Lipid structure influences the ability of glucose monocorynomycolate to signal through Mincle

Article abstract of DOI:10.1039/c6ob01781a

Mincle (macrophage-inducible C-type lectin) is a C-type lectin receptor that provides the capacity for immune sensing of a range of pathogen- and commensal-derived glycolipids. Mincle can recognize mycolic and/or corynomycolic acid esters of trehalose, glycerol and glucose from mycobacteria and corynebacteria. While simple straight-chain long fatty acids (e.g. behenic acid) can substitute for mycolic acid on trehalose and glycerol and maintain robust signalling through Mincle, glucose monobehenate has been reported to be much less active than glucose monocorynomycolate (GMCM). We report the preparation of a range of analogues of GMCM to explore structural requirements in the lipid chain for signalling through Mincle. GMCM analogues bearing simple straight chain or branched fatty acid esters provided only weak signalling through human and mouse Mincle. A GMCM variant with a truncated (pentyl) α-chain provided attenuated signalling, whereas an analogue with an extended (tricosyl; C23) α-chain signalled as potently as GMCM. This work suggests that Mincle has the ability to survey mycolate-derived glycolipids from actinomycetes, distinguishing non-pathogenic (e.g. Rhodococcus spp.) and pathogenic (e.g. Mycobacterium tuberculosis) species on the basis of α-chain length. Finally, an α-phenyldodecyl analogue of GMCM possessed similar potency to GMCM and was only slightly less potent than trehalose dimycolate (cord factor), showing that large functional groups may be tolerated in the α-chain.

Full text of DOI:10.1039/c6ob01781a

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Lipid structure influences the ability of glucose
monocorynomycolate to signal through Mincle†
Cite this: DOI: 10.1039/c6ob01781a
Phillip L. van der Peet,a Masahiro Nagata,b Sayali Shah,a Jonathan M. White,a
Sho Yamasakib and Spencer J. Williams*a
Mincle (macrophage-inducible C-type lectin) is a C-type lectin receptor that provides the capacity for
immune sensing of a range of pathogen- and commensal-derived glycolipids. Mincle can recognize
mycolic and/or corynomycolic acid esters of trehalose, glycerol and glucose from mycobacteria and
corynebacteria. While simple straight-chain long fatty acids (e.g. behenic acid) can substitute for mycolic
acid on trehalose and glycerol and maintain robust signalling through Mincle, glucose monobehenate has
been reported to be much less active than glucose monocorynomycolate (GMCM). We report the prepa-
ration of a range of analogues of GMCM to explore structural requirements in the lipid chain for signalling
through Mincle. GMCM analogues bearing simple straight chain or branched fatty acid esters provided
only weak signalling through human and mouse Mincle. A GMCM variant with a truncated (pentyl)
α-chain provided attenuated signalling, whereas an analogue with an extended (tricosyl; C23) α-chain sig-
nalled as potently as GMCM. This work suggests that Mincle has the ability to survey mycolate-derived
glycolipids from actinomycetes, distinguishing non-pathogenic (e.g. Rhodococcus spp.) and pathogenic
(e.g. Mycobacterium tuberculosis) species on the basis of α-chain length. Finally, an α-phenyldodecyl
analogue of GMCM possessed similar potency to GMCM and was only slightly less potent than trehalose
dimycolate (cord factor), showing that large functional groups may be tolerated in the α-chain.
Received 16th August 2016,
Accepted 5th September 2016
DOI: 10.1039/c6ob01781a
www.rsc.org/obc
C-type lectin receptors (CLRs) form a large family of proteins
Upon binding of activating ligands to Mincle, dual phos-
that act as pattern recognition receptors, primarily on the phorylation of its intracellular immunoreceptor tyrosine-based
surface of myeloid cells.1,2 CLRs interact with a variety of activation motif promotes recruitment of the Fc receptor
ligands including pathogen-associated molecular patterns and γ-chain (FcRγ).10 This ultimately leads to activation of nuclear
damaged-associated molecular patterns.3,4 Mincle (macro- factor kappa-B, driving the transcription of cytokines and con-
phage-inducible C-type lectin) is a CLR capable of inducing sequently promoting the development of naïve T cells into
both pro- and anti-inflammatory responses with a unique effector helper T cell subtypes.14 Stimulation of the immune
ability to recognize microbial pathogens and altered self.4–6 system by TDM and related glycolipids is Mincle-dependent
While Mincle is notable for its ability to recognize the and leads to a pro-inflammatory response that includes stimu-
Mycobacterium tuberculosis glycolipid trehalose dimycolate lation of NO synthesis7,15 and modulation of Toll-like receptor
(TDM, 1),7 and is essential for the ability of TDM to produce 2-mediated interleukin (IL)-10 and IL-12p40 responses.16
granulomas,7 it has also been implicated in the development
Immunological studies of trehalose mono- and dimycolates
of autoimmune diseases of the eye,8 immune suppression in rely upon materials extracted from various mycobacteria that
pancreatic oncogenesis,9 recognition of dead cells,10 and is are hetereogeneous in their lipid components. Studies with
important for host immune responses to Malassezia spp. simplified homogeneous analogues of TDM have cast some
fungi11–13 (Fig. 1).
light on the influence of lipid structure upon signalling through
Mincle. Studies of simple fatty acid mono- and diesters of tre-
halose reveal that long chain lipids are required for signalling
through Mincle,17–19 with strong activity seen for the C22-ana-
logues trehalose mono- and dibehenate (TDB, 2).7,14 Trehalose
diesters were found to be superior to monoesters for activation
of macrophages as well as induction of Th1/Th17 immune
responses upon immunization.20 Synthetic trehalose mono-
and dicorynomycolates (TDCM, 3), which maintain the α-alkyl-
β-hydroxy structure of authentic mycolates, but are shorter in
aSchool of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Parkville, Victoria, 3010 Australia.
E-mail: sjwill@unimelb.edu.au
bDivision of Molecular Immunology, Medical Institute of Bioregulation, Kyushu
University, Fukuoka 812-8582, Japan
†Electronic supplementary information (ESI) available. CCDC 960227. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c6ob01781a
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to incorporate a hexenyl chain into the lactate-derived ketone,
with the alkyl group of this chain providing a means for altera-
tion of the side chain subsequent to assembly of the mycolic
acid. Reaction of 5-hexenylmagnesium bromide with the (S)-
lactate derived Weinreib amide 9 21 afforded the ketone 10,
which was converted to the required benzoate 11 by treatment
with benzoic anhydride/EtNiPr2. Boron-mediated aldol reaction
of 11 with palmitaldehyde according to Paterson et al.24 pro-
ceeded in excellent yield and provided 12 that was obtained as a
single stereoisomer after a single recrystallization (see ESI†).
The absolute stereochemistry of 12 was confirmed by X-ray
crystallography, which was determined relative to the known
stereochemistry of the (S)-lactate derived stereocentre (Fig. 2a).
The high crystallinity of 12 was unexpected, given the inherent
molecular flexibility of this molecule. Fig. 2b shows a partial
packing diagram for 12, which reveals an antiparallel arrange-
ment of the fully extended alkyl chain. The crystal packing is
characterised by a combination of hydrogen bonding between
the methanol solvate which links the molecules into a 1-D
chain extending down the y direction in the crystal. Parallel
chains are held together by a combination of interdigitation of
the C15 chains and π–π interactions. The resulting strip lies
parallel to the 101 plane. Within the X-ray structure, the α- and
β-protons are oriented anti (the torsional angle is 179.5°) such
that the α-alkenyl and β-alkyl groups are gauche; by contrast,
1
the H NMR spectrum of 12 in CDCl3 reveals a coupling con-
Fig. 1 Structures of trehalose, glycerol and glucose glycolipids based
on esters of α-mycolic acid, (+)-corynomycolic acid and behenic acid.
stant of Jα,β = 6.3 Hz, suggesting that α-alkenyl and the β-alkyl
groups are anti in solution. Conversion of 12 to the 5-pentenyl-
length and lack the functionalization in the lipid chains seen
in mycobacterial mycolates, also signal through Mincle with
similar potency to TDM and TDB.21 In the case of glycolipids
with a glycerol head group, which signal more weakly than
TDM and signal selectively through human, but not mouse
Mincle, the glycerol monobehenate (GroMB, 5) and monocory-
nomycolate (GroMCM, 6) analogues appear to signal as effec-
tively as glycerol monomycolate (GroMM, 4).22 However,
significant differences have been observed for signalling by
glucose monobehenate (GMB, 7) versus glucose monocoryno-
mycolate (GMM, 8) with only the former providing robust sig-
nalling through Mincle.21 Interestingly,
a
GMM from
Rhodococcus ruber (Nocardia rubra) caused production of granu-
lomas in mice, a process known to be Mincle-dependent.23
Accordingly, in the present work we sought to investigate in more
detail the effect of lipid structure on the ability of analogues of
glucose monocorynomycolate to signal through Mincle.
