Organic chemical synthesis
Synthesis of (R)-9-cis-13,14-dihydroretinol (R)-(2) and of the corresponding acetate (R)-(3) (Fig. 1a): (R)-9-cis-13,14-dihydroretinol (R)-(2) was synthesized by DIBAL-H reduction of the already described ethyl (R)-9-cis-13,14-dihydroretinoate (R)-(1) [5] in THF at -78 ºC in 95% yield. (R)-9-cis-13,14-dihydroretinyl acetate (R)-(3) was prepared in 86% yield by acetylation of (R)-9-cis-13,14-dihydroretinol (R)-(2) with acetic anhydride and pyridine in the presence of dimethylaminopyridine (DMAP).
(3R,4E,6Z,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol (R)-(2): To a cooled (-78°C) solution of ethyl (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trienoate (R) (1) [ethyl (R)-9-cis-13,14-dihydroretinoate] (32.0 mg, 0.097 mmol) in THF (1.0 mL), DIBAL-H (0.242 mL, 0.242 mmol, 1.0 M in hexanes) was added and the resulting mixture was stirred for 30 min at -78°C. The mixture was allowed to warm up to -20°C in 2.5 h. H2O was added and the mixture was extracted with Et2O (3x). The combined organic layers were dried (Na2SO4) and the solvent removed. Flash chromatography of the residue (silica gel, first neutralized with 98:2 hexane/Et3N, then gradient from 95:5 hexane/EtOAc to EtOAc) afforded (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol (R) (2) (26.5 mg, 95%) as a colorless oil. 1H-NMR (400.13 MHz, C6D6): δ 6.93 (d, J = 16.0 Hz, 1H), 6.69 (dd, J = 14.9, 11.1 Hz, 1H), 6.29 (d, J = 16.0 Hz, 1H), 6.01 (d, J = 11.1 Hz, 1H), 5.46 (dd, J = 15.0, 8.3 Hz, 1H), 3.35 (t, J = 6.6 Hz, 2H), 2.27 (dt, J = 14.0, 6.7 Hz, 1H), 1.98 – 1.92 (m, 2H), 1.93 (s, 3H), 1.79 (s, 3H), 1.63 – 1.52 (m, 2H), 1.51 – 1.43 (m, 2H), 1.36 (q, J = 6.7 Hz, 2H), 1.11 (s, 6H), 0.91 (d, J = 6.7 Hz, 3H) ppm. 13C-NMR (100.62 MHz, C6D6): δ 140.0 (d), 138.6 (s), 132.5 (s), 130.9 (d), 129.6 (d), 129.2 (s), 128.1 (d), 124.9 (d), 60.9 (t), 40.2 (t), 39.9 (t), 34.5 (s), 34.3 (d), 33.2 (t), 29.21 (q), 29.18 (q), 22.0 (q), 20.9 (q), 20.7 (q), 19.7 (t) ppm.
(3R,4E,6Z,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-yl Acetate (R)-(3): To a solution of (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-ol (R)-(2) (5.7 mg, 0.020 mmol) in CH2Cl2 (0.5 mL) were sequentially added Ac2O (0.009 mL, 0.099 mmol), pyridine (0.008 mL, 0.099 mmol) and DMAP (0.5 mg, 0.004 mmol). The resulting mixture was stirred for 1 h at 25°C. Then, Et2O was added and the resulting solution was washed with a saturated aqueous solution of CuSO4 (2x). The organic layer was dried (Na2SO4) and evaporated. Flash chromatography of the residue (silica gel, gradient from hexane to 85:15 hexane/EtOAc) afforded (3R,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-4,6,8-trien-1-yl acetate (R)-(3) (5.6 mg, 86%) as a colorless oil. 1H-NMR (400.13 MHz, C6D6): δ 6.92 (d, J = 16.0 Hz, 1H), 6.67 (dd, J = 15.0, 10.8 Hz, 1H), 6.29 (d, J = 16.0 Hz, 1H), 5.98 (d, J = 11.1 Hz, 1H), 5.39 (dd, J = 15.0, 8.2 Hz, 1H), 4.11 – 3.92 (m, 2H), 2.16 (dq, J = 14.4, 7.2 Hz, 1H), 1.99 – 1.90 (m, 2H), 1.93 (s, 3H), 1.79 (d, J = 1.0 Hz, 3H), 1.67 (s, 3H), 1.62 – 1.53 (m, 2H), 1.50 – 1.41 (m, 4H), 1.11 (s, 6H), 0.86 (d, J = 6.8 Hz, 3H) ppm.
Synthesis of (R)-9-cis-13,14-dihydro-β,β-carotene (Fig. 1b, including numeration)
(2-Iodoethoxy)-triisopropylsilane (5). To a cooled (0°C) solution of 2-iodoethanol (4) (2 g, 11.63 mmol) in DMF (31.4 mL) were sequentially added imidazole (1.27 g, 18.61 mmol) and iPr3SiCl (3.2 mL, 15.12 mmol). After stirring for 1 h at 25°C, the reaction was quenched with a saturated aqueous solution of NaCl and extracted with Et2O (3x). The combined organic layers were washed with H2O (3x) and dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 98:2 hexane/EtOAc) to afford 2.33 g (92%) of a yellow oil identified as (2-iodoethoxy)-triisopropylsilane 5. 1H-NMR (400.16 MHz, CDCl3): δ 3.91 (t, 2H, 2H1), 3.22 (t, 2H, 2H2), 1.07-1.01 (m, 21H, SiCH(CH3)2) ppm. 13C-NMR (101.63 MHz, CDCl3): δ 64.7 (t), 18.1 (1, 6x), 12.1 (d, 3x), 6.9 (t) ppm. IR (NaCl): ν 2943 (m, C-H), 2865 (m, C-H), 1462 (m), 1379 (m), 1094 (m), 881 (m) cm-1. HRMS (ESI+): Calcd. for C11H26IOSi ([M+H]+), 329.0790; found, 329.0792.
