Preference for Glucose over Inositol Headgroup during Lysolipid

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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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ACS Chemical Neuroscience

Preference for glucose over inositol head group during lysolipid activation of G protein-coupled receptor 55

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 • structureactivity relationship • cannabinoid receptor related receptor

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 system[4]. GPR55 knockout (KO) mice have been reported to show defects in nociception[5] and in models of inflammation and neuropathic pain[6]. Early cell-based assays[7] 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 2arachidonoyl lysophosphatidylinositol, but not cannabinoids, induce ERK1/2 activation and increase intracellular Ca2+ via the Gα13-RhoA-ROCK cascade.[14] Recently it has 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.



Results and discussion Chemistry. Synthetic access to LysoPtdGlc and its analogs was established via an Hphosphonate 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. 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



and 5. The galacto configured analog 2 was accessed as described previously[15,19] utilizing a chemo-enzymatic approach.

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 (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 etherLysoPtdGlc (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



axon turning is not only independent of the GPR55 signalling cascade, but also seemingly indifferent to small variations in FA length and saturation.

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 Gα13 was inserted on the cytosolic side of GPR55, based on adenosine A2A receptor cocrystalized with GαS. The assembly was embedded in a raft like membrane,[30] 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 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



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



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.

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.

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 etherLysoPtdGlc 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.



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 the plasma membrane[35]. 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 60 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-benzylβ-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 (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, 89% in 2 steps) as a white amorphous solid. 1H NMR (600 MHz, CDCl3): δ 7.36-7.17 (m, 15H, Bn Harom), 5.69 (d, 1H, J=4.8, H-1), 4.68 (d, 1H, J=12.0, Bn CH2), 4.58 (d, 1H, J=12.0, Bn CH2), 4.53 (d, 2H, J=12.0, Bn CH2), 4.50 (d, 1H, J=12.0, Bn CH2), 4.41-4.40 (m, 1H, H-2), 4.35 (d, 1H, J=12.0, Bn CH2), 4.12 (q, 6H, J=7.2, NEt3 CH2), 3.89 (dd, 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 CH3), 1.32 (s, 9H, tBu CH3), 1.26 (t, 9H, J=7.2, NEt3 CH3) ppm;

13C

NMR (150 MHz, CDCl3): δ 138.15, 137.98, 137.90 (3C, Bn Cipso),

128.45, 128.31, 127.89, 127.73, 127.56 (15C, Bn CH2), 120.93 (1C, Ac), 97.63 (1C, C-1), 77.84 (1C, C-3), 75.27 (1C, C-4), 74.75 (1C, tBu Cquart), 74.28 (1C, C-2), 73.34, 72.48, 71.82 (3C, Bn CH2), 70.22 (1C, C-5), 69.25 (1C, C-6), 60.38 (3C, NEt3 CH2), 30.07 (3C; tBu CH3), 25.60 (1C, Ac CH3), 14.20 (3C; NEt3 CH3) ppm.



(2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl) triethylammonium

salt

(8).

1,2-tert-butyl

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hydrogenphosphonate orthoacetyl-3,4,6-tri-O-benzyl-β-D-

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 (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 (CHCl3/MeOH/NEt3 = 15:1:0.16) to give 8 (4.52 g, 6.88 mmol, 82%) as a syrup. 1H NMR (600 MHz, CDCl3): δ 7.32-7.18 (m, 15H, Bn Harom), 6.94 (d, 1H, J=640.2, HP), 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 CH2), 4.79 (d, 1H, J=11.4, Bn CH2), 4.66 (d, 1H, J=11.4, Bn CH2), 4.57 (d, 1H, J=11.4, Bn CH2), 4.57 (d, 1H, J=11.4, Bn CH2), 4.49 (d, 1H, J=11.4, Bn CH2), 3.76 (dd, 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), 3.60-3.57 (m, 1H, H-5), 2.94 (q, 6H, J=7.2, NEt3 CH2), 1.96 (s, 3H, Ac CH3), 1.24 (t, 9H, J=7.2, NEt3 CH3) ppm;

13C

NMR (150 MHz, CDCl3): δ 169.92 (1C, Ac CO) 138.19,

138.11, 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 CH2), 73.90 (d, 1C, J=7.2, C-2), 73.38 (1C, Bn CH2), 68.56 (1C, C-6), 45.42 (3C; NEt3 CH2), 21.01 (1C, Ac CH3), 8.67 (3C; NEt3 CH3) ppm; MALDI-TOF MS calcd for C29H32O9P [M-H]- m/z 555.18, found 555.53. (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-3,4,6tri-O-benzyl-β-D-glucopyranosyl)hydrogenphosphonate triethylammonium salt (3) (2.0 g,