Results and discussion
The Paterson group has reported a powerful methodology for
the construction of anti aldols by the reaction of sterically-
demanding boron enolates with ketones derived from 2-O-
benzoyl lactate.24 We previously reported an asymmetric aldol
approach to the synthesis of (+)-corynomycolic acid using this
Fig. 2 (a) Thermal ellipsoid plot for 12, including the H-bonded metha-
nol solvate, determined by single-crystal X-ray crystallography.
Ellipsoids are at the 30% probability level. (b) Partial packing diagram for
12 displaying the 1-D hydrogen bonded chains interdigitated by the
methodology.21 In the present work we modified this approach C-15 substituents from parallel chains.
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Scheme 1 Preparation of alkene-containing mycolic acid fragment 15.
mycolic acid 15 was achieved by reduction of the ketone
(NaBH4, Et2O/MeOH) to the triols 13, glycol cleavage (NaIO4/
SiO2)25 to give aldehyde 14, and Lindgren oxidation26 using
the Kraus modification27 (Scheme 1).
The first modified mycolic acid, the α-pentyl derivative 16,
was generated simply by hydrogenation of 15 using H2 and
Pd/C (Scheme 2). For construction of additional derivatives, we
sought to convert the alkene of 15 to an aldehyde to enable
Julia–Kocienski cross-coupling reactions. Protection of the
acid and alcohol groups of 15 was achieved as an acetonide by
treatment with methyl propenyl ether and PPTS, and ozonolysis
of the alkene afforded the aldehyde 17.
Two extended side chains were installed by Julia–Kocienski
olefination using N-phenyltetrazole-derived sulfones.28
Oxidation of sulfides 18 and 19 with H2O2/ammonium molyb-
date29 afforded sulfones 20 and 21 (Scheme 3). Treatment of
aldehyde 17 with either 20 or 21 in the presence of LiHDMS
under Barbier-type conditions afforded the alkenes 22 and 23,
which were saturated using H2 and Pd/C, with cleavage of the
acetonide during silica gel chromatography, to afford 24 and 25.
Esterification of glucose was achieved by SN2 substitution,
facilitated by the cesium effect.30–32 Thus treatment of tosylate
Scheme 3 Preparation of functionalized mycolic acids.
2633 with α-pentyl acid 16, α-phenyldodecyl acid 25, α-tricosyl
acid 24 and the simplified branched fatty acid 27,34 in the
presence of CsHCO3 in THF/DMF, afforded the protected
derivatives 28–31 (Table 1). Finally, debenzylation by hydro-
genolysis (H2, Pd(OH)2/C) afforded the 6-O-acyl derivatives 32–35.
The ability of the analogues 32–35, as well as previously
studied compounds TDM (1), TDB (2) and GMB (7) to signal
through Mincle was assessed using a reporter cell assay in
which green fluorescent protein (GFP) is under the control of
nuclear factor of activated T cells.7 Activating ligands bind to
Mincle resulting in signalling dependent upon the FcRγ
adaptor molecule. Glycolipids were coated onto plates and cul-
tured in the presence of the reporter cell line expressing
mouse or human Mincle. GFP-positive cells were quantified by
flow cytometry.
As can be seen from the data in Fig. 3, TDM (1) and TDB (2)
exhibit almost identical potency in signalling through mouse
and human Mincle. Compared to these control compounds,
GMCM (8) exhibits similar potency, albeit with slightly weaker
signalling at the lowest (0.001 nmol) concentration. GlcMB (7)
signals weakly through Mincle at only the highest concen-
Scheme 2 Preparation of short-chain mycolic acid 16 and protected
aldehyde 17.
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Table 1 Preparation of glucose monoesters 32–35
through Mincle demonstrates that this receptor requires
certain structural features in the lipid chain.
Compounds 32 and 33 explore the effect of altering the
length of the α-chain of GMCM (8). The α-pentyl derivative (32)
signals much more weakly through Mincle than GMCM, which
possesses a C14 α-chain. On the other hand the α-C23 derivative
(33) signals with similar potency to GMCM. Compound 34
investigated the effect of including an aryl group into the
α-chain. This derivative also provides signalling of similar
potency to GMCM (8), showing that inclusion of functional
groups distal to the glycolipid headgroup does not affect sig-
nalling through Mincle.
Glucose monomycolates constitute a unique glycolipid sig-
nature as they are derived during infection from mycobacterial
lipid and host glucose.36 This work provides the first detailed
analysis of the structural requirements of signalling through
Mincle by the lipidic portion of GMCM. In contrast to previous
work that demonstrated that a behenate chain can substitute
for a mycolate chain in TDM7 and GroMM,22 the present work
reveals a more nuanced dependence on lipid structure for sig-
nalling by GMCM analogues. Trehalose-based mono- and
diacyl derivatives signal through Mincle more potently than
those based on glucose.21 Crystal structures of human and
bovine Mincle reveal the presence of two sugar-binding sub-
sites,37,38 and as trehalose has been shown to have an affinity
36-fold greater for the carbohydrate recognition domain (CRD)
of Mincle than for the monosaccharide methyl α-D-glucopyra-
noside,37 the higher potency of trehalose-based glycolipids is
presumably a result of already strong binding through both
sugar residues of the trehalose head-group, which is further
potentiated by the presence of a lipid. By making fewer con-
tacts in the CRD, binding of glucose to Mincle is expected to
be much weaker, and thus potentiation of binding is especially
sensitive to the lipid structure. Simple straight- or branched-
tration. Previously we reported that this compound does not
signal through Mincle;21 this data extends the previous work
by assessing signalling at a higher concentration where signal-
ling is now clearly evident. Compound 35, which contains a
branched achiral fatty acid inspired by a modified trehalose
dimycolate derivative (Vizantin) optimized for immunostimu-
lation that signals through the TLR-4/MD-2 complex,35 signals
only very weakly through Mincle. Taken together, the failure of
GlcMB (7) and compound (35) to provide robust signalling
Fig. 3 NFAT-GFP reporter cells expressing either human Mincle/FcRγ or mouse Mincle/FcRγ, as well as those expressing FcRγ alone were tested for
their reactivity to plate-bound TDM (1), TDB (2), GMCM (8), GMB (7), and analogues 32–35. Assays were performed in triplicate; the mean values and
standard deviations are shown.
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chain fatty acid groups, exemplified by 7 and 35 signalled only After 30 min stirring additional 6-bromohexene (200 μL,
weakly through Mincle. Moreover, the GlcMCM analogue 32 1.5 mmol) was added and stirring was continued for 15 min. A
bearing
a truncated α-chain provided weaker signalling final batch of 6-bromohexene (840 μL 6.3 mmol) was then
through Mincle than longer α-chain mycolates such as GMCM added in portions at a rate sufficient to keep the mixture
and the analogue 33. Shorter α-alkyl chains (C8–C12) are typical refluxing. The mixture was refluxed for a further 2 h then
for certain Rhodococcus spp. derived mycolic acids,39,40 cooled to r.t. The solution was used directly in the next step.
whereas medium length α-chains (C14–C18) are typical for
(S)-8-Hydroxy-7-oxo-non-1-ene (10). A solution of 5-hexenyl-
other Rhodococcus spp.,39 Corynebacterium spp.40,41 and magnesium bromide in THF (13 mL, ∼1.0 M) was added drop-
Nocardia spp.,42 while long α-chains (C22–C24) typify patho- wise over 10 min to a stirring solution of (S)-2-hydroxy-N-
genic mycobacteria such as M. tuberculosis, Mycobacterium methoxy-N-methylpropionamide 9 21 (470 mg, 3.52 mmol) in
avium, and Mycobacterium bovis.43,44 These results suggest that dry THF at −30 °C under N2. The mixture was kept at −30 °C
Mincle provides the capacity to survey and discriminate certain for 20 min, then −15 °C for 30 min before being warmed to r.t.
pathogenic and non-pathogenic actinomycete species on the and left to stir for 18 h. Sat. aq. NH4Cl (30 mL) was added and
basis of α-chain length of glucose monomycolates. Consistent the mixture was stirred for 20 min. The mixture was extracted
with these suggestions, Yano and coworkers demonstrated with CH2Cl2 (4 × 10 mL), and the combined organic extracts
that glucose monomycolate bearing a mycolic acid derived were dried (MgSO4), filtered, and concentrated in vacuo. The
from Rhodococcus ruber (Nocardia rubra), most likely with an residue was purified by flash chromatography (15–20% EtOAc/
intermediate length C14 α-chain,39 had the capacity to form pet. spirits) to give 10 as a colourless liquid (449 mg, 81%).
