Preference for Glucose over Inositol Headgroup during Lysolipid Activation of G Protein-Coupled Receptor 55

Article abstract of DOI:10.1021/acschemneuro.8b00505

G protein-coupled receptor 55 (GPR55) is highly expressed in brain and peripheral nervous system. Originally deorphanized as a cannabinoid receptor, recently GPR55 has been described as a lysophospholipid-responsive receptor, specifically toward lysophosphatidylinositol and lysophosphatidyl-β-d-glucoside (LysoPtdGlc). To characterize lysolipid-GPR55 interaction, synthetic access to LysoPtdGlc and selected analogues was established utilizing a phosphorus(III)-based chemical approach. The biological activity of each synthetic lipid was assessed using a GPR55-dependent chemotropism assay in primary sensory neurons. Combined with molecular dynamics simulations the potential ligand entry port and binding pocket specifics are discussed. These results highlight the preference for gluco- over inositol- and galacto-configured headgroups.

Full text of DOI:10.1021/acschemneuro.8b00505

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Article
Preference for glucose over inositol head group during
lysolipid activation of G protein-coupled receptor 55
Adam T Guy, Koki Kano, Junpei Ohyama, Hiroyuki Kamiguchi,
Yoshio Hirabayashi, Yukishige Ito, Ichiro Matsuo, and Peter Greimel
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00505 • Publication Date (Web): 15 Oct 2018
Downloaded from http://pubs.acs.org on October 20, 2018
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Preference for glucose over inositol head group during lysolipid activation of G
protein-coupled receptor 55
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Adam T. Guy, Koki Kano, Jyunpei Ohyama, Hiroyuki Kamiguchi, Yoshio Hirabayashi,
Yukishige Ito, Ichiro Matsuo, and Peter Greimel*
Abstract: G protein-coupled receptor 55 (GPR55)
is highly expressed in brain and peripheral nervous
system. Originally de-orphanized as a cannabinoid
receptor, recently GPR55 has been described as a
lysophospholipid responsive receptor, specifically
towards
lysophosphatidylinositol
and
lysophosphatidyl--D-glucoside (LysoPtdGlc). To
characterize lysolipid-GPR55 interaction, synthetic
access to LysoPtdGlc and selected analogs was established utilizing a phosphorus(III)-
based chemical approach. The biological activity of each synthetic lipid was assessed
using a GPR55-dependent chemotropism assay in primary sensory neurons. Combined
with molecular dynamics simulations the potential ligand entry port and binding pocket
specifics are discussed. These results highlight the preference for gluco over inositol and
galacto configured head groups.
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lysolipids • glycolipids • G-protein coupled receptor 55 • homology model • structure-
activity relationship • cannabinoid receptor related receptor
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Introduction
GPR55 is a Class A G protein-coupled receptor (GPCR) widely distributed in the body
including central and peripheral nervous systems,[1,2,3] gastrointestinal tract and immune
[4]
system . GPR55 knockout (KO) mice have been reported to show defects in
nociception[5] and in models of inflammation and neuropathic pain . Early cell-based
[6]
[7]
assays suggested GPR55 involvement in the physiological effects of cannabis products
or endocannabinoids not mediated by the canonical cannabinoid receptors CB1 or
CB2.[8,9] However, in vitro studies of GPR55 activation by cannabinoids have produced
many inconsistent results.[10,11] Based on the higher amino acid sequence homology to
GPR23/LPAR4 (30%) compared to CB1 (13.5%) and CB2 (14.4%) it was later
reported[12,13] that lysophosphatidylinositol (LysoPtdIns, 3) and its regioisomer 2-
arachidonoyl lysophosphatidylinositol, but not cannabinoids, induce ERK1/2 activation
and increase intracellular Ca2+ via the Gα -RhoA-ROCK cascade.
[14]
Recently it has
13
been demonstrated that GPR55 is an essential component to regulate the central
projection of nociceptive sensory afferents during embryonic development[15] in a
lysophosphatidyl-β-D-glucoside (LysoPtdGlc, 1)-dependent manner.
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Phosphatidyl-β-D-glucoside (PtdGlc), the metabolic precursor of 1, was first identified in
human umbilical cord red blood cells.[16] PtdGlc features exclusively arachidic (C20:0)
and stearic (18:0) acid esters at the secondary and primary hydroxy function of the
glycerol moiety respectively. Natural PtdGlc exists as a mixture of both stereoisomers at
the glycerol backbone,[17] and both isomers can be visualized by the monoclonal antibody,
DIM21.[18,19] PtdGlc has been reported as a marker for radial glial progenitors in the
embryonic rat cortex.[20]
To probe the structural requirements for GPR55 activation we envisaged a series of
LysoPtdGlc (1) analogs featuring modifications of the head group and sn-1 ester region
(Fig. 1). The biological activity of each lysolipid was evaluated against endogenously
expressed GPR55 in primary sensory neurons, while the conformational difference as
well as lysolipid-GPR55 interaction were assessed by molecular dynamics simulation.
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Results and discussion
Chemistry. Synthetic access to LysoPtdGlc and its analogs was established via an H-
phosphonate head group intermediate (Scheme 1). Briefly, after swapping the acetyl
protecting groups of the readily accessible acetoxy ortho ester 6[17] to benzyl groups (7),
acid catalyzed ortho-ester opening afforded the desired H-phosphonate 8. To prevent
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ester migration and concomitant loss of enantiomeric purity at the glycerol sn-2 position
during condensation of 8 with a protected monoacylglycerol building block, we opted for
pivaloyl chloride activated condensation with S-(+)-1,2-isopropylideneglycerol, followed
by immediate oxidation to the desired phosphate diester. Additionally, this approach
provides simplified access to LysoPtdGlc derivatives featuring various fatty acid (FA)
residues in the future. The isopropylidene protecting group was cleaved in the presence of
acetic acid, without affecting the glycosidic linkage, followed by ammonium mediated
removal of the sole remaining acetate group yielding intermediate 9. Subsequently, the
stearoyl residue was selectively introduced to the sole primary hydroxy function by
DMAP catalyzed carbodiimide coupling, followed by removal of the benzyl groups under
reducing conditions, yielded desired 1.
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In case of analog 4 and 5 the glycerol building block was assembled prior to
condensation with previously reported[21] β-H-phosphonate 12, as both, ether and amide
linked alkyl groups, are not prone to undergo migration under the employed conditions.
To this end, R-(-)-solketal (10) was condensed with octadecane bromide, followed by
+
Amberlyst H mediated cleavage of the acetal protection. Subsequent tritylation of the
primary hydroxy function, followed by benzoyl protection of the sn-2 hydroxy function
at elevated temperature and acid mediated removal of the trityl group afforded desired
glycerol building block 11. In case of 5, (R)-3-aminopropane-1,2-diol (13) was
condensed with stearoyl chloride, followed by an analogous tritylation, benzoylation and
acid treatment sequence as above, yielding glycerol building block 14. Subsequently,
each of the glycerol building blocks was condensed with 12, affording the desired
phosphate diesters. Final deprotection was achieved by hydrazine treatment, yielding 4
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and 5. The galacto configured analog 2 was accessed as described previously[15,19]
utilizing a chemo-enzymatic approach.
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Biological Results. Next we assessed the biological activity of synthetic LysoPtdGlc (1)
and its analogs 2, 4, and 5. Cell-based receptor activation assays utilising heterologous
over-expression of GPCRs may produce data that are difficult to interpret.[21] Therefore
we tested our synthetic compounds by axon turning assay[22] with dissociated primary
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sensory neurons derived from chicken embryonic dorsal root ganglion (Fig. 2). These
cells endogenously express GPR55 and show a strong negative chemotropic response to
LysoPtdGlc,[15] providing a simple biological readout of activation of GPR55 and
associated downstream pathways. As expected, our synthetic LysoPtdGlc (1) induced the
same chemorepulsion response as we previously reported for natural LysoPtdGlc (Fig. 3).
