L. Bonnac et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1512–1515
1515
4. Yamasaki, M.; Churchill, G. C.; Galione, A. FEBS
J. 2005, 272, 4598.
(11 mg, sodium salt) was obtained as a white solid (yield
32%). H NMR (D2O, 600 MHz) d 9.35 (s, 1H), 9.21 (d,
1
5. Dowden, J.; Berridge, G.; Moreau, C.; Yamasaki, M.;
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J = 6.0 Hz, 1H), 8.87 (d, J = 7.8 Hz, 1H), 8.38 (s, 1H),
8.27-8.22 (m, 1H), 8.19 (s, 1H), 6.32 (d, J = 6.6 Hz, 1H),
6.10 (d, J = 5.4 Hz, 1H), 4.60 (pseudo t, J = 6.6 Hz, 1H),
4.58–4.55 (m, 1H), 4.51 (pseudo t, J = 5.4 Hz, 1H),
4.48–4.44 (m, 2H), 4.40 (ddd, J = 11.8, 4.0, 2.6 Hz, 1H),
4.36 (ddd, J = 11.7, 5.1, 2.7 Hz, 1H), 4.31–4.25 (m, 2H),
4.19–4.15 (m, 1H). 31P NMR (D2O, 243 MHz) d ꢀ10.13
(d, J = 20.6 Hz), ꢀ10.56 (d, J = 21.1 Hz). MS ESI calcd
(MꢀH)ꢀ 662.1018, found 662.0819.
20. NAD kinase14,15 activity was determined by an HPLC
assay. The assay mixture (150 lL) consisted of 50 mM
Tris/HCl, pH 8.0, 20 mM MgCl2, 5 mM ATP, 1 mM
NAD, and an appropriate amount of inhibitor and
enzyme. After incubation for 30 min at 37 ꢁC, an aliquot
(70 lL) was withdrawn and added to ice-cold 1.2 M
HClO4 (35 lL) and kept for 10 min. The mixture was
centrifuged for 1 min at 16,000g. An aliquot (100 lL) of
the HClO4 supernatant was neutralized by addition of
0.8 M K2CO3 (28 lL), kept at ꢀ20 ꢁC for 15 min, and
centrifuged. An aliquot of the supernatant was loaded
onto a Supelcosil LC-18 reversed-phase column (250 ·
4.6 mm, 5 lm). The elution conditions: 9 min at 100%
buffer A (0.1 M potassium phosphate, pH 6.0), 6 min at up
to 12% buffer B (buffer A, containing 20% methanol),
2.5 min at up to 45% buffer B, 2.5 min at up to 100% buffer
B, and hold at 100% buffer B for 5.5 min; finally the
gradient returns to 100% buffer A in 5 min. The column
was flushed with buffer A for 4.5 min prior to the next run.
The flow rate was 1.3 ml/min, and the column was
thermostated at 25 ꢁC. The eluate absorbance was mon-
itored at 260 nm.
14. Zerez, C. R.; Moul, D. E.; Gomez, E. G.; Lopez, V. M.;
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19. NAD analogues 1–3 were prepared by coupling of the
50-phosphoimidazolides of the corresponding adenine
sugars with nicotinamide mononucleotide (NMN) and
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1
characterized by H, 31P NMR and MS. The synthesis of
P1-(nicotinamide-ribos-50-yl)-P2(adenine-arabinofuranos-
50-yl) pyrophosphate (3) serves as an example: NMN
(82 mg, 0.24 mmol) was added to a 0.2 M MnCl2/form-
amide solution (1.5 mL, 0.30 mmol), which was dried with
molecular sieves for several days before use. To this
mixture were added adenine-arabinofuranosyl 50-phos-
phoimidazolide (101 mg, 0.20 mmol) and anhydrous
MgSO4 (50 mg, 0.42 mmol). The reaction mixture was
stirred for 24 h at rt and the crude product was precip-
itated by adding CH3CN (6 mL). After washing with
CH3CN, the solid was dissolved in 1 M TEAB solution
(10 mL), filtered, and the filtrate was used directly for
purification by RP-HPLC. The resulting Et3N salt was
applied on a column of Dowex50WX8-200 (Na+ form) at
4 ꢁC and eluted with water to give the desired sodium salt.
From about one-fourth of the crude product compound 3
30. Woenckhaus, C.; Bieber, E.; Jeck, R. Prog. Clin. Biol. Res.
1987, 232, 53.