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3.04 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 (CHCl3/MeOH/NEt3 = 15:1:0.16) to give S1 (1.56 g, 1.98 mmol, 65%) as a white amorphous solid. 1H NMR (600 MHz, CDCl3): δ 7.32-7.17 (m, 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 CH2), 4.66 (d, 1H, J=11.4, Bn CH2), 4.57 (d, 1H, J=11.4, Bn CH2), 4.56 (d, 1H, J=11.4, Bn CH2), 4.47 (d, 1H, J=11.4, Bn CH2), 4.28-4.26 (m, 1H, H-sn-2), 4.01 (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, NEt3 CH2), 1.98 (s, 3H, Ac CH3), 1.36 (s, 3H, iPr CH3), 1.31 (s, 3H, iPr CH3), 1.23 (t, 9H, J=7.2, NEt3 CH3) ppm; 13C

NMR (150 MHz, CDCl3): δ 170.02 (1C, Ac CO), 138.22, 138.14, 138.00 (3C, Bn

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 CH2), 74.88 (1C, C5), 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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68.68(1C, C-6), 67.04 (1C, C-sn-1), 5.85 (d, 1C, J=5.9, C-sn-3),45.25 (3C, NEt3 CH2), 26.86 (1C, iPr CH3), 25.39 (1C, iPr CH3), 21.10 (1C, Ac CH3), 8.47 (3C, NEt3 CH3) ppm; MALDI-TOF MS calcd for C35H42O12P [M-H]- m/z 685.24, found 685.60. (sn-glycer-3-yl) salt

(9).

(3,4,6-tri-O-benzyl-β-D-glucopyranosyl)phosphate

(2,3-O-isopropylidene-sn-glycer-3-yl)

triethylammonium

(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 (CHCl3/MeOH/NEt3 = 5:1:0.06) yielding 9 (424 mg, 0.60 mmol, 51% yield). 1H NMR (600 MHz, MeOH-d4): δ 7.37-7.13 (m, 15H, Bn Harom), 4.99 (d, 1H, J=11.4, Bn CH2), 4.93 (t, 1H, J=7.8, J =7.8 Hz, H-1), 4.77 (d, 1H, J=11.4, Bn CH2), 4.75 (d, 1H, J=11.4, Bn CH2), 4.59 (d, 1H, J=11.4, Bn CH2), 4.52 (d, 1H, J=11.4, Bn CH2), 4.49 (d, 1H, J=11.4, Bn CH2), 4.04-4.00 (m, 1H, H-sn-3a), 3.98-3.94 (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, 1H, 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), 3.16 (q, 6H, J=7.2 Hz, NEt3 CH2), 1.29 (t, 9H, J=7.2, NEt3 CH3) ppm;

13C

NMR (150

MHz, MeOH-d4): δ 140.24, 139.73, 139.47 (3C, Bn- Cipso), 129.39, 129.24, 129.20, 129.02, 128.89, 128.72, 128.60, 128.51 (15C, Bn Carom), 99.82 (d, 1C, J=5.7, C-1), 86.20 (1C, C-3), 78.46 (1C, C-4), 76.55 (d, 1C, J=8.6, C-2), 76.34 (1C, C-5), 76.23, 75.78, 74.40 (3C, Bn CH2), 72.57 (d, 1C, J=7.2, C-sn-2), 69.77 (1C, C-6), 67.97 (d, 1C,


Page 19 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J=5.9, C-sn-1), 47.66 (3C, NEt3 CH2), 9.17 (3C, NEt3 CH3) ppm; MALDI-TOF MS calcd for C30H36O11P [M-H]- m/z 603.20, found 603.61. (1-O-stearyl-sn-glycer-3-yl)


triethylammonium salt (S2). A solution of (sn-glycer-3-yl) (3,4,6-tri-O-benzyl-β-Dglucopyranosyl)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 (CHCl3/MeOH/NEt3 = 20:1:0.02) followed by gel filtration (LH-20, CHCl3/MeOH/NEt3 = 20:1:0.02) to give S2 (95.6 mg, 0.098 mmol, 57% yield) as a white waxy solid. 1H NMR (600 MHz, CDCl3): δ 7.39-7.13 (m, 15H; Bn Harom), 5.03 (d, 1H, J=11.4, Bn CH2), 5.0 (t, 1H, J=7.8, J=7.8, H-1), 4.82 (d, 1H, J=11.4, Bn CH2), 4.80 (d, 1H, J=11.4, Bn CH2), 4.55 (d, 1H, J=11.4, Bn CH2), 4.49 (d, 2H, J=11.4, Bn CH2), 4.15-4.07 (m, 3H, H-sn-1a/b, H-sn3a), 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, NEt3 CH2), 2.30-2.26 (m, 2H, Stea CH2), 1.63-1.56 (m, 2H, Stea CH2), 1.28 (t, 9H, J=7.2, NEt3 CH3), 1.27-1.24 (m, 28H, Stea CH2), 0.878 (t, 3H, J=7.2, Stea CH3) ppm;