Likewise, ether-LysoPtdGlc (4) also induced negative turning, whilst amide-LysoPtdGlc
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(5) and LysoPtdGal (2) did not cause growth cone turning, suggesting these compounds
do not activate the GPR55 signalling cascade. To ensure that the observed ether-
LysoPtdGlc (4) activity is GPR55-dependent, neuronal axons were challenged with 4 in
turning assays where ML193, a specific GPR55 inhibitor,[23] was added to the culture
medium. Treating neurons with ML193 abolished ether-LysoPtdGlc (4)-induced
chemorepulsion (Fig. 3) to the same degree we observed with LysoPtdGlc (1, Fig. S4),
demonstrating the involvement of the GPR55 signalling cascade. The ability of
LysoPtdIns (3), featuring an arachidonic acid on the sn-1 position, to induce axon turning
was not surprising as LysoPtdIns, featuring a stearic acid at sn-1 position, has been
reported previously[12,13] as a GPR55 ligand. However, since GPR55-independent cellular
signalling by LysoPtdIns has been reported[24,25] we investigated this in our turning assay
by treating neurons with ML193 before challenging with LysoPtdIns (3). Treatment with
M193 did not affect the repulsive turning response induced by LysoPtdIns (3), strongly
indicating that LysoPtdIns (3)-induced axon turning occurs via a GPR55-independent
mechanism. This corroborates our previous data[15] demonstrating the ability of
LysoPtdIns to induce axon turning in gpr55 knockout neurons. In that case, LysoPtdIns
featured a saturated stearic acid at sn-1 position. Taken together, LysoPtdIns-induced
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axon turning is not only independent of the GPR55 signalling cascade, but also seemingly
indifferent to small variations in FA length and saturation.
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Molecular Dynamics Simulation. To date, structural information on GPR55 is limited
to homology models.[26,27,28] Utilizing iTasser threading service[29] and previously
suggested alignment,[26,27] a GPR55 homology model based on the human δ-opioid,
human adenosine A2A and CXCR4 receptor was established. To retain the active R*
structure of the intracellular loop region during dynamics simulations, helix 5 (H5) of
Gα was inserted on the cytosolic side of GPR55, based on adenosine A2A receptor co-
13
[30]
crystalized with Gα . The assembly was embedded in a raft like membrane,
S
parameterised and equilibrated in the presence of LysoPtdIns featuring a saturated stearic
acid, while gradually reducing molecular restraints. Next, the computer model of PtdGlc,
LysoPtdGlc (1), LysoPtdGal (2), ether-LysoPtdGlc (4) and amide-LysoPtdGlc (5) were
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created in the CHARMM36 force field. No new parameters were required for the
anomeric β-phosphate moiety as suitable parameters have been reported previously.[31]
Additionally, the preferential phosphate geometry of model PtdGlc matched previously
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reported NMR data in hydrophobic and increasingly hydrated environments.[32,33]
Each ligand was subjected to a total of 70 ns (50 ns equilibration and 20 ns production
run) unrestrained molecular dynamics (MD) simulation at 37°C, utilizing identical
starting positions. The overall structure of GPR55 after production run as well as close
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contact of LysoPtdGlc (1) with key amino acids, such as K802.60 and E983.29, was
homologous to previously reported models (Fig. 4). Ligand-GPR55 interaction was
assessed based on the insertion depth of the FA terminal methyl group and the
carbohydrate head group of the ligand (Fig. 5A). LysoPtdGlc (1) not only moved closer
to the membrane centre compared to its initial position but also significantly extended its
FA tail towards the centre of GPR55. ether-LysoPtdGlc (4) exhibited a similar vertical
movement towards the membrane centre compared to LysoPtdGlc (1), albeit to a lesser
extent. In contrast, the head group of amide-LysoPtdGlc (5) and LysoPtdIns as well as
LysoPtdGal (5) as a whole moved in the opposite direction towards the extracellular
space. In all cases, the previously[28] identified key residues for GPR55 activation, K802.60
and E983.29, formed a stable salt bridge and remained in close contact with the phosphate
moiety of all ligands (data not shown). Taken together, ligands which settled closer to the
membrane centre correlate well with the above reported GPR55-dependent axon turning
activity.
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While inositol phosphate (InsP) and glucose phosphate (GlcP) head groups share
identical sum formula as well as the same number and equatorial orientation of hydroxy
groups, their biophysical properties and phosphate geometry, such as the dihedral angle φ,
are markedly different.[32] For example, the InsP head group in a phospholipid bilayer as
well as fully solubilised as lysolipid in water exhibit two distinct maxima of φ at -85° and
-145° with a ratio of 3:1 (Fig. 5B). Under the same conditions, GlcP head group featuring
lipids strongly favour only a single φ of -85°. Inside the GPR55 ligand binding pocket
LysoPtdGlc (1) retains its preferred geometry. In contrast, LysoPtdIns exhibits an
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increased population of its less favoured dihedral angle, associated with an energy
penalty, to adapt to the geometrical requirements of the ligand binding pocket, inferring
at least a reduced binding affinity.
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To date, LysoPtdIns has been primarily associated with intracellular signalling, while
LysoPtdGlc (1) has been demonstrated to act as an intercellular signalling molecule.[15]
Consequently, each lysolipid likely prefers a different entry direction, such as lateral via
the lipid bilayer or directly from the extracellular matrix, to facilitate GPR55 binding.
Throughout the simulation, LysoPtdGlc (1) and ether-LysoPtdGlc (2) retained the
initially closed GPR55 conformation (Fig. 6A, 6B). In contrast, ligand movement
towards the extracellular space resulted in loss of interaction between the N-terminus and
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extra cellular loop 2 (Fig. 6A, C, D), as illustrated by the distance between D13 and K165.
Loss of D13-K165 interaction during simulation was also observed in the absence of the
C10-C260 disulfide bridge (data not shown) irrespective of the employed ligand. This is
in good agreement with the reported[28] 10-fold decrease in GPR55 activity of C10A and
C260A point mutants.
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Conclusion
The ligand-binding pocket of GPR55 favours lipids featuring a carbohydrate head group
with equatorial configuration at the hydroxy functions, as evidenced by the failure of both
LysoPtdGal (2) and previously reported LysoPtdMan[15] to activate GPR55 signalling.
The GPR55-specific activity of ether-LysoPtdGlc (4) we observed suggests that the FA
carbonyl function is not essential for receptor activation. On the other hand, the reduced
flexibility of the amide bond in amide-LysoPtdGlc (5) compared to an ester or ether bond
is sufficient to prevent GPR55 activation. These in vitro experimental results are in good
agreement with our MD simulations, especially the ability of LysoPtdGlc (1) and ether-
LysoPtdGlc to remain inserted in the putative GPR55 binding pocket. The low polarity of
the binding pocket had little effect on the LysoPtdGlc (1) head group conformation. In
contrast to lipids with GlcP head group, the InsP head group has been demonstrated to
adopt a radically altered phosphate conformation in a hydrophobic environment
compared to a hydrophilic environment.[32] This intrinsic tendency of the LysoPtdIns (3)
head group consequently reduces the probability of LysoPtdIns-GPR55 interaction and
may explain why the growth cone turning activity induced by LysoPtdIns that we
observed is not dependent on GPR55.
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The metabolic precursor of LysoPtdIns, phosphatidylinositol, is predominantly associated
with the intracellular leaflet of the plasma membrane.[34] Conversely, the metabolic
precursor of LysoPtdGlc (1), PtdGlc, is exclusively localized to the extracellular leaflet of
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[35]
the plasma membrane . This distinct difference in membrane localization, together with
the orientation of the ligand entry port toward the extracellular space and the
demonstrated ability of LysoPtdGlc (1) to act as intercellular signal in vivo[15] strongly
point toward LysoPtdGlc as the specific endogenous ligand of GPR55.
Methods
Chemical synthesis.
