Synthesis of α-Glucosyl Diacylglycerides as potential adjuvants for Streptococcus pneumoniae vaccines

Article abstract of DOI:10.1016/j.carres.2020.107951

α-Glucosyl diacylglycerols (αGlc-DAGs) play an important role in providing protective immunity against Streptococcus pneumoniae infection through the engagement of the Macrophage inducible C-type lectin (Mincle). Herein, we efficiently synthesised αGlc-DAGs containing C12, C14, C16 and C18 acyl chains in 7 steps and 44–47% overall yields, and demonstrated that Mincle signaling was dependent on lipid length using mMincle and hMincle NFAT-GFP reporter cells. The greatest production of GFP in both cell types was elicited by C14 αGlc-DAG. Accordingly, C14 αGlc-DAG has potential to act as an adjuvant to augment the immune response against S. pneumoniae antigens.

Full text of DOI:10.1016/j.carres.2020.107951

Journal Pre-proof
Synthesis of α-Glucosyl Diacylglycerides as potential adjuvants for Streptococcus
pneumoniae vaccines
Ayesha Khan, Femke Hollwedel, Ulrich A. Maus, Bridget L. Stocker, Mattie S.M.
Timmer
PII:
S0008-6215(19)30745-1
DOI:
https://doi.org/10.1016/j.carres.2020.107951
CAR 107951
Reference:
To appear in: Carbohydrate Research
Received Date: 29 December 2019
Revised Date: 8 February 2020
Accepted Date: 10 February 2020
Please cite this article as: A. Khan, F. Hollwedel, U.A. Maus, B.L. Stocker, M.S.M. Timmer, Synthesis
of α-Glucosyl Diacylglycerides as potential adjuvants for Streptococcus pneumoniae vaccines,
Carbohydrate Research (2020), doi: https://doi.org/10.1016/j.carres.2020.107951.
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Synthesis of α-Glucosyl Diacylglycerides as Potential Adjuvants for Streptococcus
pneumoniae Vaccines
Ayesha Khan,a,b Femke Hollwedel,c,d Ulrich A. Maus,c,d Bridget L. Stocker,a,b* Mattie S. M.
Timmera,b*
Author Affiliations
* Corresponding Author
a School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600,
Wellington, New Zealand, E-mail: bridget.stocker@vuw.ac.nz, mattie.timmer@vuw.ac.nz
b Centre for Biodiscovery, Victoria University of Wellington, P. O. Box 600, Wellington 6140,
New Zealand.
c Department of Experimental Pneumology, Hannover Medical School, Hannover, Germany
d German Center for Lung Research, partner site BREATH, Hannover, Germany
Graphical Abstract
1

Abstract
α-Glucosyl diacylglycerols (αGlc-DAGs) play an important role in providing protective
immunity against Streptococcus pneumoniae infection through the engagement of the
Macrophage inducible C-type lectin (Mincle). Herein, we efficiently synthesised αGlc-DAGs
containing C12, C14, C16 and C18 acyl chains in 7 steps and 44-47% overall yields, and
demonstrated that Mincle signaling was dependent on lipid length using mMincle and
hMincle NFAT-GFP reporter cells. The greatest production of GFP in both cell types was
elicited by C14 αGlc-DAG. Accordingly, C14 αGlc-DAG has potential to act as an adjuvant
to augment the immune response against S. pneumoniae antigens.
Introduction
Pneumococcal disease is an infection caused by the gram-positive bacteria Streptococcus
pneumoniae. People can harbour S. pneumoniae in their nose and throat without becoming ill
(termed carriage), however infections with the bacteria can lead to sinusitis, otitis media
and/or bronchitis, while lower airway infections can lead to pneumococcal pneumonia, which
can progress to invasive pneumococcal disease (IPD), septicemia and meningitis.1,2
Pneumococcal infection is more frequently observed in the young and elderly, with
approximately 500,000 infants dying from the disease annually, and with adults greater than
65 years of age being more likely to suffer from significant morbidity and mortality resulting
from community-acquired pneumonia or pneumococcal bacteremia and meningitis.1,2,3,4
Out of the >90 serotypes of S. pneumoniae known so far, approximately 23 are responsible
for more than 90% of pneumococcal diseases. The most frequently employed vaccine against
S. pneumoniae is Pneumovax®, which is composed of capsular polysaccharides purified
from the 23 clinically prevalent pneumococcal serotypes, while Prevnar®, a conjugate
vaccine containing purified polysaccharides of the clinically most prevalent pneumococcal
serotypes coupled to carrier proteins, provides young children (<2 years) with more effective
protection against S. pneumoniae.5 Vaccination has certainly decreased the incidence of S.
pneumoniae infection, although the efficacy of commercially available vaccines and their
ability to generate herd immunity varies according to geographic location and age group. This
has been attributed to changes in the prevalence and type of capsular polysaccharide antigens
found on different S. pneumoniae strains, and also to the inability of S. pneumoniae-derived
2

polysaccharides, which behave as T-cell-independent antigens, to induce recall responses
following revaccination or infection.5,6
Global efforts toward the development of more effective pneumococcal vaccines include
increasing the repertoire of S. pneumoniae-derived capsular polysaccharides used, or
incorporating pneumococcal proteins as antigens.1,7,8 The use of adjuvants to enhance the
efficacy of the immune response to either protein- or carbohydrate-based S. pneumoniae
antigens has also been deemed a promising strategy,9 particularly as the engagement of
pattern recognition receptors (PRRs) on antigen presenting cells (APCs) may augment
antibody boosting and memory responses to polysaccharide pneumococcal antigens. While
Toll-like receptor (TLR) agonists do not increase antibody responses when co-administered
with polysaccharide antigen (though do if given several days after the antigen),10,11
Macrophage inducible C-Type Lectin (Mincle) agonists,12 such as trehalose dimycolate
(TDM) and trehalose dibehenate (TDB), show promise in this respect.9,13
In 2016, we determined that α-glucosyl diacylglycerols (αGlc-DAGs, 1, Figure 1), a class of
glycolipid that we structurally elucidated in 2012,14 played a key role in providing protective
immunity against S. pneumoniae infection through engagement of Mincle.15 Here, αGlc-DAG
containing dipalmitoyl acyl chains was the major Mincle agonist isolated from S.
pneumoniae. Subsequent studies by Yamasaki and co-workers also confirmed the role of
αGlc-DAGs in assisting with the suppression of Streptococcus infection.16 Given the
potential of Mincle ligands to augment host immunity to S. pneumoniae, and the knowledge
that lipid length affects the immunostimulatory activity of other classes of Mincle
agonist,17,18,19,20,21 we thus sought to synthesise αGlc-DAGs containing C12 (1a), C14 (1b),
C16 (1c) and C18 (1d) acyl chains so as to investigate their ability to signal through murine
and human Mincle. The results from these findings would then inform further studies into the
use of αGlc-DAGs as adjuvants for S. pneumoniae vaccines.
3