(R)-2-Methyl-4-((triisopropylsilyl)oxy)-butanal (R) (7). To a cooled (0°C) suspension of anhydrous LiCl (5.42 g, 127.93 mmol) in THF (56.1 mL) and N,N-diisopropylamine (9 mL, 63.97 mmol) was added n-BuLi (23.9 mL, 2.5 M in hexanes, 59.7 mmol) and the mixture was stirred for 15 min at 0°C and for 20 min at 25°C. The mixture was cooled down to -78°C, a solution of N-((2R,3R)-3-hydroxy-3-phenylpropan-2-yl)-N-methylpropionamide (6) (6.6 g, 29.85 mmol) in THF (96.9 mL) was added and the resulting reaction mixture was stirred for 45 min at – 78°C, 15 min at 0°C and 15 min at 25°C. After cooling down to -78°C, (2-iodoethoxy)-triisopropylsilane (5) (7 g, 21.32 mmol) was added and the resulting mixture was stirred for 17 h at 0°C. The reaction mixture was quenched with a saturated aqueous solution of NH4Cl and extracted with EtOAc (3x). The combined organic layers were washed with brine (3x) and dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 80:20 hexane/EtOAc) to afford 7.5 g (84%) of a yellow oil, which was used in the next step. To a cooled (0°C) solution of the compound obtained above (7.5 g, 17.87 mmol) in THF (128 mL), Red-Al® (11 mL, 3.5 M in toluene, 26.8 mmol) was added. After stirring for 20 min, TFA (1.4 mL, 17.87 mmol) and HCl (1.8 mL, 1 M) were added. The reaction mixture was stirred for 1 h at 50°C. The resulting mixture was then cooled down to 25°C, Et2O and a 1M HCl solution (50 mL, 1:1 v/v) were added and the mixture was extracted with Et2O (3x). The combined organic layers were washed with an aqueous solution of NaHCO3 (1x), dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 90:10 hexane/EtOAc) to afford 3.2 g (69%) of a yellow oil identified as (R)-2-methyl-4-((triisopropylsilyl)oxy)-butanal (7). [α]D17 -1.81° (c 0.61, MeOH). 1H-NMR (400.16 MHz, C6D6): δ 9.42 (s, 1H, H1), 3.67 – 3.39 (m, 2H, 2H4), 2.30 – 2.10 (m, 1H, H2), 1.79 – 1.67 (m, 1H, H3A), 1.37 – 1.25 (m, 1H, H3B), 1.06 – 1.04 (m, 21H, SiCH(CH3)2), 0.84 (d, J = 7.1 Hz, 3H, C2-CH3) ppm. 13C-NMR (101.63 MHz, C6D6): δ 203.2 (d), 61.0 (t), 43.6 (d), 34.1 (t), 18.3 (q, 6x), 18.1 (d, 3x), 12.3 (q) ppm. IR (NaCl): ν 2940 (m, C-H), 2867 (m, C-H), 2717 (m), 1727 (s, C=O), 1462 (m), 1463 (m), 1105 (m), 883 (m) cm-1. HRMS (ESI+): Calcd. for C14H31IOSi ([M+H]+), 259.2087; found, 259.2087.
(R,E)-Triisopropyl((3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pent-4-en-1-yl)silane (8). To a solution of chromium chloride (1.37 g, 11.14 mmol) in THF (8 mL) were added sequentially a solution of (R)-2-methyl-4-((triisopropylsilyl)oxy)-butanal (7) (0.36 g, 1.39 mmol) and 2-dichloromethyl-4,4,5,5-tetramethyl-1,2,3-dioxoborolane (0.59 g, 2.28 mmol) in THF (8 mL) and a solution of LiI in THF (8 mL). The resulting mixture was stirred for 15 h at 25°C. The mixture was poured into ice/H2O and the aqueous layer was extracted with Et2O (3x). The combined organic layers were dried with Na2SO4 and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 98:2 hexane/EtOAc) to afford 0.48 g (90%) of a yellow oil identified as (R,E)-triisopropyl((3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pent-4-en-1-yl)silane (8). [α]D21 -14° (c 0.56, MeOH). 1H-NMR (400.16 MHz, CDCl3): δ 6.55 (dd, J = 18.0, 7.2 Hz, 1H, H4), 5.40 (d, J = 18.0 Hz, 1H, H5), 3.68 (t, J = 6.7 Hz, 2H, 2H1), 2.47 – 2.34 (m, 1H, H3), 1.70 – 1.56 (m, 1H, H2A), 1.26 (s, 12H, -OC(CH3)2C(CH3)2O-), 1.26 – 1.24 (m, 1H, H2B), 1.06 – 1.04 (m, 21H, SiCH(CH3)2), 1.02 (d, J = 6.8 Hz, 3H, C3-CH3) ppm. 13C-NMR (101.63 MHz, CDCl3): δ 159.9 (d), 122.1 (d), 83.1 (q, 2x), 61.5 (t), 39.3 (d), 36.1 (t), 24.9 (2x), 24.3 (q, 2x), 19.6 (q), 18.2 (q, 6x), 12.2 (d, 3x) ppm. IR (NaCl): ν 2968 (m, C-H), 2938 (m, C-H), 2866 (m, C-H), 1636 (m), 1462 (w), 1339 (s), 1144 (m) cm-1. HRMS (ESI+): Calcd. for C21H44BO3Si ([M+H]+), 383.3144; found, 383.3151.