13C

NMR (150 MHz, CDCl3): δ

173.80 (1C, Stea CO), 138.89, 138.15, 137.95 (3C, Bn Cipso), 128.36, 128.30, 128.02, 127.98, 127.93, 127.70, 127.67, 127.50 (15C, Bn Carom), 96.38 (d, 1C, J=4.7, C-1), 84.81 (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 CH2), 69.13 (d, 1C, J=2.9, C-sn-2), 68.96 (1C, C-6), 67.83 (d, 1C,



J=5.7, C-sn-3), 64.93 (1C, C-sn-1), 45.64 (3C, NEt3 CH2), 34.41, 34.16, 31.93, 29.71, 29.67, 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, NEt3 CH3) ppm; MALDI-TOF MS calcd for C48H70O12P [M-H]- m/z 869.46, found 869.73. (1-O-stearyl-sn-glycer-3-yl)-β-D-glucopyranosyl phosphate triethylammonium salt (1). (1-O-stearyl-sn-glycer-3-yl)


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, CHCl3/MeOH/NEt3 = 1:1:0.02) yielding 1 (22.1 mg, 0.031 mmol, 34% yield) as a white waxy solid. 1H NMR (600 MHz, DMSO-d6): δ 5.85 (bs,1H, 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, NEt3 CH2) 3.03 (m, 1H, Glc H-4) 3.00 (t, 1H, J=8.4, Glc H-2), 2.29 (t, 2H, J=7.5, Stea CH2), 1.51 (m, 2H, Stea CH2), 1.26 (m, 28H, Stea CH2), 1.17 (t, 9H, J=7.2, NEt3 CH3), 0.854 (t, 3H, J=6.9, Stea CH3) ppm; 13C NMR (150 MHz, DMSO-d6): δ 172.99 (1C, Stea CO), 97.78 (d, 1C, J=2.85, Glc C-1), 77.42 (1C, Glc C-5), 76.79 (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, NEt3 CH2), 33.49, 31.34, 29.08, 28.97, 28.79, 28.76, 28.54, 24.50,


Page 20 of 51

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22.15, 14.01 (17C, Stea), 8.57 (3C, NEt3 CH3) ppm; 31P NMR (600 MHz, DMSO-d6): δ 0.65 (1P) ppm.

Synthetic

procedure

for

((octadecyloxy)methyl)-1,3-dioxolane

ether-LysoPtdGlc (S3).

(4).

(R)-2,2-dimethyl-4-

(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 = 15:1) to give S3 (1.85 g, 4.8 mmol, 63% yield) as a white amorphous solid. 1H NMR (400 MHz, CDCl3): δ 4.26 (m, 1H, Gly H-sn-2), 4.05 (dd, 1H, J=8.3, J=6.3, Gly H-sn-1a), 3.72 (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), 1.57 (n.r., 2H, Stea CH2), 1.42 (s, 3H, i-Pr CH3), 1.36 (s, 3H, i-Pr CH3), 1.25 (n.r., 30H, Stea CH2), 0.87 (t, 3H, J=6.7, Stea CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 109.6 (1C, 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 CH3), 23.2 (1C, Stea C-17), 14.6 (1C, Stea C-18) ppm. (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



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 1:1) yielding S4 (1.23g, 3.6 mmol, 81% yield) as a white waxy solid. 1H NMR (400 MHz, CDCl3): 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), 3.62 (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 H1a/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 (n.r., 2H, Stea CH2), 1.24 (n.r., 30H, Stea CH2), 0.85 (t, 3H, J=5.8, Stea CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 72.8 (1C, Gly C-sn-2), 72.2 (1C, Stea C-1), 70.8 (1C, Gly Csn-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


