Test compounds

All chemicals were purchased from Sigma-Aldrich if not stated otherwise. 2-AG, AEA, ARA-S, N-arachidonoyl dopamine (NADA), N-arachidonoyl-γ-aminobutyric acid (NAGABA), arachidonoyl serinol (ARA-Serinol), and N-oleoyl-L-serine (OLE-S) were bought from Cayman Chemicals. Retigabine dihydrocloride was bought from Alomone Labs. N-docosahexaenoyl-L-serine (DOC-S), N-linoleoyl-L-serine (LIN-S), N-arachidonoyl-D-serine, and N-arachidoyl-L-serine (arachidoyl-S) were synthesized in house. The synthesis methods have been partly described previously (Silverå Ejneby et al., 2018). Most of the reagents for synthesis of new compounds were from Sigma-Aldrich except O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, which was from Fluka (Fisher Scientific GTH AB). Preparative liquid chromatography (LC) was run on a Waters system with an XSELECT Phenyl-Hexyl column (250 × 19 mm, 5 μm), under neutral condition using gradient CH₃CN/water as eluent (A, water phase: 95: 5 water/CH₃CN, 10 mM NH₄OAc; B, organic phase: 90: 10 CH₃CN/water, 10 mM NH₄OAc). NMR spectra were recorded on a Varian Avance 300 MHz with solvent indicated. Chemical shift was reported in ppm on the δ scale and referenced to solvents peak (CDCl₃: δ_H = 7.26 ppm, δ_C = 77.16 ppm; methanol-d4: δ_H = 3.30 ppm, δ_C = 49.50 ppm).

To the saturated or unsaturated aliphatic acid and 2.20 equiv triethylamine in acetonitrile (20 ml) and/or dimethylformamide (DMF; 2–4 ml) was added 1.05 equiv O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, and the reaction mixture was stirred at room temperature (rt) for ∼1 h. D-serine or L-serine (1.20 equiv) was added at rt and stirred over 1–7 nights. The solution was concentrated, and 5 ml water was added. The mixture was extracted with ethyl acetate (EA; 5 ml × 3). The organic layers were combined, concentrated, and purified using preparative LC (40–100%). The desired fractions were combined and concentrated to remove most of the acetonitrile. Another 5 ml water was added to the residue. The resulting solution was adjusted to pH at ∼5 using 1 N HCl aqueous solution and extracted with EA (15 ml × 2). The organic layers were concentrated to give the desired product.

Following the general procedure with docosahexaenoic acid (100 mg, 0.304 mmol), L-serine as starting materials, and DMF (2 ml) as solvent, the reaction mixture was stirred overnight at rt, filtered, and purified using preparative LC without extraction to give the product as a syrup (16.8 mg, 13% yield). ¹HNMR (CDCl₃/CD₃OD 3:2, 300 MHz): δ 5.43–5.14 (m, 12H), 4.48 (t, J = 4.2 Hz, 1H), 3.89 (dd, J = 11.4, 4.2 Hz, 1H), 3.76 (dd, J = 11.4, 4.2 Hz, 1H), 2.85–2.57 (m, 12H), 2.39–2.30 (m, 2H), 2.28–2.20 (m, 2H), 2.06–1.92 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H). ¹³CNMR (CDCl₃/CD₃OD 3:2, 75 MHz): δ 173.4, 172.2, 131.9, 129.2, 128.4, 128.2, 128.15, 128.1, 128.0, 127.96, 127.9, 127.8, 126.9, 62.4, 54.4, 35.9, 25.5, 25.43, 25.41, 23.1, 20.4, 14.1. MS (ESI⁻): m/z calcd for C₂₅H₃₆NO₄ (M-H⁻) 414.26, found 414.51.

Following the general procedure with linoleic acid (280 mg, 0.998 mmol), L-serine (126.1 mg, 1.200 mmol) as starting materials, and acetonitrile (20 ml) as solvent, the reaction mixture was stirred over seven nights at rt, purified using preparative LC to give the product as a syrup (11.1 mg, 3%). ¹HNMR (CDCl₃, 300 MHz): δ 6.65 (br d, J = 6.6 Hz, 1H, NH), 5.45–5.25 (m, 4H), 4.50 (m, 1H), 4.14 (dd, J = 11.4, 3.6 Hz, 1H), 3.85 (dd, J = 11.4, 3.6 Hz, 1H), 2.82–2.70 (m, 2H), 2.30 (d, J = 7.5 Hz, 2H), 2.15–2.00 (m, 4H), 1.70–1.60 (m, 2H), 1.41–1.20 (m, 14H), 0.87 (t, J = 6.9 Hz, 3H). ¹³CNMR (CD₃OD/CDCl₃ 2:1, 75 MHz): δ 175.8, 173.1, 130.7, 130.6, 128.75, 128.69, 62.8, 55.5, 36.8, 32.3, 30.4, 30.11, 30.05, 30.0, 29.9, 27.9, 26.5, 26.3, 23.3, 14.3. MS (ESI⁻): m/z calcd for C₂₁H₃₆NO₄ (M-H⁻) 366.26, found 366.41.

Following the general procedure with arachidic acid (312.5 mg, 1.000 mmol), L-serine (126.1 mg, 1.200 mmol) as starting materials, acetonitrile (20 ml) and DMF (4 ml) as solvents, the reaction mixture was stirred over seven nights at rt. The solubility of this compound is poor. Part of the reaction mixture was purified using preparative LC to give the product as a white solid (5.0 mg). The yield was not calculated. ¹HNMR (CDCl₃, 300 MHz): δ 4.41 (dd, J = 4.8, 4.2 Hz, 1H), 3.89–3.75 (m, 2H), 2.29–2.22 (m, 2H), 1.35–1.25 (m, 34H), 0.89 (t, J = 7.2 Hz, 3H). MS (ESI⁻): m/z calcd for C₂₃H₄₄NO₄ (M-H⁻) 398.33, found 398.67.

Following the general procedure with docosahexaenoic acid (50 mg, 0.164 mmol), D-serine (20.7 mg, 0.197 mmol) as starting materials, and DMF (4 ml) as solvent, the reaction mixture was stirred overnight at rt, filtered, and purified using preparative LC without extraction to give the product as a syrup (21.5 mg, 33% yield). ¹HNMR (CDCl₃:CD₃OD 85:15, 300 MHz): δ 5.40–5.20 (m, 8H), 4.49 (t, J = 3.6 Hz, 1H), 3.89 (dd, J = 11.1, 3.6 Hz, 1H), 3.77 (dd, J = 11.1, 3.6 Hz, 1H), 2.82–2.68 (m, 6H), 2.21 (t, J = 7.8 Hz, 2H), 2.10–1.90 (m, 4H), 1.72–1.58 (m, 2H), 1.36–1.16 (m, 6H), 0.82 (t, J = 6.6 Hz, 3H). ¹³CNMR (CDCl₃:CD₃OD 85:15, 75 MHz): δ 174.1, 172.4, 130.5, 129.0, 128.8, 128.6, 128.3, 128.2, 127.9, 127.5, 62.6, 54.5, 35.7, 31.5, 29.3, 27.2, 26.7, 25.6, 25.0, 22.5, 14.0. MS (ESI⁻): m/z calcd for C₂₃H₃₆NO₄ (M-H⁻) 390.26, found 390.60.

All compounds were either delivered as or diluted as ethanol stock solutions except for 2-AG, which was delivered in acetonitrile. Stock solutions were stored at −20°C except for 2-AG, which was stored at −80°C.