OH
O
O
HO
HO
O
n
HO
O
O
n
O
1
a, n = 2 (C12:0)
b, n = 4 (C14:0)
c, n = 6 (C16:0)
d, n = 8 (C18:0)
Figure 1. α-Glucosyl diacylglycerols (αGlc-DAGs)
Results and Discussion:
To synthesise αGlc-DAGs 1a-d a retrosynthetic plan was proposed whereby the target
glycolipids could be obtained via esterification of benzyl protected 1-O-α-glucosyl-sn-
glycerol 2 with carboxylic acids 3a-d and subsequent hydrogenolysis of the benzyl groups
(Scheme 1). Benzyl-protected glucosylglycerol 2 would in turn be prepared via the
glucosylation of TMS-protected glucopyranose 4,22,23 which is converted in situ to the
corresponding glucosyl iodide,22,23 with commercially available isopropylidene-protected
glycerol 5, followed by protecting group manipulations. Thus, while a change from TMS to
benzyl protecting groups is proposed in this retrosynthetic plan to allow for the selective
removal of the isopropylidine group under acidic conditions, the glycosyl iodide
glycosylation methodology, as developed by Gervay-Hague,24 provides excellent α-
selectivity when using glucose donors, and thus should provide an efficient synthetic route to
the target compounds.
4

OH
O
O
HO
HO
O
n
HO
O
O
n
O
1
a, n = 2 (C12:0)
b, n = 4 (C14:0)
c, n = 6 (C16:0)
d, n = 8 (C18:0)
OBn
O
O
BnO
BnO
+
OH
HO
BnO
n
O
OH
3a-d
2
OTMS
O
O
TMSO
TMSO
+
HO
O
TMSO
OTMS
5
4
Scheme 1. Retrosynthetic plan
With a retrosynthetic strategy in place, the synthesis of TMS-protected glucose was
undertaken. Here, per-silylation of -glucose 6 was achieved under the agency of TMSCl and
triethyl amine to produce 1,2,3,4,6-penta-O-trimethylsilyl-α- -glucopyranose 4 in 98% yield
D
D
(Scheme 2).23,24 Next, glucopyranose 4 was converted into the glucosyl iodide in situ, with
the subsequent addition of isopropylidene-protected glycerol 5, DiPEA and TBAI then
providing the TMS-protected glucoside. In situ deprotection then yielded 1-O-α-glucosyl-sn-
glycerol 7 in excellent yield (83%) and as only the α-anomer, as determined by 1HNMR
analysis whereby the anomeric center was observed at δ = 4.8 ppm with J1-2 = 3.7 Hz. Once
determined, the established procedure proved to be very robust with the glucosylation
reaction being undertaken on a gram scale without affecting α-selectivity or yield.
5

OH
O
1) T
TB
MS
AI,
I, th
0 C
en
- r.t.
5,
DiPE
, 48
A
h
OTMS
O
OH
O
o
HO
HO
TMSCl, Et3N
TMSO
TMSO
HO
HO
O
HO
0 °C - r.t., 4h
98%
2) Dowex/H+, MeOH
1 h, 83%
O
O
TMSO
HO
OTMS
OH
7
6
4
1) BnBr, NaH, DMF
6 h, 74%
2) AcOH/H2O (4:1)
80 °C, 1 h, quant.
OBn
H2, Pd/C
EDCI, DMAP
OBn
O
O
O
BnO
BnO
BnO
BnO
1
O
OH
EtOH, 18 h
3a-d, Toluene
n
BnO
BnO
O
O
70 oC, 12 h
O
OH
n
8
O
2
a, n = 2 (82%)
b, n = 4 (85%)
c, n = 6 (85%)
d, n = 8 (84%)
a, n = 2 (90%)
b, n = 4 (91%)
c, n = 6 (89%)
d, n = 8 (90%)
Scheme 2. Synthesis of αGlc-DAGs (1a-d)
In optimising the glucosylation conditions it was observed that reaction time and temperature
influenced α:β-selectivity and product yields. As reported by Gervay-Hague,22,23,24 in situ
formation of per-O-TMS glucosyl iodide from glucopyranose 4 was normally undertaken at
room temperature in CH2Cl2 with a reaction time of 10 minutes before the addition of the
acceptor of choice, stirring for an additional 48 h, and in situ removal of the TMS groups. In
our hands, when using glycerol 5 as an acceptor, these conditions resulted in a modest (37%)
yield of 1-O-α-glucosyl-sn-glycerol 7 and good α:β-selectivity (10:1). Cooling the reaction to
0 °C and a 20 min reaction time for formation of per-O-TMS glucosyl iodide followed by
warming to room temperature for the glucosylation reaction led to improved α:β-selectivity
(70:1), however, the reaction yield remained modest (48%). A longer (30 min) and warmer (0
°C – r.t.) protocol for formation of the per-O-TMS glucosyl iodide prior to the addition of
glycerol 5 at 0 °C with warming to room temperature further improved product yield (76%)
and α:β-selectivity (96:1), with the optimal product yield (83%) and exclusive α-selectivity
(>100:1, α:β) being obtained after formation of per-O-TMS glucosyl iodide at 0 °C with
warming to room temperature over the course of 30 min, followed by the addition of glycerol
5 at room temperature with stirring for 48 h. In all circumstances, freshly prepared TMS-I,25 a
highly reactive and hygroscopic reagent, led to better product yields.
6

With 1-O-α-glucosyl-sn-glycerol 7 in hand, this was then subjected to benzyl protection at
the 2-, 3-, 4- and 6-positions via reaction with benzyl bromide in the presence of sodium
hydride, followed by isopropylidene deprotection under the agency of acetic acid and water
(4:1) to give diol 2 in 74% yield over the two steps. Esterification of 2 with the corresponding
commercially available carboxylic acids 3a-d was then performed in the presence of EDCI
and DMAP to give benzyl protected glycolipids 8a-d in 82-85% yield. The HMBC between
the carbonyl carbons (C11 and C11') and the protons of glycerol (H-8 and H-9a+b) confirmed
the installation of two lipids at the desired positions. Finally, the benzyl protecting groups on
8a-d were removed under the agency of Pearlman’s catalyst and H2 to give the target αGlc-
DAGs 1a-d in excellent yields (89-91%). Thus, in sum, the total syntheses of αGlc-DAGs 1a-
g were achieved in 7 steps and 44-47% overall yield. Of compounds 2a-d, only αGlc-DAG
2d has previously been synthesized in 5 steps and 36% yield from 1-O-p-nitrobenzoyl-
2,3,4,6-tetra-O-benzyl-α-D
-glucopyranose, with the later prepared in 4 steps from glucose.26
With the glycolipids in hand, the ability of these analogues to signal through murine Mincle
(mMincle) was assessed using αGlcDAG-coated plates and NFAT-GFP reporter cells.27,28
Here, Mincle signalling would be monitored by the expression of GFP, as detected by flow
cytometry, with TDB as the positive control and FcRγ only as the negative control.
Previously, mMincle agonist studies have revealed that increasing lipid length typically leads
to better Mincle signalling.12,21 In our studies, all compounds signaled through mMincle
(Figure 2), however, the optimum lipid length for mMincle signalling by αGlc-DAGs
centered around glycolipids containing C14 acyl chains. This enhanced Mincle signaling was
observed by αGlc-DAG 1b at both concentrations of glycolipid tested (1 and 4 nmol/well),
with a greater level of GFP being produced at the higher concentration of αGlc-DAG 1b.
This concentration dependence was less pronounced for the other glycolipids, and indeed for
αGlc-DAG 1d, a higher concentration of glycolipid resulted in decreased GFP production,
which may indicate that the level of coating has reached saturation.29
7

