Supplemental Material and thods Synthesis of α-vitamin E analogs All racemic α-tocopherol (synthetic compound) was purchased from Acros (Morris Plains, NJ) and was used to synthesize the all-racemic α-tocopherol analogs (VES, VEBSA, VEPSA). R,R,R-α-Tocopherol, used to synthesize the R,R,R-α-tocopherol analogs (RRR- VEBSA, RRR-VEPSA, RRR-VEBPA, RRR-VEPPA) was obtained from the hydrolysis of the succinate ester of (+)-α-tocopherol succinate, which was purchased from Sigma-Aldrich (St. Louis, M). All-racemic α-tocopherol succinate (rac-ves) was obtained in one step via esterification of α-tocopherol with succinic anhydride in the presence of 4-(dimethylamino)pyridine (DMAP) (eq.1) The sulfonic acid analogs, VEBSA and VEPSA, were synthesized from α-tocopherol by alkylation of the alkoxide of α-tocopherol with 1,3-propane sultone and 1,4-butane sultone, respectively (eq.2). The phosphonic acid analogs, RRR-VEBPA and RRR-VEPPA, were synthesized from R,R,R-α-tocopherol via 5-step sequences involving alkylation with 3- bromopropanol or 4-bromobutanol, respectively, followed by conversion of the terminal alcohol to an iodide. The iodide was displaced with triethylphosphite and the resulting phosphonates were subsequently hydrolyzed to afford the phosphonic acids (eq.3). 1
H all racemic α-tocopherol DMAP, CH 2 Cl 2, 23 C H all-racemic VES (1) H 1) NaH, THF, then 3) HCl 2 S n H 2 S n n = 1, R,R,R-VEPSA n = 2, R,R,R-VEBSA (2) H 1) NaH, THF, then BrCH 2 (CH 2 ) n CH 2 H 2) MsCl, Et 3 N 3) NaI 4) (Et 3 )P, Δ 5) HCl, Δ (H) 2 P n n = 1, R,R,R-VEPPA n = 2, R,R,R-VEBPA (3) Experiments for the Synthesis of Vitamin E Analogs General Experimental Information: All reagents used were purchased from Aldrich or Acros. Solvents were purified using a solvent filtration system purchased from Contour Glass Co. (Irvine, California). R,R,R-α-Tocopherol was obtained in gram quantities by saponification (Kt-Bu, H) of commercially available (Sigma Aldrich) (+)-α-tocopherol succinate. 1 H NMR data were obtained in CDCl 3 (using 7.26 ppm for reference of residual CHCl 3 ) at 300, 400, or 500 MHz using Varian instruments. 13 C NMR data in CDCl 3 (using 77.0 ppm as internal reference) were obtained at 75.5 MHz. ptical Rotations were obtained using a JASC DIP-370 digital polarimeter. UV/Vis spectra were obtained in chloroform and ethanol using a Hewlett Packard 8452A Diode Array spectrophotometer. IR spectra were obtained neat from thin films using a Nicolet Avatar 330 FTIR. High resolution mass spectra were obtained at SUNY, Buffalo s Mass Spec. facility on a ThermoFinnigan MAT XL spectrophotometer purchased by a National Science Foundation grant to the center (NSF CHE0091977). The melting points reported are uncorrected. 2