Reagents and instrumentation. All solvents and reagents were purchased from
commercial suppliers as reagent grade and used as is. Reactions with anhydrous solvents
(purchased from Kanto Chemical Co., Inc) were performed under nitrogen atmosphere.
Flash column chromatography was performed with Kanto Chemical Co., Ltd. silica gel
6
0 N (40-100 mesh) and indicated solvent systems, while analytical thin layer
chromatography was performed on Merck silica gel 60 F256 plates Mass spectra were
recorded either on a Shimazu AXIMA-Performance or a Jeol AccuTOF JMST700LCK
utilizing TFA as an internal standard for high resultion spectral data. NMR spectra were
obtained either on a JEOL EX-400, JEOL ECX-400, JEOL ECX-500 or JEOL ECA-600
NMR spectrometer in the indicated solvents or solvent mixtures at 25 °C, utilizing
residual non-deuterated solvent signals as chemical shift references.
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Synthetic procedure for LysoPtdGlc (1). 1,2-tert-butyl orthoacetyl-3,4,6-tri-O-benzyl-
β-D-glucopyranose (7). 1,2-tert-butyl orthoacetyl-3,4,6-tri-O-acetyl-β-D-glucopyranose
(6) (10.0 g, 24.7 mmol) was dissolved in an anhydrous tetrahydrofuran/methanol mixture
1
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(4:1, 125 mL), treated with methanolic sodium methoxide solution (1M, 3.0 mL) at RT
and stirred at 45 °C for 3 h. The reaction mixture was concentrated under reduced
pressure, suspended in anhydrous N,N-dimethylformamide (100 mL) and treated with
NaH (5.93 g, 148 mmol, 60% dispersion in Paraffin Liquid) and BnBr (17.7 mL, 148
mmol) on ice. Subsequently, the reaction mixture was stirred for 3 h and allowed to
gradually reach RT. Next the reaction was quenched with triethylamine, diluted with
ethyl acetate and washed with water. The organic phase was dried over magnesium
sulfate and concentrated under reduced pressure. The residue was purified by flash
chromatography on silica gel (hexane/ethyl acetate = 10:1) to give 7 (12.1 g, 22.0 mmol,
1
89% in 2 steps) as a white amorphous solid. H NMR (600 MHz, CDCl ): δ 7.36-7.17 (m,
3
1
5H, Bn Harom), 5.69 (d, 1H, J=4.8, H-1), 4.68 (d, 1H, J=12.0, Bn CH ), 4.58 (d, 1H,
2
J=12.0, Bn CH ), 4.53 (d, 2H, J=12.0, Bn CH ), 4.50 (d, 1H, J=12.0, Bn CH ), 4.41-4.40
2
2
2
(
m, 1H, H-2), 4.35 (d, 1H, J=12.0, Bn CH ), 4.12 (q, 6H, J=7.2, NEt CH ), 3.89 (dd,
2 3 2
J=3.0, J=3.0, H-3), 3.80-3.78 (m, 1H, H-5), 3.71 (dd, 1H, J=3.0, J =9.6, H-4), 3.67-3.61
m, 2H, H-6a/b), 1.75 (s, 3H, Ac CH ), 1.32 (s, 9H, tBu CH ), 1.26 (t, 9H, J=7.2, NEt
(
3
3
3
1
3
CH ) ppm; C NMR (150 MHz, CDCl ): δ 138.15, 137.98, 137.90 (3C, Bn Cipso),
3
3
128.45, 128.31, 127.89, 127.73, 127.56 (15C, Bn CH ), 120.93 (1C, Ac), 97.63 (1C, C-1),
2
7
7
7.84 (1C, C-3), 75.27 (1C, C-4), 74.75 (1C, tBu Cquart), 74.28 (1C, C-2), 73.34, 72.48,
1.82 (3C, Bn CH ), 70.22 (1C, C-5), 69.25 (1C, C-6), 60.38 (3C, NEt CH ), 30.07 (3C;
2
3
2
tBu CH ), 25.60 (1C, Ac CH ), 14.20 (3C; NEt CH ) ppm.
3
3
3
3
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(
2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl)
hydrogenphosphonate
orthoacetyl-3,4,6-tri-O-benzyl-β-D-
triethylammonium
salt
(8).
1,2-tert-butyl
glucopyranose (2) (4.56 g, 8.35 mmol) was suspended in anhydrous tetrahydrofuran and
treated with phosphonic acid solution (0.5 M in anhydrous tetrahydrofuran, 4.11 g, 50.1
mmol) at RT. The mixture was stirred at RT for 5 minutes, quenched with triethylamine
0
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(16.5 ml, 117 mmol) on ice and stirred for 1 h. The resulting mixture was concentrated
under reduced pressure. The residue was purified by flash chromatography on silica gel
1
(CHCl /MeOH/NEt = 15:1:0.16) to give 8 (4.52 g, 6.88 mmol, 82%) as a syrup. H
3
3
NMR (600 MHz, CDCl ): δ 7.32-7.18 (m, 15H, Bn Harom), 6.94 (d, 1H, J=640.2, HP),
3
5.17 (dd, 1H, J=7.8, J=9.0, H-1), 4.99 (dd, 1H, J=7.8, J=9.0, H-2), 4.79 (d, 1H, J=11.4,
Bn CH ), 4.79 (d, 1H, J=11.4, Bn CH ), 4.66 (d, 1H, J=11.4, Bn CH ), 4.57 (d, 1H,
2
2
2
J=11.4, Bn CH ), 4.57 (d, 1H, J=11.4, Bn CH ), 4.49 (d, 1H, J=11.4, Bn CH ), 3.76 (dd,
2
2
2
1H, J=9.0, J =9.0, H-4), 3.75-3.73 (m, 2H, H-6a/b), 3.69 (dd, 1H, J=9.0, J=9.0, H-3),
.60-3.57 (m, 1H, H-5), 2.94 (q, 6H, J=7.2, NEt CH ), 1.96 (s, 3H, Ac CH ), 1.24 (t, 9H,
3
3
2
3
1
3
J=7.2, NEt CH ) ppm; C NMR (150 MHz, CDCl ): δ 169.92 (1C, Ac CO) 138.19,
3
3
3
1
38.11, 137.95 (3C, Bn Cipso), 128.43, 128.35, 128.02, 127.87, 127.84, 127.73, 127.62
15C, Bn Carom), 95.18 (d, 1C, J=4.2, C-1), 82.82 (1C, C-3), 77.76 (1C;,C-4), 75.17 (1C,
C-5), 75.04, 75.02 (2C, Bn CH ), 73.90 (d, 1C, J=7.2, C-2), 73.38 (1C, Bn CH ), 68.56
(
2
2
(1C, C-6), 45.42 (3C; NEt CH ), 21.01 (1C, Ac CH ), 8.67 (3C; NEt CH ) ppm;
3 2 3 3 3
-
MALDI-TOF MS calcd for C H O P [M-H] m/z 555.18, found 555.53.
