Cholesteryl 6-: O-acyl-α-glucosides from diverse Helicobacter spp. signal through the C-type lectin receptor Mincle

Article abstract of DOI:10.1039/d0ob01776k

Helicobacter spp. are Gram-negative bacteria that cause a spectrum of disease in the gut, biliary tree and liver. Many Helicobacter spp. produce a range of cholesteryl α-glucosides that have the potential to act as pathogen associated molecular patterns. We report a highly stereoselective α-glucosylation of cholesterol using 3,4,6-tri-O-acetyl-2-O-benzyl-d-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate, which allowed the synthesis of cholesteryl α-glucoside (αCG) and representative Helicobacter spp. cholesteryl 6-O-acyl-α-glucosides (αCAGs; acyl = C12:0, 14:0, C16:0, C18:0, C18:1). All αCAGs, irrespective of the nature of their acyl chain composition, strongly agonised signalling through the C-type lectin receptor Mincle from human and mouse to similar degrees. By contrast, αCG only weakly signalled through human Mincle, and did not signal through mouse Mincle. These results provide a molecular basis for understanding of the immunobiology of non-pylori Helicobacter infections in humans and other animals. This journal is

Full text of DOI:10.1039/d0ob01776k

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21 December 2017
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Page 1 of 21
Organic & Biomolecular Chemistry
Cholesteryl 6-O-acyl--glucosides from diverse Helicobacter spp. signal through theVCiew-Article Online
DOI: 10.1039/D0OB01776K
type lectin receptor Mincle
Dylan G.M. Smith,1 Emi Ito,2,3 Sho Yamasaki,2,3 Spencer J. Williams*1
1 School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University
of Melbourne, Parkville, Victoria, Australia, 3010
2 Department of Molecular Immunology, Immunology Frontier Research Center, Osaka
University, Suita 565-0871, Japan
3 Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka
University, Suita 565-0871, Japan
sjwill@unimelb.edu.au
Keywords: innate immunity, glycolipid, Helicobacter pylori, glycosylation
Abstract
Helicobacter spp. are gram-negative bacteria that cause a spectrum of disease in the gut, biliary
tree and liver. Many Helicobacter spp. produce a range of cholesteryl -glucosides that have
the potential to act as pathogen associated molecular patterns. We report a highly
stereoselective -glucosylation of cholesterol using 3,4,6-tri-O-acetyl-2-O-benzyl-D-
glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate, which allowed the synthesis of
cholesteryl -glucoside (CG) and representative Helicobacter spp. cholesteryl 6-O-acyl--
glucosides (CAGs; acyl = C12:0, 14:0, C16:0, C18:0, C18:1). All CAGs, irrespective of the
nature of their acyl chain composition, strongly agonised signalling through the C-type lectin
receptor Mincle from human and mouse to similar degrees. By contrast, CG only weakly
signalled through human Mincle, and did not signal through mouse Mincle. These results
provide a molecular basis for understanding of the immunobiology of non-pylori Helicobacter
infections in humans and other animals.
.
1

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DOI: 10.1039/D0OB01776K
Introduction
Helicobacter spp. are gram-negative bacteria with a characteristic helical shape.1 Many
Helicobacter are responsible for disease, most notably Helicobacter pylori, which infects an
estimated 50% of the human population and is a cause of gastric ulcers and stomach cancer.2
Non-pylori helicobacter species include gastric and enterohepatic colonizers, which inhabit
assorted mammals and birds, and which can cause a spectrum of diseases.3, 4 Non-pylori
helicobacter species have been detected in humans and have been implicated in various
diseases of the gut, biliary tree and liver.5 Non-pylori helicobacter species are important models
of helicobacter pathology owing to the small number and limitations of animal models
available for the study of human H. pylori infection.6
Helicobacter spp. typically, but not entirely characteristically,7 produce a range of
cholesteryl -glucosides, which may be modified at the glucose 6-position with esters or
phosphatidyl groups.8, 9 Cholesterol is not produced by Helicobacter spp., and is instead
extracted from the host cell membranes or from culture media.10 Cholesteryl glucosides can
comprise up to 33% of total lipids in Helicobacter spp.7 In the case of H. pylori, 25% of total
lipids are assorted cholesteryl glucosides, and more than one half of these is cholesteryl -
glucoside (CG) itself, and a further one quarter are cholesteryl 6-O-acyl--glucosides
(CAGs), comprised almost exclusively of the C14:0 ester, along with traces of the C16:0 and
C18:0 esters.9 CAGs appear to be highly restricted natural products with limited reports
suggesting their possible existence in Acholeplasma laidlawii,11 and Acholeplasma
axanthum,12 and Borrelia hermsii.13 Table 1 summarizes the isolation of CG and CAGs
from various Helicobacter spp.8
2

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Table 1. Abundance of cholesteryl -glucoside (CG) and cholesteryl 6-O-acyl--glucosides
DOI: 10.1039/D0OB01776K
(CAGs) from assorted Helicobacter spp., and their fatty acid composition.
OH
O
OR
O
H
H
HO
HO
HO
HO
H
H
H
H
HO
HO
O
O
CG

CAGs

% of total lipids
Fatty acid composition of CAGs (R)
Organism
C12:0
C14:0 C16:0 C18:0 C18:1
CG
14.1
2.5
CAGs
6.5
H. pylori9
H.
–
–
98.5
100
trace
–
trace
–
–
–
20.4
nemestrinae8
H. rappini8
H. felis7
2.0
23.1
9.7
84
–
8
100
–
5
–
–
–
–
–
3
–
–
23.4
12.5
H. muridarum7
12.1
100
Recently, Yamasaki and co-workers demonstrated that crude cell extracts of H. pylori
could agonise signalling through the macrophage inducible C-type lectin (Mincle) receptor,14-
17
and the effects were shown to be caused by CG and CAGs.18 H. pylori specific T cell
responses and gastritis were attenuated in Mincle knockout mice, and a H. pylori strain lacking
CAGs was impaired in its ability to induce gastritis. Collectively these results demonstrated
that CAGs are an important contributor to the pathology of H. pylori, through signalling
through Mincle.
Synthetic access to homogeneous CAGs is necessary to understand how each
lipoform contributes to activity. Early efforts to synthesize these compounds suffered from
poor stereoselectivity in the glucosylation of cholesterol.19 Gervay-Hague and co-workers
developed a halide-ion catalyzed glycosylation utilizing glucosyl iodides enabling the
streamlined stereoselective synthesis of CG and CAGs.20-23 While glucosyl iodides
deployed in halide-ion catalyzed glycosylations provide high stereoselectivity in the formation
of the -linkage (typically 40:1, /β), they are very water-sensitive reagents that can be
technically challenging to handle.
Here, we undertook the synthesis of a range of CAGs representative of the main
species found in a range of Helicobacter spp. We defined a chemically stable, highly -
3