(Z)-3-Iodo-2-methylprop-2-en-1-ol (10). CuI (0.34 g, 1.78 mmol) was added to a solution of prop-2-yn-1-ol (9) (1.00 g, 11.84 mmol) in Et2O (13.2 mL). A solution of MeMgBr (17.8 mL, 3 M in Et2O, 53.51 mmol) was added at -20°C. After stirring at this temperature for 2 h, the solution was allowed to reach 25°C and further stirred for 12 h. A solution of I2 (9.1 g, 35.67 mmol) in Et2O (40 mL) was added at 0°C and the cooling bath was removed. After stirring at 25°C for 24 h, the resulting mixture was cooled down to 0°C and ice was added. The organic layer was washed with a saturated aqueous solution of Na2S2O4 (3x) and then filtered through a pad of Celite®. The residue was purified by distillation (0.2 mm Hg, 60°C) to afford 2.1 g (60%) of a yellow oil identified as (Z)-3-iodo-2-methylprop-2-en-1-ol 10. 1H-NMR (400.16 MHz, C6D6): δ 5.49 (s, 1H, H2), 3.90 (s, 2H, 2H1), 1.55 (s, 3H, C2-CH3) ppm.13C-NMR (101.63 MHz, C6D6): δ 146.6 (s), 74.1 (d), 67.5 (t), 20.9 (q) ppm. HRMS (ESI+): Calcd. for C4H8IO ([M+H]+), 198.9609; found, 198.9614.
(Z)-3-Iodo-2-methylacrylaldehyde(11). To a solution of (Z)-3-iodo-2-methylprop-2-en-1-ol (10) (0.21 g, 1.06 mmol) in CH2Cl2 (53 mL) were sequentially added MnO2 (0.93 g, 10.65 mmol) and Na2CO3 (1.13 g, 10.65 mmol) and the reaction mixture was stirred for 1 h at 25°C. Then, the mixture was filtered through a pad of Celite® washing with CH2Cl2. The solvent was evaporated to afford 0.15 g (73%) of a yellow oil identified as (Z)-3-iodo-2-methylacrylaldehyde (11). The spectroscopic data are identical to those previously reported [7].
2-(1E,3Z)-4-Iodo-3-methylbuta-1,3-dien-1-yl)-1,3,3-trimethylcyclohex-1-ene (13). To a cold (-30°C) solution of phosphonium salt (12) [8] (0.15 g, 0.31 mmol) in THF (2.5 mL), nBuLi (0.9 mL, 2.36 M in hexanes, 0.36 mmol) was added and the mixture was stirred at 0°C for 45 min. After cooling down to -30°C, a solution of (Z)-3-iodo-2-methylacrylaldehyde (11) (0.07 g, 0.36 mmol) in THF (2.5 mL) was added and the reaction mixture was stirred for 1.5 h. Water (5 mL) was added and the mixture was extracted with hexane (3x). The organic extracts were dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (C18-silica gel, CH3CN) to afford 65 mg (64%) of a yellow oil identified as 2-(1E,3Z)-4-iodo-3-methylbuta-1,3-dien-1-yl)-1,3,3-trimethylcyclohex-1-ene (13). 1H-NMR (400.16 MHz, C6D6): δ 6.80 (d, J = 16.1 Hz, 1H), 6.32 (d, J = 16.1 Hz, 1H), 5.80 (s, 1H), 1.90 – 1.86 (m, 2H), 1.78 (s, 3H, CH3), 1.66 (s, 3H, CH3), 1.57 – 1.48 (m, 2H), 1.44 – 1.39 (m, 2H), 1.07 (s, 6H, 2xCH3) ppm.13C-NMR (101.63 MHz, C6D6): δ 142.3 (s), 137.5 (s), 135.1 (d), 132.1 (d), 130.8 (s), 78.6 (d), 39.7 (t), 34.0 (s), 33.1 (t), 29.1 (q, 2x), 22.0 (q), 20.9 (q), 19.3 (t) ppm. UV (MeOH): λmax 266 nm. IR (NaCl): ν 2925 (s, C-H), 2861 (m, C-H), 1441 (m), 741 (s) cm-1. HRMS (ESI+): Calcd. for C14H21I ([M+H]+), 316.0688; found, 316.0684.