2
9
32
9
(2,3-O-isopropylidene-sn-glycer-3-yl)
(2-O-acetyl-3,4,6-tri-O-benzyl-β-D-
glucopyranosyl) phosphate triethylammonium salt (S1). A mixture of (2-O-acetyl-3,4,6-
tri-O-benzyl-β-D-glucopyranosyl)hydrogenphosphonate triethylammonium salt (3) (2.0 g,
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.04 mmol) and (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (495 μL, 3.80 mmol) in
an anhydrous tetrahydrofuran (75 mL) was treated with pyridine (12.4 mL). The
condensation was initiated by addition of pivaloyl chloride (936 μL, 7.60 mmol) and the
resulting mixture was stirred for 15 minutes at RT. Subsequently, the reaction mixture
was treated with iodine solution (0.197 M iodine, 1.93 g, 7.60 mmol iodine in
pyridine/water = 95:5) at RT and stirred for 50 min. The reaction was quenched with
aqueous sodium thiosulfate solution (1M) and the organic layer was diluted with
chloroform. After phase separation, the organic phase was washed with brine, followed
by triethylamine-acetate buffer (pH = 8.5) and dried over magnesium sulfate. The
combined organic phase was concentrated under reduced pressure and purified by flash
chromatography on silica gel (CHCl /MeOH/NEt = 15:1:0.16) to give S1 (1.56 g, 1.98
1
1
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mmol, 65%) as a white amorphous solid. H NMR (600 MHz, CDCl ): δ 7.32-7.17 (m,
3
15H, Bn Harom) 5.19 (dd, 1H, J=8.4, J=8.4, H-1), 4.99 (dd, 1H, J=8.4, J=8.4, H-2), 4.79
(d, 2H, J=11.4, Bn CH ), 4.66 (d, 1H, J=11.4, Bn CH ), 4.57 (d, 1H, J=11.4, Bn CH ),
2 2 2
4
4
.56 (d, 1H, J=11.4, Bn CH ), 4.47 (d, 1H, J=11.4, Bn CH ), 4.28-4.26 (m, 1H, H-sn-2),
2 2
.01 (dd, 1H, J=6.6, J=8.4, H-sn-1a), 3.98-3.95 (m, 1H, H-sn-3a), 3.83 (dd, 1H, J=6.0,
J=8.4, H-sn-1b), 3.82-3.78 (m, 1H, H-sn-3b), 3.77-3.72 (m, 3H; H-4, H-6a/b), 3.70 (dd,
J=8.4, J=8.4, H-3), 3.60-3.58 (m, 1H; H-5), 2.97 (q, 6H, J=7.2, NEt CH ), 1.98 (s, 3H,
3
2
Ac CH ), 1.36 (s, 3H, iPr CH ), 1.31 (s, 3H, iPr CH ), 1.23 (t, 9H, J=7.2, NEt CH ) ppm;
3
3
3
3
3
1
3
C NMR (150 MHz, CDCl ): δ 170.02 (1C, Ac CO), 138.22, 138.14, 138.00 (3C, Bn
3
Cipso), 128.43, 128.35, 128.01, 127.85, 127.68, 127.61 (15C, Bn Carom), 96.05 (d, 1C,
J=4.4, C-1), 82.91 (1C, C-3), 77.76 (1C, C-4), 75.91, 74.99 (2C, Bn CH ), 74.88 (1C, C-
2
5), 74.74 (d, 1H, J=10.1, C-sn-2), 73.82 (d, 1C, J=8.6, C-2), 73.33 (1C, Bn CH2),
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8.68(1C, C-6), 67.04 (1C, C-sn-1), 5.85 (d, 1C, J=5.9, C-sn-3), 45.4 (3C, NEt CH ),
3
2
6.86 (1C, iPr CH ), 25.39 (1C, iPr CH ), 21.10 (1C, Ac CH ), 8.47 (3C, NEt CH ) ppm;
3
3
3
3
3
-
MALDI-TOF MS calcd for C H O P [M-H] m/z 685.24, found 685.60.
3
5
42 12
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(sn-glycer-3-yl) (3,4,6-tri-O-benzyl-β-D-glucopyranosyl)phosphate triethylammonium
salt (9). (2,3-O-isopropylidene-sn-glycer-3-yl) (2-O-acetyl-3,4,6-tri-O-benzyl-β-D-
glucopyranosyl) phosphate triethylammonium salt (S1) (917 mg, 1.16 mmol) was
dissolved in 50% acetic acid aqueous solution (10 mL) and stirred at RT for 5 h. The
crude mixture was concentrated under reduced pressure and the residue was dissolved in
tetrahydrofuran (10 mL). The slurry was treated with aqueous ammonia (20 mL) and
sonicated for 3 h, prior to concentration under reduced pressure. Subsequently, the
solvent was removed under reduced pressure and the residue was purified by flash
chromatography on silica gel (CHCl /MeOH/NEt = 5:1:0.06) yielding 9 (424 mg, 0.60
3
3
1
mmol, 51% yield). H NMR (600 MHz, MeOH-d ): δ 7.37-7.13 (m, 15H, Bn Harom),
4
4
.99 (d, 1H, J=11.4, Bn CH ), 4.93 (t, 1H, J=7.8, J =7.8 Hz, H-1), 4.77 (d, 1H, J=11.4,
2
Bn CH ), 4.75 (d, 1H, J=11.4, Bn CH ), 4.59 (d, 1H, J=11.4, Bn CH ), 4.52 (d, 1H,
2
2
2
J=11.4, Bn CH ), 4.49 (d, 1H, J=11.4, Bn CH ), 4.04-4.00 (m, 1H, H-sn-3a), 3.98-3.94
2
2
(m, 1H, H-sn-3b), 3.80-3.77 (m, 1H, H-sn-2), 3.75-3.70 (m, 2H, H-6a/b), 3.63-3.60 (m,
1
3
H, H-sn-1a), 3.58-3.53 (m, 4H; H-3, H-4, H-5, H-sn-1b), 3.46 (t, 1H, J=7.8, J=7.8, H-2),
1
3
.16 (q, 6H, J=7.2 Hz, NEt CH ), 1.29 (t, 9H, J=7.2, NEt CH ) ppm; C NMR (150
3
2
3
3
MHz, MeOH-d ): δ 140.24, 139.73, 139.47 (3C, Bn- Cipso), 129.39, 129.24, 129.20,
4
1
29.02, 128.89, 128.72, 128.60, 128.51 (15C, Bn Carom), 99.82 (d, 1C, J=5.7, C-1),
6.20 (1C, C-3), 78.46 (1C, C-4), 76.55 (d, 1C, J=8.6, C-2), 76.34 (1C, C-5), 76.23,
8
75.78, 74.40 (3C, Bn CH ), 72.57 (d, 1C, J=7.2, C-sn-2), 69.77 (1C, C-6), 67.97 (d, 1C,
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J=5.9, C-sn-1), 47.66 (3C, NEt CH ), 9.17 (3C, NEt CH ) ppm; MALDI-TOF MS calcd
3
2
3
3
-
for C H O P [M-H] m/z 603.20, found 603.61.
3
0
36 11
(1-O-stearyl-sn-glycer-3-yl)
(3,4,6-tri-O-benzyl-β-D-glucopyranosyl)phosphate
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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triethylammonium salt (S2). A solution of (sn-glycer-3-yl) (3,4,6-tri-O-benzyl-β-D-
glucopyranosyl)phosphate triethylammonium salt (4) (121 mg, 0.17 mmol), stearic acid
(51.3 mg, 0.18 mmol) and N,N’-dimethylpyridin-4-amine (6.3 mg, 0.052 mmol) in
anhydrous dichloromethane (6 mL) was treated with N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide hydrochloride (32.8 mg, 0.17 mmol) at 0 °C. After 6 h at RT, the
reaction mixture was quenched with methanol and concentrated under reduced pressure.