granulomas and stimulate the production of macrophage [α]2D2 56.6 (c 1.02, CHCl3); 1H NMR (400 MHz, CDCl3)
chemotactic factors in mice.23 Finally, the potent signalling δ 1.36–1.43 (5 H, m, CH2CH2CHvCH2,CH3), 1.60–1.68 (2 H,
provided by the phenylalkyl derivative 34, which was similar to m, CH2CH2CvO), 2.03–2.09 (2 H, m, CH2CHvCH2), 2.47 (2 H,
GMCM (8) and only slightly less potent than TDM (1) shows ddt, J 7.3, 17.0 Hz, CH2CvO), 4.23 (1 H, J 7.1 Hz, CHOH),
that large groups may be tolerated in the α-chain. This infor- 4.93–5.03 (2 H, m, CHvCH2), 5.78 (1 H, ddt, J 6.7, 10.3, 17.0
mation should assist in the design of functional chemical Hz, CHvCH2); 13C NMR (100 MHz, CDCl3) δ 19.8 (CH3), 23.0,
probes such as dye-labelled analogues for more detailed 28.4, 33.4, 37.3 (4 C, CH2), 72.6 (CHOH), 114.8, 138.2 (CvC),
studies of Mincle.
212.6 (CvO); HRMS (ESI+): m/z 157.1227 [M + H]+ (calcd
[C9H17O2]+ 157.1223).
(S)-7-Oxonon-1-en-8-yl
Diisopropylethylamine (5.1 mL, 29.3 mmol) was added to a
stirring mixture of 10 (2.29 g, 14.6 mmol), benzoic anhydride
benzoate
(11).
N,N-
Experimental
General
Proton nuclear magnetic resonance spectra (1H NMR, 400 or (4.96 g, 21.9 mmol), DMAP (179 mg, 1.46 mmol) and CH2Cl2
500 MHz) and proton decoupled carbon nuclear magnetic (50 mL) at r.t. under N2. After stirring for 16 h the mixture was
resonance spectra (13C NMR, 100 or 125 MHz) were obtained washed with H2O (50 mL) and the aqueous layer extracted with
in deuterochloroform or methanol-d4 (CD3OD) with residual CH2Cl2 (2 × 10 mL). The combined organic extracts were
protonated solvent or solvent carbon signals as internal stan- washed with sat. aq. NaCl (25 mL), dried (MgSO4), filtered, and
dards. Abbreviations for multiplicity are s, singlet; d, doublet; concentrated in vacuo. The residue was purified by flash
t, triplet; q, quartet. Flash chromatography was carried out chromatography (3–5% EtOAc/pet. spirits) to give the benzoate
on silica gel 60 according to the procedure of Still et al.45 11 as a colourless oil (2.97 g, 78%). [α]D22 16.0 (c 1.00, CHCl3);
Analytical thin layer chromatography (t.l.c.) was conducted 1H NMR (400 MHz, CDCl3) δ 1.40 (2 H, overlapping dt, 7.6 Hz,
on aluminium-backed 2 mm thick silica gel 60 F254 and CH2CHvCH2), 1.52 (3 H, d, J 7.0 Hz, CH3), 1.60–1.68 (2 H, m,
chromatograms were visualized with ceric ammonium molyb- CH2), 2.02–2.08 (2 H, m, CH2CH2CvO), 2.49 (1 H, dt, J 7.3,
date (Hanessian’s stain), potassium permanganate or 5% 17.5 Hz, CH2CvO), 2.62 (2 H, dt, J 7.3, 17.5 Hz, CH2CvO),
H2SO4/MeOH, with charring as necessary. Melting points 4.92–5.02 (2 H, m, CH2vCH), 5.33 (1 H, q, J 7.0 Hz, CHCH3),
were obtained using a hot-stage or capillary apparatus and are 5.78 (1 H, ddt, J 6.7, 10.3, 17.0, CHvCH2), 7.46 (2 H, m, Ph),
corrected. High resolution mass spectra (HRMS) were obtained 7.59 (1 H, m, Ph), 8.08 (2 H, m, Ph); 13C NMR (100 MHz,
using an ESI-TOF-MS; all samples were run using 0.1% formic CDCl3) δ 16.4 (CH3), 22.7, 28.4, 33.5, 38.0 (4 C, CH2), 75.2
acid. Dry CH2Cl2, THF, and Et2O were obtained from a dry (CHOBz), 114.7 (CH2vCH), 128.5, 129.5, 129.3, 133.4 (6 C,
solvent apparatus (Glass Contour of SG Water, Nashua, USA) Ph), 138.4 (CHvCH2), 165.9 (COPh), 207.8 (CH2CvO); HRMS
as per the procedure of Pangborn et al.46 Dry DMF was dried (ESI+): m/z 261.1479 [M + H]+ (calcd [C16H21O3]+ 261.1485).
over 4 Å molecular sieves. Pet. spirits refers to petroleum ether,
boiling range 40–60 °C.
(S)-8-Hydroxy-7-oxo-non-1-ene (10)
(2S,4R,5R)-5-Hydroxy-3-oxo-4-(pent-4-enyl)eicosan-2-yl ben-
zoate (12). The ketone 11 in dry Et2O (660 mg, 2.54 mmol,
10 mL) was added by cannula to a stirring mixture of dicyclo-
5-Hexenylmagnesium bromide. Magnesium turnings (452 mg, hexylchloroborane47 (1.0 mL, 4.56 mmol), dimethylethylamine
18.6 mmol) were added to a dry flask under N2 then ground (412 µL, 3.80 mmol) at −78 °C under N2. After 15 min, the
with vigorous magnetic stirring overnight. Dry THF (13 mL) reaction was warmed to r.t. for 1 h, then cooled to −78 °C,
was added, followed by 6-bromohexene (200 μL, 1.5 mmol). before the addition of hexadecanal48 (2.74 g, 11.4 mmol) in
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dry Et2O (50 mL) by cannula. The mixture was stirred at to the method of Zhong and Shing,25 was added to a stirred
−78 °C for 1 h, then was moved to a freezer (−17 °C) overnight. mixture of the triols 13 (369 mg, 0.926 mmol) and CH2Cl2
The mixture was warmed to 0 °C with stirring and aq. (10 mL) under N2. The mixture was stirred at r.t. for 1 h, then
NaH2PO4/Na2HPO4 (pH 7, 0.1 M, 40 mL) and MeOH (40 mL) additional of 21% w/w NaIO4/SiO2 (191 mg, 0.185 mmol) was
were added, followed by 30% aq. H2O2 (40 mL). The mixture added and stirring was continued for 30 min. The mixture was
was stirred at 0 °C for 1 h then extracted with CH2Cl2 (100 mL, filtered through a sintered glass frit and evaporated to dryness
3 × 50 mL). The combined organic extracts were dried under reduced pressure. The crude aldehyde 14 was dissolved
(MgSO4), filtered and concentrated in vacuo. The residue was in t-BuOH (10 mL) then water (2 mL), cis-2-butene (730 μL,
purified by flash chromatography (20% EtOAc/pet. spirits) to 8.33 mmol), NaH2PO4 (192 mg, 1.39 mmol) and NaClO2
give the title compound along with a small amount of an un- (314 mg, 2.78 mmol) were added at 0 °C under N2 atmosphere.
identified diastereoisomer (1.00 g, 79%, 9 : 1 12/diastereo- The resulting mixture was stirred for 0.5 h at 0 °C then 70 min
isomer). Pure 12 was obtained as white needles at r.t. before being concentrated in vacuo. The residue was par-
(m.p. 62–64 °C) by crystallization from hot MeOH. [α]2D3 43.8 titioned between CH2Cl2 (80 mL) and H2O (40 mL). Sat. aq.