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selective glucosyl donor for the glycosylation of cholesterol to give CG and converted this to
DOI: 10.1039/D0OB01776K
a range of saturated and unsaturated CAGs. Finally, we report the ability of these CAGs to
agonize signalling through Mincle.
Results and Discussion
Development of a highly stereoselective -glucosylation of cholesterol
We sought to explore a range of glycosyl donors for the stereoselective glycosylation of
cholesterol that varied their remote protecting groups, and then elaborate the resulting
cholesteryl glucoside to provide the CAGs reported from a variety of Helicobacter spp.
Conventionally, glycosylations can be achieved by activation of a chemically-stable glycosyl
donor, nominally proceeding via an oxocarbenium ion intermediate. However, in the absence
of a suitable directing group, stereoselectivity is often poor, although it can be influenced by
the reaction conditions, activator and protecting group patterns on the glycosyl donor and
acceptor.24 We elected to explore the effect of systematically varying acetyl/benzyl groups
while maintaining a common 2-O-benzyl group.
Various combinations of acetyl/benzyl groups have been shown to have dramatic
effects on rates and stereoselectivity of glucosylation.25 Tetra-O-benzylglucosyl donors have
been applied to glucosylation of cholesterol with greatly varying stereoselectivities.26, 27
Frechét noted that 6-O-acetyl-2,3,4-tri-O-benzyl glucosyl bromides often provide high -
stereoselectivity.28, 29 30 Nifantiev reported that 3-O-acetyl groups, especially in combination
with a 6-O-acetyl group, can promote enhanced -selectivity.31-33 van Boom reported that
3,4,6-tri-O-acetyl-2-O-benzylglucosyl donors provide excellent -selectivity in the
glucosylation of primary alcohols, which typically react with poor stereoselectivity owing to
their high reactivity.34-36 Based on these literature precedents, we elected to prepare the
corresponding Yu phenyltrifluoroacetimidates37 and examine their potential as -selective
glycosyl donors for glycosylation of cholesterol. Reaction of the precursor hemiacetals 1, 2,31,
38
331 and 4 with phenyltrifluoroacetimidoyl chloride (PTFAI-Cl; prepared from
phenyltrifluoroacetamide and dichlorotriphenylphosphorane)40 and pyridine afforded the
phenyltrifluoroacetimidates 5,41 6,42 7 31 and 8 (Figure 1).†
4

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DOI: 10.1039/D0OB01776K
OBn
O
OAc
O
BnO
BnO
BnO
BnO
BnO
BnO
OR
OR
1
5
2
6
R = H
R = C(NPh)CF3
R = H
R = C(NPh)CF3
OAc
O
OAc
O
BnO
AcO
AcO
AcO
BnO
BnO
OR
OR
3
7
4
8
R = H
R = C(NPh)CF3
R = H
R = C(NPh)CF3
Figure 1. Glycosyl donors and their precursor hemiacetals used in this study.
Cholesterol is a highly crystalline compound with relatively poor solubility in toluene
and ether, particularly at temperatures below 0 °C. Glycosylation of cholesterol with
tetrabenzyl donor 5 in CH2Cl2 or toluene at 0 °C, activated by TfOH (Table 2), provided no
selectivity (1:1 /), while ether at 0 °C afforded a 4:1 /β ratio. Consequently, we chose to
perform subsequent glycosylations in ether at 0 °C, initiated with catalytic TfOH. Ether has
been proposed to provide nucleophilic participation, although no direct evidence in support of
this has been reported.43 The remaining glucosyl donors showed improving selectivity under
these conditions. The monoacetyl donor 6 gave an 8:1 /β ratio, the diacetyl donor 7 gave a
10:1 /β ratio, and the triacetyl donor 8 was the best, with a 14:1 /β ratio. In this last case,
flash chromatography, followed by recrystallization afforded pure -12 in 88% yield. In all
cases the stereochemical outcome was unrelated to the anomeric configuration of the glycosyl
donors.
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DOI: 10.1039/D0OB01776K
Table 2. Glucosylation of cholesterol by donors 5–8.
OR6
O
cholesterol
OR6
O
H
R4O
R3O
R4O
R3O
TfOH, Et2O, 0 oC
H
H
BnO
OC(NPh)CCF3
BnO
O
Donor
R6
R4
R3
Product
Yield (%)
ratio (/β)
4:1
5
6
7
8
Bn
Ac
Ac
Ac
Bn
Bn
Bn
Ac
Bn
Bn
Ac
Ac
9
91%
97%
93%
88%
10
11
12
9:1
10:1
14:1
To confirm that Et2O was providing a directing effect, we repeated the glycosylation
using 8 but in CH2Cl2 at 0 °C, which provided only a 3:1 /β ratio. As cholesterol exhibits
higher solubility in CH2Cl2 we lowered the temperature to -40 °C, which provided an improved
stereoselectivity of 10:1 /β, but failed to surpass that of the reaction performed in Et2O at 0
°C, highlighting the beneficial role played by Et2O.
Synthesis of CG and a range of CAGs
The benzyl group of -12 was removed using transfer hydrogenation
(ethanol/cyclohexene, Pd(OH)2/C),26 which maintained the alkene of the steroid, affording
alcohol 10 in 91% yield (Scheme 1). Deacetylation under Zemplèn conditions afforded CG
14. The CAGs were prepared in a four step process from CG. Trimethylsilylation using
TMSCl and Et3N afforded the per-TMS ether 15, followed by selective deprotection of the 6-
position using NH4OAc in CH2Cl2/MeOH22 afforded the alcohol 16. Acylation with lauroyl,
myristoyl, palmitoyl, oleoyl or stearyl chloride in pyridine/CH2Cl2, followed by treatment with
acidic ion-exchange resin in methanol/chloroform, afforded CAG-C12:0, CAG-
C14:0, CAG-C16:0, CAG-C18:0, and CAG-C18:1 in yields of 63-79%.
6

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DOI: 10.1039/D0OB01776K
OH
O
OAc
O
H
H
NaOMe, MeOH
HO
AcO
AcO
HO
H
H
H
H
92%
HO
RO
O
O
CG (14)

cyclohexene
ethanol, Pd(OH)2/C
88%
12
- R = Bn
13
R = H
1. RCOCl, DMAP,
pyr/CH2Cl2
CAG-C12:0
CAG-C14:0
CAG-C16:0
CAG-C18:0
CAG-C18:1