The residue was purified first by flash chromatography on silica gel (CHCl /MeOH/NEt
3
3
=
20:1:0.02) followed by gel filtration (LH-20, CHCl /MeOH/NEt = 20:1:0.02) to give
3
3
1
S2 (95.6 mg, 0.098 mmol, 57% yield) as a white waxy solid. H NMR (600 MHz,
CDCl ): δ 7.39-7.13 (m, 15H; Bn Harom), 5.03 (d, 1H, J=11.4, Bn CH ), 5.0 (t, 1H,
3
2
J=7.8, J=7.8, H-1), 4.82 (d, 1H, J=11.4, Bn CH ), 4.80 (d, 1H, J=11.4, Bn CH ), 4.55 (d,
2
2
1
H, J=11.4, Bn CH ), 4.49 (d, 2H, J=11.4, Bn CH ), 4.15-4.07 (m, 3H, H-sn-1a/b, H-sn-
2 2
3
a), 4.01-3.97 (m, 2H, H-sn-2, H-sn-3b), 3.72-3.65 (m, 2H, H-6a/b), 3.63-3.62 (m, 2H,
H-2, H-5), 3.57-3.52 (m, 2H, H-3, H-4), 3.02 (q, 6H, J=7.2, NEt CH ), 2.30-2.26 (m, 2H,
3
2
Stea CH ), 1.63-1.56 (m, 2H, Stea CH ), 1.28 (t, 9H, J=7.2, NEt CH ), 1.27-1.24 (m,
2
2
3
3
1
3
2
8H, Stea CH ), 0.878 (t, 3H, J=7.2, Stea CH ) ppm; C NMR (150 MHz, CDCl ): δ
2 3 3
173.80 (1C, Stea CO), 138.89, 138.15, 137.95 (3C, Bn Cipso), 128.36, 128.30, 128.02,
1
8
27.98, 127.93, 127.70, 127.67, 127.50 (15C, Bn Carom), 96.38 (d, 1C, J=4.7, C-1),
4.81 (1C, C-5), 77.14 (1C, C-3), 75.69 (d, 1C, J=5.9, C-2), 75.22 (1C, C-4), 75.13,
74.96, 73.36 (3C, Bn CH ), 69.13 (d, 1C, J=2.9, C-sn-2), 68.96 (1C, C-6), 67.83 (d, 1C,
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J=5.7, C-sn-3), 64.93 (1C, C-sn-1), 45.64 (3C, NEt CH ), 34.41, 34.16, 31.93, 29.71,
3
2
2
9.67, 29.54, 29.51, 29.38, 29.32, 29.26, 29.19, 25.07, 24.90, 22.70, 14.13 (17C, Stea),
-
8.55 (3C, NEt CH ) ppm; MALDI-TOF MS calcd for C H O P [M-H] m/z 869.46,
3
3
48 70 12
0
1
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0
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found 869.73.
(1-O-stearyl-sn-glycer-3-yl)-β-D-glucopyranosyl phosphate triethylammonium salt (1).
(1-O-stearyl-sn-glycer-3-yl) (3,4,6-tri-O-benzyl-β-D-glucopyranosyl)phosphate
triethylammonium salt (S2) (90.0 mg, 0.093 mmol) was suspended in ethanol (18 mL)
and stirred for 16 h at 35 °C in the presence of Pd/C (102.2 mg) under a hydrogen
atmosphere. The reaction mixture was filtered over Celite and the solvent was removed
under reduced pressure. The residue was subjected to flash chromatography on silica gel
(
IATROBEZES 6RS-806, CHCl /MeOH/NEt = 1:1:0.02) yielding 1 (22.1 mg, 0.031
3 3
1
mmol, 34% yield) as a white waxy solid. H NMR (600 MHz, DMSO-d ): δ 5.85 (bs,1H,
6
Glc OH-2), 5.66 (bs, 1H, Gly OH-sn-2), 4.91 (m, 2H, Glc OH-3/OH-4), 4.67 (t, 1H,
J=7.8, Glc H-1), 4.56 (bs, 1H, Glc OH-6), 3.99 (dd, 1H, J=4.2, J=10.8, Gly H-sn-1a),
3
.90 (dd, 1H, J=6.0, J=11.4, Gly H-sn-1b), 3.76 (m, 1H, Gly H-sn-2), 3.72 (m, 2H, Gly
H-sn-3a/b), 3.65 (m, 1H, Glc H-6a), 3.44 (m, 1H, Glc H-6b), 6.28 (m, 1H, Glc H-3) 3.08
m, 1H, Glc H-5) 3.05 (m, 6H, NEt CH ) 3.03 (m, 1H, Glc H-4) 3.00 (t, 1H, J=8.4, Glc
(
3
2
H-2), 2.29 (t, 2H, J=7.5, Stea CH ), 1.51 (m, 2H, Stea CH ), 1.26 (m, 28H, Stea CH ),
2
2
2
1
3
1
.17 (t, 9H, J=7.2, NEt CH ), 0.854 (t, 3H, J=6.9, Stea CH ) ppm; C NMR (150 MHz,
3 3 3
DMSO-d ): δ 172.99 (1C, Stea CO), 97.78 (d, 1C, J=2.85, Glc C-1), 77.42 (1C, Glc C-5),
6
7
6.79 (1C, Glc C-3), 74.95 (d, 1C, J=4.35, Glc C-2), 69.75 (1C, Glc C-4), 68.20 (d, 1C,
J=4.35, Gly C-sn-2), 66.18 (d, 1C, J=5.7, Gly C-sn-3), 65.33 (1C, Gly C-sn-1), 61.03 (1C,
Glc C-6), 45.49 (3C, NEt CH ), 33.49, 31.34, 29.08, 28.97, 28.79, 28.76, 28.54, 24.50,
3
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2.15, 14.01 (17C, Stea), 8.57 (3C, NEt CH ) ppm; P NMR (600 MHz, DMSO-d ): δ -
3
3
6
.65 (1P) ppm.
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Synthetic
procedure
for
ether-LysoPtdGlc
(S3).
(4).
(R)-2,2-dimethyl-4-
((octadecyloxy)methyl)-1,3-dioxolane
(R)-(-)-2,2-Dimethyl-1,3-dioxolae-4-
methanol (10) (1.0 g, 7.57 mmol, 1.0 Eq.) was suspended in anhydrous DMF (50 mL)
and treated on ice with 1-bromooctadecane (2.84 mL, 2.78 g, 8.33 mmol, 1.1 Eq.). Next
the reaction mixture was heated to 30 °C, stirred for 15 min and treated twice with
sodium hydride (60% suspension in oil, 0.35 g, 8.7 mmol, 1.15 Eq.) during 4 h. The
reaction was quenched with sodium bicarbonate (saturated in water, 20 mL) and
extracted with chloroform (80 mL). The organic phase was washed with water (20 mL),
dried over sodium sulphate and the solvent was removed under reduced pressure. The
remaining slurry was purified by flash chromatography on silica gel (hexane/ethyl acetate
1
=
15:1) to give S3 (1.85 g, 4.8 mmol, 63% yield) as a white amorphous solid. H NMR
(
400 MHz, CDCl ): δ 4.26 (m, 1H, Gly H-sn-2), 4.05 (dd, 1H, J=8.3, J=6.3, Gly H-sn-1a),
3
3
1
.72 (dd, 1H, J=8.2, J=6.5, Gly H-sn-1b), 3.46 (n.r., 4H, Gly H-sn-3a/b, Stea H-1a/b),
.57 (n.r., 2H, Stea CH ), 1.42 (s, 3H, i-Pr CH ), 1.36 (s, 3H, i-Pr CH ), 1.25 (n.r., 30H,
2
3
3
1
3
Stea CH ), 0.87 (t, 3H, J=6.7, Stea CH ) ppm; C NMR (100 MHz, CDCl ): δ 109.6 (1C,
2
3
3
i-Pr Cq.), 75.1 (1C, Gly C-sn-2), 72.3 (1C, Gly C-sn-3), 72.2 (1C, Stea C-1), 67.3 (1C,
Gly C-sn-1), 32.4 (1C, Stea C-16), 30.2, 30.1, 30, 29.9, 29.8, 27.3 (14C, Stea), 26.5, 25.9
(
2C, i-Pr CH ), 23.2 (1C, Stea C-17), 14.6 (1C, Stea C-18) ppm.
