Kinetics experiments were carried out at ambient temperature (24 o -26 o C) on a 250 MHz Bruker

Transcription

1 Experimental Materials and Methods. All 31 P NMR and 1 H NMR spectra were recorded on 250 MHz Bruker or DRX 500 MHz instruments. All 31 P NMR spectra were acquired using broadband gated decoupling. 31 P Chemical shifts are reported in parts per million using 1% triphenylphosphine oxide in benzene-d 6 as the coaxial reference (triphenylphosphine oxide/toluene-d 8 has a chemical shift of ppm relative to 85% phosphoric acid). 31 P NMR Kinetics experiments were carried out at ambient temperature (24 o -26 o C) on a 250 MHz Bruker NMR. 1 H Chemical shifts are reported in parts per million from tetramethylsilane. Flash chromatography using silica gel grade 60 ( mesh) was carried out for the chromatographic purification of phosphoramidate ester 19. Thin layer chromatography was performed using Analtech glass plates precoated with silica gel (250 microns). Visualization of the plates was accomplished using UV and p-anisaldehyde dip (1.85% p-anisaldehyde, 20.5% sulfuric acid, 0.75% acetic acid in 95% EtOH) followed by heating. The LAH reduction of N- methylamido alcohol 15 was monitored by silica gel TLC using I 2 for visualization. The chromatographic separation of phosphate coupling reaction mixtures was carried out using C18 (40 µm) preparatory LC packing (Fisher) or pre-packed Maxi-Clean C18 cartridges (Alltech). Cation exchange was carried out using DOWEX 50W-X8, conditioned to the ammonium form. β-d-glucose 1-phosphate (di(cyclohexylammonium) salt) and thymidine were purchased from Sigma. Benzyl alcohol and γ-butyrolactone were purchased from Aldrich. All reactions were carried out under an atmosphere of argon unless otherwise specified or if reagents containing water were used. N-Methyl-N-(4-chlorobutyl)amine hydrochloride 17 was dried by coevaporation with acetonitrile prior to use. Thymidine, β-d-glucose 1-phosphate, and tetrabutylammonium chloride were coevaporated with pyridine prior to use. Benzyl alcohol and 4-(N-methylamino)butan-1-ol 16 were distilled prior to use.

2 Chemistry N-Methyl-4-hydroxybutyramide (15): Methylamine (6.0 ml, 130 mmol) was condensed in a pressure tube at 78 o C. γ-butyrolactone (1.0 ml, 13 mmol) was added to the condensed methylamine slowly with stirring at 78 o C. The mixture was sealed and warmed to room temperature, and stirring was continued for 1.5 h. Excess methylamine was evaporated under atmospheric pressure, and the crude reaction mixture was further concentrated under reduced pressure to afford N-methylamido alcohol 15 (1.5g, 99%) as a viscous oil. Product 15 was used without further purification. 1 H NMR (CDCl 3 ): (see attached spectra) δ 5.99 (bs, 1H), 3.70 (t, 2H), 2.82 (d, 3H), 2.72 (bs, 1H), 2.37 (t, 2H), 1.88 (m, 2H). 4-(N-Methylamino)butan-1-ol (16): N-methylamido alcohol 15 (5.0g, 43 mmol) was dissolved in THF (100 ml) under an atmosphere of argon in a two-necked flame-dried flask equipped with a reflux condenser. Lithium aluminum hydride (1.0 M in diethyl ether, 86 ml, 86 mmol) was added slowly with stirring at room temperature. The resulting exothermic reaction brought the mixture to reflux. The reaction mixture was allowed to cool to room temperature following the addition of LAH and stirred for 2 h. The reaction was quenched by the slow addition of Na 2 SO 4.10H 2 O (27.5g, 86.0 mmol), and the resulting mixture was filtered. The solid cake was rinsed several times with CH 2 Cl 2 and the filtrate was concentrated to afford amino alcohol 16 (2.9g, 66%) as a colorless oil. 1 H NMR (CDCl 3 ): (see attached spectra) δ 3.64 (bs, 1H), 3.56 (t, 2H), 2.61 (t, 2H), 2.42 (s, 3H), 1.65 (m, 4H). N-Methyl-N-(4-chlorobutyl)amine hydrochloride (17): Amino alcohol 16 (1.0g, 9.7 mmol) was dissolved in CH 2 Cl 2 (20 ml). Anhydrous HCl (g) was bubbled through the mixture until the mixture turned litmus paper red. The mixture was cooled to 0 o C and thionyl chloride ( 0.78 ml,