, 73%
, 79%
, 67%
, 72%
, 63%
OR
O
TMSCl
Et3N
H
TMSO
TMSO
2. Dowex 50 H+
H
H
TMSO
O
NH4OAc
CH2Cl2/MeOH
68% over two steps
15
R = TMS
R = H
16
Scheme 1. Synthesis of CG and CAGs.
Signalling of CG and CAGs through mouse and human Mincle
The ability of CG and the CAGs to induce signalling through Mincle was
investigated using a nuclear factor of activated T cells–green fluorescent protein (NFAT-GFP)
T cell hybridoma that is activated by signalling through Mincle. These cell lines have been
developed for both human (h) and mouse (m) Mincle, and which co-express Mincle and the
adaptor protein FcR.44 As previously reported,18 we found that CG caused weak activation
of the hMincle reporter cell line, and failed to activate the mMincle reporter cell line (Figure
2). All acyl forms of CAGs activated the reporter cells by signalling through both hMincle
and mMincle, with levels of activation broadly similar across the suite of compounds.
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DOI: 10.1039/D0OB01776K
Figure 2. Synthetic CG and CAGs signal through mouse and human Mincle. NFAT-GFP
2B4 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 TDB and
cholesteryl glucosides. Quantities denote the amount in nmol; assays were performed in
triplicate; the mean values and standard deviations are shown.
Conclusions
The cholesteryl glucosides of Helicobacter spp. are prominent glycolipids in the cell wall of
Helicobacter spp., and considerable effort has been directed at understanding their effects on
the immune system.23, 45 As gram-negative bacteria they produce the classical pathogen
associated molecular pattern (PAMP) lipopolysaccharide (LPS), however, H. pylori modifies
LPS by under-acylation, allowing it to evade detection by the classical LPS Toll-like receptor
(TLR) 4.46, 47 Our findings agree with contemporaneous work18 and shows that CAGs are
PAMPs that signal through the C-type lectin receptor Mincle.
Cholesteryl glucosides are unique signatures of host infection, as they are derived from
bacterial glucosylation of host cholesterol. In this regard they are reminiscent of glucose
monomycolate, another potent agonist of Mincle signalling,48, 49 which is derived from acyl
transfer by Mycobacterium tuberculosis to host glucose.50 While this work focussed on the
synthesis of CAGs that have been isolated from mono-cultures of Helicobacter spp.,
metabolic labelling methods have shown that the lipid composition of CAGs is altered when
co-cultured with human gastric cells, leading to the incorporation of longer and further
unsaturated lipid chains, including C18:2 and C20:4.51 Longer-chain CAGs have been
implicated in the delivery of the virulence factor cytotoxin-associated gene A into the host
cell.51 The recent cloning of hp0499 as the H. pylori CG acyltransferase has facilitated the
8

