Reaction #1125481

ord-7748f721448b4529b1d51962b9a31472

Reaction equation

c1ccc2scnc2c1
benzothiazole
NC(=O)c1ccc[n+]([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)c1
NADP+
c1ccc2scnc2c1
benzothiazole
c1ccc2scnc2c1
benzothiazole
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
O=C1C=Cc2n/c(=C3\N[C@@H](C(=O)O)CS3)sc2=C1
luciferin
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
[Cl-].[Cl-].[Mg+2]
MgCl2
O=C([O-])CC(O)(CC(=O)[O-])C(=O)[O-].[Na+].[Na+].[Na+]
sodium citrate
N[C@H](CS)C(=O)O
D-cysteine
Cl.NC(CO)(CO)CO
Tris-HCl
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
→
c1ccc2scnc2c1
benzothiazole
N#Cc1nc2ccc(O)cc2s1
6-hydroxybenzo[d]thiazole-2-carbonitrile

Reactants

c1ccc2scnc2c1
benzothiazole
—
KPO4
—
KPO4
NC(=O)c1ccc[n+]([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)c1
NADP+
c1ccc2scnc2c1
benzothiazole
c1ccc2scnc2c1
benzothiazole
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
O=C1C=Cc2n/c(=C3\N[C@@H](C(=O)O)CS3)sc2=C1
luciferin
O=P(O)(O)OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O
glucose-6-phosphate
[Cl-].[Cl-].[Mg+2]
MgCl2
O=C([O-])CC(O)(CC(=O)[O-])C(=O)[O-].[Na+].[Na+].[Na+]
sodium citrate
—
P450
N[C@H](CS)C(=O)O
D-cysteine
Cl.NC(CO)(CO)CO
Tris-HCl
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH

Reagents

None

Solvents

—
KPO4
CS(C)=O
DMSO

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    workup.ADDITIONwas added to each reaction
  2. 2
    workup.WAITto incubate at room temperature for 20 minutes

Procedure

Stock solutions of the benzothiazole compounds 3016, 3019, 3026, 3806, 3814, 3820, 3821, 3833, 3835, 3866, and 3868 were made at 50 mM in DMSO. One picomole of each P450 enzyme (Supersomes™, BD Bioscience) was incubated with 50 mM of each of the compounds in a 50 μl, reaction in KPO4 buffer pH 7.4 (25 mM KPO4 for CYP2C9, 50 mM KPO4 for CYP2B6, -2C8, -2C19, -4F2, -4F3A and -4F3B, 100 mM KPO4 for CYP1A1, -1A2, -1B1, -2D6, -2E1, -3A5, -3A7, -2J2, -4F12, -19 and minus P450 control, 200 mM KPO4 for CYP3A4) or 100 mM Tris-HCl, pH 7.5 (for CYP2A6, -2C18 and -4A11). Reactions were initiated by adding an NADPH regenerating system (final concentrations of the regenerating system components in the assay were: 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, 0.4 U/mL glucose-6-phosphate dehydrogenase and 0.05 mM sodium citrate). Reactions were incubated for 30 minutes at 37° C. After incubation, 50 μl, of P450-Glo™ luciferin detection reagent (available from Promega Corp.) supplemented with 6.6 mM D-cysteine was added to each reaction, allowed to incubate at room temperature for 20 minutes, and luminescence was detected on a Veritas luminometer. FIGS. 14-25 demonstrate that the benzothiazole compounds can also be used to detect CYP450 enzyme activity as light output in this assay scheme. Each compound was active with one or more P450 enzymes and the profile of P450 activity varied across the panel of enzymes depending on the compound structure. The light output specific to a given P450 enzyme can be due to oxidation of the benzothiazole compound by the P450 enzyme to yield 6-hydroxybenzo[d]thiazole-2-carbonitrile (a benzothiazole derivative). The 6-hydroxybenzo-[d]thiazole-2-carbonitrile can then react with D-cysteine to form a D-luciferin derivative that in turns reacts with luciferase in the luciferin detection reagent to generate light in proportion to the amount of D-luciferin present.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US08551721B2uspto-grants-2013_10