Supplementary Method Synthesis of 2-alkyl-MPT(R) General information (R) enantiomer of 2-alkyl (18:1) MPT (hereafter designated as 2-alkyl- MPT(R)), was synthesized as previously described 1, with some modifications as described below. All commercially available reagents were used as received. Dry THF and DCM (Kanto Chemical Co.) were obtained by passing commercially available predried, oxygen-free formulations. H was distilled from iodide and magnesium turnings. DMF was purchased from Wako (for peptide synthesis, grade: 99.5%). All reactions in solution-phase were monitored by thin-layer chromatography carried out on Merck silica gel plates (0.2 mm, 60F-254) with UV light, and visualized with anisaldehyde, or 10% ethanolic phosphomolybdic acid. Silica gel 60N (Kanto Chemical Co. 100~210 µm) was used for column chromatography, and Merck silica gel plate (2.0 mm, 60F-254) was used for preparative thin layer chromatography. 1 H NMR spectra (400 MHz) and 13 C NMR spectra (100 MHz) were recorded on JEL JNM- AL400 spectrometers in the indicated solvent. Chemical shifts (δ) were measured for solutions in CDCl 3, and are reported in parts per million (ppm) relative to the signal of internal tetramethylsilane (0 ppm for 1 H). NMR spectral data are reported as follows: chloroform (7.26 ppm for 1 H) or chloroform-d (77.0 ppm for 13 C) when internal standard is not indicated. Multiplicities are reported by the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet), dt (double triplet), WWW.NATURE.CM/NATURE 1
ddd (double double doublet), ddt (double double triplet), dddd (double double double doublet), brs (broad singlet), brd (broad doublet), J (coupling constants in Hertz). Mass spectra and high-resolution mass spectra were measured on Thermo Scientific Exactive Plus rbitrap Mass Spectrometer (ESI) instruments. IR spectra were recorded on a Shimadzu FTIR-8400. nly the strongest and/or structurally important absorption are reported as the IR data afforded in cm -1. The specific rotations were measured on a JASC P-1010 polarimeter. Melting points were measured on Round Science RFS-10, and are not corrected. (S)-3-Methoxy-1-(triphenylmethyloxy)propan-2-ol TrtCl, NEt 3 CH 2 Cl 2 rt, 12 h, quant To a solution of (R)-3-methoxypropane-1,2-diol (ref2; 1.22 g, 11.5 mmol, 1.0 equiv) and triethylamine (2.39 ml, 17.2 mmol, 1.5 equiv) in dry CH 2 Cl 2 (17 ml, 1.5 ml/mmol) was added chlorotriphenylmethane (4.18 g, 15.0 mmol, 1.3 equiv) at 0 C under argon. After being stirred at room temperature for 12 h, the reaction mixture was quenched with saturated aqueous NH 4 Cl at 0 C. The organic layer was separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated aqueous NaHC 3, brine, dried over MgS 4 and filtered. The filtrate was concentrated in vacuo and then the resulting residue was purified by column chromatography on silica gel (eluted with hexane/ethyl acetate = 8/1 to 3/1) to afford trityl ether (4.01 g, 11.5 mmol, quant) as a white solid. WWW.NATURE.CM/NATURE 2
[α] 24 D 3.87 (c 1.08, CHCl 3 ), mp 87.4 88.1 C 1 H NMR (400 MHz, CDCl 3 ) δ 7.44 7.21 (m, 15H), 3.99 3.91 (m, 1H), 3.49 (dd, 1H, J = 4.0, 9.6 Hz), 3.43 (dd, 1H, J = 6.6, 9.6 Hz), 3.34 (s, 3H), 3.20 3.18 (m, 2H), 2.45 (d, 1H, J = 4.4 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 143.8, 128.6, 127.8, 127.0, 86.7, 74.1, 69.8, 64.7, 59.1; FT-IR (neat) 3420, 3070, 2924, 2876, 1597, 1450, 1220, 1075, 969, 902 cm - 1 ; HRMS[ESI] calcd for C 23 H 24 3 Na [M+Na] + 371.1618, found 371.1609. (R)-3-Methoxy-1-(triphenylmethyloxy)propan-2-ol 4-nitrobenzoic acid DEAD, PPh 3 THF 0 C, 12 h Ar Ar=4-N 2 C 6 H 4 K 2 C 3 H rt, 1 h 2 steps 76% To a solution of alcohol (2.66 g, 7.65 mmol, 