Silyl Ether (14). To a solution of 2-(1E,3Z)-4-iodo-3-methylbuta-1,3-dien-1-yl)-1,3,3-trimethylcyclohex-1-ene (13) (0.16 g, 0.51 mmol) in THF (0.2 mL) was added Pd(PPh3)4 (45 mg, 0.039 mmol). After stirring for 5 min at 25°C, a solution of (R)-alkenylborane (8) (150 mg, 0.392 mmol) in THF (1.0 mL) and a 10% aqueous solution of TlOH (4.26 mL) were added and the reaction mixture was stirred for 2 h. The mixture was extracted with Et2O (3x) and the combined organic layers were washed with brine (3x) and dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 95:5 hexane/EtOAc) to afford 0.12 g (71%) of a colourless oil identified as silyl ether (14). [α]D20 15.8° (c 0.25, MeOH).1H-NMR (400.16 MHz, C6D6): δ 6.94 (d, J = 16.0 Hz, 1H, H7), 6.76 (dd, J = 14.9, 11.1 Hz, 1H, H11), 6.27 (d, J = 16.0 Hz, 1H, H8), 6.04 (d, J = 11.1 Hz, 1H, H10), 5.52 (dd, J = 14.9, 8.3 Hz, 1H, H12), 3.69 – 3.65 (m, 2H, 2H15), 2.55 – 2.46 (m, 1H, H13), 1.99 – 1.93 (m, 2H, 2H4), 1.92 (s, 3H, CH3), 1.81 (s, 3H, CH3), 1.64 – 1.57 (m, 2H, 2H3), 1.55 – 1.51 (m, 1H, H14A), 1.52 – 1.45 (m, 2H, 2H2), 1.14 – 1.11 (m, 21H, 3xSiiPr3), 1.11 (s, 6H, 2xCH3), 1.08 – 1.02 (m, 1H. H14B), 1.00 (d, J = 6.8 Hz, 3H, CH3) ppm. 13C-NMR (101.63 MHz, C6D6): δ 139.9 (d), 138.6 (s), 132.4 (s), 131.0 (d), 129.6 (d), 129.1 (s), 127.9 (s), 125.2 (s), 61.7 (t), 40.6 (t), 39.9 (t), 34.5 (s), 34.2 (d), 33.3 (t), 29.2 (q), 22.0 (q), 21.2 (q), 20.7 (q), 19.8 (t), 18.4 (q, 6x), 12.4 (d, 3x) ppm. IR (NaCl): ν 2971 (s, C-H), 2864 (s, C-H), 1462 (m, C-H), 1365 (m) 1105 (s), 965 (s) cm-1. HRMS (ESI+): Calcd. for C29H53OSi ([M+H]+), 445.3846; found, 445.3860.
(R)-9-cis-13,14-Dihydroretinol (2). To a cooled (0°C) solution of silyl ether (14) (91.2 mg, 0.212 mmol) in THF (3.5 mL) was added nBu4NF (0.32 mL, 1M in THF, 0.32 mmol) and the mixture was stirred for 0.5 h at 25°C. A saturated aqueous solution of NaHCO3 was added and the mixture was extracted with Et2O (3x). The combined organic layers were washed with brine (3x) and dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (gradient from 95:5 to 70:30 hexane/EtOAc) to afford 41 mg (67%) of a colorless oil identified as (R)-9-cis-13,14-dihydroretinol (2). [α]D21 -9.4° (c 0.37, MeOH). 1H-NMR (400.16 MHz, C6D6): δ 6.93 (d, J = 16.0 Hz, 1H), 6.69 (dd, J = 15, 11.1 Hz, 1H), 6.29 (d, J = 16.0 Hz, 1H), 6.01 (d, J = 11.1 Hz, 1H), 5.46 (dd, J = 15.0, 8.3 Hz, 1H), 3.35 (t, J = 6.6 Hz, 2H), 2.34 – 2.19 (m, 1H), 1.99 – 1.89 (m, 2H), 1.93 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.62 – 1.53 (m, 2H), 1.50 – 1.44 (m, 2H), 1.36 (q, J = 6.7 Hz, 2H), 1.11 (s, 6H, 2xCH3), 0.91 (d, J = 6.7 Hz, 3H, CH3) ppm. 13C-NMR (101.63 MHz, C6D6): δ 140.0 (d), 138.6 (s), 132.5 (s), 130.9 (d), 129.6 (d), 129.2 (s), 128.0 (d), 124.9 (d), 60.9 (t), 40.2 (t), 39.9 (t), 34.5 (s), 34.3 (d), 33.2 (t), 29.21 (q), 29.19 (q), 22.0 (q), 20.9 (q), 20.7 (q), 19.7 (t) ppm. IR (NaCl): ν 3500 - 3300 (br, O-H), 2926 (s, C-H), 1451 (m, C-H), 965 (s) cm-1. HRMS (ESI+): Calcd. for C20H33O ([M+H]+), 289.2584; found, 289.2525.
