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The 19F NMR signal of 1 was silent with CAI and EZA over 24 h in vitro
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The 19F NMR signal of 1 was silent with CAI and EZA over 24 h in vitro.
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53
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79955670299
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Chemically synthesized FBOH in buffer solution also showed a signal at -62.9 ppm (Fig. S2, ESI†)
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Chemically synthesized FBOH in buffer solution also showed a signal at -62.9 ppm (Fig. S2, ESI†).
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54
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79955669757
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Note
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Since we set the experimental conditions where all of the labeling reagents are complexed with CAI, the determined values of kL and kHcomplex are considered as the rates from the complexed state between CAI and the reagents.
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-
-
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55
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79955672350
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Note
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It should be noted that because of the need for a relatively high concentration of FB-CAI (20 mM) for 19F NMR measurements, we were unable to determine the precise affinity values of FB-CAI towards the strong inhibitors, EZA, AAZ and NBS.
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-
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56
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79955649949
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Note
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The labeling site of FB-eCAI was shown to be at His67, which is identical to the site identified for FB-CAI prepared in vitro.
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57
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79955647589
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Note
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Under the conditions using less than 400 M of 1, we did not observe any toxicity for RBCs over the time period of the assay (over 48 h).
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-
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58
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79955655042
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Note
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The validity of this mole number was also supported by in-cell 19F NMR measurements, in which the intracellular concentration of 1 can be determined from an integral of the signal at -62.3 ppm corresponding to the 1-eCAI complex (Fig. 4b). The mole number estimated in this manner was 194 ± 2 nmol.
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79955647411
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Note that following mixing of the RBC suspension with 1, the supernatant is replaced with fresh buffer before starting incubation (also see the Methods section)
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Note that following mixing of the RBC suspension with 1, the supernatant is replaced with fresh buffer before starting incubation (also see the Methods section).
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