MgS 4, filtered, and concentrated in vacuo. Flash chromatography of the crude oil on Si 2 (10-60% EtAc/hexanes gradient) afforded 1.26 g (85%) of all-racemic α-tocopherol succinate as viscous yellow oil. IR (neat, thin film) υ 3522-2587 (br), 2927, 1755, 1715, 1461, 1377, 1151, 1109 cm -1 ; 1 H NMR (CDCl 3, 300 MHz) δ 0.83-0.88 (12H, m), 1.00-1.62 (24H, m), 1.70-1.86 (2 H, m), (±) Succinic acid mono-[2,5,7,8-tetramethyl-2-(4,8,12-trimethyl-tridecyl)- chromone-6-yl) ester (all racemic-α-tocopheryl succinate, Rac-VES). Racemic α-tocopherol (1.20 g, 2.79 mmol) was dissolved in anhydrous CH 2 Cl 2 (15 ml) and was treated with succinic anhydride (0.28 g, 2.79 mmol, 1 equiv) and 4-dimethylaminopyridine (DMAP, 0.34 g, 2.79 mmol, 1 equiv) at 23 C. After being stirred for 3 days, the mixture was diluted with Et 2 (60 ml) and aqueous HCl (3 N, 20 ml) and the organic and aqueous layers were separated. The aqueous layer was extracted with Et 2 (30 ml). The combined organic layers were dried over 1.97 (3H, s), 2.01 (3H, s), 2.08 (3H, s), 2.56-2.60 (2H, m), 2.80-2.85 (2H, m), 2.91-2.96 (2H, m); 13 C NMR (CDCl 3, 75.5 MHz) δ 11.8, 12.0, 12.9, 19.68, 19.74, 20.6, 21.0, 22.6, 22.7, 24.4, 24.8, 28.0, 28.6, 28.9, 31.1, 32.7, 32.8, 37.3, 37.4, 37.5, 39.4, 75.1, 117.4, 123.0, 124.9, 126.7, 140.4, 149.5, 170.8, 177.4; HRMS (ESI) calculated for C 33 H 55 5 [M+H] + : 531.4044, found 531.4034. 4-[2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyl-tridecyl)-chroman-6-yloxy]-butyric acid (RRR-α-tocopheryl succinate ether analog, RRR-TSE). RRR-TSE was produced as previously report (15, 16). 3-[2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethy-tridecyl)-3,4-dihydro-2H-chroman- 6-yloxy]-propane-1-sulfonic acid (R,R,R-α-Tocopheryloxypropyl sulfonic acid, RRR- VEPSA). RRR-α Tocopherol (RRR-Toc) (1.00 g, 2.32 mmol), and NaH (0.111 g, 4.64 mmol), 3
were combined in a 25 ml two neck round bottom flask in THF (20 ml, 0.12 M) at 0 o C under argon. 1, 3-Propane sultone (0.34 g, 2.79 mmol) was added to the reaction mixture after stirring at room temperature for 50 min. The reaction was then heated at reflux for 24 h. Upon completion the reaction was quenched with conc. HCl, extracted with a 3:1 mixture of EtAc: iprh, washed with acidified brine, then dried over anhydrous Na 2 S 4. The organic layer was then concentrated in vacuo, to form a brown solid which was then subjected to flash chromatography using 1 1 / 2 inch silica gel [gradient of EtAc: hexane from (1: 4) to 100% EtAc] followed by EtAc:HAc (20 : 1)], to yield (0.792g, 60 %) as a tan / brown waxy solid. [α] 25 D = + 4.99 (c 1.0, chloroform); λ max /nm (UV/Vis, chloroform): 288; (UV/Vis, ethanol): 212, 288; R f ( 1:1 EtAc: i-prh): 0.2; IR (neat, thin film) υ 2925 (s), 1461 (m), 1376 (m), 1187 (s), 1091 (m), 1056 (s) (m) cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 0.86 0.88 (12 H, m), 1.08 1.38 (18 H, m), 1.26 (3 H, s), 1.53 1.54 (4 H, m), 1.68 1.83 (2 H, m), 2.05 (3 H, s), 2.08 (3 H, s), 2.11 (3 H, s), 2.36 (2 H, m), 2.53 (2 H, m), 3.48 (2 H, m), 3.78 (2 H, m,); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.7, 11.8, 12.7, 19.6, 19.7, 20.6, 21.0, 22.6, 22.7, 23.5, 24.5, 24.8, 25.2, 28.0, 29.7, 31.3, 32.7, 32.8, 37.3, 37.5, 37.6, 39.4, 40.4, 49.0, 70.8, 74.7, 117.4, 122.8, 125.6, 127.6, 147.6, 147.9; HRMS (ESI) for C 32 H 55 Na 2 5 S [M + 2Na + - H + ] + calculated 597.3566, found 597.3577. 3-[2,5,7,8-Tetramethyl-2-(4,8,12-trimethy-tridecyl)-3,4-dihydro-2H-chroman-6- yloxy]-propane-1-sulfonic acid (Rac-α-Tocopheryloxypropyl sulfonic acid, Rac-VEPSA). All racemic α-tocopherol (Rac-Toc) (1.01 g, 2.32 mmol) was converted to all racemic VEPSA (1.32 g, 99%) using the same procedure as used for the synthesis of R,R,R-VEPSA. All racemic VEPSA was isolated as brown waxy solid. R f (1:1 EtAc: iprh): 0.2; IR (neat, thin film) υ 2925 (s), 1460 (m), 1376 (m), 1190 (s), 1092 (m), 1056 (s), 739 (s); 1 H NMR (300 4