3 11 mmol) was added dropwise with stirring. The reaction mixture was warmed to room temperature and stirred 4 h. The mixture was then concentrated under reduced pressure and coevaporated (3x) with CH 2 Cl 2 to remove dissolved HCl. Amine hydrochloride 17 was obtained as a white solid (1.46g, 96%) and was used without further purification. 1 H NMR (CDCl 3 ): (see attached spectra) δ 9.57 (bs, 1H), 3.59 (t, 2H), 2.99 (m, 2H), 2.70 (t, 3H), 1.98 (m, 4H). Benzyl N-methyl-N-(4-chlorobutyl) thymidyl phosphoramidate (19): Benzyl alcohol (1.28 ml, 8.26 mmol) was dissolved in CH 2 Cl 2 (25 ml) and cooled to 78 o C. Phosphorus trichloride (6.19 ml, 2.0 M in CH 2 Cl 2, 12.4 mmol) was added slowly followed by the dropwise addition of diisopropylethylamine (3.23 ml, 18.6 mmol). The reaction mixture was allowed to stir at 78 o C for 15 min. N-Methyl-N-(2-chlorobutyl)amine hydrochloride 17 (1.94 g, 12.4 mmol) was dissolved in anhydrous CH 2 Cl 2 (10 ml) and added dropwise to the reaction mixture. Diisopropylethylamine (6.47 ml, 37.1 mmol) was added dropwise, and the reaction mixture was warmed to 60 o C and stirred for 20 min. Thymidine (2.0 g, 8.3 mmol) was coevaporated several times with anhydrous pyridine (8 40 ml) and then dissolved in pyridine (20 ml) and cooled to 45 o C. The mixture of thymidine in pyridine was then titrated with the reaction mixture containing intermediate 18 until thymidine disappeared. The disappearance of thymidine was monitored by TLC (90:10 CHCl 3 :MeOH). tert-butyl hydroperoxide (2.5 ml, M in decane, > 12.5 mmol) was added dropwise to the reaction mixture at 45 o C, and the mixture was warmed to 0 o C over 30 min. Saturated aqueous NH 4 Cl (100 ml) was added, and the aqueous layer was extracted with CH 2 Cl 2 (3 50 ml). The combined organic layers were dried over Na 2 SO 4 and concentrated to a volume of 20 ml. The crude reaction mixture was purified by chromatography on silica gel (CHCl 3 to remove excess tbuooh and pyridine, then 5:95 MeOH:CHCl 3 ) to afford phosphoramidate 19 as a white foam (2.94g, 70%) and a

4 1:1mixture of diastereomers. R f = 0.27 (5:95 MeOH:CHCl 3 ). 1 H NMR (CDCl 3 ): (see attached spectra) δ 9.56 and 9.50 (s, 1H, 1:1 mixture), 7.34 (m, 6H), 6.30 (q, 1H), 4.99 (m, 2H), 4.47 (m, 1H), 4.13 (m, 3H), 3.52 (t, 2H), 3.26 (bs, 1H), 3.02 (m, 2H), 2.62 and 2.61 (d, 3H, 1:1 mixture), 2.40 (m, 1H), 2.08 (m, 1H), 1.86 and 1.80 (s, 3H, 1:1 mixture), 1.67 (m, 4H). 31 P NMR (CDCl 3 ): δ and (1:1 mixture). HRMS (C 22 H 31 ClN 3 O 7 P) theor (M+H) + ; found: Thymidine 5 -diphospho-β-d-glucose (TDP-Glc): Phosphoramidate 19 (166mg, mmol) was dried by coevaporation with anhydrous CH 3 CN and then dissolved in anhydrous THF (3 ml). Pd/C (10%, 10 mg) was suspended in THF (1 ml) and transferred to the flask containing phosphoramidate 19. The flask was equipped with a balloon filled with hydrogen, and the reaction mixture was stirred for 1.5 h at room temperature. β-d-glucose 1-phosphate, di(cyclohexylammonium) salt (59.1 mg, mmol) and TBAC (71.7 mg, mmol) were combined under argon and dried by coevaporation with anhydrous pyridine. The mixture was dissolved in dry pyridine (3 ml) and added dropwise to the hydrogenolysis reaction mixture at room temperature. The reaction mixture was concentrated under reduced pressure to a volume of 3 ml, and stirring was continued for 1 h. The catalyst was removed by filtration, and water (2 ml) was added to the filtrate. The aqueous layer was washed with CH 2 Cl 2 (5 x 2 ml) to remove excess TBAC and amine hydrochloride, and the aqueous layer was concentrated. Conversion of the crude product mixture to ammonium salts was accomplished on DOWEX 50W-X8 purchased as the hydrogen form and converted to the ammonium form using NH 4 OH. Subsequent separation of reactants and products was accomplished using C18 chromatography (50 mm NH 4 OAc, ph5.5). Unreacted β-d-glucose 1-phosphate was eluted from the column in the void volume. The desired product TDP-Glc was then eluted. TMP and the self-condensation