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study of the role of CAGs, showing that secreted CG acyltransferase produces CDAOGI: 1s0.i1n03V9hie/wDo0AsrOtticBl0e1O7n7l6inKe
cells, which promote clustering of lipid rafts, the gathering of adhesion molecules, and bacterial
adhesion.52 The consistent Mincle signalling agonism seen for CAGs bearing variation in the
acyl chain suggest that these other host lipid-modified CAGs will also be able to signal
through Mincle.
While the best studied agonists of Mincle are mycobacterial lipids such as trehalose
dimycolate and glucose monomycolate, and related species, there are reports of other classes
of agonist molecules more closely related to the cholesteryl glucosides.16 Crystalline
cholesterol signals through human Mincle, but does not signal through mouse Mincle.53
Mutagenesis studies demonstrated that signalling by cholesterol involves the cholesterol
recognition site (CRAC) of human Mincle, a feature that is absent in rodent Mincle. Notably,
alkylation of cholesterol abrogates its ability to signal through Mincle.53 CG and CAGs
signal through both human and mouse Mincle, and so binding is unlikely to require the CRAC
site of human Mincle. The abilities of CAGs to agonize Mincle signalling is reminiscent of
simplified agonists that were developed by appending a cholesteryloxyacetyl group to O6 of
octyl and lauryl glucosides, where the cholesteryl group was concluded to act as a surrogate
for a lipid group.54 The results presented here are consistent with broad structure activity trends
espoused by Decout that for effective Mincle signalling antagonists, at least three of four key
structural motifs (two sugars, two alkyl chains) must be present, with the cholesterol in place
of an alkyl chain.48
The ability of non-pylori CAGs to signal through Mincle is significant as non-pylori
Helicobacter are often found in humans as well as non-human animals. Therefore, the human
immune responses to H. pylori driven by CG and CAGs signalling through Mincle may
allow a better understanding of the pathobiology of non-pylori Helicobacter infections in
humans and other animals.
Experimental
All reactions were conducted under a dry nitrogen atmosphere. Pyridine was distilled over
KOH. Dry Et2O, CH2Cl2 and THF were obtained from a Grubbs solvent purification apparatus
(Glass Contour, USA) as described by Pangborn et al. DMF was dried over 4 Å molecular
sieves. All other reagents were commercial grade and were used without further purification.
Thin layer chromatography (TLC) was performed on aluminium backed Merck Silica Gel 60
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sheets, detection was achieved by irradiation with UV light and/or by staining with ceric
DOI: 10.1039/D0OB01776K
ammonium molybdate. Flash chromatography was performed according to the methodology
13
of Still et al. using Scharlau Silica Gel 60.55 1H and C NMR spectra were recorded using
Agilent 400, 500 or 600 MHz instruments. Chemical shifts were referenced to residual solvent
1
13
1
signals. Signal assignment in H and C NMR spectra was made using COSY, and H–13C
HSQC techniques. Melting points were obtained using a Reichert-Jung hot-stage apparatus.
2,3,4,6-Tetra-O-benzyl-D-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate (5)
K2CO3 (165 mg, 1.20 mmol) was added to a solution of 2,3,4,6-tetra-O-benzyl-α-D-
glucopyranose 1 (324 mg, 0.60 mmol) and PTFAI-Cl40 (0.099 ml, 0.90 mmol) in acetone (6
ml). The mixture was stirred for 3 h and then was filtered through a bed of Celite and the filtrate
concentrated. The residue was purified by flash chromatography (Et3N/EtOAc/hexanes) to
afford an anomeric mixture (assigned as primarily ) of the imidate 5 (406 mg, 95% yield) as
a yellow oil. 1H NMR (600 MHz, CDCl3) δ 7.39–7.05 (20 H, m, Ph), 6.72 (2 H, d, J = 6.1 Hz,
NPh), 6.52 (1 H, s, H1), 4.99 (1 H, d, J = 10.9 Hz, PhCH2), 4.86 (2 H, d, J = 10.9 Hz, PhCH2),
4.75 (2 H, s, PhCH2), 4.61 (1 H, d, J = 12.1 Hz, PhCH2), 4.52 (1 H, d, J = 10.7 Hz, PhCH2),
4.50 (1 H, d, J = 12.1 Hz, PhCH2), 4.06–3.65 (6 H, m, H2,3,4,5,6a,6b); 13C NMR (151 MHz,
CDCl3) δ 143.6, 138.6, 138.0, 137.8, 137.8, 128.7, 128.45, 128.41, 128.39, 128.38, 128.0,
127.92, 127.90, 127.84, 127.82, 127.75, 127.63, 127.62 (Ph), 124.1, 119.4 (NPh), 81.5 (C1),
79.2, 76.8, 75.7, 75.3, 73.5, 73.3, 73.1 (C2,3,4,5,PhCH2), 68.1 (C6).
6-O-Acetyl-2,3,4-tri-O-benzyl-α-D-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate
(6)
K2CO3 (362 mg, 1.75 mmol) was added to a solution of 6-O-acetyl-2,3,4-tri-O-benzyl-α-D-
glucopyranose31 2 (430 mg, 0.87 mmol) and PTFAI-Cl (0.192 ml, 1.75 mmol) in acetone (8.7
ml). The mixture was stirred for 3 h and then filtered through Celite and the filtrate
concentrated. The residue was purified by flash chromatography (Et3N/EtOAc/hexanes) to
afford an anomeric mixture (0.5:1 /) of the imidate 6 (550 mg, 95% yield) as a yellow oil.
Data for -anomer: 1H NMR (600 MHz, CDCl3) δ 7.39–7.05 (18 H, m, Ph), 6.73 (1 H, d, J =
6.5 Hz, NPh), 6.46 (1 H, br s, H1), 5.02 (1 H, d, J = 10.8 Hz, PhCH2), 4.90 (1 H, d, J = 10.8
Hz, PhCH2), 4.87 (1 H, d, J = 10.8 Hz, PhCH2), 4.80–4.71 (2 H, m, PhCH2), 4.60 (1 H, d, J =
10.7 Hz, PhCH2), 4.37–3.54 (6 H, m, H2,3,4,5,6a,6b), 2.04 (3 H, s, Ac); 13C NMR (151 MHz,
CDCl3) δ 170.6 (C=O), 143.5, 138.3, 137.6, 137.5, 128.7, 128.53, 128.50, 128.4, 128.2, 128.1,
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128.0, 127.9, 127.8, 127.6 (Ph), 124.2, 119.3 (NPh), 93.0 (C1), 81.4 (C3), 79.2 (C2D),O7I: 610..410(3C9/D40)O,B01776K
75.8 (PhCH2), 75.3 (PhCH2), 73.3 (PhCH2), 71.4 (C5), 62.6 (C6), 20.8 (CH3).
3,6-Di-O-acetyl-2,4-di-O-benzyl-α-D-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate
(7)
K2CO3 (297 mg, 2.14 mmol) was added to a solution of 3,6-di-O-acetyl-2,4-di-O-benzyl-α-D-
glucopyranose31 3 (477 mg, 1.07 mmol) and PTFAI-Cl (0.177 ml, 1.61 mmol) in acetone (10
ml). The mixture was stirred for 3 h and then filtered through a bed of Celite and the filtrate
concentrated. The residue was purified by flash chromatography (Et3N/EtOAc/hexanes) to
afford an anomeric mixture (0.5:1 /) of the imidate 7 (626 mg, 95% yield) as a yellow oil.
1H NMR (500 MHz, CDCl3) δ 7.40–7.06 (19 H, m, Ph), 6.82 (2 H, d, J = 7.6 Hz, NPh), 6.73
(1 H, d, J = 7.1 Hz, NPh), 6.49 (0.5 H, br s, H1), 5.59 (1 H, br s, H1), 5.59 (0.5 H, t, J =
9.6 Hz, H3), 5.26 (1 H, br s, H3), 4.81 (1 H, d, J = 11.7 Hz, PhCH2), 4.70 (0.5 H, d, J =
12.4 Hz, PhCH2), 4.65–4.47 (4.5 H, m, 3  PhCH2, 3  PhCH2), 4.39–4.02 (3.5 H, m,
H5,6,6), 3.39–3.83 (4 H, m, H2,2,4,4,5), 2.07, 2.05, 2.01, 1.91 (9 H, 4  s, Ac); 13C
NMR (126 MHz, CDCl3) δ 170.50, 170.48, 169.7, 169.6 (C=O), 143.2, 137.4, 137.1, 128.8,
128.59, 128.57, 128.52, 128.49, 128.2, 128.12, 128.06, 128.05, 128.03, 127.7 (Ph), 124.5,
119.3, 119.2 (NPh), 96.8 (br, C1, C1 not seen), 77.9, 76.3, 75.33, 75.29, 75.2, 74.6, 74.4,
74.3, 73.4, 73.0, 72.9, 71.2 (C2,3,4,5,2,3,4,5,PhCH2), 62.4 (C6,6), 21.0, 20.9,
20.8 (CH3).
3,4,6-Tri-O-acetyl-2-O-benzyl-D-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate (8)
K2CO3 (286 mg, 2.07 mmol) was added to a solution of 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-
glucopyranose35 4 (410 mg, 1.03 mmol) and PTFAI-Cl (0.170 ml, 1.55 mmol) in acetone (10
ml). The mixture was stirred for 3 h, filtered through a bed of Celite and the filtrate
concentrated. The residue was purified by flash chromatography (Et3N/EtOAc/hexanes) to
afford an anomeric mixture (0.5:1 /) of the imidate 8 (552 mg, 94% yield) as a yellow oil.
1H NMR (500 MHz, CDCl3) δ 7.41–7.07 (15 H, m), 6.83 (1 H, d, J = 7.7 Hz, NPh), 6.74 (0.5
H, d, J = 7.5 Hz, NPh), 6.49 (0.5 H, br s, H1), 5.73 (1 H, br s, H1), 5.47 (0.5 H, t, J = 9.7
Hz, H3), 5.26–5.14 (1 H, m, H3), 5.10–4.99 (1.5 H, m, H4,4), 4.82 (1 H, d, J = 11.7 Hz,
PhCH2), 4.71–4.59 (2 H, m, 2  PhCH2,PhCH2), 4.33–4.21 (1.5 H, m, H6a,6a), 4.18–
4.04 (2 H, m, H5,6b,6b), 3.81–3.64 (2.5 H, m, H2,2,5), 2.08, 2.06, 2.04, 2.01, 2.00,
1.94 (13.5 H, 6  s, Ac); 13C NMR (126 MHz, CDCl3) δ 170.54, 170.51, 170.0, 169.7, 169.6
11

