylation, the title compound 3f was obtained as a white solid
36 mg, 0.0375 mmol, 80%). R
¼ 0.3 (EtOAc/MeOH, 9:1, v/v);
N); IR (film) ¼ 3329, 2917, 2849, 1727,
467, 1383, 1215, 1154, 1102, 1023, 984, 937, 806, 753, 665,
Acknowledgements
(
f
The authors would like to thank the Marsden Fund (VUW1401)
and the Health Research Council of New Zealand (Hercus Fellow-
ship, BLS, 2013/33).
1
7:3
½a
ꢅ
¼ þ49 (c ¼ 1.0, C
5 5
H
D
1
ꢂ1
1
5
1
J
77 cm ; H NMR (500 MHz, C
), 5.14e5.09 (m, 2H, H-5), 5.05e5.0 (m, 2H, H-6a), 4.85 (dd,
6a,6b ¼ 11.7 Hz, J5,6b ¼ 5.4 Hz, 2H, H-6b), 4.77 (t, J2,3 ¼ J3,4 ¼ 9.5 Hz,
5
H
5
N)
d
5.92 (d, J1,2 ¼ 3.7 Hz, 2H, H-
Appendix A. Supplementary data
2
H, H-3), 4.33 (dd, J2,3 ¼ 9.5 Hz, J1,2 ¼ 3.7 Hz, 2H, H-2), 4.19 (t,
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2017.11.076.
J
3,4 ¼ J4,5 ¼ 9.5 Hz, 2H, H-4), 2.40e2.29 (m, 4H, H-8), 1.64 (p,
J8,9 ¼ J9,10 ¼ 7.5 Hz, 4H, H-9), 1.55e1.46 (m, 2H, H-25), 1.37e1.13 (m,
13
6
0H, H-10eH-24), 0.88 (d, J25,26 ¼ 6.6, 12H, H-26); C NMR
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Please cite this article in press as: Khan A, et al., Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity,
Tetrahedron (2017), https://doi.org/10.1016/j.tet.2017.11.076