(R)-9-cis-13,14-Dihydroretinal (15). To a solution of (R)-9-cis-13,14-dihydroretinol (2) (25 mg, 0.087 mmol) in CH2Cl2 (3.7 mL) at 0°C were added Dess-Martin periodinane (73.5 mg, 0.173 mmol) and pyridine (0.014 mL, 0.173 mmol) and the reaction mixture was stirred for 30 min at 0°C and for 1 h at 25°C. Then, Et2O (5 mL), a saturated aqueous solution of NaHCO3 (3 mL) and a saturated aqueous solution of Na2S2O3 (3 mL) were added. The layers were separated, the aqueous layer was extracted with Et2O (3x), the combined organic layers were washed with a saturated aqueous solution of NaHCO3 (2x) and brine (2x), dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, gradient from 95:5 to 70:30 hexane/EtOAc) to afford 14 mg (57%) of a colorless oil identified as (R)-9-cis-13,14-dihydroretinal (15). [α]D21 1.5° (c 0.30, MeOH). 1H-NMR (400.16 MHz, C6D6): δ 9.27 (t, J = 2.0 Hz, 1H, CHO), 6.91 (d, J = 16.0 Hz, 1H, H7), 6.60 (dd, J = 14.8, 11.0 Hz, 1H, H11), 6.30 (d, J = 16.0 Hz, 1H, H8), 5.93 (d, J = 11.0 Hz, 1H, H10), 5.36 (dd, J = 15.1, 7.5 Hz, 1H, H12), 2.53 – 2.43 (m, 1H, H13), 1.98 – 1.93 (m, 2H3), 1.91 (s, 3H, CH3), 1.88 (dd, J = 6.7, 1.9 Hz, 1H, H14A), 1.81 (s, 3H, CH3), 1.81 – 1.77 (d, J = 2.1 Hz, 1H, H14B), 1.63 – 1.53 (m, 2H, 2H2), 1.50 – 1.44 (m, 2H, 2H4), 1.38 (s, 3H, CH3), 1.12 (s, 6H, 2xCH3) ppm. 13C-NMR (101.63 MHz, C6D6): δ 200.1 (s), 138.6 (s), 137.7 (d), 133.2 (s), 130.8 (d), 129.4 (d), 129.1 (s), 128.7 (d), 125.1 (d), 50.4 (t), 39.9 (t), 34.5 (s), 33.3 (q), 31.9 (d), 30.3 (t), 29.2 (t), 22.1 (q), 20.7 (q), 20.4 (q), 19.7 (t), 14.4 (q) ppm. IR (NaCl): ν 2920 (s, C-H), 2854 (s, C-H), 1458 (w), 965 (w) cm-1. HRMS (ESI+): Calcd. for C20H31O ([M+H]+), 287.2362; found, 287.2369.
(2E,4E,6E,8E)-(3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl)triphenyl-phosphonium Chloride (17). To a solution of all-trans-retinol (16) (1.37 g, 4.78 mmol) in MeOH (2.7 mL) was added PPh3 (1.44 g, 5.5 mmol) and a 4 M solution of HCl in dioxane (1.4 mL) and the reaction mixture was stirred for 2 h at 25°C. Then, the mixture was poured into water and extracted with Et2O (2x). The aqueous layer was extracted with EtOAc (3x), the combined organic layers were dried (Na2SO4) and the solvent was evaporated. The light orange residue was triturated three times with EtOAc to afford 1.39 g (52%) of a yellow solid identified as (2E,4E,6E,8E)-(3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl)triphenyl-phosphonium chloride (17). 1H-NMR (400.16 MHz, CDCl3): δ 7.94 – 7.83 (m, 6H), 7.82 – 7.74 (m, 3H), 7.73 – 7.63 (m, 6H), 6.49 (m, 1H), 6.26 – 5.88 (m, 4H), 5.39 (m, 1H), 5.09 – 4.97 (m, 2H), 2.01 (d, J = 6.1 Hz, 2H, CH2), 1.91 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.63 (s, 6H, 2xCH3), 1.48 (m, 4H, 2xCH2), 1.01 (s, 3H, CH3) ppm. 13C-NMR (101.63 MHz, CDCl3): δ 143.8 (s, J C-P = 2.9 Hz, 3x), 137.7 (s), 137.5 (s), 135.0 (d, JC-P = 2.9 Hz, 3x), 133.9 (d, JC-P = 9.7 Hz, 6x), 132.0 (d, J-P = 12.4 Hz), 130.3 (d, JC-P = 12.4 Hz, 6x), 129.5 (d, JC-P = 2.9 Hz), 128.5 (d, JC-P = 12.2 Hz), 127.5 (d), 126.4 (d, JC-P = 5.2 Hz), 118.7 (s), 117.8 (s), 114.2 (d, JC-P = 12.1 Hz), 39.6 (t), 34.2 (s), 33.0 (t), 28.9 (q, 2x), 25.4 (t), 24.9 (t), 19.2 (q), 13.1 (q), 12.8 (q) ppm. HRMS (ESI+): Calcd. for C38H44P ([M-Cl]+), 531.3175; found, 531.3166.