Organic & Biomolecular Chemistry
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(C=O), 143.2, 143.1, 137.19, 137.17, 128.8, 128.6, 128.5, 128.2, 128.14, 128.13,D1O2I: 710..7103(9P/Dh0)O,B01776K
124.5, 124.4, 119.3, 119.2 (NPh), 96.7 (C1), 92.4 (C1), 77.6 (C2), 75.8 (C2), 74.7
(PhCH2), 73.7 (C3), 73.3 (PhCH2), 72.4 (C5), 71.5 (C3), 70.0 (C5), 68.0 (C4), 67.8
(C4), 61.6 (C6,6), 20.8, 20.67, 20.66, 20.61, 20.59 (CH3); HRMS: [M + Na]+ calcd for
C27H28F3NO9Na m/z 590.1608; found 590.1595.
General Glycosylation Procedure
A solution of a cholesterol (1 equiv) and N-phenyltrifluoracetimidoyl donor (1.2 equiv) in
anhydrous solvent (8 mL/1 mmol of the donor) was stirred with activated 3 Å molecular sieves
at rt for 20 min. The mixture was cooled to 0 °C, and TfOH (0.2 equiv) was added. The reaction
mixture was stirred at 0 °C for 15 min, at which stage TLC showed that the reaction was
completed. Triethylamine (2.3 equiv) was added at 0 °C to quench the reaction. The resulting
mixture was diluted with CH2Cl2, and the solids were removed by filtration through a pad of
Celite, with rinsing with additional CH2Cl2. The combined filtrates were washed with aq. sat.
NaHCO3 and then water. The organic solution was dried (MgSO4), and the solvent was
removed under reduced pressure to give a crude residue. Purification by flash chromatography
followed by 1H NMR was used determine the ratio of anomers. Yields were calculated on the
basis of the limiting reagent, cholesterol.
Cholesteryl 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-glucopyranoside (12)
Cholesterol (69.8 mg. 0.188 mmol) and imidate 8 (123 mg, 0.216 mmol) in dry Et2O (1.5 ml)
and powdered 3 Å molecular sieves were treated with TfOH (3.2 µl) according to the General
Glycosylation Procedure. The crude residue was purified by flash chromatography
(EtOAc/hexanes) to afford an anomeric mixture (14:1 /) of the cholesteryl glucoside (122
mg, 88% yield) as a white solid. This solid was recrystallised from ethanol to afford the pure
-anomer 12, mp = 178–181 °C; [α]D20 +69.0 (c 0.94, CHCl3); 1H NMR (600 MHz, CDCl3) δ
7.29–7.18 (5 H, m, Ph), 5.37 (1 H, t, J = 9.7 Hz, H3'), 5.29–5.24 (1 H, m, H6), 4.91–4.84 (2 H,
m, H1',4'), 4.56 (1 H, d, J = 12.4 Hz, PhCH2), 4.51 (1 H, d, J = 12.4 Hz, PhCH2), 4.17 (1 H,
dd, J = 12.2, 4.8 Hz, H6a'), 4.03 (1 H, ddd, J = 10.3, 4.7, 2.1 Hz, H5'), 3.97 (1 H, dd, J = 12.2,
2.0 Hz, H6b'), 3.48 (1 H, dd, J = 10.0, 3.6 Hz, H2), 3.38–3.30 (1 H, m, H3), 2.40–2.31 (1 H,
m), 2.23 (1 H, ddd, J = 13.3, 4.7, 1.8 Hz), 1.99, 1.95, 1.94 (9 H, 3  s, Ac), 1.98–1.87 (2 H, m),
1.85–0.75 (24 H, m), 0.94 (3 H, s), 0.85 (3 H, d, J = 6.5 Hz), 0.80 (3 H, d, J = 2.5 Hz), 0.79 (3
13
H, d, J = 2.5 Hz), 0.61 (3 H, s); C NMR (101 MHz, CDCl3) δ 170.6, 170.1, 169.9 (C=O),
12