Figure 2. NFAT-GFP 2B4 reporter cells expressing mMincle + FcRγ, or FcRγ-only were
stimulated using αGlcDAG-coated plates (1 nmol/well or 4 nmol/well) for 24 h. The cells
were then harvested and examined for NFAT-GFP expression. Data reported is representative
of three independent experiments performed in duplicate (mean ± SEM).
While there is a high degree of homology between murine and human Mincle,30 species-
specific activity has been observed for select classes of Mincle ligands.12 Accordingly, we
also sought to explore the ability of αGlc-DAGs 1a-d to signal through hMincle using
glycolipid concentrations of 0.1 nmol/well and 1 nmol/well (Figure 3). In agreement with our
murine data, all αGlc-DAGs signaled through hMincle, with C14 αGlc-DAG (1b) eliciting
the most potent response. In general, an increase in lipid length has been associated with
increased hMincle signalling,12 although in a recent study by Evans and co-workers which
investigated the effect of the lipid length of α-branched trehalose diesters on mMincle and
hMincle agonist activity, those compounds containing chain lengths of 5-14 carbons led to
greater cytokine production by HEK cells transfected with hMincle than analogues
containing lipid chains of longer or shorter length.21 However, Evans and co-workers
observed a species-specific response, with longer lipids leading to enhanced mMincle agonist
activity,21 while we observed similar relative signalling capacities by both mMincle and
hMincle reporter cells following stimulation with the αGlc-DAGs.
8

Figure 3. NFAT-GFP 2B4 reporter cells expressing hMincle + FcRγ, or FcRγ-only were
stimulated using αGlcDAG-coated plates (0.1 nmol/well or 1 nmol/well) for 18 h. The cells
were then harvested and examined for NFAT-GFP expression. Data reported is a
representative of two independent experiments performed in duplicate (mean ± SEM).
Thus, in summary, we have demonstrated that C14 αGlc-DAG 1b contains the optimum
glycolipid length for murine and human Mincle signaling and is thus our lead αGlc-DAG
agonist. Moreover, the synthetic route is efficient and the glucosylation reaction can be
performed on the gram scale without affecting the yield or α:β-selectivity. Further studies
into the potential of αGlc-DAG 1b to augment the immune response against capsular
polysaccharide antigens from S. pneumoniae will be investigated in due course.
Conclusions
In conclusion we developed an efficient 7-step route for the synthesis of αGlc-DAGs and
determined that αGlc-DAGs 1a-d signal through both murine and human Mincle. Mincle
signaling was dependent on lipid length, with the greatest production of GFP by the NFAT-
GFP reporter cells being elicited by αGlc-DAG 2b, which contains C14 acyl chains, rather
than those derivatives containing longer or shorter acyl chains. Given the essential role of
9

αGlc-DAGs in providing Mincle-mediated protective immunity against S. pneumoniae
infection, αGlc-DAG 2b thus shows promise as an adjuvant to enhance immunity against S.
pneumoniae antigens.
Experimental
Chemistry General. All reactions were performed under an argon atmosphere unless stated
otherwise. Prior to use the following solvents were distilled: toluene (ROMIL), acetone
(Fisher Scientific), ethyl acetate (Fisher Scientific) and petroleum ether (Merck), DCM
(Fisher Chemicals), methanol (Fisher Scientific). Lauric acid (Fisher Scientific), palmitic acid
(Fulka), myristic acid (BDH), stearic acid (Fisher Scientific),
D-glucose (Applichem),
diisopropylethylamine (Sigma Aldich), trimethylsilyl chloride (Sigma Aldrich),
hexamethyldisilane (BDH), iodine (Univar), MgSO4 (Pure Science), NaCl (Chem Solute),
Et2O (LabServ), DMAP (Lab Supply), EDCI (Chem Impex), acetic acid (Scharlau), Et3N
(Sigma), NaHCO3 (Pure Science), KMnO4 (AnalR), CDCl3 (Aldrich), CD3OD (Apollo
Scientific), C5D5N (Apollo Scientific), and Dowex 50WX8-200, were used as received.
Reactions were monitored by TLC analysis by dipping in 10% H2SO4 in EtOH followed by
charring or dipping in a solution of KMnO4 (0.05 M), K2CO3 (0.4 M) and NaOH (0.06%) in
water. Column chromatography was performed using Pure Science silica gel (40-63 µm). All
solvents were removed by evaporation under reduced pressure. High resolution mass spectra
were recorded on an Agilent 6530 Q-TOF mass spectrometer utilising a JetStreamTM electro-
spray ionisation (ESI) source in positive or negative mode. Optical rotations were recorded
on an Autopol II (Rudolph Research Analytical) at 589 nm (sodium D-line). Infrared (IR)
spectra were recorded as thin films using a Bruker Platinum-ATR spectrometer and are
reported in wave numbers (cm-1). Nuclear magnetic resonance spectra were obtained at 20 °C
in CDCl3, CD3OD or C5D5N using a Varian INOVA operating at 500 MHz. Chemical shifts
are given in ppm (δ) relative to the solvent residual peak. NMR peak assignments were made
using COSY, HSQC, and HMBC 2D experiments.
Compound data
1,2,3,4,6-Penta-O-trimethylsilyl-D-glucose (4). D-Glucose (2.0 g, 0.011 mol) was co-
evaporated with DMF (3 × 25 mL) and then suspended in dry DMF (50 mL). Et3N (8.6 mL,
10

0.061 mol) was added and the resulting solution was cooled to 0 °C before TMSCl (1.10 g,
3.96 mmol) was added. The mixture was stirred for 4 hours at r.t., after which point hexanes
(100 mL) and crushed ice (ca. 50 mL) were added. The aqueous layer was extracted with
hexane (3 x 25 mL) and the combined organic layers were washed with water (2 x 50 mL)
and brine (3 x 50 mL), dried over MgSO4, filtered, and concentrated in vacuo to give the
titled compound 4 as a colorless oil. The data obtained matched that reported in literature.31
3-O-(α-D-Glucopyranosyl)-1,2-O-isopropylidene-sn-glycerol (7). TBAI (550 mg, 1.52
mmol), (S)-1,2-O-isopropylideneglycerol (132 mg, 1.0 mmol) and DIPEA (0.40 mL, 2.02
mmol) were added to an oven-dried flask containing activated 4 Å molecular sieves in dry
CH2Cl2 (7 mL) and the solution was stirred at r.t. for 30 min. TMS-protected D-glucose 4
(550 mg, 1.01 mmol) was azeotroped with anhydrous toluene (2 × 5 mL) and then dissolved
in dry CH2Cl2 (2 mL) and cooled to 0 °C. Freshly prepared TMSI (0.14 mL, 1.1 mmol),
which was generated by refluxing iodine (2.2 g, 8.7 mmol) over dry distilled
hexamethyldisilane (2.0 mL, 9.8 mmol) for 30 minutes followed by distillation, was
transferred by syringe into the flask containing TMS-protected glucose. The ice bath was then
removed and the solution was stirred at r.t. for 30 min, after which time the in situ generated
TMS-glucosyl iodide was cannulated into the acceptor flask and the resulting solution stirred
for 48 hours at r.t. The reaction mixture was then filtered to remove the molecular sieves, and
the flask was rinsed with hexane:EtOAc (1:1, 5 mL). The resulting solution was cooled to 0
°C and TBAI removed by filtration. The solvent was concentrated in vacuo and the residue
was dissolved in MeOH (5 mL). Dowex/H+ (300 mgs) and the resulting suspension stirred at
r.t. until no TMS protected sugar was observed by TLC (9:1, PE:EtOAc, v/v). The suspension
was then filtered to remove the resin, neutralised by the addition of DIPEA (pH = 7) and then
concentrated in vacuo to give a yellow oil. The product was purified using silica gel flash
column chromatography (EtOAc to EtOAc/MeOH, 9:1, v/v) to give the title compound 7 as a
colorless oil (247 mg, 0.84 mmol, 83% yield over two steps). Rf = 0.30 (EtOAc/MeOH, 4:1,
v/v); [ꢀ]ꢂꢁꢃ.ꢄ = +58 (c = 1, CH2Cl2); IR (film) = 3385, 2495, 1647, 1450, 1377, 1213, 1154,
1
1115, 1029, 971, 834, 463 cm-1; H NMR (500 MHz, CD3OD) δ 4.83 (d, J1,2 = 3.7 Hz, 1H,
H-1), 4.60 (s, 2H, OH), 4.36 (p, J8,7 = J8,9 = 5.91, 1H, H-8), 4.10 (dd, J9a,9b = 8.31, J9a,8 = 6.53
Hz, 1H, H-9a), 3.81 (dd, J6a,6b = 11.9 Hz, J6a,5 = 2.4 Hz, 1H, H-6a), 3.77 (dd, J9b,9a = 6.64 Hz,
J9b,8 = 2.22 Hz, 1H, H-9b), 3.76–3.74 (m, 1H, H-7b), 3.67 (t, J6a,6b = J6b,5 = 5.9 Hz, 1H, H-
6b), 3.64 (t, J3,2 = J3,4 = 8.8 Hz, 1H, H-3), 3.62–3.57 (m, 1H, H-5), 3.51 (dd, J7a,7b = 10.4, J7a,8
= 6.2 Hz, 1H, H-7a), 3.39–3.37 (m, 1H, H-2), 3.28 (t, J3,4 = J4,5 = 9.3 Hz, 1H, H-4), 1.41 (s,
11