(c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (3 H, t, J 6.80 NaCl (10 mL) was added to break the emulsion formed, the
Hz, CH3CH2), 1.18–1.54 (30 H, m, 15 × CH2), 1.55 (3 H, d, J 7.1 organic layer was separated and the aqueous layer extracted
Hz, CH3CH), 1.65–1.81 (2 H, m, CH2), 2.08 (2 H, q, J 7.1 Hz, with CH2Cl2 (2 × 20 mL). The combined organic extracts were
CH2CHvCH2), 2.49 (1 H, br s, OH), 2.84 (1 H, q, J 6.3 Hz, washed with H2O (2 × 30 mL) then sat. aq. NaCl (30 mL) and
CHCvO), 3.74 (1 H, br s, CHOH), 4.95–5.04 (2 H, m, dried (MgSO4). The solvent was evaporated under reduced
CH2vCH), 5.47 (1 H, q, J 7.0 Hz, CHOBz), 5.79 (1 H, ddt, J 6.7, pressure to afford an oil which was purified by flash chromato-
10.3, 17.0, CHvCH2), 7.46 (2 H, m, Ar), 7.59 (1 H, m, Ar), 8.08 graphy (88 : 10 : 2 pet. spirits/EtOAc/AcOH) to give 15 as an
1
(2 H, m, Ar); 13C NMR (125 MHz, CDCl3) δ 14.3, 16.0 (2 C, amorphous solid (220 mg, 82%). [α]D22 9.8 (c 1.02, CHCl3); H
CH3), 22.8, 26.0, 26.7, 28.8, 29.5, 29.67, 29.73, 29.74, 29.79, NMR (500 MHz, CDCl3) δ 0.89 (3H, t, J 7.0 Hz, CH3), 1.58–1.19
29.80, 29.81, 29.84, 32.1, 33.9, 35.7 (17 C, CH2), 52.7 (CHCO), (32 H, m, CH2), 1.59–1.82 (2 H, m, CH2), 2.02–2.16 (2 H, m,
75.4 (CHOBz), 115.1 (CH2vCH), 128.6, 129.61, 130.0, 133.5 CH2CvO), 2.41–2.53 (1 H, m, OH), 3.73 (1 H, s, CHOH),
(6 C, Ph), 138.3 (CHvCH2), 165.8 (PhCvO), 212.4 (CHCvO); 4.90–5.11 (2 H, m, CHvCH2), 5.79 (1 H, ddt, J 6.7, 10.2, 16.9
HRMS (ESI+): m/z 501.3941 [M + H]+ (calcd [C32H48O4]+ Hz, CHvCH2); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.7, 25.7,
501.3938).
26.5, 28.8, 29.4, 29.5, 29.59, 29.61, 29.66, 29.70, 31.9, 33.5,
(2S,4R/S,5R)-4-(Pent-4-en-1-yl)eicosane-2,3,5-triol (13).
A
35.4, 50.9, 72.2, 115.0, 138.1, 180.3; HRMS (ESI+): m/z 391.3193
mixture of 12 (566 mg, 1.13 mmol), NaBH4 (85.5 mg, [M + Na]+ (calcd [C23H44O3Na]+ 391.3183).
2.26 mmol), MeOH (3 mL) and Et2O (6 mL) was stirred under
(2R,3R)-3-Hydroxy-2-pentyloctadecanoic acid (16). A mixture
N2 for 30 min at r.t. The mixture was poured into sat. aq. of 15 (28.6 mg, 0.0776 mmol), 10% Pd/C (30 mg), EtOH
NaHCO3 (40 mL), diluted with water (20 mL) and extracted (0.5 mL) and EtOAc (4.5 mL) was stirred under an H2 atmo-
with Et2O (4 × 10 mL). The combined organic extracts were sphere for 20 h. The mixture was filtered through Celite, con-
washed sequentially with sat. aq. NaHCO3 (20 mL), H2O centrated in vacuo and the residue purified by flash
(20 mL), and sat. aq. NaCl (20 mL), then dried (MgSO4). The chromatography (88 : 10 : 2 pet. spirits/EtOAc/AcOH) to afford
solvent was evaporated under reduced pressure to afford an 16 (28.2 mg, 98%). [α]2D5 12.2 (c 1.38, CHCl3); 1H NMR
oil. The crude oil was dissolved in THF (6 mL) and treated (400 MHz, CDCl3) δ 0.80–0.96 (6 H, m, 2 × CH3), 1.13–1.79
with NaOMe/MeOH (1 M, 3 mL), and the mixture was stirred (36 H, m, 18 × CH2), 2.36–2.53 (1 H, m, CHCO2H), 3.71 (1 H,
under N2 at r.t. for 90 min. The mixture was poured into sat. m, CHOH); 13C NMR (100 MHz, CDCl3) δ 14.0, 14.1 (2 C, CH3),
aq. NH4Cl and extracted with Et2O (3 × 20 mL). The combined 22.4, 22.7, 25.7, 27.0, 29.3, 29.4, 29.5, 29.57, 29.59, 29.64, 29.7,
organic extracts were washed with H2O (20 mL), dried 31.6, 31.9, 35.4 (27 C, CH2), 51.0 (CHCO2H), 72.2 (CHOH),
(MgSO4), filtered, and concentrated under reduced pressure. 180.4 (CvO); HRMS (ESI+): m/z 388.3773 [M + NH4]+ (calcd
The crude residue was purified by flash chromatography (30% [C23H40NO3]+ 338.3785).
EtOAc/pet. spirits) to give 13 (378 mg, 89%) as a mixture of dia-
4-((5R,6R)-2,2-Dimethyl-4-oxo-6-pentadecyl-1,3-dioxan-5-yl)
stereoisomers. Partial 1H NMR (500 MHz, CDCl3) δ 0.88 (3 H, t, butanal (17)
J 7.0 Hz, CH3CH2), 1.10–1.67 (36 H, m, 16 × CH2, CH3CH(OH),
Acetonide formation. A mixture of 15 (18.8 mg, 0.051 mmol),
CH(CH2)3CHvCH2), 1.94–2.14 (2 H, m, CH2CHvCH2), 2.85 pyridinium p-toluenesulfonate (0.7 mg), 2-methoxypropane
(1 H, s, OH), 3.13–3.59 (2 H, m, 2 × OH), 3.64–3.99 (3 H, m, 3 × (19.6 μL, 0.204 mmol) and dry CH2Cl2 (3 mL) was stirred
CHOH), 4.93–5.03 (2 H, m, CHvCH2), 5.72–5.84 (1 H, m, under an N2 atmosphere. After 70 min the mixture was concen-
CHvCH2); 13C NMR (125 MHz, CDCl3) δ 14.2, 18.2, 19.3, 22.8, trated to dryness under reduced pressure, resuspended in dry
24.7, 25.6, 25.9, 26.4, 27.2, 28.4, 29.5, 29.76, 29.79, 29.83, 32.0, CH2Cl2 (5 mL), and powdered NaHCO3 (2.1 mg, 0.025 mmol)
34.1, 34.3, 35.6, 35.9, 42.8, 44.3, 68.8, 69.1, 73.2, 73.4, 76.4, was added and the mixture was stirred vigorously for 20 min.
77.1, 114.8, 115.0, 138.5, 138.7; HRMS (ESI+): m/z 421.3661 The mixture was again concentrated to dryness under reduced
[M + Na]+ (calcd [C25H50O3Na]+ 421.3652).
pressure and resuspended in dry CH2Cl2 (5 mL) to give a
(2R,3R)-3-Hydroxy-2-(pent-4-en-1-yl)octadecanoic acid (15). mixture containing 5-((5R,6R)-2,2-dimethyl-4-oxo-6-pentadecyl-
21% w/w NaIO4/SiO2 (1.91 g, 1.85 mmol), prepared according 1,3-dioxan-5-yl)pent-1-ene, which was subject to ozonolysis as
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outlined below. HRMS (ESI+): m/z 431.3485 [M + Na]+ (calcd (2 H, m, CH2), 6.57–7.65 (3 H, m, Ph), 7.66–7.72 (2 H, m, Ph);
[C26H48O3Na]+ 431.3496). 13C NMR (100 MHz, CDCl3) δ 14.3 (CH3), 22.1, 22.8, 28.3, 29.1,
Ozonolysis. The mixture from above was cooled to −78 °C 29.4, 29.5, 29.6, 29.7, 29.78, 29.81, 29.83, 29.85, 32.1, 56.2 (18
and O2/O3 was bubbled through with stirring until a blue C, CH2), 125.2, 129.8, 131.6, 133.2, 153.6 (Ar); HRMS (ESI+):
colour persisted for 5 min. N2 was bubbled through the m/z 499.3084 [M + Na]+ (calcd [C26H45N4O2SNa]+ 499.3077).