MHz, CDCl 3 ) δ 0.85 0.88 (12 H, m), 1.08 1.39 (18 H, m), 1.26 (3 H, s), 1.48 1.59 (4 H, m), 1.61 1.75 (2 H, m), 1.97 (3 H, s), 1.98 (3 H, s), 2.01 (3 H, s), 2.30 (2 H, m), 2.43 (2 H, m), 3.34 (2 H, m), 3.63 (2 H, m); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.6, 12.6, 19.6, 19.7, 19.8, 20.5, 21.1, 22.6, 22.7, 23.1, 24.5, 24.8, 25.7, 28.0, 29.7, 31.3, 32.8, 37.3, 37.5, 39.4, 40.9, 48.6, 71.1, 74.4, 117.1, 122.5, 125.6, 127.6, 147.5, 147.9; HRMS (ESI) for [M + H] + C 32 H 57 5 S: calculated 553.3921, found 553.3937. 4-[2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyl-tridecyl)-3,4-dihydro-2H-chroman- 6-yloxy]-butane-1-sulfonic acid (R,R,R-α-Tocopheryloxybutyl sulfonic acid, RRR- VEBSA). RRR-α Τocopherol (RRR-Toc) (1.00 g, 2.32 mmol), and NaH (0.111 g, 4.64 mmol), were combined in a 25 ml two neck round bottom flask in THF (20 ml, 0.12 M) at 0 0 C under argon. 1, 4-Butane sultone (0.38 g, 2. 79 mmol) was added to the reaction mixture after stirring at room temperature for 50 min. The reaction was refluxed for 24 h. Upon completion, the reaction was quenched with conc. HCl, extracted with a 3:1 mixture of EtAc: iprh, washed with acidified brine, then dried over anhydrous Na 2 S 4. The crude organic layer was then concentrated, using a rotovap, to form a brown solid. This was then subjected to flash chromatography using 1 1 / 2 inch silica gel [gradient of EtAc: Hexane from (1: 4) to 100% EtAc] followed by EtAc : HAc (20 : 1)], to yield (1.02g, 76%) as light brown waxy solid. [α] 25 D = + 3.59 ( c 1.0, chloroform); λ max /nm (UV/Vis, chloroform): 288, (UV/Vis, ethanol): 214, 276; R f (1:1 EtAc: iprh): 0.19; IR (neat, thin film) υ 2924 (s), 1459 (m), 1377 (m), 1169 (s), 1090 (m), 1054 (s) cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 0.86 0.88 (12 H, m), 1.09 1.45 (20 H, m), 1.27 (3 H, s), 1.49 1.55 (4 H, m), 1.69 1.74 (2 H, m), 1.82 1.92 (2 H, m), 2.03 (3 H, s), 2.06 (3 H, s), 2.09 (3 H, s), 2.50 (2 H, m), 3.16 (2 H, m), 3.609 (2 H, m); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.7, 11.8, 12.7, 19.6, 19.7, 20.6, 21.0, 21.4, 22.6, 22.7, 23.4, 24.5, 5
24.8, 28.0, 29.2, 31.3, 32.7, 32.8, 37.3, 37.5, 37.5, 37.6, 39.4, 40.6, 51.3, 72.2, 74.6, 117.3, 122.6, 125.7, 127.6, 147.7, 148.1; HRMS (EI) for C 33 H 58 5 S [M] + calculated 566.4005, found 566.4002. 4-[2,5,7,8-Tetramethyl-2-(4,8,12-trimethyl-tridecyl)-3,4-dihydro-2H-chroman-6- yloxy]-butane-1-sulfonic acid (Rac-α-Tocopheryloxybutyl sulfonic acid, Rac-VEBSA). All racemic α-tocopherol (1.00 g, 2.32 mmol) was converted to all-racemic VEBSA (1.03 g, 76%) using the same procedure as for R,R,R-VEBSA. All racemic VEBSA was isolated as a brown solid. R f (1:1 EtAc: iprh): 0.2; IR (neat, thin film) υ 2925 (s), 1460 (m), 1377 (m), 1188 (s), 1090 (m), 1054 (s), 736 (w) cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 0.85 0.89 (12 H, m), 1.09 1.38 (23 H, m), 1.48 1.59 (4 H, m), 1.60 1.75 (2 H, m), 1.76 1.90 (2 H, m), 1.90 2.10 (9 H, m), 2.44 (2 H, m), 3.08 (2 H, m), 3.54 (2 H, m); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.7, 11.8, 12.7, 19.6, 19.7, 20.6, 21.0, 21.4, 22.6, 22.7, 23.4, 24.5, 24.8, 28.0, 29.2, 31.3, 32.7, 32.8, 37.3, 37.47, 37.51, 37.6, 39.4, 40.6, 51.3, 72.2, 74.6, 117.3, 122.6, 125.7, 127.6, 147.7, 148.1; HRMS (ESI) for [M + 2Na H + ] + C 33 H 57 Na 2 5 S + : calculated 611.3722, found 611.3693. 