Page 22 of 51

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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. Purification by flash chromatography on silica gel (hexane/ethyl acetate = 3:1) gave 11 (0.49 g, 0.64 mmol, 44% yield over 3 steps) as a colorless oil. 1H NMR (500 MHz, CDCl3): δ 8.07 (d, 2H, J=8.6, Bz Hortho), 7.57 (dd, 1H, J=7.4, J=7.4, Bz Hpara), 7.44 (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 CH2-1a), 3.48 (dd, 1H, J=9.2, J=6.9, Stea CH2-1b), 1.57 (m, 4H, Stea CH2), 1.28 (n.r., 28H, Stea CH2), 0.88 (t, 3H, J=7.2, Stea CH3) ppm;

13C

NMR (125 MHz, CDCl3): δ

166.7 (1C, Bz CO), 133.5 (1C, Bz Cpara), 130.4 (1C, Bz Cipso), 130.1 (2C, Bz Cortho), 128.7 (2C, Bz Cmeta), 74.1 (1C, Gly C-sn-2), 72.3 (1C, Stea C-1), 70.5 (1C, Gly C-sn-1), 63.5 (1C, Gly C-sn-3), 32.3 (1C), 30.1 (10C), 29.9 (1C), 29.8 (1C), 29.7 (1C), 26.4 (1C), 23 (1C, Stea), 14.5 (1C, Stea C-18) ppm. 3

(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-tetraO-acetyl-β-D-glucopyranosylphosphonate (12) (126 mg, 0.25 mmol, 1.25Eq.) dissolved



Page 24 of 51

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 (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 (CHCl3/MeOH/NEt3 = 25:1:0.1) to give S5 (125 mg, 0.13 mmol, 67% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.07 (d, 2H, J=8.3, Bz Hortho), 7.54 (dd, 1H, 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-sn2), 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, 1H, 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 H6a/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, 2H, Stea H-1a/b), 2.93 (q, 6H, J=7.2, NEt3 CH2), 2.03 (s, 3H, Ac CH3), 2.02 (s, 3H, Ac CH3), 2.01 (s, 3H, Ac CH3), 1.98 (s, 3H, Ac CH3), 1.49 (n.r., 2H, Stea CH2), 1.24 (, 30H, Stea CH2), 1.19 (t, 9H, J=7., NEt3 CH3), 0.87 (t, 3H, J=6.5, Stea CH3) ppm;

13C

NMR

(100 MHz, CDCl3): δ 170.7 (1C, Ac CO), 170.2 (1C, Ac CO), 169.8 (1C, Ac CO), 169.7 (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), 73.2 (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),


Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


62.2 (1C, Glc C-6), 45.8 (3C, NEt3 CH2), 32.4, 30.2, 30.1, 29.9, 29.8, 27.8, 26.5 (16C, Stea), 23.2 (1C, Ac CH3), 21.3 (1C, Ac CH3), 21.2 (1C, Ac CH3), 21.1 (1C, Ac CH3), 14.6 (1C, Stea C-18), 8.9 (3C, NEt3 CH3) ppm; MS (ESI, neg): calc. for C42H66O16P [MHNEt3]– m/z 857.41, found 857.36. (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 (CHCl3/MeOH/NEt3 = 3:1:0.025) yielding 4 (12 mg, 0.017 mmol, 75% yield) as a white waxy solid. 1H NMR (600 MHz, CDCl3/MeOH-d4=2:1): δ 4.82 (dd, 1H, 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 Hsn-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, NEt3 CH2), 1.50 (m, 2H, Stea CH2), 1.23 (t, 9H, J=7.3, NEt3 CH3), 1.20 (n.r., 30H, Stea), 0.82 (t, 3H, J=7.1, Stea CH3) ppm;

31P

NMR (500 MHz, MeOH-d4): δ -0.26 (1P) ppm; HR-MS (ESI, neg): calc. for

C27H54O11P [M-HNEt3]– m/z 585.34037, found 585.33705.



Synthetic

procedure

for

amide-LysoPtdGlc

Page 26 of 51

(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, 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 ethanol, yielding S6 (5.136 g, 14.4 mmol, 90% yield) as a white waxy solid. 1H NMR (500 MHz, CDCl3/MeOH-d4=10:1): δ 3.72 (m, 1H, Gly H-sn-2), 3.50 (dd, 1H, J=11.5, 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 CH2), 1.59 (m, 2H, Stea CH2), 1.24 (n.r., 28H, Stea CH2), 0.84 (t, 3H, J=6.9, Stea CH3) ppm;

13C

NMR (500 MHz, CDCl3/MeOH-d4=10:1): δ 176.6 (1C, Stea CO), 70.7 (1C,

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, 29.7, 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.) 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. 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. 1H

NMR (400 MHz, CDCl3): δ 8.04 (d, 2H, J=7.8, Bz Hortho), 7.59 (dd, 1H, J=7.6,

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, 1H, 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), 1.62 (n.r., 4H, Stea CH2), 1.25 (n.r., 26H, Stea CH2), 0.88 (t, 3H, J=6.8, Stea CH3) ppm; 13C

NMR (400 MHz, CDCl3): δ 175.3 (1C, Stea CO), 160.4 (1C, Bz CO), 133.6 (1C, Bz

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.