mixture with stirring for 5 min and the mixture was warmed to
1-Phenyl-5-((8-phenyloctyl)thio)-1H-tetrazole (19). DIAD
r.t. Ph3P (26.8 mg, 0.102 mmol) was added and the mixture (429 µL, 2.18 mmol) was added to a stirred mixture of Ph3P
was stirred at r.t. for 2 h. The mixture was concentrated under (571 mg, 2.18 mmol), 1-phenyl-1H-tetrazole-5-thiol (388 mg,
reduced pressure and the residue taken up in 10 : 1 pet. 2.18 mmol), 8-phenyl-1-octanol (375 mg, 1.82 mmol), pow-
spirits/EtOAc containing 1.2% Et3N and loaded onto a silica dered dried 4 Å molecular sieves (13 g) and dry THF (10 mL) at
gel column through a pipette containing a cotton plug in 0 °C under N2. The mixture was allowed to warm to r.t. and
order to remove solid Ph3PO. The column was eluted with stirred for 18 h. The mixture was filtered through Celite and
10 : 1 pet. spirits/EtOAc containing 1.2% Et3N to afford pure 17 the solvent evaporated under reduced pressure. The residue
(18 mg, 86%). [α]2D4 1.7 (c 1.965, CHCl3); 1H NMR (500 MHz, was purified by flash chromatography (30% EtOAc/pet. spirits)
CDCl3) δ 0.88 (3 H, t, J 6.9 Hz, CH3), 1.07–1.90 (32 H, m, 16 × to give 19 (631 mg, 95%). 1H NMR (400 MHz, CDCl3)
CH2), 2.32 (1 H, ddd, J 3.5, 5.2, 10.1 Hz, CHC(O)OR), 2.39–2.60 δ 1.28–1.38 (6 H, m, 3 × CH2), 1.39–1.48 (2 H, m, CH2),
(2 H, m, CH2), 3.91 (1 H, ddd, J 2.3, 8.9, 11.0 Hz, CHOC 1.55–1.65 (2 H, m, CH2), 1.82 (2 H, tt, J 7.4, 7.8 Hz, CH2), 2.59
(CH3)2); 13C NMR (125 MHz, CDCl3) δ 14.2 (CH3), 19.3, 22.8, (2 H, t, J 7.6 Hz, CH2), 3.69–3.76 (2 H, t, J 7.22 Hz, CH2),
25.3, 25.4, 26.7, 29.4, 29.49, 29.53, 29.69, 29.72, 29.77, 29.79, 7.13–7.20 (3 H, m, Ph), 7.22–7.32 (2 H, m, Ph), 7.49–7.63 (5 H,
29.80, 29.83, 32.1, 33.8 (CH2), 44.0, 45.5 (C(CH3)2), 70.8 (CHC m, Ph); 13C NMR (100 MHz, CDCl3) δ 28.9, 29.0, 29.15, 29.24,
(O)OR), 105.7 (CHOC(CH3)2), 170.9 (CHCvO), 201.9 (HCvO); 29.4, 31.5, 33.4, 36.0 (8 C, CH2), 123.9, 125.7, 128.3, 128.4,
HRMS (ESI+): m/z 433.3290 [M + Na]+ (calcd [C25H46O4Na]+ 129.8, 130.1, 133.8, 142.8, 154.6 (13 × Ar); HRMS (ESI+): m/z
433.3288).
367.1962 [M + H]+ (calcd [C21H27N4S]+ 367.1951).
5-(Nonadecylthio)-1-phenyl-1H-tetrazole (18). A mixture of
1-Phenyl-5-((8-phenyloctyl)sulfonyl)-1H-tetrazole
(21).
1-phenyl-1H-tetrazole-5-thiol (141 mg, 0.791 mmol), bromono- Compound 19 (518 mg, 1.41 mmol) was treated, as described
1
nadecane (250 mg, 0.722 mmol) and K2CO3 (149 mg, for the preparation of 20, to give 21 (518 mg, 92%). H NMR
1.08 mmol) in acetone (10 mL) was stirred at 60 °C for 16 h. (400 MHz, CDCl3) δ 1.35 (6 H, br s, 3 × CH2), 1.44–1.54 (2 H,
The mixture was cooled to r.t. and the solvent was evaporated m, CH2), 1.63 (1 H, pentet, J 7.5 Hz, CH2), 1.90–2.00 (2 H, m,
under reduced pressure. The residue was partitioned between CH2), 2.56–2.69 (2 H, t, J 7.8 Hz, CH2), 3.73 (2 H, t, J 8.0 Hz,
CH2Cl2 (10 mL) and water (10 mL), the organic phase separ- CH2), 7.15–7.23 (3 H, m, Ph), 7.23–7.33 (2 H, m, Ph), 7.55–7.66
ated and the aqueous layer extracted with CH2Cl2 (2 × 10 mL). (3 H, m, Ph), 7.66–7.74 (2 H, m, Ph); 13C NMR (100 MHz,
The combined organic extracts were dried (MgSO4) and the CDCl3) δ 22.0, 28.1, 28.9, 29.09, 29.11, 31.4, 36.0, 56.0 (8 C,
solvent evaporated under reduced pressure. The residue was CH2), 125.2, 125.7, 128.3, 128.4, 129.8, 131.5, 133.1, 142.7,
purified by flash chromatography (10% EtOAc/pet. spirits) to 153.6 (13 C, Ar); HRMS (ESI+): m/z 422.1666 [M + Na]+ (calcd
give 18 as an amorphous solid (310 mg, 97%). 1H NMR [C21H26N4SO2Na]+ 422.1669).
(400 MHz, CDCl3) δ 0.88 (3 H, t, J 7.3 Hz, CH3), 1.11–1.51 (32
(2R,3R)-3-Hydroxy-2-(12-phenyldodec-4-en-1-yl)octadecanoic
H, m, 16 × CH2), 1.81 (2 H, pentet, J 7.5 Hz, CH2), 3.39 (2 H, t, acid (23). LiHMDS in THF (1 M, 202 µL, 202 µmol) was added
J 7.3 Hz, CH2), 7.51–7.63 (5 H, m, Ph); 13C NMR (125 MHz, to a stirring mixture of the aldehyde 17 (41.5 mg, 101 µmol),
CDCl3) δ 14.3 (CH3), 22.8, 28.8, 29.18, 29.22, 29.5, 29.6, 29.7, sulfone 21 (80.6 mg, 202 µmol) and dry THF at −10 °C under
29.76, 29.80, 29.84, 32.1, 33.5 (18 C, CH2), 124.0, 129.9, 130.2, N2. The mixture was warmed to r.t. and was allowed to stir
133.9 (6 × Ph), 154.7 (tetrazole-C); HRMS (ESI+): m/z 555.3879 until the aldehyde was consumed (t.l.c.). The mixture was
[M + Na]+ (calcd [C29H56O8Na]+ 555.3867).
poured into sat. aq. NH4Cl (20 mL) and extracted with Et2O
5-(Nonadecylsulfonyl)-1-phenyl-1H-tetrazole (20). An ice-cold (3 × 10 mL). The combined organic extracts were washed with
solution of (NH4)Mo7O24 (96 mg, 0.0778 mmol) in 30% aq. water (20 mL) then sat. aq. NaCl (20 mL), dried (MgSO4), and
H2O2 (1.0 mL, 9.8 mmol) was added to a stirring mixture of 18 the solvent evaporated under reduced pressure. Flash chrom-
(116 mg, 0.233 mmol), THF (9 mL) and EtOH (9 mL) at 5 °C. atography of the crude material (5–20% EtOAc/pet. spirits with
1
After 1.5 h additional of (NH4)Mo7O24 (96 mg, 0.0778 mmol) 1% AcOH) gave 23 (35.6 mg, 65%). H NMR (400 MHz, CDCl3)
in 30% aq. H2O2 (1.0 mL, 9.8 mmol) was added and the δ 0.88 (3 H, t, J 6.8 Hz, CH3), 1.14–1.79 (42 H, m, 21 × CH2),
mixture was stirred at r.t. for 19 h. The mixture was poured 1.87–2.14 (4 H, m, CH2), 2.41–2.50 (1 H, m, CHCO2H), 2.60
into H2O (200 mL) and extracted with CH2Cl2 (1 × 20 mL, 2 × (2 H, t, J 7.6 Hz, CH2), 3.71 (1 H, br s, CHOH), 5.28–5.46 (2 H,
10 mL). The combined organic extracts were washed with H2O m, CHvCH), 7.12–7.20 (3 H, m, Ph), 7.23–7.31 (2 H, m, Ph);
(50 mL), dried (MgSO4), filtered and concentrated in vacuo. 13C NMR (100 MHz, CDCl3) δ 14.3 (CH3), 22.8, 25.9, 27.1, 27.3,
Flash chromatography (30–50% EtOAc/pet. spirits) of the 27.4, 27.5, 29.0, 29.1, 29.3, 29.4, 29.46, 29.52, 29.6, 29.68,
residue gave 20 (91.9 mg, 83%). 1H NMR (400 MHz, CDCl3) 29.73, 29.76, 29.77, 29.82, 29.9, 31.7, 32.1, 32.5, 32.7, 35.6, 36.1
δ 0.88 (3 H, t, J 6.8 Hz, CH3), 1.09–1.43 (30 H, m, 15 × CH2), (24 × CH2), 51.0 (CHCO2H), 72.3 (CHOH), 125.7, 128.3, 128.5,
1.40–1.54 (2 H, m, CH2), 1.87–2.06 (2 H, m, CH2), 3.69–3.76 128.94, 129.4, 130.8, 131.4, 143.03, 143.05 (Ph), 180.2 (CvO);
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HRMS (ESI+): m/z 565.4584 [M + Na]+ (calcd [C36H62O3Na]+ chromatography (10–20% EtOAc/pet. spirits) giving 28 as a col-
1
565.4591).
