The aqueous layer was evaporated again, and dissolved in 10 mL of 1 1 aqueous TFA solution. induced by methanol and purified by phenylsepharose chromatography and cation exchange chromatography (SP column, GE Healthcare, Little Chalfont, UK). All the chemicals used in the enzymatic assays were purchased from Sigma (Santa Clara, CA, USA). 3.2. Chemistry General Process: Coupling Reaction to Synthesize NAD Analogues The related lyophilized analogue of NMN (0.l mmol, 1.0 eq.) was dissolved in dried DMF (0.5 mL). Carbonyldiimidazole (CDI, 114 mg, 0.7 mmol, 7.0 eq.) was added under argon atmosphere. The reaction combination was stirred at space heat and monitored by HPLC. After 3 h, all the starting material had been consumed and a new peak appeared. A small amount of methanol (50 L) was added to hydrolyze the excess CDI. The solvent of the reaction combination was evaporated after 30 min, and then the additional nucleoside monophosphate (0.12 mmol, 1.2 eq) which was dissolved in anhydrous DMF (1.5 mL) containing tri-(3). Compound 2 [26] (564 mg, 1.5 mmol, 1.0 eq.) was dissolved in dichloromethane (DCM, 4 mL) under an argon atmosphere. The perfect solution is was cooled to ?25 C, and PPh3 (555 mg, 2.1 mmol, 1.4 eq.) in DCM (3 mL) were added, stirred for 15 min, then CBr4 (750 mg, 2.29 mmol, 1.5 eq.) in DCM (2 mL) was added. After reacting for 0.5 h at ?17 C, silica gel (900 mg) was added to the combination, which was filtered and washed with DCM. The combined filtrates were concentrated under reduced pressure and the residue were purified by column chromatography (petroleum ether-ethyl acetate = 150:1) to give 3 like a colorless oil (, 350 mg, 53%). 1H-NMR (400 MHz, CDCl3) 8.21C7.98 (m, 4H), 7.68C7.38 (m, 6H), 6.34 (s, 1H), 5.30C5.27 (m, 1H), 4.87 (m, 1H), 4.77 (dd, = 12.5, 3.2 Hz, 1H), 4.63 (dd, = 12.5, 4.5 Hz, 1H), 1.72 (d, = 21.5 Hz, 3H). (5)Compound 3 (330 mg, 0.76 mmol, 1.0 eq.) was dissloved in anhydrous acetonitrile (MeCN, 3 mL), nicotinamide (463 mg, 0.38 mmol, 5.0 eq.) was added and the combination YIL 781 was refluxed over night. The solvent of the reaction combination was evaporated to TNF give a yellow oil. The combination was dissolved in MeOH (4 mL), K2CO3 (126 mg, 0.91 mmol, 1.2 eq.) was added and the combination stirred for 2 h at space temperature. The combination was concentrated under reduced pressure and the residue were purified by column chromatography (DCM-MeOH = 3:1), to give compound 5 (250 mg, 94%) like a pale yellow vesicular solid. 1H-NMR (400 MHz, D2O) 9.32 (s, 1H), 9.11 (d, = 6.3 Hz, 1H), 8.99 (d, = 8.2 Hz, 1H), 8.26 (t, = 7.2 Hz, 1H), 6.52 (d, = 17.1 Hz, 1H), 4.63C4.56 (m, 1H), 4.30 (m, 1H), 4.05C3.97 (m, 1H), 3.78 (dd, = 13.1, 4.3 Hz, 1H), 1.58 (d, = 22.8 Hz, 3H); 19F-NMR (376 MHz, D2O) ?172.73. (6). Compound 5 (176 mg, 0.50 mmol, 1.0 eq.) was dissolved in trimethyl phosphate (TMP, 2.5 mL), and POCl3 (0.23 mL, 2.50 mmol, 5.0 eq.) was added slowly to the reaction combination under snow bath chilling. The combination was stirred for 2 h at 0 C, aqueous sodium hydroxide was then added to neutralize excess acidity to a final pH of 7. The perfect solution is was reduced to dryness, and then the gummy residue was dissolved in water (10 mL) and extracted with ethyl acetate (3 10 mL). The YIL 781 aqueous coating was evaporated again, and dissolved in 10 mL of 1 1 aqueous TFA answer. After purification by HPLC and lyophilization, 6 (160 mg, 69%) was generated like a white cotton-shaped solid. 1H-NMR (400 MHz, YIL 781 D2O) 9.32 (s, 1H), 9.10 (d, = YIL 781 6.1 Hz, 1H), 8.98 (d, = 8.1 Hz, 1H), 8.24 (dd, = 7.7, 6.7 Hz, 1H), 6.53 (d, = 17.2 Hz, 1H), 4.70C4.66 (m, 1H), 4.39 (dd, = 24.0, 9.3 Hz, 1H), 4.32 (ddd, = 12.2, 5.9, 2.0 Hz, 1H), 4.16C4.08 (m, 1H), 1.56 (d, = 22.8 Hz, 3H); 31P-NMR (162 MHz, D2O) 0.02; 19F-NMR (376 MHz, D2O) ?75.63, ?173.38. (2-CH3-2-F NAD, 7). Compound 6 (TFA salt, 0.10 mmol, 46 mg, 1.0 eq.) and AMP.