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, HRMS (m/z): [M+H] + calcd for C26H22N3O3, 424.1661, found, 424.1607. 2,8-Dibenzyl-6-(4-(benzyloxy)phenyl)imidazo[1,2-a]pyrazin-3-yl acetate (20bA): Obtained as an off-white solid (0.44 g) pure enough (traces of THF and acetic acid) to be used directly in the next step, Obtained as an off-white solid (4.02 g, 72%) after a recrystallization in cyclohexane. 1 H NMR (CDCl3): 7.92 (m, 2H), 7.83 (s, 1H), 7.63 (m, 2H), 7.47 (m, 2H), 7.42-7.30 (m, 4H), 7.23 (m, 1H), 6.35 (m, 1H), vol.1

, -(benzyloxy)phenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3-yl acetate

H. Nmr, 36 (dd, 1H, J = 3.1, 1.9 Hz), 6.17 (dd, 1H, CDCl3): 7.83 (s, 1H), 7.61 (m, 3H), 7.49 (m, 3H), 7.45-7.21 (m, 8H), 7.02 (m, 1H), vol.6

, HRMS

, Obtained as a white solid (0.5 g, 77%) after a recrystallization in cyclohexane. 1 H NMR (CDCl3): 7.79 (s, 1H), 7.61 (m, 3H), 7.51-7.30 (m, 14H), 7.23 (3H), 7.01 (m, 1H), 6.94 (m, 2H), vol.5

, Benzyl-2-(furan-2-ylmethyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3-yl acetate

H. Nmr, DMSO-d6): 9.65 (s, 1H), 8.55 (s, 1H), 7.90 (m, 2H), 7.55 (m, 1H), 7.47 (m, 2H), 7.27 (m, 2H), 7.22 (m, 1H), 6.88 (m, 2H), vol.6, p.39

H. Nmr, HRMS (m/z): [M+H] + calcd for C28H24N3O4: 466.1767, found, 466.1758. the cells by centrifugation (1.5 L), pellet was resuspended in 50 mM Tris-HCl pH 8.0, 50 mM NaCl with protease inhibitors (Sigma) and lysozyme (0.1 mg/mL). Cells were disrupted by freezing-thawing cycle lysis method. DNase I (Sigma-Aldrich) was then added to remove DNA from the sample. The crude extract was centrifuged 30 min at 1250 g. The supernatant was collected and NaCl (500 mM), imidazole (20 mM) and Triton X100 (0.1%) were added. Then this cleared lysate was loaded on a His-Trap HP column (5 mL, GE-Healthcare) at a flow rate of 4 mL/min using an AKTA pure chromatography system (GE-Healthcare), DMSO-d6) 9.65 (s, 1H), 9.20 (s, 1H), 8.53 (s, 1H), 7.89 (m, 2H), 7.48 (m, 2H), 7.24 (m, 3H), 7.07 (m, 2H), 6.87 (m, 2H), 6.69 (m, 2H), 4.46 (s, 2H), vol.3, p.5

, The nanoKAZ was eluted with a gradient of imidazole from 20 mM to, p.50

. Ge-heath, -benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one). The fractions of high activity were pooled, and loaded on a HiTrap Q column (1 mL, GE-Healthcare) equilibrated in 50 mM Tris-HCl pH 8.0, NaCl 50 mM. The protein was eluted in 50 mM MES pH 6.5, 50 mM NaCl at 1 mL/min at stain-free SDS gel (4-15% Mini-PROTEAN® TGX Stain-Free? Protein Gels, Bio-Rad). The gel was activated by UV trans-illumination for 5 min (Bio-Gel Doc XR Imaging System) and fluorescence of tryptophan in protein bands were imaged, Catalytic activity of fractions was profiled using a luminometer Hydex by diluting 10 7 folds the fractions in PBS with 27 M of furimazine

, As depicted in figure S5, the light emission intensity decreases with time: steep for "flash" (5dB) and slow (3/5aB) for "glow" substrates. For verifying if the reaction rate indicated by the photon emission (RLU/s) decreases with time, the same enzyme amount, pp.62-72

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, 5R)-3-benzyl-5-phenylpiperazin-2-one (15a, vol.3, p.57

, 5S)-3-benzyl-5-phenylpiperazin-2-one (15a, second dia, vol.3, p.59

, 5S)-3-benzyl-5-phenylpiperazin-2-one (15a, second dia, vol.3, p.65

, -(benzyloxy)phenyl)-5-oxo-2,3,4,5-tetrahydropyrazine 1-oxide (17b, p.85