3
(
S)-3-(octadecyloxy)propane-1,2-diol (S4). S3 (1.7 g, 4.4 mmol, 1.0 Eq.) was dissolved
in chloroform (20 mL) and methanol (20 mL), treated with washed Amberlyst IR-120 ion
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exchange resin (2 g) and stirred for 6h at RT. The reaction mixture was filtered over a
Celite plug and the solvent was removed under reduced pressure. The residue was
subjected to flash chromatography on silica gel (gradient of hexane/ethyl acetate = 2:1 to
0
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2
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0
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1
1
:1) yielding S4 (1.23g, 3.6 mmol, 81% yield) as a white waxy solid. H NMR (400 MHz,
CDCl ): δ 3.84 (m, 1H, Gly H-sn-2), 3.69 (ddd, 1H, J=11.2, J=7.2, J=3.5, Gly H-sn-1a),
3
3
1
.62 (ddd, 1H, J=11.4, J=7.1, J=5.4, Gly H-sn-1b), 3.46 (n.r., 4H, Gly H-sn-3a/b, Stea H-
a/b), 2.76 (d, 1H, J=5.1, Gly HO-sn-2), 2.37 (dd, 1H, J=6.7, J=5.5, Gly HO-sn-1), 1.55
1
3
(
n.r., 2H, Stea CH ), 1.24 (n.r., 30H, Stea CH2), 0.85 (t, 3H, J=5.8, Stea CH ) ppm; C
2
3
NMR (100 MHz, CDCl ): δ 72.8 (1C, Gly C-sn-2), 72.2 (1C, Stea C-1), 70.8 (1C, Gly C-
3
sn-1), 64.6 (1C, Gly C-sn-3), 32.4 (1C), 30.2 (1C, Stea C-16), 30.1 (1C), 30 (8C), 29.9
(1C), 29.8 (1C), 26.6 (1C), 23.2 (1C, Stea), 14.6 (1C, Stea C-18) ppm.
(S)-1-hydroxy-3-(octadecyloxy)propan-2-yl benzoate (11). S4 (0.5 g, 1.45 mmol, 1.0 Eq.)
and 4,4’-dimethyoxytrityl chloride (0.62 g, 1.7 mmol, 1.2 Eq.) were suspended in
anhydrous pyridine (5 mL). The mixture was gently heated until a clear solution formed
and stirred for 5 h at RT. Subsequently, N,N’-dimethylpyridin-4-amine (0.22 g, 1.8 mmol,
1
.25 Eq.) was added under Schlenk conditions, the reaction volume was increased with
anhydrous pyridine (5 mL) and the resulting mixture was stirred for 15min at RT prior to
treatment with benzoyl chloride (0.26 mL, 0.31 g, 2.2 mmol, 1.5 Eq.). After 24 h at RT
the reaction was quenched by addition of ethanol (1 mL) and the solvent was removed
under reduced pressure. The remaining slurry was twice resuspended in toluene (5 mL)
prior to solvent removal under reduced pressure. Finally, the mixture was dissolved in
chloroform (20 mL), extracted against HCl (5% in water, 10 mL), followed by extraction
against sodium bicarbonate (saturated in water, 5 mL) and brine (5 mL). The organic
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phase was dried over sodium sulphate and the solvent was removed under reduced
pressure to give a waxy, white solid.
The crude solid (1.1 g) was dissolved in dichloromethane (10 mL), treated with washed
Amberlyst IR-120 ion-exchange resin (2 g) and stirred for 2 h at RT. The ion-exchange
resin was filtered off via a Celite plug and the filtrate was extracted against sodium
bicarbonate (saturated in water, 10 mL) and water (10 mL). The resulting organic phase
was dried over sodium sulphate and the solvent was removed under reduced pressure.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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1
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8
9
0
Purification by flash chromatography on silica gel (hexane/ethyl acetate = 3:1) gave 11
1
(
0.49 g, 0.64 mmol, 44% yield over 3 steps) as a colorless oil. H NMR (500 MHz,
CDCl ): δ 8.07 (d, 2H, J=8.6, Bz Hortho), 7.57 (dd, 1H, J=7.4, J=7.4, Bz Hpara), 7.44
3
(dd, 2H, J=8., J=7.4, Bz Hmeta), 5.24 (m, 1H, Gly H-sn-2), 3.97 (dd, 1H, J=11.9, J=3.9,
Gly H-sn-3a), 3.94 (dd, 1H, J=12., J=4.6, Gly H-sn-3b), 3.79 (dd, 1H, J=10.6, J=4.9, Gly
H-sn-1a), 3.74 (dd, 1H, J=10.9, J=5.2, Gly H-sn-1b), 3.52 (dd, 1H, J=9.5, J=6.6, Stea
CH -1a), 3.48 (dd, 1H, J=9.2, J=6.9, Stea CH -1b), 1.57 (m, 4H, Stea CH2), 1.28 (n.r.,
2
2
1
3
2
1
1
6
2
3
8H, Stea CH ), 0.88 (t, 3H, J=7.2, Stea CH ) ppm; C NMR (125 MHz, CDCl ): δ
2 3 3
66.7 (1C, Bz CO), 133.5 (1C, Bz Cpara), 130.4 (1C, Bz Cipso), 130.1 (2C, Bz Cortho),
28.7 (2C, Bz Cmeta), 74.1 (1C, Gly C-sn-2), 72.3 (1C, Stea C-1), 70.5 (1C, Gly C-sn-1),
3.5 (1C, Gly C-sn-3), 32.3 (1C), 30.1 (10C), 29.9 (1C), 29.8 (1C), 29.7 (1C), 26.4 (1C),
3 (1C, Stea), 14.5 (1C, Stea C-18) ppm.
(2-O-benzoyl-1-O-stearyloxy-sn-glycer-3-yl)
(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)phosphate triethylammonium salt (S5). A solution of 11 (150 mg, 0.19
mmol, 1.0 Eq.) in anhydrous tetrahydrofurane (5 mL) was added rapidly to 2,3,4,6-tetra-
O-acetyl-β-D-glucopyranosylphosphonate (12) (126 mg, 0.25 mmol, 1.25Eq.) dissolved
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in anhydrous pyridine (0.8 mL) at 0 °C under Schlenk conditions. The resulting mixture
was immediately treated with pivaloyl chloride (60 μL, 0.49 mmol, 2.5 Eq.), the ice bath
was removed and the reaction heated to RT. After 5 min at RT, phosphonate oxidation
was induced by addition of iodine solution (0.197 M in pyridine/water = 95:5, 1.3 mL)
and the reaction mixture was stirred for 30 min at RT. The reaction was quenched with
aqueous sodium thiosulfate solution (1 M, 12 mL) and extracted with chloroform (35
mL). The organic phase was washed with triethylammonium hydrogen carbonate solution
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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0
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9
0
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2
3
4
5
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8
9
0
(1 M, 10 mL), dried over sodium sulphate and the solvent was removed under reduced
pressure. The residue was purified by flash chromatography on silica gel
(CHCl /MeOH/NEt = 25:1:0.1) to give S5 (125 mg, 0.13 mmol, 67% yield) as a
3
3
1
colorless oil. H NMR (400 MHz, CDCl ): δ 8.07 (d, 2H, J=8.3, Bz Hortho), 7.54 (dd, 1H,
3
J=7.8, J=6.8, Bz Hpara), 7.43 (dd, 2H, J=7.8, J=7.3, Bz Hmeta), 5.39 (n.r., 1H, Gly H-sn-
2), 5.30 (dd, 1H, J=8.1, J=7.8, Glc H-1), 5.20 (dd, 1H, J=9.6, J=9.2, Glc H-3), 5.10 (dd,
1
6
2
H, J=12.6, J=6.5, Glc H-4), 5.00 (dd, 1H, J=11.2, J=5.9, Glc H-2), 4.19 (n.r., 4H, Glc H-
a/b, Gly H-sn-3a/b), 3.78 (m, 1H, Glc H-5), 3.67 (d, 2H, J=4.6, Gly H-sn-1a/b), 3.41 (m,
H, Stea H-1a/b), 2.93 (q, 6H, J=7.2, NEt CH ), 2.03 (s, 3H, Ac CH ), 2.02 (s, 3H, Ac
3
2
3
CH ), 2.01 (s, 3H, Ac CH ), 1.98 (s, 3H, Ac CH ), 1.49 (n.r., 2H, Stea CH ), 1.24 (, 30H,
3
3
3
2
1
3
Stea CH ), 1.19 (t, 9H, J=7., NEt CH ), 0.87 (t, 3H, J=6.5, Stea CH ) ppm; C NMR
2
3
3
3
(
100 MHz, CDCl ): δ 170.7 (1C, Ac CO), 170.2 (1C, Ac CO), 169.8 (1C, Ac CO), 169.7
3
(1C, Ac CO), 166.2 (1C, Bz CO), 133.1 (1C, Bz Cpara), 130.6 (1C, Bz Cipso), 130 (2C,
Bz Cortho), 128.5 (2C, Bz Cmeta), 96.1 (d, 1C, J=16.6, Glc C-1), 73.4 (1C, Glc C-3),
7
3.2 (d, 1C, J=33.2, Gly C-sn-2), 72.3 (d, 1C, J=26.5, Glc C-2), 72.2 (1C, Glc C-5), 72
(1C, Stea C-1), 69.7 (1C, Gly C-sn-1), 68.6 (1C, Glc C-4), 65 (d, 1C, J=19.9, Gly C-sn-3),
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2.2 (1C, Glc C-6), 45.8 (3C, NEt CH ), 32.4, 30.2, 30.1, 29.9, 29.8, 27.8, 26.5 (16C,
3
2
Stea), 23.2 (1C, Ac CH ), 21.3 (1C, Ac CH ), 21.2 (1C, Ac CH ), 21.1 (1C, Ac CH ),
3
3
3
3
14.6 (1C, Stea C-18), 8.9 (3C, NEt CH ) ppm; MS (ESI, neg): calc. for C H O P [M-
3
3
42 66 16
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2
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–
HNEt ] m/z 857.41, found 857.36.