1.0 equiv), 4-nitrobenzoic acid (1.41 g, 8.41 mmol, 1.1 equiv) and PPh 3 (2.21 g, 8.41 mmol, 1.1 equiv) in dry THF (31 ml, 4.0 ml/mmol) was added diethylazodicarboxylate (40% in toluene, 1.41 g, 8.41 mmol, 1.1 equiv) dropwise at 0 C under argon. After being stirred at the same temperature for 12 h, the reaction mixture was concentrated in vacuo. The crude nitrobenzoate was used for the next reaction without further purification. To a solution of crude nitrobenzoate (1.0 equiv) in H THF (1:1, 77 ml, 10 ml/mmol) was added K 2 C 3 (996 mg, 7.65 mmol, 1.0 equiv) at 0 C. After being stirred at room temperature for 1 h, the reaction mixture was filtered through a pad of Celite, and the filtrate was diluted with water. The aqueous layer was extracted twice with ethyl acetate and the organic layer was washed with brine, dried over MgS 4 and filtered. The WWW.NATURE.CM/NATURE 3
filtrate was concentrated in vacuo and then the resulting residue was purified by column chromatography on silica gel (eluted with hexane/ethyl acetate = 8/1 to 3/1) to afford trityl ether (2.03 g, 5.83 mmol, 2 steps 76%) as a white solid. [α] 25 D +4.64 (c 1.05, CHCl 3 ), mp 87.2 87.8 C HRMS[ESI] calcd for C 23 H 24 3 Na [M+Na] + 371.1618, found 371.1610. (R)-leyl ether leyl chloride NaH, TBAI DMF 70 C, 12 h, 71% To a solution of alcohol (500 mg, 1.44 mmol, 1.00 equiv) in dry DMF (5.8 ml, 4.0 ml/mmol) was added NaH (55% in mineral oil, 272 mg, 3.74 mmol, 2.6 equiv) at 0 C under argon. After the suspension was stirred at the same temperature for 1 h, oleyl chloride (710 µl, 2.16 mmol, 1.5 equiv) and tetrabutylammonium iodide (266 mg, 0.72 mmol, 0.5 equiv) were added at 0 C. After being stirred at 70 C for 12 h, the reaction mixture was quenched with aqueous 1 M HCl at 0 C. The organic layer was separated and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were washed twice with saturated aqueous NaHC 3, brine, dried over MgS 4 and filtered. The filtrate was concentrated in vacuo and then the resulting residue was purified by column chromatography on silica gel (eluted with hexane/ethyl acetate = 30/1 to 20/1) to afford oleyl ether (611 mg, 1.02 mmol, 71%) as a colorless oil. [α] 27 D +7.88 (c 1.02, CHCl 3 ) WWW.NATURE.CM/NATURE 4
1 H NMR (400 MHz, CDCl 3 ) δ 7.45 (d, 6H, J = 7.6 Hz), 7.31 7.20 (m, 9H), 5.38 5.31 (m, 2H), 3.53 3.49 (m, 5H), 3.33 (s, 3H), 3.18 (d, 2H, J = 4.8 Hz), 2.07 1.95 (m, 4H), 1.60 1.52 (m, 2H), 1.36 1.23 (m, 22H), 0.88 (t, 3H, J = 6.8 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 144.1, 129.89, 129.84, 128.7, 127.7, 126.9, 86.6, 78.2, 73.2, 70.7, 63.4, 59.2, 31.9, 30.1, 29.8, 29.6, 29.5, 29.47, 29.3, 29.27, 27.2, 26.1, 22.7, 14.1; FT-IR (neat) 2925, 2854, 1449, 1110, 1076, 706 cm -1 ; HRMS[ESI] calcd for C 41 H 58 3 Na [M+Na] + 621.4278, found 621.4266. (R)-leyl ether phosphorothioate 1) 0.01 M HCl CHCl 3 -H (1:1) 0 C, 18 h 2) Phosphoramidite 1H-tetrazole CH 2 Cl 2, rt, 1 h; sulfur, CS 2 -Pyridine rt, 12 h, 2 steps 72% P S CN CN To a solution of trityl ether (525 mg, 0.877 mmol, 1.0 equiv) in dry CHCl 3 H (1:1, 67 ml, 76 ml/mmol) was added conc HCl (73 µl, 0.877 mmol) at 0 C. After being stirred at the same temperature for 18 h, the reaction mixture was diluted with water at 0 C. The organic layer was separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over MgS 4 and filtered. The filtrate was concentrated in vacuo and then the resulting residue was purified by short-pass column chromatography on silica gel (eluted with hexane/ethyl acetate = 30/1 to 20/1) to afford alcohol. The alcohol was used for next reaction without further purification. WWW.NATURE.CM/NATURE 5