(2-O-benzoyl-1-N-stearamido-sn-glycer-3-yl)

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(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-tetraO-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 (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 (CHCl3/MeOH/NEt3 = 25:1:0.02) to give S7 (123 mg, 0.13 mmol, 97% yield) as a slightly yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.00 (d, 2H, J=8.3, Bz Hortho), 7.54 (m, 1H, Bz Hpara), 7.41 (dd, 2H, J=7.8, J=7.6, Bz Hmeta), 7.12 (m, 1H, NH), 5.29 (dd, 1H, 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, NEt3 CH2), 2.17 (m, 2H, Stea CH2), 2.05 (s, 3H, Ac CH3), 2.02 (s, 3H, Ac CH3), 2.01 (s, 3H, Ac CH3), 1.98 (s, 3H, Ac CH3), 1.59 (m, 2H, Stea CH2), 1.25 (n.r , 28H, Stea CH2), 1.21 (t, 9H, J=7.3, NEt3 CH3), 0.87 (t, 3H, J=6.8, Stea CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 173.7 (1C, Stea


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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), 166.1 (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), 64.2 (d, 1C, J=26.5, Gly C-sn-3), 62.2 (1C, Glc C-6), 62.2, 46.1 (3C, NEt3 CH2), 39.5, 39 (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), 21.3 (1C, Ac CH3), 21.2 (1C, Ac CH3), 21.1 (1C, Ac CH3), 14.6 (1C, Stea C-18), 9.5 (3C, NEt3 CH3) ppm; MS (ESI, neg): calc. for C42H65NO16P [M-HNEt3]– m/z 870.40, found 870.44. (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 (CHCl3/MeOH/NEt3 = 4:1:0.025) yielding 5 (18 mg, 0.03 mmol, 59% yield) as a white waxy solid. 1H NMR (600 MHz, CDCl3/MeOH-d4=2:1): δ 4.82 (dd, 1H, J=7.7, J=7.1, 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), 3.80 (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, 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, 1H, J=9.3, J=8.2, Glc H-2), 3.08 (q, 6H, J=7.3, NEt3 CH2), 2.14 (t, 2H, J=7.7, Stea CH2), 1.54 (m, 2H, Stea CH2), 1.28 (t, 9H, J=7.4, NEt3 CH3), 1.20 (n.r., 28H, Stea), 0.82 (t, 3H,



J=6.9, Stea CH3) ppm;

31P

NMR (600 MHz, CDCl3/MeOH-d4=2:1): δ -0.28 (1P) ppm.

HR-MS (ESI, neg): calc. for C27H53NO11P [M-HNEt3]– m/z 598.33562, found 598.33208. Data detailing characterization of all intermediates is available in the Supporting Information.

Biological assay Animals and cell culture. All protocols using animals were approved by the RIKEN Wako Animal Experiments Committee. Fertilized Boris Brown chicken eggs were purchased from a local supplier (Inoue Poultry Farm, Sagamihara, Japan) and incubated at 38 °C until the embryos developed to Hamburger and Hamilton stage 36.[36] Dissection and culture of nociceptive sensory neurons from dorsal root ganglia was carried out as previously described.[15]

Turning assay. Axon turning assays[22] were performed as previously described[15] with minor modifications. LysoPtdGlc (1) and synthetic analogs 2, 4 and 5 were dissolved in a vehicle of PBS (Life Technologies, CA) and used at an intrapipette concentration of 10 µM. LysoPtdIns (3, 1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphoinositol, Avanti Polar Lipids, Inc., Alabama, US) was dissolved in PBS and used at a concentration of 20 µM. When used, ML193[25] (Tocris Bioscience, Bristol, UK) was dissolved in DMSO and added to the culture medium to produce a final bath concentration of 10 µM at least 20 minutes before start of the turning assay. As a control for the use of ML193, an equivalent volume (0.001%, v/v) of DMSO alone was added to the culture medium. Single extending axon growth cones were challenged with a microscopic concentration


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gradient of LysoPtdGlc, synthetic analog or vehicle alone, for 45 minutes. The axonal growth cones were imaged for 10 minutes prior to start of the experiment, and only growth cones that extended in a straight line were selected for use. Growth cones were photographed at the start and end of the experiment using a QICAM Fast 1394 (QImaging, Burnaby, Canada) CCD digital camera controlled by Metavue software (version 7.8.2.0, Molecular Devices, CA). The turning angle, defined as the angle between the axonal growth cone’s initial trajectory and its position 45 minutes later, and axon extension were calculated using Metavue (Fig. 2). Growth cones that collapsed or failed to extend more than 10 µm during the assay were discounted.