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Organic & Biomolecular Chemistry
140.5 (C5), 137.8, 128.4, 128.0, 127.8 (Ph), 122.0 (C6), 95.0 (C1'), 78.2 (C3), 76.5 (C2'),V7ie2w.A7rticle Online
DOI: 10.1039/D0OB01776K
(PhCH2), 71.9 (C3'), 68.9 (C4'), 67.2 (C5'), 62.1 (C6'), 56.7, 56.1, 50.1, 42.3, 39.9, 39.7, 39.5,
37.0, 36.7, 36.2, 35.8, 31.9, 31.8, 28.2, 28.0, 27.6, 24.2, 23.8, 22.8, 22.5, 21.0, 20.8 (CH3),
20.73 (CH3), 20.68 (CH3), 19.4, 18.7, 11.8; HRMS: [M + NH4]+ calcd for C46H72NO9 m/z
782.5202; found 782.5219.
Cholesteryl 3,4,6-tri-O-acetyl-α-D-glucopyranoside (13)
A mixture of Pd(OH)2 (20% on carbon, 100 mg) and compound 12 (100 mg, 0.13 mmol) in
ethanol/cyclohexene (2:1, 1.5 ml) was stirred at reflux overnight then filtered through Celite
and concentrated under reduced pressure. The residue was purified by flash chromatography
(EtOAc/hexanes) to afford the triester 13 (77 mg, 88% yield) as a white solid. [α]D20 +90.2 (c
0.89, CHCl3). 1H NMR (500 MHz, CDCl3) δ 5.35 (1 H, m, H6), 5.21 (1 H, t, J = 9.7 Hz, H3'),
5.04 (1 H, d, J = 4.0 Hz, H1'), 4.98 (1 H, t, J = 9.7 Hz, H4'), 4.27–4.20 (1 H, m, H6a'), 4.10–
4.03 (2 H, m, H5',6b'), 3.64 (1 H, ddd, J = 11.6, 9.9, 4.0 Hz, H2'), 3.54–3.46 (1 H, m, H3), 2.37
(2 H, d, J = 7.7 Hz), 2.07 (6 H, s, 2  Ac), 2.02 (3 H, s, Ac), 2.04–1.77 (7 H, m), 1.61–0.82 (20
H, m), 1.01 (3 H, s), 0.90 (3 H, d, J = 6.5 Hz), 0.86 (3 H, d, J = 2.2 Hz), 0.85 (3 H, d, J = 2.2
13
Hz), 0.67 (3 H, s); C NMR (126 MHz, CDCl3) δ 171.0, 170.6, 169.6 (C=O), 140.1 (C5),
122.4 (C6), 97.0 (C1'), 79.2 (C3), 73.6 (C3'), 70.7 (C2'), 68.2 (C4'), 67.7 (C5'), 62.2 (C6'), 56.7,
56.1, 50.1, 42.3, 40.1, 39.7, 39.5, 36.9, 36.6, 36.2, 35.8, 31.9, 31.8, 28.2, 28.1, 28.0, 24.3, 23.8,
22.8, 22.5, 21.0, 20.9 (CH3), 20.7 (CH3), 20.6 (CH3), 19.3, 18.7, 11.8; HRMS: [M + Na]+ calcd
for C39H62O9Na m/z 697.4286; found 697.4298.
Cholesteryl α-D-glucopyranoside (αCG, 14)
NaOMe solution (1 M, 0.10 ml) was added to a solution of triester 13 (100 mg, 0.13 mmol) in
MeOH/THF (3:2, 2.5 ml). The mixture was stirred at rt for 2 h and then was quenched with
Dowex resin (H+ form), filtered and concentrated under reduced pressure to afford αCG (66
mg, 92% yield) as a white solid. mp 220–224 °C (lit.20 220.1−221.8 °C); [α]D20 +51.8 (c 0.5,
DMSO) (lit.20 [α]D23 +30.2 (c 0.54, CHCl3:MeOH)); 1H NMR (400 MHz, DMSO-d6) δ 5.25 (1
H, m, H6), 4.78 (1 H, d, J = 5.4 Hz, OH), 4.73 (1 H, d, J = 3.7 Hz, H1'), 4.65 (1 H, d, J = 4.8
Hz, OH), 4.42 (1 H, d, J = 6.7 Hz, H6a'), 4.37 (1 H, t, J = 5.7 Hz, OH), 3.60–3.51 (1 H, m,
H6b'), 3.38 (3 H, m, H3',5',3), 3.14–3.07 (1 H, m, H2'), 3.00 (1 H, td, J = 9.0, 5.5 Hz, H4'), 2.46
(1 H, dt, J = 3.5, 1.8 Hz), 2.35–2.24 (1 H, m), 2.19 (1 H, t, J = 11.5 Hz), 2.00–1.64 (5 H, m),
1.55–0.73 (34 H, m), 0.61 (3 H, s); 13C NMR (101 MHz, DMSO-d6) δ 140.7 (C5), 121.1 (C6),
13

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97.0 (C1'), 76.5 (C3), 73.2 (C3'), 72.9 (C5'), 71.9 (C2'), 70.4 (C4'), 61.0 (C6'), 56.2, 55.6, 49.6,
DOI: 10.1039/D0OB01776K
41.9, 40.4, 40.1, 39.0, 38.9, 36.6, 36.2, 35.7, 35.2, 31.45, 31.38, 27.8, 27.4, 23.9, 23.2, 22.7,
22.4, 20.6, 19.1, 18.6, 11.7; HRMS: [M + NH4]+ calcd for C33H60NO6 m/z 566.4415; found
566.4442.
Cholesteryl 2,3,4-tri-O-trimethylsilyl-α-D-glucopyranoside (16)
Cholesteryl -glucoside 14 (195 mg, 0.355 mmol) was suspended in CH2Cl2 (5 ml) followed
by the addition of TMSCl (266 l, 1.78 mmol) and Et3N (260 l, 1.85 mmol). The mixture was
stirred overnight and was then diluted sequentially with pentane and sat. NaHCO3. The organic
phase was separated and washed with satd. aq. NaHCO3, water and brine, then dried (Na2SO4)
and concentrated under reduced pressure. The residue was dissolved in CH2Cl2/MeOH (1:1, 4
ml) and NH4OAc (61 mg, 0.79 mmol) was added. The solution was stirred for 48 h then was
concentrated and purified by flash chromatography (silica gel was pre-treated with 4%
Et3N/hexanes; eluted with EtOAc/hexanes) to afford the alcohol 16 (185 mg, 68%) as a white
glass. 1H NMR (600 MHz, CDCl3) δ 5.34–5.31 (1 H, m, H6), 4.82 (1 H, d, J = 3.6 Hz, H1'),
3.76 (2 H, m, H3',6a'), 3.73–3.66 (2 H, m, H5',6b'), 3.47–3.43 (1 H, m, H4'), 3.40 (1H, dd, J =
9.2, 3.6 Hz, H2'), 3.40–3.34 (1 H, m, H3), 2.41–2.33 (1 H, m), 2.32–2.27 (1 H, m), 2.03–1.93
(2 H, m), 1.88–1.79 (3 H, m), 1.61–0.81 (34 H, m), 0.67 (3 H, s), 0.20–0.16 (9 H, m, TMS),
13
0.16–0.14 (9 H, m, TMS), 0.14–0.10 (9 H, m, TMS); C NMR (151 MHz, CDCl3) δ 141.1,
121.8, 97.7, 77.9, 74.6, 73.7, 72.2, 72.0, 62.0, 56.9, 56.3, 50.3, 42.5, 40.3, 40.0, 39.7, 37.3,
36.9, 36.4, 35.9, 32.1, 32.0, 28.4, 28.2, 28.0, 24.4, 24.0, 23.0, 22.7, 21.2, 19.6, 18.9, 12.0, 1.5,
1.1, 0.7.
Cholesteryl 6-O-dodecanoyl-α-D-glucopyranoside (CAG-C12:0)
DMAP (5.2 mg, 0.047 mmol) was added to a solution of alcohol 16 (36 mg, 0.047 mmol) in
pyridine/CH2Cl2 (3:100, 1.2 ml) at 0 °C. Lauryl chloride (22 ul, 0.094 mmol) was added and
the solution was stirred at rt for 4 h. MeOH (50 l) was added and the solution was stirred for
30 min. The solution was concentrated under reduced pressure to afford a colourless residue.
The residue was dissolved in MeOH/CHCl3 (1:1, 2 ml) and Dowex 50WX8-200 (H+ form,
approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate
concentrated under reduced pressure. The residue was purified by flash chromatography
20
(EtOAc/hexanes/MeOH) to afford CAG-C12:0 (25 mg, 73% yield) as a white solid. [α]D
+32.2 (c 0.95, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.37–5.33 (1 H, m, H6), 5.01 (1 H, d, J
14