(R)-9-cis-13,14-Dihydro-β,β-carotene(18). To a cooled (-78°C) solution of phosphonium chloride (17) (24.9 mg, 0.044 mmol) in THF (0.2 mL) was added nBuLi (0.027 mL, 1.6 M in hexanes, 0.044 mmol) and the reaction mixture was stirred for 30 min. Then a solution of (R)-9-cis-13,14-dihydroretinal (15) (9.0 mg, 0.031 mmol) in THF (0.13 mL) was added and the mixture was stirred for 1 h at -78°C and for 30 min at 25°C. Water was added and the mixture was extracted with Et2O (3x). The combined organic layers were washed with a saturated aqueous solution of NaCl (2x) and dried (Na2SO4). The solvent was evaporated and the residue was purified by column chromatography (C18-silica gel, CH3CN) to afford 11.3 mg (67%) of a red foam identified as (R)-9-cis-13,14-dihydro-β,β-carotene (18). It was observed that this product was very unstable and easily degraded. 1H-NMR (400.16 MHz, acetone-d6): δ 6.69 – 6.60 (m, 2H), 6.56 (dd, J = 14.4, 11.2 Hz, 1H, H11’), 6.48 (dd, J = 14.9, 11.1 Hz, 1H, H15), 6.34 (d, J = 15.1 Hz, 1H), 6.21 – 6.13 (m, 4H), 6.12 (dd, J = 11.5, 7.8 Hz, 1H), 5.94 (d, J = 11.7 Hz, 1H, H10’), 5.81 – 5.71 (m, 1H, H15’), 5.62 (dd, J = 15.0, 8.2 Hz, 1H, H12’), 2.44 – 2.33 (m, 1H, H13’), 2.25 – 2.19 (m, 2H, 2H14’), 2.05 – 2.00 (m, 4H), 1.96 (s, 3H, CH3), 1.92 (s, 3H, CH3), 1.91 (s, 3H, CH3), 1.72 (s, 3H, CH3), 1.70 (s, 3H, CH3), 1.67 – 1.56 (m, 4H), 1.50 – 1.45 (m, 4H), 1.04 (s, 3H, CH3), 1.03 (s, 12H, 4xCH3) ppm. 13C-NMR (101.63 MHz, acetone-d6): δ 140.3 (d), 139.1 (d), 139.0 (s), 138.9 (s), 138.7 (d), 134.9 (d), 134.8 (s), 133.1 (s), 132.8 (d), 132.2 (d), 131.3 (d), 130.0 (d, 2x), 129.8 (s), 129.7 (s), 129.6 (d), 128.5 (d), 127.1 (d), 126.2 (s), 125.4 (d), 41.6 (t, 2x), 40.6 (t), 40.7 (t), 38.3 (d), 35.1 (s), 35.0 (s), 30.9 (t, 2x), 30.8 (q), 29.5 (q, 4x), 22.2 (q), 22.1 (q), 20.8 (q), 20.7 (q), 20.1 (t), 12.9 (q) ppm. UV (MeOH): λmax 324 nm. HRMS (ESI+): Calcd. for C40H59 ([M+H]+), 539.4604; found, 539.4611.
Synthesis of 9-cis-β,β-carotene (Fig. 1c, including added numeration)
Ethyl 9-cis-Retinoate (21). To a cooled (0°C) solution of ethyl (E)-4-(diethoxyphosphoryl)-3-methylbut-2-enoate (20) (0.227 g, 1.10 mmol) in THF (2.0 mL) was added nBuLi (0.63 mL, 1.00 mmol, 1.6 M in hexane) and DMPU (0.15 mL, 1.24 mmol). After stirring for 1 h, the reaction was cooled down to -78°C and a solution of (2Z,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-2,4-dienal (19) (0.1 g, 0.46 mmol) in THF (2.5 mL) was added and the mixture was stirred for 2 h. Water was added and the mixture was extracted with Et2O (3x). The combined organic layers were dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, 95:5 hexane/EtOAc) to afford 0.144 g (96%) of a yellow solid identified as ethyl 9-cis-retinoate (21). The spectroscopic data are identical to those previously reported [8].
9-cis-Retinol (22). To a cooled (-78°C) solution of ethyl 9-cis-retinoate (21) (0.154 g, 0.47 mmol) in THF (2.3 mL) was added DIBAL-H (1.08 mL, 1.08 mmol, 1.0 M in toluene) and the reaction was stirred for 2 h. Water was added and the mixture was extracted with EtOAc (3x). The combined organic layers were dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (silica gel, from 95:5 to 80:20 hexane/EtOAc) to afford 0.08 g (60%) of a yellow oil identified as 9-cis-retinol (22). The spectroscopic data are identical to those previously reported [9].
9-cis-Retinal (23). To a solution of 9-cis-retinol (22) (65 mg, 0.23 mmol) in CH2Cl2 (9.1 mL) were added MnO2 (197 mg, 2.27 mmol) and Na2CO3 (240 mg, 2.27 mmol) and the reaction mixture was stirred for 1.5 h at 25 °C. The mixture was filtered through a pad of Celite® washing with CH2Cl2. The solvent was evaporated to afford 38 mg (58%) of a yellow oil identified as 9-cis-retinal (23). The spectroscopic data matched those previously reported [10].