Statistics. Statistical analyses were carried out using GraphPad Prism for Mac (version 6.0c, GraphPad Software Inc, CA). Turning assay datasets for turning angle and axon extension were analyzed by Mann-Whitney test or Kruskal-Wallis test with Dunn’s multiple comparisons post-test.

Molecular dynamics simulation Ligand parameterisation and topology file. The LysoPtdIns ligand (residue: 1LPI) was derived from SAPI residue provided with the CHARMM36 lipid force field (toppar_all36_lipid_inositol.str) by removing the sn-2 arachidonic acid residue. Similar, SAPI was utilized as a template to create the PtdGlc (residue: 12PG), by exchanging the arachidonic acid with arachidic acid as well as converting the inositol head group to the required glucose head group. Parameterisation of the ligand was based on a combination of the CHARMM36 lipid[37] and carbohydrate[38] force field. The equatorial phosphate



parameters for carbohydrates were adopted from Mallajosyula et al[31]. After ligand generation and energy minimisation with CHARMM v35b6, residue 12PG was used as a template to construct the LysoPtdGlc (residue: 1LPG) and LysoPtdGal (residue: LPGA) residues. The missing parameters for ether-LysoPtdGlc (residue: 1EPG) and amideLysoPtdGlc (residue: 1APG) were adopted from the Ether35 and CGenFF36 force fields, respectively. All ligands were generated and energy minimized prior to insertion into the final assembly. To probe the phosphate geometry of hydrated PtdGlc (residue: PPPG) and PtdIns (residue: DPPI) bilayers, a membrane patch for each lipid was generated and equilibrated for 200 ns, prior to geometry analysis. Solution state of LysoPtdGlc (residue: XLPG) and LysoPtdIns (residue: 1XPI) head group geometry was probed by 50 ns dynamics simulation of a single lysolipid molecule in a large box of water. The FA chains were truncated to comprehensively probe the phosphate geometry. The resulting streamfile ‘toppar_all36_lipid_ptdglc.str’ is available in the Supporting Information.

Homology model. Sequence alignment with template structures was based on the previously identified conserved class A patterns reported in human GPR55, as reported by Kotsikorou et al[26]. All gaps were placed in the loop regions during model generation by iTASSER[29] server utilizing template structures and template-target alignment restraints. Trans membrane helices (TMH) 1 to TMH5, including extracellular loop (EC) 2 were derived from the human delta opioid receptor (PDB: 4N6H), TMH6 and 7 were derived form the agonist bound, thermostabilised human adenosine A2a Receptor (PDB: 2YDV), while the C-terminal and the location of both proposed disulfide bridges were


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derived from antagonist bound CXCR4 chemokine receptor (PDB: 2ODU). The Nterminus (MSQQNTSGDCLFDGVNELM) was submitted to iTASSER server without restraints, while the intracellular loop (IC) 3 (LLGRRDHTQDWVQ) was submitted the iTASSER server with distance restraints. The final model was assembled in VMD[39], submitted to H++ server[40] to predict protonation state at a pH of 6.8 and salinity of 100 mM. The resulting model was converted to CHARMM36 force field and equilibrated with NAMD 8.12[41] for 100 ps while restraining the amino acid backbone atoms of the TMH regions. Subsequently, LysoPtdIns was inserted with its phosphate moiety in close proximity to K80 and E98, as reported previously.[28] The GPR55-ligand assembly was equilibrated for 50 ps utilizing harmonic restraints on the TMH region as well as on the inositol ring to maintain its chair conformation. To generate and maintain active state conformation R* of the GPR55 models intracellular region, the C-terminal 26 amino acids, primarily helices 5, of its downstream effector Gα13 was inserted. To this end, the TMH region of adenosine A2A receptor cocrystallized with GαS (PDB: 5G53) was superimposed on the GPR55 model and the matching region of GαS was extracted and point mutated to match the Gα13 sequence by UCSF Chimera.[42] The generated Gα13 section was inserted on the intracellular side of the GPR55 model, based on the above detailed superpositioning. The completed assembly was protonated by H++ server, converted to CHARMM36 force field and was allowed to relax for 2 ns while maintaining harmonic restraints on parts of the TMH backbones region, Gα13 helices and inserted ligand. The occurrence of clashes mainly between GPR55 and Gα13 were periodically checked and manually resolved if needed.