3-[2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyl-tridecyl)3,4-dihydro-2H-chroman- 6-yloxy]-propylphosphonic acid (R,R,R-α-Tocopheryloxypropyl phosphonic acid, RRR- VEPPA). RRR-α-Tocopheryloxypropyl diethylphosphonate (0.177 g, 0.291 mmol), and 9 N HCl (2 ml) were combined in a 25 ml round bottom flask. The reaction mixture was refluxed at 100 0 C for 24 h. Upon completion the reaction was extracted with CH 2 Cl 2 and dried (Na 2 S 4 ). Concentration in vacuo yielded RRR-VEPPA as a brown oil (0.1576 g, 89%) that was not subjected to flash chromatography due to its highly polar nature. [α] 25 D = + 0.79 0 (c 1.0, chloroform); λ max /nm (UV/Vis, chloroform): 282, (UV/Vis, ethanol): 214, 286; R f (1:1 EtAc: iprh): 0.14; IR (neat, thin film) υ 2924 (s), 2358 (w), 1459 (m), 1259 (m), 1088 (s), 1015 (s), 6
799 (s); 1 H NMR (400 MHz, CDCl 3 ) δ 0.83 0.87 (12 H, m), 1.06 1.53 (25 H, m), 1.25 (3 H, s), 1.72 1.77 (2 H, m), 2.06 (3 H, s), 2.09 (3 H, s), 2.14 (3 H, s), 2.24 (2 H, s), 2.45 2.60 (2 H, m), 3.69 (2 H, m); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.8, 11.9, 12.7, 19.7, 19.8, 20.6, 21.0, 22.6, 22.7, 23.2, 23.8, 24.4, 24.8, 28.0, 29.7, 31.3, 32.7, 32.8, 37.3, 37.5, 37.5, 39.4, 40.2, 72.2, 74.8, 117.5, 122.9, 125.8, 127.7, 129.3, 147.9, 147.9; HRMS (ESI) for C 32 H 58 5 P + [M + H] + : calculated 553.4022, found 553.4009. 3-[2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyl-tridecyl)3,4-dihydro-2H-chroman- 6-yloxy]-propylphosphonic acid (R,R,R-α-Tocopheryloxypropyl phosphonic acid, RRR- VEPPA). RRR-α-Tocopheryloxypropyl diethylphosphonate (0.177 g, 0.291 mmol), and 9 N HCl (2 ml) were combined in a 25 ml round bottom flask. The reaction mixture was refluxed at 100 0 C for 24 h. Upon completion the reaction was extracted with CH 2 Cl 2 and dried (Na 2 S 4 ). Concentration in vacuo yielded RRR-VEPPA as a brown oil (0.1576 g, 89%) that was not subjected to flash chromatography due to its highly polar nature. [α] 25 D = + 0.79 0 (c 1.0, chloroform); λ max /nm (UV/Vis, chloroform): 282, (UV/Vis, ethanol): 214, 286; R f (1:1 EtAc: iprh): 0.14; IR (neat, thin film) υ 2924 (s), 2358 (w), 1459 (m), 1259 (m), 1088 (s), 1015 (s), 799 (s); 1 H NMR (400 MHz, CDCl 3 ) δ 0.83 0.87 (12 H, m), 1.06 1.53 (25 H, m), 1.25 (3 H, s), 1.72 1.77 (2 H, m), 2.06 (3 H, s), 2.09 (3 H, s), 2.14 (3 H, s), 2.24 (2 H, s), 2.45 2.60 (2 H, m), 3.69 (2 H, m); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.8, 11.9, 12.7, 19.7, 19.8, 20.6, 21.0, 22.6, 22.7, 23.2, 23.8, 24.4, 24.8, 28.0, 29.7, 31.3, 32.7, 32.8, 37.3, 37.5, 37.5, 39.4, 40.2, 72.2, 74.8, 117.5, 122.9, 125.8, 127.7, 129.3, 147.9, 147.9; HRMS (ESI) for C 32 H 58 5 P + [M + H] + : calculated 553.4022, found 553.4009. 4-[2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyl-tridecyl)-3,4-dihydro-2H-chromen- 6-yloxy]-butylphosphonic acid (RRR-α-Tocopheryloxybutyl phosphonic acid, RRR- 7