ourless gum (26.7 mg, 57%). [α]2D5 0.4 (c 1.05, CHCl3); H NMR
(2R,3R)-3-Hydroxy-2-(12-phenyldodecyl)octadecanoic
acid (400 MHz, CDCl3) δ 0.78–0.92 (6 H, m, 2 × CH3), 1.06–1.75
(25). H2 was bubbled through a mixture of compound 23 (36 H, m, 18 × CH2), 2.39–2.53 (2 H, m, OH, CHCO2),
(34.6 mg, 63.7 µmol) and 10% Pd/C (35 mg) in 1 : 1 EtOAc/ 3.44–3.54, 3.60–3.71 (5 H, 2 × m, H2,3,4,5, CHOH), 4.16–4.24
EtOH (6 mL) for 1 h, and the mixture was then stirred under (1 H, m, H6a), 4.48–4.55 (2 H, m, H1,6b), 4.55–4.64 (2 H, m,
an H2 atmosphere for 16 h. The mixture was filtered through CH2Ph), 4.69 (1 H, d, J 10.9 Hz, CH2Ph), 4.77 (1 H, d, J 10.9 Hz,
Celite and the plug washed with copious quantities of EtOAc/ CH2Ph), 4.85–4.97 (4 H, m, J 10.9 Hz, CH2Ph), 4.87–4.97 (4 H,
EtOH. The filtrate was concentrated to dryness under reduced m, 2 × CH2), 7.22–7.37 (20 H, m, Ph); 13C NMR (100 MHz,
pressure and the residue purified by flash chromatography CDCl3) δ 14.2, 14.3 (2 C, CH3), 22.6, 22.8, 26.0, 27.3, 29.5, 29.7,
(5–20% EtOAc/pet. spirits with 1% AcOH) to give 25 (34.0 mg, 29.75, 29.80, 29.82, 29.9, 31.9, 32.1, 35.8 (18 C, CH2), 51.5
98%). [α]2D5 8.6 (c 1.58, CHCl3); 1H NMR (400 MHz, CDCl3) (CHCO2), 63.0, 71.3 (2 C, CH2), 72.5, 73.0 (2 C, CH), 75.1, 75.3,
δ 0.88 (3 H, t, J 6.8 Hz, CH3), 1.14–1.79 (46 H, m, 23 × CH2), 75.9 (3 C, CH2), 78.0, 82.4, 84.7 (3 C, C2,3,4), 102.5 (C1), 127.8,
2.38–2.48 (1 H, m, CHCO2H), 2.60 (1 H, t, J 7.6 Hz, CH2), 3.71 128.00, 128.02, 128.1, 128.2, 128.3, 128.5, 128.56, 128.57,
(1 H, br s, CHOH), 5.28–5.46 (2 H, m, CHvCH); 7.12–7.20 (3 128.7, 137.3, 137.8, 138.4, 138.5 (20 C, Ph), 175.4 (CvO);
H, m, Ph), 7.23–7.31 (2 H, m, Ph); 13C NMR (100 MHz, CDCl3) HRMS (ESI+): m/z 910.6181 [M + Na]+ (calcd [C57H80O8Na]+
δ 14.3 (CH3), 22.8, 25.86, 27.5, 29.51, 29.53, 29.6, 29.69, 29.70, 910.6191).
29.76, 29.79, 29.81, 29.82, 29.84, 29.9, 31.7, 32.1, 35.6, 36.2
6-O-((2R,3R)-3-Hydroxy-2-(pentyl)octadecanoyl)-D-glucopyra-
(CH2), 51.3 (CHCO2H), 72.4 (CHOH), 125.7, 128.3, 128.5, 143.1 nose (32). A mixture of 28 (18.0 mg, 0.0201 mmol) and
(Ph), 180.4 (CvO); HRMS (ESI−): m/z 1087.9618 [2M – H]− Pd(OH)2/C (20 mg) in MeOH (3 mL) and THF (3 mL) was
(calcd [C72H127O6]− 1087.9638).
stirred under H2 (50 psi) for 24 h. The reaction was filtered
(2R,3R)-3-Hydroxy-2-(tricosanyl)octadecanoic
acid
(24). through a Celite plug and the plug rinsed sequentially with
LiHMDS in THF (1 M, 118 µL, 118 µmol) was added to a stir- MeOH/THF (3 : 2, 6 mL), MeOH (6 mL) and THF (6 mL). The
ring mixture of the aldehyde 17 (26.8 mg, 65.3 µmol), sulfone solvent was evaporated from the combined filtrates under
20 (62.2 mg, 130 µmol) and dry THF (3 mL) at −10 °C under reduced pressure and the residue was purified by flash chrom-
N2. The mixture was warmed to r.t. and allowed to stir until atography (90 : 9 : 1 CHCl3/MeOH/H2O) to give 32 as an amor-
the aldehyde was consumed (t.l.c.). The mixture was poured phous solid (5.7 mg, 53%, 3 : 2 α : β). [α]2D3 31 (c 0.15, CH3OH);
into sat. aq. NH4Cl (20 mL) and extracted with Et2O (3 × 1H NMR (400 MHz, CD3OD/CDCl3 1 : 1) δ 0.87 (6 H, m, 2 ×
10 mL). The combined organic extracts were washed with CH3), 1.08–1.67 (36 H, m, 18 × CH2), 2.42 (1 H, ddd, J 3.6, 7.2,
water (20 mL) then sat. aq. NaCl (20 mL), dried (MgSO4) and 10.2 Hz, CHCO2), 3.08–3.22 (0.4 H, m, H2β), 3.27–3.37 (1.6 H,
the solvent evaporated under reduced pressure. The residue m, H2α,4α,4β), 3.48 (0.45 H, ddd, J 2.2, 6.1, 8.8 Hz, H5β),
was purified by flash chromatography (5–20% EtOAc/pet. 3.62–3.75 (2 H, m, CHOH, H3α,3β), 3.96 (0.55 H, ddd, J 2.2,
spirits with 1% AcOH) to give slightly impure (2R,3R)-3- 5.0, 10.1 Hz, H5α), 4.18 (0.4 H, dd, J 6.0, 12.0 Hz, H6aβ), 4.21
hydroxy-2-(tricos-4-en-1-yl)octadecanoic acid 22 (23.4 mg, (0.6 H, dd, J 5.0, 12.0 Hz, H6aα), 4.39–4.518 (1.4 H, m, H6b,
58%), which was used without further purification; HRMS H1β), 5.08 (0.6 H, d, J 3.7 Hz, H1α); 13C NMR (125 MHz,
(ESI+): m/z 643.5995 [M + Na]+ (calcd [C41H80O3Na]+ 643.6000).
CD3OD) δ 14.26, 14.28, 14.4 (2 C, CH3), 23.11, 23.14, 23.4, 26.2,
Compound 22 (30.3 mg, 0.0488 mmol) was treated, as 26.3, 27.9, 29.5, 30.1, 30.3, 30.36, 30.39, 32.47, 32.49, 32.6,
described for the preparation of 25, to give 24 (26.6 mg, 88%). 35.41, 35.44 (14 C, CH2), 53.8 (CHCO2), 64.2, 64.5 (C6α,β), 70.2,
[α]2D5 7.0 (c 0.20, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 71.3, 71.4, 73.2, 73.3, 74.3, 74.8, 75.6, 77.4 (C2,3,4,5), 93.4
(6 H, t, J 6.8 Hz, 2 × CH3), 1.11–1.67 (72 H, m, 36 × CH2), 2.31 (C1β), 97.7 (C1α), 175.97, 176.04 (CvO); HRMS (ESI+): m/z
(1 H, br s, CHCO2H), 3.62 (1 H, br s, CHOH); 13C NMR 555.3879 [M + Na]+ (calcd [C29H56O8Na]+ 555.3867).