Membrane insertion and dynamics simulation. Next, the complex was inserted in the centre of a fully equilibrated (200 ns MD simulation with NAMD) membrane patch composed equimolar of palmitoylsphingomyelin and cholesterol, while lipids overlapping the protein complex were removed. The resulting scene, consisting of GPR55, Gα13 H5, LysoPtdIns as ligand and the lipid membrane, was hydrated with TIP3, neutralized with potassium chloride at 100 mM and equilibrated for 2 ns in the NPT ensemble with NAMD to allow closing of the membrane-protein gap while preventing water from entering the gap and maintaining harmonic restraints on the backbone of TMH region, Gα13 H5 and the inositol ring conformation. The resulting scene was utilized as starting position for the molecular dynamics simulations with LysoPtdGlc (residue: 1LPG), LysoPtdGal (residue: LPGA), etherLysoPtdGlc (residue: 1EPG) and amide-LysoPtdGlc (residue: 1APG) respectively. In all cases, the respective ligand was placed at exactly the same coordinates as LysoPtdIns. The resulting 4 scenes were each subjected to a 50 ns equilibration and 20 ns production run in the NPT ensemble on the NAMD package with 2 fs steps, 1 atm and 310 K. During the initial 2 ns the harmonic restraints on the GPR55 TMH and Gα13 H5 backbones, ligand head group chair conformation and position were stepwise removed. The results were visualized with VMD, analyzed with proprietary scripts and the graphs were generated with GNU Plot 4.6.

Associated content The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxxx/xxx.xx


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Spectroscopic data (PDF) CHARMM36 residue topology file (STR)

Author information Corresponding Author * Phone: +81-(0)48-462-1111; Fax: +81-(0)48-462-4681; E-mail: [email protected] ORCID Peter Greimel: 0000-0002-4931-6183 Adam T. Guy: 0000-0002-8826-7433 Hiroyuki Kamiguchi: 0000-0003-1802-6324 Yukishige Ito: 0000-0001-6251-7249 Ichiro Matsuo: 0000-0003-0740-1184

Author Contributions The study was conceived by YH and PG. Synthesis of 1 was performed by KK and JO under supervision of IM and PG. Synthesis of 2, 4 and 5 was conducted by PG under supervision by YI. ATG conducted the biological assay under supervision of HK. Molecular model was conducted by PG. The manuscript was written by ATG and PG. All authors approved the final version of the manuscript.

Funding This work was in part supported by Integrated Lipidology Program of RIKEN (PG, YH), AMED-Crest Grant JP18gm0910006 (HK), Grants-in Aid for Scientific Research



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16H06290 (YI), 16K08259 (PG) and 17K14970 (ATG) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Mizutani Foundation for Glycoscience (PG).

Notes The authors declare no competing financial interest.

Acknowledgements We are grateful to M. Inoue for technical assistance. Mass spectral data were acquired at the mass spectrometry facility run by Molecular Structure Characterization Unit (RIKEN CSRS, Wako). Computer simulation was partially conducted on HOKUSAI at RIKEN.

Abbreviations PtdGlc,

phosphatidyl-β-D-glucoside;

LysoPtdGlc,

lysophosphatidyl-β-D-glucoside;

LysoPtdGal, lysophosphatidyl-β-D-galactoside; LysoPtdIns, lysophosphatidylinositol; GlcP, β-D-glucopyranosyl-1-phosphate; InsP, myo-inositol-1-phosphate; Gpr55, G protein-coupled receptor 55; LPAR4, lysophosphatidic acid receptor 4; CB1, cannabinoid receptor type 1; CB2, cannabinoid receptor type 1; ERK1, extracellular signal–regulated kinase 1; ERK2, extracellular signal–regulated kinase 2; RhoA, ras homolog gene family, member A; ROCK, Rho-associated protein kinase; DMAP, N,N-dimethylaminopyridine; ML193,

N-[4-[[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl]phenyl]-6,8-dimethyl-2-(2-

pyridinyl)-4-quinolinecarboxamide; CXCR4, C-X-C chemokine receptor type 4; FA, fatty acid.