3
(2-O-benzoyl-1-O-stearyloxy-sn-glycer-3-yl)
β-D-glucopyranosylphosphate
triethylammonium salt (4). S5 (22 mg, 0.024 mmol, 1.0 Eq.) were dissolved in
chloroform (0.5 mL) and methanol (1.25 mL), treated with hydrazine (0.1 mL) and
stirred for 3.5 h at RT. Subsequently, the reaction mixture was increased with chloroform
(6.5 mL) and methanol (2.25 mL) and washed with triethylammonium
hydrogencarbonate solution (1 M, 10 mL), dried over sodium sulphate and the solvent
was removed under reduced pressure. The residue was purified by preparative thin layer
chromatography (CHCl /MeOH/NEt = 3:1:0.025) yielding 4 (12 mg, 0.017 mmol, 75%
3
3
1
yield) as a white waxy solid. H NMR (600 MHz, CDCl /MeOH-d =2:1): δ 4.82 (dd, 1H,
3
4
J=8.1, J=7.6, Glc H-1), 3.97 (dd, 1H, J=7.6, J=7.1, Gly H-sn-3a), 3.87 (n.r., 2H, Gly H-
sn-3b, Gly H-sn-2), 3.83 (dd, 1H, J=12.1, J=2.5, Glc H-6a), 3.58 (dd, 1H, J=12.1, J=7.1,
Glc H-6b), 3.41 (n.r., 4H, Gly H-sn-1a/b, Stea H-1a/b), 3.36 (dd, 1H, J=9.1, J=9.1, Glc
H-3), 3.33 (ddd, 1H, J=9.8, J=7.1, J=2.8, Glc H-5), 3.23 (dd, 1H, J=9.6, J=7.6, Glc H-4),
3
.21 (dd, 1H, J=9.1, J=7.6, Glc H-2), 2.98 (q, 6H, J=7.4, NEt CH ), 1.50 (m, 2H, Stea
3 2
CH ), 1.23 (t, 9H, J=7.3, NEt CH ), 1.20 (n.r., 30H, Stea), 0.82 (t, 3H, J=7.1, Stea CH )
2
3
3
3
3
1
ppm; P NMR (500 MHz, MeOH-d ): δ -0.26 (1P) ppm; HR-MS (ESI, neg): calc. for
4
–
C H O P [M-HNEt ] m/z 585.34037, found 585.33705.
2
7
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Synthetic
procedure
for
amide-LysoPtdGlc
(5).
(R)-N-(2,3-
dihydroxypropyl)stearamide (S6). A 1% solution of (S)-(-)-3-amino-1,2-propanediol (13)
(1.45 g, 15.9 mmol, 1.0 Eq) in methanol was treated with triethylamine (2.35 mL, 16.7
mmol, 1.05 Eq.) and cooled to 0 °C. Subsequently, stearoyl chloride (6 mL, 17.5 mmol,
0
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1
.1 Eq.) in THF (10 mL) was added drop-wise and the resulting reaction was stirred over
night while reaching RT. The precipitate was filtered off, washed twice with methanol
20mL each) and the solvent was removed under reduced pressure. The resulting residue
was suspended in hot ethanol, and upon reaching RT the mixture was further cooled to
(
4
°C. The resulting white precipitated was collected by filtration and was with cold
1
ethanol, yielding S6 (5.136 g, 14.4 mmol, 90% yield) as a white waxy solid. H NMR
(500 MHz, CDCl /MeOH-d =10:1): δ 3.72 (m, 1H, Gly H-sn-2), 3.50 (dd, 1H, J=11.5,
3 4
J=4.6, Gly H-sn-3a), 3.47 (dd, 1H, J=11.7, J=4.9, Gly H-sn-3b), 3.36 (dd, 1H, J=14.3,
J=4.6, Gly H-sn-1a), 3.31 (dd, 1H, J=13.7, J=6.3, Gly H-sn-1b), 2.26 (t, 2H, J=7.7, Stea
CH ), 1.59 (m, 2H, Stea CH ), 1.24 (n.r., 28H, Stea CH ), 0.84 (t, 3H, J=6.9, Stea CH )
2
2
2
3
1
3
ppm; C NMR (500 MHz, CDCl /MeOH-d =10:1): δ 176.6 (1C, Stea CO), 70.7 (1C,
3
4
Gly C-sn-2), 63.7 (1C, Gly C-sn-3), 42.8 (1C, Gly C-sn-1), 36.2, 32.2, 30, 29.9, 29.9,
9.7, 29.6, 29.6, 29.5, 26.1, 22.9 (16C, Stea), 14.3 (1C, Stea C-18) ppm.
R)-1-hydroxy-3-stearamidopropan-2-yl benzoate (14). S6 (2.09 g, 5.84 mmol, 1.0 Eq.)
2
(
and 4,4’-dimethyoxytrityl chloride (2.5 g, 7.0 mmol, 1.2 Eq.) were suspended in
anhydrous pyridine (30 mL). The mixture was gently heated until a clear solution formed
and stirred for 1 h at RT. Subsequently, N,N’-dimethylpyridin-4-amine (0.89 g, 7.3 mmol,
1
.25 Eq.) was added under Schlenk conditions and the resulting mixture was stirred for
15min at RT prior to treatment with benzoyl chloride (1.03 mL, 1.24 g, 8.76 mmol, 1.5
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Eq.). The resulting mixture was stirred for 30 min at RT, followed by 1 h at 80 °C. The
reaction was quenched by addition of ethanol (2 mL) and the solvent was removed under
reduced pressure. The remaining slurry was twice resuspended in toluene (10 mL) prior
to solvent removal under reduced pressure. Finally, the mixture was dissolved in
chloroform (50 mL), extracted against HCl (5% in water, 20 mL), followed by extraction
against sodium bicarbonate (saturated in water, 25 mL) and brine (15 mL). The organic
phase was dried over sodium sulphate and the solvent was removed under reduced
pressure to give a waxy, yellow solid.
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The crude solid (5.9 g) was dissolved in dichloromethane (30 mL), treated with washed
Amberlyst IR-120 ion-exchange resin (10 g) and stirred for 2 h at RT. The ion-exchange
resin was filtered off via a Celite plug and the filtrate was extracted against sodium
bicarbonate (saturated in water, 10 mL) and diluted brine (brine/water = 1:2, 15 mL). The
resulting organic phase was dried over sodium sulphate and the solvent was removed
under reduced pressure. Purification by flash chromatography on silica gel (hexane/ethyl
acetate = 3:1) gave 14 (1.15 g, 2.50 mmol, 43% yield over 3 steps) as a white, waxy solid.