To a solution of the crude alcohol (1.0 equiv) and 1H-tetrazole (92 mg, 1.32 mmol, 1.5 equiv) in CH 2 Cl 2 (13 ml, 10 ml/mmol) was added a solution of amidite (310 mg, 1.14 mmol, 1.3 equiv) in CH 2 Cl 2 at 0 C under argon. After the mixture was stirred at room temperature for 1 h, sulfur (0.877 mmol, 1.0 equiv) and a solution of CS 2 pyridine (1:1, 175 µl, 0.2 ml/mmol) were added at room temperature under argon. After being stirred at the same temperature for 12 h, the mixture was quenched with aqueous 1 M HCl at 0 C. The organic layer was separated and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were washed twice with saturated aqueous NaHC 3, brine, dried over MgS 4 and filtered. The filtrate was concentrated in vacuo and then the resulting residue was purified by column chromatography on silica gel (eluted with hexane/ethyl acetate = 30/1 to 20/1) to afford phosphorothioate (352 mg, 0.631 mmol, 72%) as a colorless oil. [α] 28 D 1.31 (c 1.04, CHCl 3 ) 1 H NMR (400 MHz, CDCl 3 ) δ 5.39 5.30 (m, 2H), 4.34 4.23 (m, 5H), 4.15 4.09 (m, 1H), 3.65 3.52 (m, 3H), 3.47 (d, 2H, J = 4.8 Hz), 3.37 (s, 3H), 2.77 (t, 4H, J = 6.4 Hz), 2.06 1.94 (m, 4H), 1.59 1.50 (m, 2H), 1.36 1.21 (m, 22H), 0.88 (t, 3H, J = 7.6 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 129.9, 129.8, 116.3, 71.2, 70.6, 67.9, 67.8, 62.44, 62.39, 62.35, 59.3, 32.5, 31.8, 29.9, 29.67, 29.6, 29.56, 29.4, 29.38, 29.34, 29.26, 29.22, 29.17, 29.1, 29.0, 27.1, 25.9, 22.6, 19.4, 19.3, 19.29, 14.1; FT-IR (neat) 2925, 2854, 1457, 1033, 1005, 944, 844, 796 cm -1 ; HRMS[ESI] calcd for C 28 H 51 5 PSNa [M+Na] + 581.3149, found 581.3134. WWW.NATURE.CM/NATURE 6
(R)-leyl ether phosphorothioate sodium salt P S CN CN 1) tbunh 2, BSA, MeCN rt, 48 h 2) 1 M aq Et 3 NHC 3 rt, 1 h 3) Dowex X8-200 resin H 2, rt, 1 h, 3 steps 41% P S Na To a mixture of thioate (50 mg, 0.089 mmol, 1.00 equiv) and N, - bistrimethylsilylacetamide (0.1 ml) in dry acetonitrile (0.7 ml) was added t-butylamine (0.7 ml) at room temperature. After being stirred at the same temperature for 48 h, the reaction mixture was concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (eluted with ethyl acetate/h = 8/1 to 1/1) to afford acid as a colorless oil. 1.0 M triethylammonium bicarbonate buffer (5.0 ml) was added to the acid at room temperature and the mixture was stirred for 1 h at the same temperature. The resulting cloudy solution was concentrated in vacuo and the residue was dissolved in water (2.0 ml). To this solution was added a sodium ion-exchange resin (Dowex 50WX8-200 resin, neutral Na + form) at room temperature and the resulting suspension was shaken for 1 h at the same temperature. The mixture was filtered and the resin was washed with ultrapure water (Milli-Q water). The filtrate was lyophilized to afford sodium salt (17 mg, 0.036 mmol, 41%) as a white solid. The obtained Na salt was stored at 80 C under a nitrogen atmosphere. [α] 26 D +1.99 (c 1.35, CHCl 3 ) 1 H NMR (400 MHz, CDCl 3 ) (acid form) δ 5.39 5.29 (m, 2H), 4.27 3.36 (m, 10H), 2.06 1.94 (m, 4H), 1.59 1.54 (m, 2H), 1.37 1.18 (m, 22H), 0.88 (t, 3H, J = 6.8 Hz); FT-IR WWW.NATURE.CM/NATURE 7
(neat) 2924, 2853, 2362, 1668, 1466, 1243, 1097, 1021, 772 cm -1 ; HRMS[ESI] calcd for C 22 H 46 6 PS [M+H] + 453.2798, found 453.2799. References 1. Jiang, G., Inoue, A., Aoki, J. & Prestwich, G. D. Phosphorothioate analogs of sn- 2 radyl lysophosphatidic acid (LPA): metabolically stabilized LPA receptor agonists. Bioorg. Med. Chem. Lett. 23, 1865 9 (2013). 2. Jakubowska, A., Żuchowski, G. & Kulig, K. Cyclic sulfates as useful tools in the asymmetric synthesis of 1-aminocyclopropane-1-carboxylic acid derivatives. Tetrahedron: Asymmetry 26, 1261 1267 (2015). WWW.NATURE.CM/NATURE 8