3H, H-1'), 1.35 (s, 3H, H-1'); 13C NMR (125 MHz, CD3OD) 109.25 (C-2'), 99.07 (C-1), 74.62
(C-8), 73.67 (C-3), 72.44 (C-5), 72.22 (C-2), 70.33 (C-4), 68.59 (C-7), 66.24 (C-9), 61.24 (C-
6), 25.64 (C-1'), 24.24 (C-1'); HRMS (ESI) m/z calculated for [C12H22NaO8]+: 317.1206,
found 317.1229.
3-O-(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-sn-glycerol
(2).
3-O-(α-D-
Glucopyranosyl)-1,2-O-isopropylidene-sn-glycerol 7 (416 mg, 1.41 mmol) was co-
evaporated with DMF (3 × 5 mL), suspended in dry DMF (2 mL) and then BnBr (1.0 mL,
8.48 mmol) was added to the reaction mixture. The resulting solution was cooled to 0 °C,
NaH [201 mg (60% suspension in mineral oil, 8.48 mmol)] was added, and then the reaction
was allowed to warm to r.t. After stirring for 6 hours, the reaction was quenched with
methanol (2 mL), concentrated in vacuo, re-dissolved in Et2O (10 mL), washed with water
(10 mL) and brine (10 mL), dried over MgSO4, filtered, and concentrated under reduced
pressure. The resulting residue was purified by silica gel flash column chromatography (PE to
PE/EtOAc, 8:1, v/v) to give 1,2-O-isopropylidene-3-O-(2,3,4,6-tetra-O-benzyl-α-D-
glucopyranosyl)-sn-glycerol as a colorless oil (641 mg, 1.05 mmol, 74%). Rf = 0.35
(PE/EtOAc, 4:1, v/v); [ꢀ]ꢅꢁꢆ.ꢆ = +32 (c = 1, CH2Cl2); IR (film) = 3029, 2984, 1496, 1379,
1
1209, 1155, 1027, 910, 796, 605, 515, 405 cm-1; H NMR (500 MHz, CDCl3) δ 7.42–7.24
(m, 18H, CHarom), 7.18-7.11 (d, J = 7.1 Hz, 2H, CHarom), 5.01 (d, Ja,b = 11.8 Hz, 1H, CHa
3-O-Bn), 4.90–4.87 (m, 2H, CHa 4-O-Bn, H-1), 4.84 (d, Ja,b = 11.8 Hz, 1H, CHb 3-O-Bn),
4.80 (d, Ja,b = 11.9 Hz, 1H, CHa 2-O-Bn), 4.68 (d, Ja,b = 12.1 Hz, 1H, CHb 2-O-Bn), 4.63 (d,
Ja,b = 12.2 Hz, 1H, CHa 6-O-Bn), 4.52–4.46 (m, 2H, CHb 6-O-Bn, CHb 4-O-Bn), 4.38 (p, J7,8
= J8,9 = 6.2 Hz, 1H, H-8), 4.12–4.06 (t, J9,8 = 7.6 Hz, 1H, H-9a), 3.99 (t, J3,2 = J3,4 = 9.3 Hz,
1H, H-3), 3.85-3.74 (m, 3H, H-5, H-6a, H-9b), 3.69–3.62 (m, 3H, H-4, H-7a, H-6b), 3.61–
3.56 (m, 2H, H-2, H-7b), 1.45 (s, 3H, H-1'), 1.39 (s, 3H, H-1'); 13C NMR (125 MHz, CDCl3)
δ 138.89 (Ci, 3-O-Bn), 138.33 (Ci, 4-O-Bn), 138.30 (Ci, 2-O-Bn), 137.95 (Ci, 6-O-Bn),
128.51, 128.45, 128.43, 128.09, 128.01, 127.98, 127.95, 127.76, 127.65 (CHarom), 109.50
(C-2'), 97.56 (C-8), 81.99 (C-3), 80.04 (C-2), 77.65 (C-4), 75.76 (CH2, 3-O-Bn), 75.14 (CH2,
4-O-Bn), 74.65 (C-9), 73.54 (CH2, 2-O-Bn), 73.16 (CH2, 6-O-Bn), 70.41 (C-5), 69.09 (C-7),
68.48 (C-6), 67.10 (C-9), 26.91 (C-1'), 25.53 (C-1'); HRMS (ESI) m/z calculated for
[C40H50NO8]+: 672.3531, found 672.3572. A solution of 1,2-O-isopropylidene-3-O-(2,3,4,6-
tetra-O-benzyl-α-D-glucopyranosyl)-sn-glycerol in AcOH:H2O (10 mL, 4:1, v/v) was heated
to 80 °C. After 1 hour, the reaction mixture was concentrated in vacuo and the resulting
residue was purified using silica gel flash column chromatography (PE/EtOAc, 10:1-2:1, v/v)
12