VEBPA). Two ml 9 N HCl was added to a solution of approximately 0.5 ml acetone and RRRα-tocopheryloxybutyl diethylphosphonate (0.0482 g, 0.0774 mmol) in a 25 ml round bottom flask. The reaction mixture was refluxed at 100 C for 24 h. Upon completion the reaction was extracted with CH 2 Cl 2 and dried (Na 2 S 4 ). Concentration of product in vacuo gave RRR- VEBPA as a brown oil (0.0320 g, 73%). λ max /nm (UV/Vis, chloroform): 282, (UV/Vis, ethanol): 214, 286; R f (1:1 EtAc: iprh): 0.16; IR (neat, thin film) δ 2925 (s), 2357 (s), 1460 (m), 1378 (m), 1259 (m), 1090 (m), 1090 (m), 805 (m); 1 H NMR (400 MHz, CDCl 3 ) δ 0.83 0.87 (12 H, m), 1.07 1.54 (27 H, m), 1.26 (3 H, s), 1.73 1.86 (4 H, m), 2.07 (3 H, s), 2.10 (3 H, s), 2.15 (3 H, s), 2.45 2.60 (2 H, m), 3.64 (2 H, m); 13 C NMR (75.5 MHz, CDCl 3 ) δ 11.8, 11.9, 12.7, 14.1, 19.65, 19.74, 20.7, 21.0, 22.6, 22.7, 23.8, 24.5, 24.8, 27.1, 28.0, 29.4, 29.7, 30.0, 31.3, 31.9, 32.8, 37.3, 37.5, 39.4, 40.2, 74.7, 117.5, 122.8, 125.7, 127.7, 147.8, 147.9; HRMS (ESI) for [M + H] + C 33 H 60 5 P + : calculated 567.4178, found 567.4186. Detection of VEBSA and VES VEBSA and VES were detected by the HPLC using a reverse phase c-18 column. The mobile phase was 10 mm ammonium acetate in methanol. VEBSA and VES were detected via UV-Vis at 280 nm wavelength and 220 nm wavelength, respectively. The retention time for both VEBSA and VES was ~350 seconds. The extraction and quantification of VEBSA and VES We extracted VEBSA or VES from 300 μl serum by adding 200 μl 6M HCl and 200 μl ethyl acetate. The sample was then vortexed and centrifuged for 1 min at 12,000 rpm. The organic layer was separated and filtered through a c-18 sep-pack column filter and then 8
concentrated. This sample was then re-dissolved in 40 μl of methanol and detected on the HPLC as described above. The area under the curve was then determined and the standard calibration curve was used to determine the concentration of VEBSA (or VES) in methanol. Standard solutions of 0 μm, 20 μm, 30 μm, 50 μm, 100 μm, 150 um, and 200 μm of VEBSA (or VES) were prepared in methanol and chromatographed on the HPLC. Each sample was run three times. The area under the curve (AUC) for each sample was then calculated using GraphPad Prism software ver 5.01. A standard calibration curve was then plotted based on the average of the AUC of VEBSA (or VES) versus the standard concentrations. 9
Supplemental Figure Legend: Supplemental Figure 1: The HPLC detection graph of detection of VEBSA and VES in pooled mouse serum collected from mice after oral administration of VEBSA, VES or mock for one month. Detection of VEBSA in pooled mouse serum collected from mice after oral administration of VEBSA (A) and mock (B). Detection of VES in pooled mouse serum collected from mice after oral administration of VES (C) and mock (D). The area under the curve (AUC) for each sample was then calculated using GraphPad Prism software ver 5.01. to determine the concentration of VEBSA (or VES) in methanol. Each sample was run three times. A B C D 10