9-cis-β,β-Carotene (24). To a solution of phosphonium salt (17) (24.9 mg, 0.044 mmol) in THF (0.20 mL) at -78°C was added nBuLi (0.027 mL, 1.6 M in hexane, 0.044 mmol) and the resulting mixture was stirred for 30 min. Then, a solution of 9-cis-retinal (23) (9.0 mg, 0.031 mmol) in THF (0.13 mL) was added and the mixture was stirred for 1 h at -78°C and for 30 min at 25°C. Water was added and the mixture was extracted with Et2O (3x). The combined organic layers were washed with brine (2x) and dried (Na2SO4) and the solvent was evaporated. The residue was purified by column chromatography (C18-silica gel, CH3CN) to afford 11.3 mg (67%) of a red foam identified as 9-cis-β,β-carotene (24).1H-NMR (400.16 MHz, CDCl3): δ 6.74 (dd, J = 14.8, 11.6 Hz, 2H), 6.68 (d, J = 7.9 Hz, 1H), 6.66 – 6.54 (m, 3H), 6.28 (d, J = 14.9 Hz, 1H), 6.26 – 6.19 (m, 2H), 6.19 – 6.08 (m, 4H), 6.05 (d, J = 11.5 Hz, 1H), 2.08 – 1.99 (m, 4H), 1.97 (s, 3H), 1.95 (s, 6H), 1.76 (s, 3H), 1.71 (s, 3H), 1.66 – 1.59 (m, 4H), 1.51 – 1.39 (m, 4H), 1.25 (s, 3H), 1.04 (s, 6H), 1.03 (s, 6H) ppm. 13C-NMR (101.63 MHz, CDCl3): δ 138.4 (s), 138.1 (s), 137.8 (d), 136.7 (d), 136.4 (s, 2x), 136.1 (s), 134.6 (d), 130.9 (d), 132.4 (d), 130.2 (d, 3x), 130.0 (d), 129.6 (s, 2x), 129.5 (d, 2x), 128.5 (d), 127.1 (s), 127.0 (d), 123.8 (d), 39.7 (t, 2x), 34.4 (s, 2x), 33.3 (t, 2x), 29.1 (q, 2x), 28.6 (q, 2x), 22.0 (q), 21.9 (q), 20.9 (q), 19.4 (t, 2x), 13.0 (q), 12.9 (q) ppm. UV (MeOH): λmax 397 nm. HRMS (ESI+): Calcd. for C40H56 ([M]+), 536.4375; found, 536.4376. The spectroscopic data matched those previously reported [11, 12].
Samples used for LC-MS analysis
Food samples: Beef liver (n=3) was bought at a local butcher in Vigo, Spain. Conserves of peaches in can (n=3; Metades, Pessago em calda, Auchan/Alcampo-home-brand / 420 g can) were purchased at Alcampo, Vigo, Spain.
Human serum samples (n=3) were obtained from the blood of healthy volunteers with all the subjects’ written informed consent.
Animal experiments
Animals: Wild-type (WT) C57BL6N male mice (Charles River, France) used for metabolic analyses were housed in groups of 4-5 mice per cage in a 7 am-7 pm light/dark cycle in individually ventilated cages (Techniplast, Italy). RXRγ-/- and control WT mice were raised on mixed C57BL6N and 129SVpas genetic background as described [13]. Food (standard chow diet, D04 from SAFE, France) and water were freely available. All animal care and experimentation were carried out in accordance with European Union Council (2010/63/EU) and the French Ministry of Agriculture (87848) guidelines for the use of laboratory animals in behavioural studies. French National Ethics Committees approved all experimental protocols under specific authorization (No. 2016022411354542). Accordingly, animal experimentation and statistical analyses were planned to minimize the number of animals used, within the constraints of necessary power.
Animal treatments: ATROL, 9CDHROL and 9-cis-13,14-dihydro-β,β-carotene (9CDHBC) were dissolved in ethanol, and then mixed with sunflower oil, so that the final solution contained 3% ethanol. Vehicle treatments consisted of 3% ethanol solution in sunflower oil. Treatments were administered per os as a single dose at 40 mg/kg for each substance and volume/weight ratio 3 ml/kg for chemical analyses. Mice were always treated during active dark phase of the Light/Dark cycle at 10pm and samples were collected 11 h later in the light protected conditions, weighted, frozen in liquid nitrogen and stored at -80°C until analyses. Serum was prepared and immediately stored in brown vials at -80°C until further analysis. For memory tests (see below) n=8 for WT and n=5 for RXRγ-/- mice were injected single dose of 40 mg/kg of 9CDHROL at volume/weight ratio of 3 ml/kg and 9-12 h before testing. Such a long latency was selected to allow not only the most complete metabolic conversion of 9CDHROL to 9CDHRA, but also induction of transcriptional programs controlled by RXRs including translation and maturation of transcribed proteins prior to testing. To establish whether vehicle treatment may affect animal performance we analyzed memory in mice treated with vehicle alone. This control testing session took place 48 h before testing session with 9CDHROL treatment.
Memory analysis: Working memory analysis was carried out in the Institute Clinique de la Souris (http://www.ics-mci.fr/) according to standard operating procedures. 12-week old male mice (n=10 WT and n=8 RXRγ-/-) were trained in the DNMTP in the T-maze using 5 trials per day according to a protocol previously described [14] with modifications to facilitate pharmacological tests [13]. Briefly, all mice were first trained over 10 days to acquire procedural memory in delayed non-match to place (DNMTP) paradigm in the T-maze. Each day mice were running for a reward of 25% sucrose during 5 trials. Each trial was composed of learning phase (only one arm was opened and baited) and testing phase (two arms opened, but only newly opened arm was baited) separated by minimal (15sec) inter-trial interval (ITI). The percent of correct choices (choice of rewarded arm) was calculated for each animal every day. Minimum 4 correct choices out of total of 5 choices (80%) over 3 consecutive days was considered as criterion of acquisition and mice which did not acquire this criterion were excluded from the study (n=2 WT and n=3 RXRγ-/-). Thus final number of mice taken into account for analyses was n=8 for WT and n=5 for RXRγ-/- group. Since the results were significant and consistent with previous observations [13], the cohort numbers were not increased reducing thus the number of utilized animals. After training period of 10 days the ITIs were extended from 3 to 24 min in order to establish the minimum time at which mouse performed at chance (60% of correct choices or less). These ITIs were next used for testing promnemonic activities of treatments.