to give the title compound 2 as a colorless oil (599 mg, 1.046 mmol, quant.). Rf = 0.4
(PE/EtOAc, 1:1, v/v); [ꢀ]ꢂꢁꢅ.ꢇ = +36 (c = 1, CH2Cl2); IR (film) = 3363, 2918, 1453, 1379,
1
1259, 1155, 1054, 838, 734, 605, 433 cm-1; H NMR (500 MHz, CDCl3) δ 7.41–7.22 (m,
15H, CHarom), 7.17–7.10 (m, 2H, CHarom), 4.95 (d, Ja,b = 10.9 Hz, 1H, CHa 3-O-Bn), 4.85
(d, Ja,b = 11.3 Hz, 1H, CHb 3-O-Bn), 4.84–4.79 (m, 2H, CHa 4-O-Bn, CHa 2-O-Bn), 4.73 (d,
J1,2 = 3.7 Hz, 1H, H-1), 4.65 (d, Ja,b = 11.9 Hz, 1H, CHb 2-O-Bn), 4.60 (d, Ja,b = 12.1 Hz, 1H,
CHa 6-O-Bn), 4.50–4.45 (m, 2H, CHb 4-O-Bn, CHb 6-O-Bn), 3.97 (t, J2,3 = J3,4 = 9.3 Hz, 1H,
H-3), 3.89–3.82 (m, 2H, H-5, H-8), 3.80 (dd, J7a,b = 10.4 Hz, J7a,8 = 3.5 Hz, 1H, 7a), 3.66–
3.61 (m, 3H, H-9a,b, H-6a), 3.60–3.56 (m, 2H, H-6b, H-2), 3.46 (dd, J7a,b = 10.5 Hz, J7b,8
=
6.8 Hz, 1H, H-7b); 13C NMR (125 MHz, CDCl3) δ 138.72 (Ci, 3-O-Bn), 138.16 (Ci, 4-O-Bn),
137.93 (Ci, 2-O-Bn), 137.78 (Ci, 6-O-Bn), 128.70, 128.55, 128.53, 128.52, 128.29, 128.24,
128.18, 128.12, 128.02, 128.00, 127.93, 127.88, 127.79 (CHarom), 98.62 (C-1), 82.18 (C-3),
80.12 (C-2), 77.89 (C-4), 79.95 (CH2, 3-O-Bn), 75.20 (CH2, 4-O-Bn), 73.81 (CH2, 2-O-Bn),
73.64 (CH2, 6-O-Bn), 71.70 (C-7), 70.63 (CH2, C-8), 70.38 (C-5), 68.53 (C-9), 63.79 (C-6);
HRMS (ESI) m/z calculated for [C37H46NO8]+: 632.3217, found 632.3264.
4.1.2 General esterification procedure: 3-O-(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-
sn-glycerol 2 (1.0 mmol, 1 equiv.) and the appropriate carboxylic acid (4.4 mmol, 4 equiv.)
were co-evaporated together with dry toluene, then suspended in dry toluene (5 mL). To the
reaction mixture, EDCI (6.6 mmol, 6.6 equiv.) and DMAP (1 mmol, 1 equiv.) were added
and the resulting suspension was heated to 70 °C for 12 h. The reaction was cooled to r.t and
diluted with Et2O (5 mL). The organic layer was then washed with water (5 mL), NaHCO3 (5
mL), and brine (5 mL). The combined aqueous phases were re-extracted with Et2O (5 mL)
and the combined organic phases were dried with anhydrous MgSO4, filtered, and
concentrated in vacuo. The product was purified using gradient silica-gel flash column
chromatography (PE to PE:EtOAc, 9:1, v/v).
1,2-di-O-Dodecanoyl-3-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-sn-glycerol (8a).
By subjecting diol 2 (110 mg, 0.179 mmol), lauric acid 3a (137 mg, 0.79 mmol), EDCI (193
mg, 1.18 mmol) and DMAP (12.4 mg, 0.18 mmol) to the general esterification procedure, the
title compound 8a was obtained as colorless oil (143 mg, 0.146 mmol, 82%). Rf = 0.55
(PE/EtOAc, 4:1, v/v); [ꢀ]ꢂꢁꢈ.ꢇ = +24 (c = 1, CH2Cl2); IR (film) = 2922, 2852, 1740, 1697,
1
1465, 1234, 1028, 907, 733, 563, 435 cm-1; H NMR (500 MHz, CDCl3) δ 7.31–7.16 (m,
17H, CHarom), 7.09–7.05 (m, 2H, CHarom), 5.19 (p, J7,8 = J8,9 = 5.3 Hz, 1H, H-8), 4.90 (d,
Ja,b = 10.8 Hz, 1H, CHa 3-O-Bn), 4.79–4.67 (m, 4H, H-1, CHb 3-O-Bn, CHa 4-O-Bn, CHa 2-
13

O-Bn), 4.60–4.51 (m, 2H, CHa 6-O-Bn, CHb 2-O-Bn), 4.41–4.38 (m, 2H, CHb 6-O-Bn, CHb
4-O-Bn), 4.35 (dd, J9a,b = 11.9 Hz, J9a,8 = 3.5 Hz, 2H, H-9a), 4.13 (dd, J9a,b = 11.5 Hz, J9a,8
=
5.7 Hz, 1H, H-9b), 3.88 (t, J2,3 = J3,4 = 9.0 Hz, 1H, H-3), 3.71–3.62 (m, 3H, H-7a, H-5, H-6a),
3.61–3.53 (m, 2H, H-4, H-6b), 3.52–3.47 (m, 2H, H-7b, H-2), 2.25–2.18 (m, 4H, H-11, H-
11'), 1.56–1.49 (m, 4H, H-12, H-12'), 1.27-1.14 (m, 36H, H-13-20, H-13'-H20'), 0.81 (t, J24,25
13
= J24',25' = 6.8 Hz, 6H, H-21, H-21'); C NMR (125 MHz, CDCl3) δ 173.53 (C-10'), 173.19
(C-10), 138.92 (Ci, 3-O-Bn), 138.42 (Ci, 4-O-Bn), 138.38 (Ci, 2-O-Bn), 137.99 (Ci, 6-O-Bn),
128.60, 128.52, 128.49, 128.09, 128.00, 127.97, 127.85, 127.81, 127.72 (CHarom), 97.89 (C-
1), 81.95 (C-3), 80.17 (C-2), 77.60 (C-4), 75.83 (CH2, 3-O-Bn), 75.20 (CH2, 4-O-Bn), 73.65
(CH2, 2-O-Bn), 73.26 (CH2, 6-O-Bn), 70.69 (C-5), 69.98 (C-8), 68.47 (C-6), 66.54 (C-7),
62.62 (C-9), 34.43 (C-11), 34.26 (C-11'), 32.06 (C-20, C-20'), 29.79, 29.78, 29.74, 29.65,
29.58, 29.50, 29.46, 29.39, 29.27, 29.21 (C-14-19, C14'-19'), 25.06 (C-12), 25.04 (C-12'),
22.83 (C-13, C-13'), 14.279 (C-21), 14.27 (C-21'); HRMS (ESI) m/z calculated for
[C61H90NO10]+: 996.6559, found 996.6560.
1,2-Di-O-tetradecanoyl-3-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-sn-glycerol
(8b). By subjecting diol 2 (110 mg, 0.179 mmol), myristic acid 3b (180 mg, 0.79 mmol),
EDCI (193 mg, 1.18 mmol) and DMAP (12.4 mg, 0.179 mmol) to the general esterification
procedure, the title compound 8b was obtained as a colorless oil (157 mg, 0.152 mmol, 85%).
Rf = 0.6 (PE/EtOAc, 4:1, v/v); [ꢀ]ꢁꢂꢈ.ꢉ = +26 (c = 1, CH2Cl2); IR (film) = 2922, 2852, 1740,
1696, 1419, 1246, 1155, 1070, 733, 557, 414 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.37–7.20
(m, 17H, CHarom), 7.15–7.09 (m, 2H, CHarom), 5.24 (p, J7,8 = J8,9 = 5.6 Hz, 1H, H-8), 4.94
(d, Ja,b = 11.2 Hz, 1H, CHa 3-O-Bn), 4.84–4.72 (m, 4H, H-1, CHb 3-O-Bn, CHa 4-O-Bn, CHa
2-O-Bn), 4.63–4.56 (m, 2H, CHa 6-O-Bn, CHb 2-O-Bn), 4.41–4.38 (m, 2H, CHb 6-O-Bn,
CHb 4-O-Bn), 4.40 (dd, J9a,b = 11.3 Hz, J9a,8 = 3.5 Hz, 2H, H-9a), 4.18 (dd, J9a,b = 11.5 Hz,
J9a,8 = 3.7 Hz, 1H, H-9b), 3.93 (t, J2,3 = J3,4 = 9.7 Hz, 1H, H-3), 3.77–3.68 (m, 3H, H-7a, H-5,
H-6a), 3.66–3.58 (m, 2H, H-4, H-6b), 3.58–3.52 (m, 2H, H-7b, H-2), 2.33–2.21 (m, 4H, H-
11, H-11'), 1.62–1.54 (m, 4H, H-12, H-12'), 1.34–1.10 (m, 48H, H-13-22, H-13'-H22'), 0.87
13
(t, J24,25 = J24',25' = 6.8 Hz, 6H, H-23, H-23'); C NMR (125 MHz, CDCl3) δ 173.50 (C-10'),
173.16 (C-10), 138.93 (Ci, 3-O-Bn), 138.43 (Ci, 4-O-Bn), 138.39 (Ci, 2-O-Bn), 138.00 (Ci,
6-O-Bn), 128.60, 128.57, 128.51, 128.48, 128.09, 128.02, 127.99, 127.97, 127.85, 127.80,
127.71 (CHarom), 97.89 (C-1), 81.95 (C-3), 80.19 (C-2), 77.60 (C-4), 75.83 (CH2, 3-O-Bn),
75.19 (CH2, 4-O-Bn), 73.65 (CH2, 2-O-Bn), 73.26 (CH2, 6-O-Bn), 70.69 (C-5), 69.98 (C-8),
68.48 (C-6), 66.54 (C-7), 62.62 (C-9), 34.43 (C-11), 34.26 (C-11'), 32.07 (C-22, C-22'),
14