(100 MHz, CDCl3) δ 13.97, 14.10 (2 × CH3), 22.40, 22.66, 25.67,
Benzyl
2,3,4-tri-O-benzyl-6-O-((2R,3R)-3-hydroxy-2-(tricosa-
26.95, 29.34, 29.38, 29.49, 29.57, 29.59, 29.64, 29.67, 31.64, nyl)octadecanoyl)-β-D-glucopyranoside (29). A mixture of the
31.90, 35.39 (27 C, CH2), 51.00 (CHCO2H), 72.17 (CHOH), tosylate 26 33 (6.6 mg, 0.0095 mmol), the acid 24 (6.5 mg,
180.38; HRMS (ESI−): m/z 621.6191 [M
−
H]− (calcd 0.0104 mmol) and CsHCO3 (9.2 mg, 0.0474 mmol) was treated
[C41H81O3]− 621.6191).
according to the procedure described for the preparation of 28
Benzyl
2,3,4-tri-O-benzyl-6-O-((2R,3R)-3-hydroxy-2-(pentyl) to give 29 (6.2 mg, 57%) as a colourless gum. [α]2D2 −3.6 (c 0.32,
octadecanoyl)-β-D-glucopyranoside (28). A mixture of the acid CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.78–0.93 (6 H, m, 2 ×
16 (19.3 mg, 0.052 mmol), the tosylate 26 33 (24.6 mg, CH3), 1.10–1.79 (72 H, m, 36 × CH2), 2.40–2.49 (2 H, m, OH,
0.0347 mmol) and CsHCO3 (33.6 mg, 0.174 mmol) in dry DMF CHCO2), 3.42–3.54, 3.58–3.71 (5 H, 2 m, H2,3,4,5, CHOH),
(0.5 mL) and dry THF (2.5 mL) was stirred at 70 °C for 48 h 4.15–4.25 (1 H, m, H6a), 4.47–4.55 (2 H, m, H1,6b), 4.55–4.64
under N2. The mixture was then cooled to r.t., poured into (2 H, m, CH2Ph), 4.69 (1 H, d, J 10.9 Hz, CH2Ph), 4.77 (1 H, d,
H2O (20 mL) and extracted with EtOAc (3 × 10 mL). The com- J 10.9 Hz, CH2Ph), 4.82–4.98 (4 H, m, J 10.9 Hz, CH2Ph),
bined organic extracts were washed with H2O (20 mL), then 4.82–4.98 (4 H, m, 2 × CH2), 7.21–7.38 (20 H, m, Ph); 13C NMR
brine (20 mL), dried (MgSO4), and the solvent evaporated (100 MHz, CDCl3) δ 14.1 (2 × CH3), 22.7, 25.8, 27.5, 29.3, 29.4,
under reduced pressure. The residue was purified by flash 29.57, 29.63, 29.7, 31.9, 35.6, (36 C, CH2), 51.3 (CHCO2), 62.8,
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71.1 (2 × CH2), 72.3, 72.8 (2 × CH), 74.9, 75.1, 75.7 (3 × CH2Ph), (4.5 mg, 72%, 3 : 2 α : β). [α]2D5 25 (c 0.15, CHCl3); 1H NMR
77.8, 82.3, 84.5 (3 C, C2,3,4), 102.3 (C1), 127.7, 127.82, 127.84, (400 MHz, CD3OD/CDCl3 1 : 1) δ 0.83 (3 H, t, J 6.7 Hz, CH3),
127.9, 128.0, 128.1, 128.3, 128.37, 128.38, 128.5, 137.1, 137.7, 1.03–1.70 (48 H, m, 24 × CH2), 2.33–2.46 (2 H, m, OH, CHCO2),
138.2, 138.3 (20 C, Ph), 175.2 (CvO); HRMS (ESI+): m/z 2.54 (2 H, t, J 7.8 Hz, CH2Ph), 3.14 (0.4 H, t, J 8.3 Hz, H2β),
1167.8576 [M + Na]+ (calcd [C76H116O8Na]+ 1167.8562).
6-O-((2R,3R)-3-Hydroxy-2-(tricosanyl)octadecanoyl)-D-gluco-
3.24–3.38 (2 H, 2 m, H2α,3β,4α,4β), 3.42–3.49 (0.4 H, ddd,
J 2.4, 6.4, 9.2 Hz, H5β), 3.63–3.74 (1.6 H, m, CHOH, H3α),
pyranose (33). Compound 28 (6.2 mg, 0.00541 mmol) was 3.90–3.99 (0.6 H, m, H5α), 4.13–4.23 (1 H, 2 overlapping dd,
hydrogenated as described for the preparation of 32 to give 33 H6aα,β), 4.36–4.47 (1.4 H, m, H6bα,β, H1β), 5.08 (0.60 H, d,
as an amorphous solid (2.6 mg, 60%). [α]2D5 22 (c 0.20, CH3OH/ J 3.7 Hz, H1α); 13C NMR (175 MHz, CD3OD) δ 13.66 (CH3),
CHCl3 1 : 1); 1H NMR (500 MHz, CD3OD/CDCl3 1 : 9) δ 0.82 22.51, 25.31, 27.28, 27.31, 28.86, 29.14, 29.21, 29.24, 29.26,
(6 H, t, J 6.9 Hz, 2 × CH3), 1.05–1.6, (72 H, m, 36 CH2), 2.42 (1 29.35, 29.38, 29.39, 29.44, 29.45, 29.46, 29.48, 29.50, 29.54,
H, ddt, J 4.8, 7.6, 10.0 Hz, CHCO2), 3.15 (0.4 H, dd, J 8.1, 8.8 31.43, 31.79, 33.35, 34.67, 35.80 (26 × CH2), 52.82, 52.89
Hz, H2β), 3.25–3.32 (0.6 H, m, H4α), 3.33–3.38 (1 H, m, (CHCO2), 63.47, 63.64 (C6α,β), 69.25, 70.40, 70.48, 72.33, 72.35,
H2α,3β), 3.39–3.48 (0.4 H, m, H5β), 3.62–3.75 (2 H, m, CHOH, 73.42, 73.84, 74.63, 76.39 (C2α,β, C3α,β, C4α,β, C5α,β, CHOH),
H3α, H4β), 3.93 (0.6 H, ddd, J 2.4, 5.5, 10.0 Hz, H5α), 4.16–4.26 92.47 (C1α), 96.70 (C1β), 125.36, 128.03, 128.22, 142.79
(1 H, m, H6aα,β), 4.36–4.42 (1 H, m, H6bα,β), 4.44 (0.4 H, d, (4 × Ar), 175.26, 175.31 (CvO); HRMS (ESI+): m/z 724.5744
J 7.8 Hz, H1β), 5.09 (0.6 H, d, J 3.7 Hz, H1α); 13C NMR [M + NH4]+ (calcd [C42H78NO8]+ 724.5722).
(125 MHz, CD3OD/CDCl3 1 : 9) δ 14.0 (CH3), 22.7, 25.5,
6-O-(3′-Nonyl)dodecanoyl-D-glucose (35). A mixture of 26 33
27.4, 29.2, 29.4, 29.5, 29.6, 29.68, 29.72, 32.0, 35.1 (36 × CH2), (12.3 mg, 0.0177 mmol), acid 27 34 (10.3 mg, 0.0356 mmol),
52.8, 52.9 (CHCO2), 63.7 (C6), 69.3, 70.4, 70.6, 72.4, 72.6, 73.7, CsHCO3 (158.3 mg, 0.079 mmol), dry THF (5 mL) and dry
73.8, 74.6 (C2,3,4,5), 92.4 (C1β), 96.7 (C1α), 175.3 (CvO); DMF (1 mL) were stirred vigorously at 80 °C under an N2 atmo-
HRMS (ESI+): m/z 807.6660 [M + Na]+ (calcd [C47H92O8Na]+ sphere for 5 h. The mixture was cooled then poured into H2O
807.6684).
(30 mL). The aqueous layer was extracted sequentially with
Benzyl
2,3,4-tri-O-benzyl-6-O-((2R,3R)-3-hydroxy-2-(12- Et2O (2 × 10 mL), EtOAc (2 × 10 mL), and CH2Cl2 (2 × 10 mL).
phenyldodecyl)octadecanoyl)-β-D-glucopyranoside
(30).