Cell cultures and pharmacological treatments: 158N mouse oligodendrocyte cells [15] were cultured in 60 of T-175 falcon flasks supplemented with 5% calf serum (Invitrogen, France). After reaching confluency of 80% cells were T75 falcon flasks treated with 10-3M ethanol solutions of 9CDHROL, 9CDHROL-ES (as an acetate-ester), 9CDHRA, 9-cis-dihydroretinoyl-ester (9CDHRA-ES, as an ethyl-ester) or ATROL, or 10-3M DMSO solutions of 9CDHBC, 9CBC and ATBC to attain final concentration of 10-6M for each compounds. Ethanol at corresponding concentration was used as control, vehicle treatment. Each treatment was performed in triplicate providing thus n=3 independent biological samples, each sample obtained by pulling cells from 3 independent flasks to produce a cell pellet of about 120-150 mg and corresponding to about 60 million cells. Briefly, cells were harvested 18 hours after treatment using isotonic, non-enzymatic cell dissociation buffer (Invitrogen, ref. 13150-016). After centrifugation and weighting all samples were frozen in liquid nitrogen and stored at -80° until analyses. All the procedures were carried out in light protected conditions to avoid photolysis of retinoids.
LC-MS analysis - combined retinoid and carotenoid analysis
Analytical procedures: Analyses were performed under dark yellow/amber light using previously sample preparation as a validated protocol [16] for RA-isomer and ROL analysis. The basic methodology [16] was improved using a more sensitive MS setup using a high performance liquid chromatography mass spectrometry (Agilent 1260 Infinity LC system; Madrid, Spain)–mass spectrometry (SCIEX Triple Quad 3500 System; Sciex, Madrid, Spain) plus and additional online diode array detector (Waters 966 DAD, Waters, Santiago de Compostella, Spain) system. In addition to the first and second eluents already mentioned in [16] a third and fourth eluent after 20 min elution time were used. Linear gradient from 20 min 20% (isopropanol : methanol : methyl-tert-butyl-ether (MTBE) / 30 : 30 : 40) - 80% (isopropanol : methanol / 50 : 50), 25 min 40% (isopropanol : methanol : MTBE / 30 : 30 : 40) - 60% (isopropanol : methanol / 50 : 50), 29 min 70% (isopropanol : methanol : MTBE / 30 : 30 : 40) - 30% (isopropanol : methanol / 50 : 50), 30 min 0% (isopropanol : methanol : MTBE / 30 : 30 : 40) - 100% (isopropanol : methanol / 50 : 50), 30.1 min 20% (isopropanol : methanol : MTBE / 30 : 30 : 40) - 80% (isopropanol : methanol / 50 : 50). For the detection of retinoic acids MS-MS settings 301 -> 205 m/z, 13,14-dihydroretinoic acid MS-MS setting 303 -> 207 m/z, for the detection of 13,14-dihydroretinol MS-MS settings 290 -> 69 m/z and for the detection of 9CDHBC 405 -> 405 / 405 -> 95 m/z were used while for 9CDHBC additionally a diode array detector at 366 nm and for 9CBC and ATBC exclusively a diode array detector at 411 nm were used. For each additional analyzed substance (ATDHRA, 9CDHRA, ATDHROL, 9CDHROL, ATBC, 9CBC and 9CDHBC) a linearization, recovery determination, intra- and inter-day variability and limit of quantification and limit of determination was performed and implemented in the concentration calculation process. The used standard compounds were synthesized as described in the manuscript earlier, obtained like described in [16] or alternatively for 9CDHRA and ATDHRA as previously published [5]. Accordingly, for sample preparation 100 mg of the material (if samples were under 100 mg, water was added up to the used standard weight: 100 mg) or 100 μl serum was diluted with a threefold volume of isopropanol, the tissues were minced by scissors, vortexed for 10 seconds, put in a ultrasonic bath for 5 minutes, shaken for 6 minutes and centrifuged at 13000 rpm in a Heraeus BIOFUGE Fresco at +4°C. After centrifugation, the supernatants were dried in a GYROZEN centrifugal vacuum concentrator equipped with an ILMAC MPC 301-Z vacuum pump (CONTROLTECNICA, Madrid, Spain) at 30°C. The dried extracts were resuspended with 30 μl of methanol – MTBE (50 : 50) and transferred into the auto sampler and 10 µl subsequently analyzed.
Statistical analysis
Statistical analyses for behavioral data were performed using two-way ANOVA or for procedural learning two-way ANOVA with repeated measures and followed by post-hoc Bonferroni test. Analyses of retinoid / carotenoid metabolism as well as ITI analyses in DNMTP behavioral test were performed using student t-test with a statistically significance accepted at p < 0.05. Significant differences are indicated in the corresponding figures and figure legends.