29.85, 29.83, 29.80, 29.66, 29.51, 29.46, 29.30, 29.27 (C-14-21, C14'-21'), 25.06 (C-12),
25.04 (C-12'), 22.84 (C-13, C-13'), 14.27 (C-23, C-23'); HRMS (ESI) m/z calculated for
[C65H98NO10]+: 1052.7185, found 1052.7170.
1,2-di-O-Hexadecanoyl-3-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-sn-glycerol
(8c). By subjecting diol 2 (110 mg, 0.18 mmol), palmitic acid 3c (202 mg, 0.78 mmol), EDCI
(193 mg, 1.18 mmol) and DMAP (12.4 mg, 0.18 mmol) to the general esterification
procedure, the title compound 8c was obtained as a white solid (166 mg, 0.15 mmol, 85%).
Rf = 0.6 (PE/EtOAc, 4:1, v/v); [ꢀ]ꢁꢂꢊ.ꢊ = +30 (c = 1, CH2Cl2); IR (film) = 2922, 2835, 1740,
1
1735, 1465, 1363, 1241, 1207, 1157, 1089, 1071, 1028, 734, 696 cm-1; H NMR (500 MHz,
CDCl3) δ 7.33–7.17 (m, 17H, CHarom), 7.09–7.05 (m, 2H, CHarom), 5.19 (p, J7,8 = J8,9 = 5.4
Hz, 1H, H-8), 4.90 (d, Ja,b = 10.8 Hz, 1H, CHa, 3-O-Bn), 4.79–4.67 (m, 4H, H-1, CHb 3-O-
Bn, CHa 4-O-Bn, CHa 2-O-Bn), 4.60–4.51 (m, 2H, CHa 6-O-Bn, CHb 2-O-Bn), 4.41–4.38 (m,
2H, CHb 6-O-Bn, CHb 4-O-Bn), 4.35 (dd, J9a,b = 11.3 Hz, J9a,8 = 3.5 Hz, 2H, H-9a), 4.13 (dd,
J9a,b = 11.5 Hz, J9a,8 = 3.7 Hz, 1H, H-9b), 3.88 (t, J2,3 = J3,4 = 9.3 Hz, 1H, H-3), 3.73–3.63 (m,
3H, H-7a, H-5, H-6a), 3.61–3.53 (m, 2H, H-4, H-6b), 3.52–3.47 (m, 2H, H-7b, H-2), 2.24–
2.17 (m, 4H, H-11, H-11'), 1.57–1.49 (m, 4H, H-12, H-12'), 1.28–1.06 (m, 47H, H-13-24, H-
13
13'-H24'), 0.83 (t, J24,25 = J24',25' = 6.8 Hz, 6H, H-25, H-25'); C NMR (125 MHz, CDCl3) δ
173.49 (C-10'), 173.16 (C-10), 138.93 (Ci, 3-O-Bn), 138.43 (Ci, 4-O-Bn), 138.39 (Ci, 2-O-
Bn), 138.00 (Ci, 6-O-Bn), 128.60, 128.51, 128.48, 128.08, 128.02, 127.99, 127.97, 127.84,
127.80, 127.71 (CHarom), 97.88 (C-1), 81.95 (C-3), 80.19 (C-2), 77.60 (C-4), 75.82 (CH2, 3-
O-Bn), 75.19 (CH2, 4-O-Bn), 73.65 (CH2, 2-O-Bn), 73.26 (CH2, 6-O-Bn), 70.69 (C-5), 69.98
(C-8), 68.49 (C-6), 66.54 (C-7), 62.62 (C-9), 34.43 (C-11), 34.26 (C-11'), 32.07 (C-24, C-
24'), 29.85, 29.83, 29.80, 29.66, 29.51, 29.46, 29.30, 29.27 (C-14-23, C14'-23'), 25.06 (C-12),
25.04 (C-12'), 22.84 (C-13, C-13'), 14.27 (C-25, C-25'); HRMS (ESI) m/z calculated for
[C69H106NO10]+: 1108.7811, found 1108.7819.
1,2-di-O-Octadecanoyl-3-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-sn-glycerol
(8d). By subjecting diol 2 (110 mg, 0.179 mmol), stearic acid 3d (224 mg, 0.79 mmol), EDCI
(192.6 mg, 1.18 mmol) and DMAP (12.4 mg, 0.18 mmol) to the general esterification
procedure, the title compound 8d was obtained as a white solid (174 mg, 0.150 mmol, 84%).
Rf = 0.6 (PE/EtOAc, 4:1, v/v); [ꢀ]ꢅꢁꢆ.ꢆ = +32 (c = 1, CHCl3); IR (film) = 3030, 2921, 1740,
1
1701, 1696, 1559, 1453, 1359, 1207, 1157, 910, 733, 601, 526, 462 cm-1; H NMR (500
MHz, CDCl3) δ 7.30–7.17 (m, 17H, CHarom), 7.08 (m, 2H, CHarom), 5.17 (p, J7,8 = J8,9
=
5.4 Hz, 1H, H-8), 4.88 (d, Ja,b = 10.8 Hz, 1H, CHa 3-O-Bn), 4.79 –4.67 (m, 4H, H-1, CHb 3-
15