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= 4.0 Hz, H1'), 4.50–4.43 (1 H, m, H6a'), 4.26 (1 H, d, J = 12.2 Hz, H6b'), 3.86 (1 H, ddd, J =
DOI: 10.1039/D0OB01776K
9.9, 4.8, 2.0 Hz, H5'), 3.72 (1 H, t, J = 9.2 Hz, H3'), 3.53–3.44 (2 H, m, H2',3), 3.33 (1H, t, J =
9.5 Hz, H4'), 3.31 (2 H, br s, 2  OH), 2.41–2.20 (5 H, m), 2.06–1.78 (5 H, m), 1.65–0.81 (54
H, m), 0.67 (3 H, s); 13C NMR (126 MHz, CDCl3) δ 174.8 (C=O), 140.4 (C5), 122.4 (C6), 97.0
(C1'), 78.6 (C3), 74.8 (C3'), 72.2 (C2'), 70.20 (C4'), 70.16 (C5'), 63.3 (C6'), 56.9, 56.3, 50.2,
42.5, 40.3, 39.9, 39.7, 37.1, 36.8, 36.3, 35.9, 34.4, 32.1, 32.0, 29.8, 29.7, 29.51, 29.46, 29.4,
28.4, 28.2, 25.1, 24.4, 24.0, 23.0, 22.8, 22.7, 21.2, 19.5, 18.9, 14.3, 12.0; HRMS: [M + NH4]+
calcd for C45H82NO7 m/z 748.6086; found 748.6077.
Cholesteryl 6-O-tetradecanoyl-α-D-glucopyranoside (CAG-C14:0)
Myristoyl chloride (24 l, 0.088 mmol) was added to a solution of DMAP (5.0 mg, 0.044
mmol) and alcohol 16 (34 mg, 0.044 mmol) in pyridine/CH2Cl2 (3:100, 1.2 ml) at 0 °C. The
solution was allowed to stir at rt for 4 h, then MeOH (50 l) was added and the mixture was
allowed to stir for 30 min. The solvent was evaporated under reduced pressure to afford a white
residue. The residue was redissolved in MeOH/CHCl3 (1:1, 2 ml) and Dowex 50WX8-200 (H+
form, approx. 100 mg) was added. After stirring for 1 h the mixture was filtered and the filtrate
concentrated under reduced pressure. The residue was purified by flash chromatography
20
(EtOAc/hexanes/MeOH) to afford CAG-C14:0 (26 mg, 79% yield) as a white solid. [α]D
+31.5 (c 1, CHCl3) (lit.21 [α]D24 +21.9 (c 0.70, CHCl3)); 1H NMR (500 MHz, CDCl3) δ 5.37–
5.33 (1 H, m, H6), 5.01 (1 H, d, J = 4.0 Hz, H1'), 4.48 (1 H, dd, J = 12.2, 4.9 Hz, H6a'), 4.25
(1 H, dd, J = 12.2, 2.0 Hz, H6b'), 3.86 (1 H, ddd, J = 9.9, 4.8, 2.1 Hz, H5'), 3.72 (1 H, t, J = 9.2
Hz, H3'), 3.54–3.44 (2 H, m, H2',3), 3.36–3.29 (1 H, m, H4'), 3.15 (1 H, br s, OH), 3.04 (1 H,
br s, OH), 2.42–2.28 (4 H, m), 2.20 (1 H, br s, OH), 2.04–1.77 (5 H, m), 1.70–0.81 (58 H, m),
0.67 (3 H, s); 13C NMR (126 MHz, CDCl3) δ 174.6 (C=O), 140.2 (C5), 122.2 (C6), 96.8 (C1'),
78.4 (C3), 74.6 (C3'), 72.1 (C2'), 70.0 (C4',5'), 63.1 (C6'), 56.7, 56.2, 50.1, 42.3, 40.1, 39.7,
39.5, 37.0, 36.6, 36.2, 35.8, 34.2, 31.9, 31.8, 29.70, 29.66, 29.6, 29.6, 29.4, 29.3, 29.2, 28.2,
28.01, 27.99, 24.9, 24.3, 23.8, 22.8, 22.7, 22.5, 21.0, 19.3, 18.7, 14.1, 11.8; HRMS: [M + NH4]+
calcd for C45H78O7 m/z 776.6399; found 776.6370.
Cholesteryl 6-O-hexadecanoyl-α-D-glucopyranoside (CAG-C16:0)
Palmityl chloride (27 l, 0.088 mmol) was added to a solution of DMAP (5.0 mg, 0.044 mmol)
and alcohol 16 (34 mg, 0.044 mmol) in pyridine/CH2Cl2 (3:100, 1.2 ml) at 0 °C. The solution
was stirred at rt for 4 h, then MeOH (50 l) was added and the solution was stirred for 30 min.
15