A
The combined organic extracts were dried (MgSO4), filtered
mixture of the acid 25 (18.0 mg, 0.0330 mmol), tosylate 26 33 and concentrated under reduced pressure. The residue was
(15.3 mg, 0.0220 mmol) and CsHCO3 (21.3 mg, 0.110 mmol) partially purified by flash chromatography to give impure
were processed according to the procedure described for the benzyl
2,3,4-tri-O-benzyl-6-O-(3′-nonyldodecanoyl)-α-D-gluco-
preparation of 28 to give 30 (17.0 mg, 72%) as a colourless oil. pyranoside (31), which was used without further purification;
1
[α]2D4 0.8 (c 0.55, CHCl3); H NMR (400 MHz, CDCl3) δ 0.88 (3 HRMS (ESI+): m/z 866.5933 [M + NH4]+ (calcd [C55H76O7
+
H, t, J 6.8 Hz, CH3), 1.16–1.79 (48 H, m, 24 × CH2), 2.42–2.51 (2 NH4]+ 866.5929). The crude material was deprotected as
H, m, CHCO2, CHOH), 2.55–2.64 (2 H, m, CH2Ph), 3.46–3.57, described for the preparation of 32 to give 35 (2.8 mg, 53%
3.61–3.73 (5 H, 2 m, H2,3,4,5, CHOH), 4.18–4.26 (1 H, m, over two steps). [α]2D4 29 (c 0.10, CH3OH/CHCl3 1 : 1); H NMR
1
H6a), 4.50–4.57 (2 H, m, H1,6b), 4.60 (1 H, d, J 10.9 Hz, (400 MHz, CD3OD) δ 0.90 (6 H, t, J 6.8 Hz, 2 × CH3), 1.14–1.42
CH2Ph), 4.63 (1 H, d, J 11.8 Hz, CH2Ph), 4.71 (1 H, d, J 10.9 Hz, (32 H, m, 16 × CH2), 1.78–1.88 (1 H, m, CH(C9H19)2), 2.21–2.33
CH2Ph), 4.79 (1 H, d, J 10.9 Hz, CH2Ph), 4.86–4.98 (4 H, m, (2 H, m, CH2CO2), 3.14 (0.4 H, dd, J 8.1, 8.8 Hz, H2β),
CH2Ph), 7.13–7.40 (25 H, m, Ph); 13C NMR (100 MHz, CDCl3) 3.25–3.37 (2 H, 2 m, H2α,3β,4α,4β), 3.44–3.50 (0.4 H, m, H5β),
δ 14.3 (CH3), 22.8, 26.0, 27.7, 29.51, 29.52, 29.6, 29.7, 29.75, 3.67 (0.6 H, t, J 9.3 Hz, H3α), 3.96 (0.6 H, ddd, J 2.1, 5.2, 10.0
29.79, 29.9, 31.7, 32.1, 35.8, 36.1 (CH2), 51.4 (CHCO2), Hz, H5α), 4.11–4.21 (1 H, 2 overlapping dd, H6a), 4.35–4.44
63.0 (C6), 71.3 (CH2Ar), 72.5, 73.0 (CH), 75.1, 75.3, 75.9 (CH2), (1 H, m, H6b), 4.47 (0.4 H, J 7.7 Hz, H1β), 5.08 (0.6 H, d, J 3.7
77.9, 82.4, 84.7 (CH), 102.5 (C1), 125.7, 127.8, 128.01, 128.03, Hz, H1α); 13C NMR (125 MHz, CD3OD) δ 14.0 (CH3), 22.7, 25.5,
128.1, 128.2, 128.29, 128.34, 128.51, 128.53, 128.6, 128.7, 27.4, 29.2, 29.3, 29.5, 29.6, 29.68, 29.72, 32.0, 35.1, 52.8, 52.9,
137.3, 137.8, 138.4, 138.5, 143.1 (Ph), 175.4 (CvO); HRMS 63.7, 69.3, 70.6, 72.4, 72.6, 73.7, 73.8, 74.6, 76.4, 92.4 (C1β),
(ESI+): m/z 1089.7149 [M + Na]+ (calcd [C70H98O8 + Na]+ 96.7 (C1α), 175.31, 175.35 (CvO); HRMS (ESI+): m/z 506.4069
1089.7154).
[M + NH4]+ (calcd [C55H80NO7]+ 506.4051).
6-O-((2R,3R)-3-Hydroxy-2-(12-phenyldodecyl)octadecanoyl)-
D-glucopyranose (34). A mixture of 30 (9.5 mg, 0.0089 mmol)
and Pd(OH)2/C (10 mg) in MeOH (3 mL) and THF (3 mL) was
Reporter cells assays
stirred under H2 (50 psi) for 24 h. A further 10 mg Pd(OH)2/C 2B4-NFAT-GFP reporter cells expressing mouse or human
was added and the reaction again stirred under H2 atmosphere Mincle together with FcRγ were prepared as previously
(50 psi) for a second 24 h. The reaction was filtered through a described.21 To stimulate reporter cells, each glycolipid dis-
Celite plug and the plug rinsed sequentially with MeOH/THF solved in chloroform methanol (2 : 1) at 1 mg ml−1 was diluted
(3 : 2, 6 mL), MeOH (6 mL) and THF (6 mL). The solvent was in isopropanol, then added to 96-well plates at 20 μl per well,
evaporated from the combined filtrates under reduced followed by the evaporation of the solvent as described pre-
pressure and the residue was purified by flash chromatography viously. Reporter cells were stimulated for 16 to 20 h. Reporter
(90 : 9 : 1 CHCl3/MeOH/H2O) to give 34 as an amorphous solid activity was determined by flow cytometry.
This journal is © The Royal Society of Chemistry 2016
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Paper
Organic & Biomolecular Chemistry
Crystallography
S. R. Almeida and G. D. Brown, Cell Host Microbe, 2011, 9,
436–443.
12 S. Yamasaki, M. Matsumoto, O. Takeuchi, T. Matsuzawa,
E. Ishikawa, M. Sakuma, H. Tateno, J. Uno, J. Hirabayashi,
Y. Mikami, K. Takeda, S. Akira and T. Saito, Proc. Natl.
Acad. Sci. U. S. A., 2009, 106, 1897–1902.
13 M. B. Richardson, S. Torigoe, S. Yamasaki and
S. J. Williams, Chem. Commun., 2015, 51, 15027–15030.
14 H. Schoenen, B. Bodendorfer, K. Hitchens, S. Manzanero,
K. Werninghaus, F. Nimmerjahn, E. M. Agger, S. Stenger,
P. Andersen, J. Ruland, G. D. Brown, C. Wells and R. Lang,
J. Immunol., 2010, 184, 2756–2760.
15 H. Schoenen, A. Huber, N. Sonda, S. Zimmermann,
J. Jantsch, B. Lepenies, V. Bronte and R. Lang, J. Immunol.,
2014, 193, 3664–3675.
16 E. C. Patin, S. Willcocks, S. Orr, T. H. Ward, R. Lang and
U. E. Schaible, Innate Immun., 2016, 22, 181–185.
17 A. A. Khan, S. H. Chee, R. J. McLaughlin, J. L. Harper,
F. Kamena, M. S. M. Timmer and B. L. Stocker,
ChemBioChem, 2011, 12, 2572–2576.
18 B. L. Stocker, A. A. Khan, S. H. Chee, F. Kamena and
M. S. Timmer, ChemBioChem, 2014, 15, 382–388.
19 M. V. Pimm, R. W. Baldwin, J. Polonsky and E. Lederer,
Int. J. Cancer, 1979, 24, 780–785.
Crystal data for compound 12. C32H52O5 (CH3OH), M =
532.77, T = 130.0 K, λ = 1.54180, monoclinic, space group P21,
a = 12.8359(8), b = 4.9611(2), c = 26.202(2) Å, β = 97.210(6)°. V =
1655.35(17)) Å3, Z = 2, Dc = 1.07 mg M−3 μ(Cu-Kα) 0.547 mm−1
,
F(000) = 588, crystal size 0.6 × 0.12 × 0.04 mm3, 11 321 reflec-
tions measured, 5581 independent reflections [R(int)
=
0.0484], the final R was 0.0658 [I > 2σ(I) 4301 data] and wR(F2)
was 0.2006 (all data) GOOF = 1.006, absolute structure para-
meter 0.1(2). This data has been deposited with the
Cambridge Crystallographic Data Centre, deposition code
CCDC 960227.
Acknowledgements
Funding was provided by the Australian Research Council, a
JSPS Grant-in-Aid for Scientific Research, a Grant-in-Aid for
Scientific Research on Innovative Areas (26110009) from MEXT,
and Grants from MHLW and the Research on Development of
New Drugs, AMED. SS thanks the Department of State
Development, Business and Innovation of Victoria for a Victoria-
India Doctoral Scholarship and the Australia India Institute.
20 A. Huber, R. S. Kallerup, K. S. Korsholm, H. Franzyk,
B. Lepenies, D. Christensen, C. Foged and R. Lang, Innate
Immun., 2016, 22, 405–418.
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