O-Bn, CHa 4-O-Bn, CHa 2-O-Bn), 4.60–4.50 (m, 2H, CHa 6-O-Bn, CHb 2-O-Bn), 4.41–4.38
(m, 2H, CHb 6-O-Bn, CHb 4-O-Bn), 4.35 (dd, J9a,b = 11.3 Hz, J9a,8 = 3.5 Hz, 2H, H-9a), 4.13
(dd, J9a,b = 11.5 Hz, J9a,8 = 3.7 Hz, 1H, H-9b), 3.88 (t, J2,3 = J3,4 = 9.3 Hz, 1H, H-3), 3.70–
3.62 (m, 3H, H-7a, H-5, H-6a), 3.61–3.53 (m, 2H, H-4, H-6b), 3.50 (m, 2H, H-7b, H-2),
2.23–2.17 (m, 4H, H-11, H-11'), 1.56–1.49 (m, 4H, H-12, H-12'), 1.28-1.06 (m, 56H, H-13-
26, H-13'-H26'), 0.83 (t, J24,25 = J24',25' = 6.8 Hz, 6H, H-27, H-27'); 13C NMR (125 MHz,
CDCl3) δ 173.52 (C-10'), 173.19 (C-10), 138.96 (Ci, 3-O-Bn), 138.46 (Ci, 4-O-Bn), 138.42
(Ci, 2-O-Bn), 138.03 (Ci, 6-O-Bn), 128.62, 128.54, 128.51, 128.11, 128.04, 127.02, 127.99,
127.87, 127.83, 127.74 (CHarom), 97.91 (C-1), 81.98 (C-3), 80.22 (C-2), 77.63 (C-4), 75.85
(CH2, 3-O-Bn), 75.22 (CH2, 4-O-Bn), 73.68 (CH2, 2-O-Bn), 73.28 (CH2, 6-O-Bn), 70.72 (C-
5), 70.01 (C-8), 68.52 (C-6), 66.57 (C-7), 62.64 (C-9), 34.46 (C-11), 34.29 (C-11'), 32.10 (C-
26, C-26'), 29.88, 29.86, 29.83, 29.69, 29.54, 29.49, 29.33, 29.30 (C-14-25, C14'-25'), 25.08
(C-12), 25.07 (C-12'), 22.87 (C-13, C-13'), 14.30 (C-27, C-27'); HRMS (ESI) m/z calculated
for [C73H114NO10]+: 1164.8437, found 1164.8438.
4.1.3 General debenzylation procedure: To
a
solution of benzyl-protected
glucosyldiacylglycerides 8a-d dissolved in EtOH:EtOAc (5 mL, 1:1, v/v) was added
Pd(OH)2/C. H2 gas was allowed to bubble through the reaction mixture for 18 hours, at which
point the suspension was diluted with hot pyridine (5 mL), filtered over celite, and
concentrated in vacuo. The product was purified using gradient silica gel flash column
chromatography (DCM to DCM:MeOH, 9:1, v/v).
1,2-Di-O-dodecanoyl-3-O-α-
D
-glucopyranosyl-sn-glycerol
(1a).
Benzyl
protected
glucosyldiacylglyceride 8a (90 mg, 0.091 mmol) and Pd(OH)2/C (70 mg) were subjected to
the conditions described in the general procedure for debenzylation to give the title
compound 1a as a white solid (51 mg, 0.082 mmol, 90%). Rf = 0.75 (DCM/MeoH, 1.5:8.5,
v/v); [ꢀ]ꢅꢁꢊ.ꢆ = +42 (c = 1, C5H5N); IR (film) = 3381, 2918, 1648, 1537, 1302, 1230, 1010,
966, 824, 587, 529 cm-1; 1H NMR (500 MHz, CDCl3) 5.67 (p, J = 5.7 Hz, 1H, H-8), 5.39 (d,
J1,2 = 3.6 Hz, 1H, H-1), 4.76 (dd, J9a,b = 11.9 Hz, J9a,8 = 3.4 Hz, 1H, 9a), 4.62 (t, J2,3 = J3,4
=
9.2 Hz, 1H, H-3), 4.57–4.50 (m, 2H, H-7a, H-6a), 4.44–4.37 (m, 2H, H-5, H-6b), 4.32–4.23
(m, 2H, H-7a, H-4), 4.18 (dd, J2,3 = 9.6 Hz, J1,2 = 3.7 Hz, 1H, H-2), 3.95 (dd, J7a,b = 10.7 Hz,
J7b,8 = 5.5 Hz, 1H, H-7b), 2.46–2.39 (m, 4H, H-11, H11'), 1.73–1.63 (m, 4H, H-12, H-12'),
1.37–1.18 (m, 36H, H-13-22, H-13'-22'), 0.88 (t, J23,22 = J23',22' = 6.9 Hz, 6H, H-23, H-23');
13C NMR (125 MHz, CDCl3)
δ 173.78 (C10'), 173.60 (C10), 101.43 (C-1), 75.76 (C-3), 75.17
(C-5), 74.21 (C-2), 72.52 (C-4), 71.16 (C-8), 66.75 (C-7), 63.41(C-9), 63.24 (C-6), 34.92
16

(C11), 34.70 (C-11'), 32.57 (C20, C20'), 30.37, 30.34, 30.24, 30.07, 29.83 (C-14-19, C14'-C-
19'), 25.70 (C12, C12'), 23.39 (C-13, C13'), 14.74 (C-21, C-21')
for [C33H62NaO10]+: 641.4235, found 641.4230.
; HRMS (ESI) m/z calculated
1,2-Di-O-tetradecanoyl-3-O-α-D-glucopyranosyl-sn-glycerol (1b). Benzyl protected
glucosyldiacylglyceride 8b (150 mg, 0.144 mmol) and Pd(OH)2/C (86 mg) were subjected to
the conditions described in the general procedure for debenzylation to give the title
compound as a white solid 1b (89 mg, 0.131 mmol, 91%). Rf = 0.8 (DCM/MeOH, 1.5:8.5,
v/v); [ꢀ]ꢅꢁꢇ.ꢃ = +54 (c = 1, C5H5N); IR (film) = 3368, 2924, 1648, 1537, 1302, 1230, 1008,
966, 886, 824, 587, 529 cm1; 1H NMR (500 MHz, CDCl3) 5.67 (p, J = 5.7 Hz, 1H, H-8), 5.39
(d, J1,2 = 4.0 Hz, 1H, H-1), 4.76 (dd, J9a,b = 11.9 Hz, J9a,8 = 3.2 Hz, 1H, 9a), 4.62 (t, J2,3 = J3,4
= 8.9 Hz, 1H, H-3), 4.56–4.50 (m, 2H, H-7a, H-6a), 4.44–4.37 (m, 2H, H-5, H-6b), 4.32–4.22
(m, 2H, H-7a, H-4), 4.18 (dd, J2,3 = 9.6 Hz, J1,2 = 3.7 Hz, 1H, H-2), 3.95 (dd, J7a,b = 10.7 Hz,
J7b,8 = 5.5 Hz, 1H, H-7b), 2.46–2.39 (m, 4H, H-11, H11'), 1.73–1.63 (m, 4H, H-12, H-12'),
1.37–1.18 (m, 36H, H-13-22, H-13'-22'), 0.88 (t, J23,22 = J23',22' = 6.9 Hz, 6H, H-23, H-23');
13C NMR (125 MHz, CDCl3)
δ 173.80 (C10'), 173.63 (C10), 101.43 (C-1), 75.77 (C-3), 75.20
(C-5), 74.22 (C-2), 72.52 (C-4), 71.17 (C-8), 66.74 (C-7), 63.44 (C-9), 63.25 (C-6), 34.95
(C11), 34.73 (C-11'), 32.63 (C22, C22'), 30.51, 30.49, 30.46, 30.44, 30.32, 30.14, 30.13,
29.89, 29.88 (C-13-21, C13'-C-21'), 25.74 (C12, C12'), 23.44 (C-13, C13'), 14.79 (C-23, C-
23'); HRMS (ESI) m/z calculated for [C37H70NaO10]+: 697.4861, found 697.4858.
1,2-Di-O-hexadecanoyl-3-O-α-D-glucopyranosyl-sn-glycerol (1c). Benzyl protected
glucosyldiacylglyceride 8c (120 mg, 0.109 mmol) and Pd(OH)2/C (80 mg) were subjected to
the conditions described in the general procedure for debenzylation to give the title
compound 1c as a white solid (71 mg, 0.097 mmol, 89%). Rf = 0.8 (DCM/MeOH, 1.5:8.5,
v/v); [ꢀ]ꢅꢁꢉ.ꢇ = +48 (c = 1, C5H5N); IR (film) = 3376, 2917, 1734, 1648, 1537, 1302, 1230,
1010, 966, 886, 824, 537, 529 cm1; 1H NMR (500 MHz, CDCl3) 5.67 (p, J = 5.6 Hz, 1H, H-
8), 5.39 (d, J1,2 = 3.6 Hz, 1H, H-1), 4.75 (dd, J9a,b = 11.6 Hz, J9a,8 = 3.4 Hz, 1H, 9a), 4.62 (t,
J2,3 = J3,4 = 9.2 Hz, 1H, H-3), 4.56–4.48 (m, 2H, H-7a, H-6a), 4.44–4.37 (m, 2H, H-5, H-6b),
4.32–4.22 (m, 2H, H-7a, H-4), 4.17 (dd, J2,3 = 9.6 Hz, J1,2 = 3.7 Hz, 1H, H-2), 3.94 (dd, J7a,b
= 10.7 Hz, J7b,8 = 5.5 Hz, 1H, H-7b), 2.48–2.39 (m, 4H, H-11, H11'), 1.74–1.64 (m, 4H, H-
12, H-12'), 1.40–1.27 (m, 52H, H-13-22, H-13'-22'), 0.89 (t, J23,22 = J23',22' = 6.9 Hz, 6H, H-
23, H-23'); 13C NMR (125 MHz, CDCl3)
δ 173.77 (C10'), 173.60 (C10), 101.41 (C-1), 75.74
(C-3), 75.17 (C-5), 74.19 (C-2), 72.50 (C-4), 71.14 (C-8), 66.72 (C-7), 63.41(C-9), 63.22 (C-
6), 34.92 (C11), 34.70 (C-11'), 32.60 (C24, C24'), 30.48, 30.47, 30.43, 30.41, 30.10, 29.86,
17