5 product can be eluted after the desired product (NH 4 OAc, ph5.5 to 20:80 CH 3 CN/ NH 4 OAc, ph5.5). HPLC retention time for the reaction products are as follows: TDP-Glc (NH 4 OAc, 50mM, ph5.5, R t = 4.1 min); TMP (NH 4 OAc, 50mM, ph5.5, R t = 7.5 min); self-condensation (15:85 CH 3 CN/NH 4 OAc, 50mM, ph5.5, R t = 8.6 and 9.1 min, 1:1 mixture of diastereomers). TDP-Glc was isolated as a white solid (59 mg, 77%, 91% based on recovered starting material). 1 H NMR (D 2 O): (see attached spectra) δ 7.61 (s, 1H), 6.22 (t, 1H, J = Hz), 4.89 (overlapping dd, 1H, J PH = 7.94 Hz, J HH = 7.93 Hz), 4.53 (m, 1H), 4.05 (m, 3H), 3.75 (d, 1H), 3.58 (dd, 1H), 3.40 (m, 2H), 3.26 (m, 2H), 2.25 (m, 2H), 1.81 (s, 3H). 31 P NMR (D 2 O): δ (d, 1P, J PP = Hz), (d, 1P, J PP = Hz). HRMS (C 16 H 26 N 2 O 16 P 2 ) theor (M-H) + ; found: Thymidine 5 -diphospho-β-l-rhamnose (TDP-Rha): TDP-Rha was prepared (as described above for TDP-Glc) as the ammonium salt from phosphoramidate 19 (263 mg, mmol) and β-l-rhamnose 1-phosphate, bis(triethylammonium) salt (91 mg, 0.20 mmol). Excess TBAC (283 mg, 1.02 mmol) was required in this case to dissolve the sugar phosphate in anhydrous pyridine. Despite efforts to dry the sugar phosphate by coevaporation with anhydrous pyridine, significant hydrolysis of the reactive pyrrolidinium ion intermediate during the phosphate coupling reaction was observed by 31 P NMR and resulted in incomplete conversion of rhamnose 1-phosphate to the desired product. TDP-Rha was isolated as a white solid (67 mg, 56%, 97% yield based on recovered starting material). The 1 H and 31 P NMR spectral data (see attached spectra) were identical to that reported by Thorson, et al (see reference 1h in manuscript).

6 1 H NMR (CDCl 3 ) of N-methyl-4-hydroxybutyramide (15): PPM 1 H NMR (CDCl 3 ) of 4-(N-methylamino)butan-1-ol (16): PPM

7 1 H NMR (CDCl 3 ) of N-methyl-N-(4-chlorobutyl)amine hydrochloride (17): PPM 1 H NMR (CDCl 3 ) of benzyl N-methyl-N-(4-chlorobutyl) thymidyl phosphoramidate (19): PPM

8 31 P NMR (CDCl 3 ) of benzyl N-methyl-N-(4-chlorobutyl) thymidyl phosphoramidate (19): PPM 1 H NMR (D 2 O) of thymidine 5 -diphospho-β-d-glucose (TDP-Glc): PPM

9 31 P NMR (D 2 O) of thymidine 5 -diphospho-β-d-glucose (TDP-Glc): PPM 1 H NMR (D 2 O) of thymidine 5 -diphospho-β-l-rhamnose (TDP-Rha): PPM

10 31 P NMR (NH 4 OAc, 50mM, ph5.5) of thymidine 5 -diphospho-β-l-rhamnose (TDP-Rha): PPM