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66x71mm (300 x 300 DPI)


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Figure 1: Structural characteristics and similarities of investigated lysophospholipids. 131x77mm (300 x 300 DPI)



Scheme 1: Synthesis of LysoPtdGlc (1) and its analogs 4 and 5. Reagents and conditions: a) (i) NaOMe, MeOH, THF, RT; (ii) NaH, BnBr, DMF, 0 °C → RT (89% 2 steps); b) (i) H3PO3, THF, RT; (ii) NEt3, 0 °C (82%); c) (i) (S)-(+)-1,3-dioxioran-2,2-dimethyl-4-methanol, PivCl, pyridine, THF, 0 °C → RT; (ii) I2, pyridine/H2O, (iii) 50% AcOH, RT, (iv) NH3 aq., sonication (33% for 3 in 4 steps); d) (i) stearic acid, EDC, DMAP, CH2Cl2, RT; (ii) Pd/C, H2, EtOH, 35 °C (19% 2 steps); e) 1-bromooctadecane, NaH, TBAI, DMF;

63%; f) Amberlyst H+, CHCl3:MeOH=1:1; 81%; g) (i) DMTCl, pyridine; (ii) BzCl, DMAP, 80˚C; h) Amberlyst

H+, CH2Cl2:MeOH=1:1; 44% 3 steps; i) (i) PivCl, pyridine, THF, 10 min, 0 °C → RT; (ii) I2, pyridine/H2O, 30 min, RT; 67%; j) N2H4:HOAc = 4:1, CHCl3:MeOH = 1:2.5; 90%; k) stearoyl chloride, NEt3,

MeOH:THF=15:1; 90%; l) (i) DMTCl, pyridine; (ii) BzCl, DMAP, 80˚C; m) Amberlyst H+, CH2Cl2:MeOH=1:1; 43% 3 steps; n) (i) PivCl, pyridine, THF, 10 min, 0 °C → RT; (ii) I2, pyridine/H2O, 30 min, RT; 97%; o) N2H4:HOAc = 4:1, CHCl3:MeOH = 1:2.5; 91%.


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110x138mm (300 x 300 DPI)



Figure 2: Axon turning assay. A: Cartoon of assay design and representative images of chemorepulsion and chemoattraction. B: Axonal growth cone showing chemorepulsive turning response to a microscopic concentration gradient of ether-LysoPtdGlc (4) at start (left panel) and end of the assay (right panel). Numbers indicate minutes after initiation of concentration gradient, arrow denotes the source. Scale bar: 10 µm. C: the same growth cone as shown in B with a single white line superimposed on the photomicrograph to indicate initial direction of growth (left panel). A second straight line was traced from the centre of the growth cone at the beginning of the assay to the centre of the growth cone at the end of the assay (right panel). The turning angle (x) between this line and the original direction of growth was calculated using Metavue. Axon extension (e) was calculated as the length of the second straight line, in microns, using the same software. 183x156mm (300 x 300 DPI)


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Figure 3: Axon turning activity of LysoPtdGlc (1), synthetic analogs, and LysoPtdIns (3). Bars show mean turning angle ± SEM, numbers in parentheses indicate the number of axons tested. **P < 0.01, *P < 0.05; Kruskal–Wallis test. Assay conditions are described in detail in materials and methods. 185x126mm (300 x 300 DPI)



Figure 4: Top (A) and side (B) view of GPR55, embedded in a pSM:Chol=1:1 membrane patch after equilibration with LysoPtdGlc (1). Ligand and membrane lipid phosphor atoms are depicted as space filling model. C, turquoise; O, red; P, gold. 121x177mm (300 x 300 DPI)


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Figure 5: Ligand and GPR55 conformation during dynamics simulation. A) Ligand position relative to membrane centre; black, head group position; gray, terminal methyl group of fatty acid residue. B) Dihedral angle distribution of LysoPtdIns and LysoPtdGlc; inner circle, membrane bound parent lipid; middle circle, short chain lysolipid in solution; outer circle, lysolipid in GPR55 ligand binding pocket during production run; light gray area, φ=-145°+/-30°; dark area, φ=-85°+/-30°; R=C (ins),O (glc). 136x138mm (300 x 300 DPI)



Figure 6: Putative ligand entry port. Distance between Asp13 and Lys165 on GPR55 with distances below 0.5 nm indicating the presence of a salt bridge (A). Snap shots of lysolipid-GPR55 assemblies at the end of the production run with LysoPtdGlc (B), LysoPtdIns (C) and LysoPtdGal (D), in all cases featuring stearic acid at the sn-1 position. Ligand, stick model; GPR55, green cartoon with section of helices 3 and 4 removed for better view; selected amino acids, space filling model. 146x126mm (300 x 300 DPI)


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Preference for Glucose over Inositol Headgroup during Lysolipid

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