29.85 (C-14-23, C14'-C-23'), 25.71 (C12, C12'), 23.42 (C-13, C13'), 14.76 (C-25, C-25');
HRMS (ESI) m/z calculated for [C41H78NaO10]+: 753.5487, found 753.5494.
1,2-Di-O-octadecanoyl-3-O-α-D-glucopyranosyl-sn-glycerol (1d). Benzyl protected
glucosyldiacylglyceride 8d (125 mg, 0.109 mmol) and Pd(OH)2/C (90 mg) were subjected to
the conditions described in the general procedure for debenzylation to give the title
compound 1d as a white solid (77 mg, 0.098 mmol, 90%). Rf = 0.8 (DCM/MeOH, 1.5:8.5,
v/v); [ꢀ]ꢂꢁꢋ.ꢆ = +58 (c = 1, C5H5N); IR (film) = 3365, 2917, 1648, 1537, 1302, 1230, 1010,
966, 824, 587, 529 cm-1; 1H NMR (500 MHz, CDCl3) 5.65 (p, J = 5.7 Hz, 1H, H-8), 5.38 (d,
J1,2 = 3.6 Hz, 1H, H-1), 4.75 (dd, J9a,b = 11.5 Hz, J9a,8 = 3.6 Hz, 1H, 9a), 4.62 (t, J2,3 = J3,4
=
9.2 Hz, 1H, H-3), 4.57–4.48 (m, 2H, H-7a, H-6a), 4.44–4.36 (m, 2H, H-5, H-6b), 4.30–4.22
(m, 2H, H-7a, H-4), 4.17 (dd, J2,3 = 9.6 Hz, J1,2 = 3.7 Hz, 1H, H-2), 3.94 (dd, J7a,b = 10.7 Hz,
J7b,8 = 5.5 Hz, 1H, H-7b), 2.45–2.38 (m, 4H, H-11, H11'), 1.72–1.62 (m, 4H, H-12, H-12'),
1.34–1.20 (m, 36H, H-13-22, H-13'-22'), 0.87 (t, J23,22 = J23',22' = 6.9 Hz, 6H, H-23, H-23');
13C NMR (125 MHz, CDCl3)
δ 173.79 (C10'), 173.62 (C10), 101.44 (C-1), 75.77 (C-3), 75.20
(C-5), 74.23 (C-2), 72.52 (C-4), 71.16 (C-8), 66.74 (C-7), 63.44 (C-9), 63.25 (C-6), 34.95
(C11), 34.73 (C-11'), 32.62 (C26, C26'), 30.52, 30.50, 30.49, 30.46, 30.43, 30.33, 30.14,
30.12, 29.89, 29.88 (C-14-25, C14'-C-25'), 25.74 (C12, C12'), 23.44 (C-13, C13'), 14.78 (C-
27, C-27'); HRMS (ESI) m/z calculated for [C45H86NaO10]+: 809.6113, found 809.6121.
Biological Methods
2B4-NFAT-GFP reporter cells assay. Synthesised αGlcDAGs 2a-d and TDB18 or TDM
(purchased) in chloroform/methanol (2:1, v/v, 1 mM) were serially diluted with isopropanol
and added to the wells of 96-well plates, followed by evaporation of the solvent. The
concentration of 2B4-NFAT-GFP reporter cells expressing mMincle + FcRγ, hMincle +
FcRγ or FcRγ was adjusted to 4×105 cells/mL and 100 µL/well was then added to glycolipid-
coated plates (0.1, 1, or 4 nmol/well) for 18 h or 24 h. The reporter cells were harvested,
stained with DAPI, and analysed for NFAT-GFP expression using flow cytometry.27 All
synthetic compounds were confirmed to be free of endotoxin at a sensitivity of ≤0.125
EU/mL by Limulus amebocyte lysate (LAL) assay using an endotoxin kit (Pyrotell, Limulus
Amebocyte Lysate).
Acknowledgements
18

The authors would like to thank The Royal Society of New Zealand, Marsden Fund
(VUW1401), the Health Research Council New Zealand (Hercus Fellowship, B.L.S.,
2013/33) for funding, and the Federal Ministry of Education and Research supporting the
German Center for Lung Research (U.A.M). Additionally, we would like to acknowledge
Professor Sho Yamasaki for providing the hMincle expressing 2B4 reporter cells and Amy J.
Foster for the graphical abstract.
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20

Synthesis of α-Glucosyl Diacylglycerides as Potential Adjuvants for Streptococcus
pneumoniae Vaccines
Ayesha Khan,a,b Femke Hollwedel,c,d Ulrich A. Maus,c,d Bridget L. Stocker,a,b* Mattie S. M.
Timmera,b*
Highlights
•
•
•
We efficiently synthesised α-glucosyl diacylglycerols (αGlc-DAGs) containing C12,
C14, C16 and C18 acyl chains in 7 steps and 44-47% overall yields
Mincle signalling was dependent on lipid length, with C14 αGlc-DAG exhibiting the
greatest signalling
C14 αGlc-DAG has potential to act as an adjuvant to augment the immune response
against Streptococcus pneumoniae antigens
1

29 December 2019
Wellington
NEW ZEALAND
I hereby declare that there are no conflicts of interests to report in relation to the
submission of our manuscript:‘Synthesis of α-Glucosyl Diacylglycerides as potential
adjuvants for Streptococcus pneumoniae vaccines.’
Regards,
Bridget L. Stocker
School of Chemical and Physical Sciences
Victoria University of Wellington
PO Box 600, Wellington
NEW ZEALAND