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The solvent was evaporated under reduced pressure to afford a white residue. TheDrOeIs: i1d0.u10e39w/Da0sOB01776K
redissolved in MeOH/CHCl3 (1:1, 2 ml) and Dowex 50WX8-200 (H+ form, approx. 100mg)
was added. After stirring for 1 h the mixture was filtered and the filtrate concentrated under
reduced pressure. The residue was purified by flash chromatography (EtOAc/hexanes/MeOH)
20
1
to afford CAG-C16:0 (23 mg, 67% yield) as a white solid. [α]D +28.9 (c 1, CHCl3); H
NMR (500 MHz, CDCl3) δ 5.37–5.32 (1 H, m, H6), 5.01 (1 H, d, J = 4.0 Hz, H1'), 4.46 (1 H,
dd, J = 12.1, 3.3 Hz, H6a'), 4.26 (1 H, d, J = 12.0 Hz, H6b'), 3.86 (1 H, ddd, J = 9.9, 4.9, 2.0
Hz, H5'), 3.72 (1 H, t, J = 9.3 Hz, H3'), 3.54–3.43 (2 H, m, H2',3), 3.33 (1 H, td, J = 9.7, 3.1
Hz, H4'), 3.27 (2 H, br s, 2  OH), 2.41–2.23 (5 H, m), 2.05–1.77 (5 H, m), 1.66–0.79 (62 H,
m), 0.67 (3 H, s); 13C NMR (126 MHz, CDCl3) δ 174.7 (C=O), 140.4 (C5), 122.4 (C6), 97.0
(C1'), 78.6 (C3), 74.8 (C3'), 72.2 (C2'), 70.21 (C5'), 70.15 (C4'), 63.3 (C6), 56.9, 56.3, 50.3,
42.5, 40.3, 39.9, 39.7, 37.1, 36.8, 36.3, 35.9, 34.4, 32.1, 32.0, 29.9, 29.8, 29.7, 29.53, 29.46,
29.4, 28.4, 28.2, 25.1, 24.4, 24.0, 23.0, 22.8, 22.7, 21.2, 19.5, 18.9, 14.3, 12.0; HRMS: [M +
NH4]+ calcd for C49H90NO7 m/z 804.6712; found 804.6725.
Cholesteryl 6-O-octadecanoyl-α-D-glucopyranoside (CAG-C18:0)
Stearyl chloride (28 l, 0.082 mmol) was added to a solution of DMAP (4.5 mg, 0.041 mmol)
and alcohol 16 (31 mg, 0.041 mmol) in pyridine/CH2Cl2 (3:100, 1.2 ml) at 0 °C. The solution
was stirred at rt for 4 h, then MeOH (50 l) was added and the solution was stirred for 30 min.
The solvent was evaporated under reduced pressure to afford a white residue. The residue was
dissolved in MeOH/CHCl3 (1:1, 2 ml) and Dowex 50WX8-200 (H+ form, approx. 100 mg) was
added. After stirring for 1 h the mixture was filtered and the filtrate concentrated under reduced
pressure. The residue was purified by flash chromatography (EtOAc/hexanes/MeOH) to afford
CAG-C18:0 (24 mg, 72% yield) as a white solid. [α]D20 +29.5 (c 1, CHCl3); 1H NMR (500
MHz, CDCl3) δ 5.35 (1 H, m, H6), 5.01 (1 H, d, J = 3.8 Hz, H1'), 4.46 (1 H, dd, J = 11.4, 5.1
Hz, H6a'), 4.26 (1 H, d, J = 12.2 Hz, H6b'), 3.86 (1 H, ddd, J = 9.9, 4.9, 2.0 Hz, H5'), 3.72 (1
H, t, J = 9.2 Hz, H3'), 3.53–3.43 (2 H, m, H2',3), 3.33 (1H, t, J = 9.5 Hz, H4'), 3.34–3.02 (2 H,
br s, 2  OH), 2.42–2.16 (5 H, m), 2.06–1.77 (5 H, m), 1.75–0.80 (66 H, m), 0.67 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ 174.7 (C=O), 140.4 (C5), 122.4 (C6), 97.0 (C1'), 78.6 (C3), 74.8
(C3'), 72.2 (C2'), 70.2 (C4',5'), 63.3 (C6'), 56.9, 56.3, 50.3, 42.5, 40.3, 39.9, 39.7, 37.1, 36.8,
36.3, 35.9, 34.4, 32.1, 32.0, 29.9, 29.83, 29.75, 29.52, 29.46, 29.4, 28.4, 28.2, 25.1, 24.4, 24.0,
23.0, 22.8, 22.7, 21.2, 19.5, 18.9, 14.3, 12.0; HRMS: [M + NH4]+ calcd for C51H94NO7 m/z
832.7025; found 832.6982.
16

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DOI: 10.1039/D0OB01776K
Cholesteryl 6-O-oleyl-α-D-glucopyranoside (CAG-C18:1)
Oleyl chloride (28 l, 0.086 mmol) was added to a solution of DMAP (5.0 mg, 0.043 mmol)
and alcohol 16 (33 mg, 0.043 mmol) in pyridine/CH2Cl2 (3:100, 1.2 ml) at 0 °C. The solution
was stirred at rt for 4 h, then MeOH (50 l) was added and the solution stirred for 30 min. The
solvent was evaporated under reduced pressure to afford a white residue. The residue was
dissolved in MeOH/CHCl3 (1:1, 2 ml) and Dowex 50WX8-200 (H+ form, approx. 100 mg) was
added. After stirring for 1 h the mixture was filtered and the filtrate concentrated under reduced
pressure. The residue was purified by flash chromatography (EtOAc/hexanes/MeOH) to afford
CAG-C18:1 (22 mg, 63% yield) as a white solid. [α]D20 +31.1 (c 1, CHCl3); 1H NMR (500
MHz, CDCl3) δ 5.40–5.29 (3 H, m, H6,9'',10''), 5.01 (1 H, d, J = 4.0 Hz, H1'), 4.47 (1 H, dd, J
= 11.8, 3.9 Hz, H6a'), 4.26 (1 H, d, J = 12.1 Hz, H6b'), 3.86 (1 H, ddd, J = 9.9, 4.8, 2.0 Hz,
H5'), 3.72 (1 H, t, J = 9.2 Hz, H3'), 3.53–3.43 (2 H, m, H2',3), 3.33 (1 H, t, J = 9.5 Hz, H4'),
3.29 (2 H, br s, 2  OH), 2.42–2.21 (5 H, m), 2.09–1.77 (9 H, m), 1.66–0.81 (60 H, m), 0.67 (3
H, s); 13C NMR (126 MHz, CDCl3) δ 174.7 (C=O), 140.4 (C5), 130.2, 129.8 (C9''',10'''), 122.4
(C6), 97.0 (C1'), 78.6 (C3), 74.8 (C3'), 72.2 (C2'), 70.2 (C4',5'), 63.3 (C6'), 56.9, 56.3, 50.3,
42.5, 40.3, 39.9, 39.7, 37.1, 36.8, 36.3, 35.9, 34.4, 32.1, 32.1, 32.0, 29.9, 29.9, 29.7, 29.49,
29.47, 29.4, 28.4, 28.2, 28.2, 27.38, 27.35, 25.1, 24.4, 24.0, 23.0, 22.8, 22.7, 21.2, 19.5, 18.9,
14.3, 12.0; HRMS: [M + NH4]+ calcd for C51H92NO7 m/z 830.6868; found 830.6900.
Mincle reporter cell signalling assays
2B4-NFAT-GFP reporter cells expressing mouse or human Mincle together with FcRγ, as well
as an FcRγ only control, were prepared as described previously.44, 49 Each glycolipid was
dissolved in chloroform methanol (2:1) at 2 mg mL−1, then diluted in isopropanol, was added
to 96-well plates at 20 μL per well, followed by the evaporation of the solvent. Reporter cells
were stimulated for 16−20 h, and activation of NFAT-GFP was monitored by flow cytometry.
Acknowledgements
We thank the Australian Research Council for funding (DP160100597, DP180101957) and
AMED (JP19gm0910010, JP19ak0101070 and JP19fk0108075).
17

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Table of contents
Stereoselective -glucosylation of cholesterol enabled synthesis of cholesteryl 6-O-acyl-
glucosides from diverse Helicobacter spp. and demonstration that they signal through the
innate immune receptor Mincle.