1
H NMR (400 MHz, CDCl ): δ 8.04 (d, 2H, J=7.8, Bz Hortho), 7.59 (dd, 1H, J=7.6,
3
J=7.3, Bz Hpara), 7.45 (dd, 2H, J=7.8, J=7.6, Bz Hmeta), 5.89 (n.r., 1H, NH), 5.09 (m,
1
H, Gly H-sn-2), 3.72 (n.r., 5H, Gly H-sn-1a/b, H-sn-3a/b, OH), 2.25 (m, 2H, Stea CH2),
.62 (n.r., 4H, Stea CH ), 1.25 (n.r., 26H, Stea CH ), 0.88 (t, 3H, J=6.8, Stea CH ) ppm;
1
2
2
3
1
3
C NMR (400 MHz, CDCl ): δ 175.3 (1C, Stea CO), 160.4 (1C, Bz CO), 133.6 (1C, Bz
3
Cpara), 130 (1C, Bz Cipso), 129.9 (2C, Bz Cortho), 128.7 (2C, Bz Cmeta), 74 (1C, Gly
C-sn-2), 60.8 (1C, Gly C-sn-3), 39.3 (1C, Gly C-sn-1), 37.1, 32.4, 30.2, 29.9, 29.8, 29.8,
29.7, 26.2, 23.2 (16C, Stea), 14.6 (1C, Stea C-18) ppm.
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(
2-O-benzoyl-1-N-stearamido-sn-glycer-3-yl)
(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)phosphate triethylammonium salt (S7). A solution of 14 (60 mg, 0.13
mmol, 1.0 Eq.) in anhydrous tetrahydrofurane (3 mL) was added rapidly to 2,3,4,6-tetra-
O-acetyl-β-d-glucopyranosylphosphonate (12) (83 mg, 0.16 mmol, 1.25 Eq.) dissolved in
anhydrous pyridine (0.5 mL) at 0 °C under Schlenk conditions. The resulting mixture was
immediately treated with pivaloyl chloride (41 μL, 0.33 mmol, 2.5 Eq.), the ice bath was
removed and the reaction heated to RT. After 5 min at RT, phosphonate oxidation was
induced by addition of iodine solution (0.197 M in pyridine/water = 95:5, 1.3 mL) and
the reaction mixture was stirred for 30 min at RT. The reaction was quenched with
aqueous sodium thiosulfate solution (1 M, 10 mL) and extracted with chloroform (30
mL). The organic phase was washed with triethylammonium hydrogen carbonate solution
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(1 M, 10 mL), dried over sodium sulphate and the solvent was removed under reduced
pressure. The residue was purified by flash chromatography on silica gel
(CHCl /MeOH/NEt = 25:1:0.02) to give S7 (123 mg, 0.13 mmol, 97% yield) as a
3
3
1
slightly yellow oil. H NMR (400 MHz, CDCl ): δ 8.00 (d, 2H, J=8.3, Bz Hortho), 7.54
3
(
m, 1H, Bz Hpara), 7.41 (dd, 2H, J=7.8, J=7.6, Bz Hmeta), 7.12 (m, 1H, NH), 5.29 (dd,
H, J=8.1, J=8.1, Glc H-1), 5.21 (n.r., 1H, Gly H-sn-2), 5.20 (dd, 1H, J=9.5, J=9.5, Glc
H-3), 5.07 (dd, 1H, J=9.8, J=9.5, Glc H-4), 5.00 (dd, 1H, J=9.5, J=8.1, Glc H-2), 4.21
n.r., 2H, Glc H-6a/b), 4.13 (n.r., 2H, Gly H-sn-3a/b), 3.80 (m, 1H, J=9.9, J=4.2, J=2.6,
Glc H-5), 3.67 (n.r., 2H, Gly H-sn-1a/b), 2.92 (q, 6H, J=7.2, NEt CH ), 2.17 (m, 2H,
1
(
3
2
Stea CH ), 2.05 (s, 3H, Ac CH ), 2.02 (s, 3H, Ac CH ), 2.01 (s, 3H, Ac CH ), 1.98 (s, 3H,
2
3
3
3
Ac CH ), 1.59 (m, 2H, Stea CH ), 1.25 (n.r , 28H, Stea CH ), 1.21 (t, 9H, J=7.3, NEt
3
3
2
2
1
3
CH ), 0.87 (t, 3H, J=6.8, Stea CH ) ppm; C NMR (100 MHz, CDCl ): δ 173.7 (1C, Stea
3
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CO), 170.7 (1C, Ac CO), 170.1 (1C, Ac CO), 169.8 (1C, Ac CO), 167.2 (1C, Ac CO),
1
66.1 (1C, Bz CO), 133.3 (1C, Bz Cpara), 130.2 (1C, Bz Cipso), 130 (2C, Bz Cortho),
128.5 (2C, Bz Cmeta), 96.3 (d, 1C, J=19.9, Glc C-1), 73.2 (1C, Glc C-3), 72.4 (1C, Glc
C-5), 72.3 (d, 1C, J=26.5, Glc C-2), 72.2 (d, 1C, J=29.8, Gly C-sn-2), 68.6 (1C, Glc C-4),
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4.2 (d, 1C, J=26.5, Gly C-sn-3), 62.2 (1C, Glc C-6), 62.2, 46.1 (3C, NEt CH ), 39.5, 39
3 2
(
1C, Gly C-sn-1), 37.3, 32.4, 30.2, 30, 29.9, 29.8, 26.3 (16C, Stea), 23.2 (1C, Ac CH3),
2
1.3 (1C, Ac CH ), 21.2 (1C, Ac CH ), 21.1 (1C, Ac CH ), 14.6 (1C, Stea C-18), 9.5 (3C,
3 3 3
–
NEt CH ) ppm; MS (ESI, neg): calc. for C H NO P [M-HNEt ] m/z 870.40, found
3
3
42 65
16
3
8
70.44.
(2-O-benzoyl-1-N-stearamido-sn-glycer-3-yl)
β-d-glucopyranosylphosphate
triethylammonium salt (5). S7 (50 mg, 0.05 mmol, 1.0 Eq.) were dissolved in anhydrous
methanol (5 mL) and treated twice with sodium methoxide (each 6 mg, 0.1 mmol, 2.0
Eq.) while being stirred for 5 h at RT. Subsequently, the solvent was removed under
reduced pressure and the residue was purified by preparative thin layer chromatography
(
CHCl /MeOH/NEt = 4:1:0.025) yielding 5 (18 mg, 0.03 mmol, 59% yield) as a white
3 3
1
waxy solid. H NMR (600 MHz, CDCl /MeOH-d =2:1): δ 4.82 (dd, 1H, J=7.7, J=7.1,
3
4
Glc H-1), 3.89 (ddd, 1H, J=11., J=7.1, J=3.8, Gly H-sn-3a), 3.84 (n.r., 1H, Gly H-sn-3b),
.80 (dd, 1H, J=12.5, J=2.3, Glc H-6a), 3.78 (m, 1H, Gly H-sn-2), 3.60 (dd, 1H, J=12.1,
3
J=6.6, Glc H-6b), 3.36 (dd, 1H, J=9.3, J=8.8, Glc H-3), 3.31 (ddd, 1H, J=9.5, J=6.5, J=2.6,
Glc H-5), 3.37 (n.r., 2H, Gly H-sn-1a/b), 3.23 (dd, 1H, J=9.3, J=9.3, Glc H-4), 3.23 (dd,
1
1
H, J=9.3, J=8.2, Glc H-2), 3.08 (q, 6H, J=7.3, NEt CH ), 2.14 (t, 2H, J=7.7, Stea CH ),
3 2 2
.54 (m, 2H, Stea CH ), 1.28 (t, 9H, J=7.4, NEt CH ), 1.20 (n.r., 28H, Stea), 0.82 (t, 3H,
2
3
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