We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.

[9-[10-(4-t-Butyl-2,6-dimethyl)phenyl]anthryl](4-bromo-2,6-dimethylphenyl)diazomethane (1-N(2)) was found to be stable enough to survive under Sonogashira and Suzuki coupling reaction conditions, and bis(diazo) compounds incorporated into the 1,4-positions of butadiyne (3-2N(2)) and the 2,5-position of thiophene (4-2N(2)) were prepared. Irradiation of those bis(diazo) compounds generated bis(carbenes), which were characterized by ESR and UV-vis spectroscopic techniques in a matrix at low temperature, as well as time-resolved UV-vis spectroscopy in solution at room temperature. These studies revealed that both of the bis(carbenes), 3 and 4, have a singlet quinoidal diradical ground state with a very small singlet-triplet energy gap of less than 1 kcal/mol. A remarkable increase in the lifetime of bis(carbenes) as opposed to that of monocarbene (1) was noted and was interpreted to indicate that bis(carbenes) are thermodynamically stabilized as a result of delocalization of unpaired electrons throughout a pi-net framework. Despite the stability, both bis(carbenes) are readily trapped by molecular oxygen to afford bis(ketones) as main products.

(2,6-dimethyl-4-tert-butylphenyl)(2,4,6-tribromophenyl)diazomethane(-N(2)) was found to be stable enough to survive under Sonogashira coupling reaction conditions, and aryldiazomethyl substituents were introduced at the 1,4-positions of butadiyne (4-2N(2)) and the 2,5-positions of thiophene(5-2N(2)). Irradiation of those bis(diazo) compounds generated bis(carbenes), which were characterized by using ESR and UV/vis spectroscopic techniques in a matrix at low temperature as well as time-resolved UV/vis spectroscopy in solution at room temperature. These studies revealed that both of the bis(carbenes), 4 and 5, have singlet quinoidal diradical ground states with a very small singlet-triplet energy gap of less than 1 kcal mol(-1). A remarkable increase in the lifetime of bis(carbenes), as opposed to that of the monocarbene (2), was noted and was interpreted to indicate that bis(carbenes) are thermodynamically stabilized as a result of delocalization of unpaired electrons throughout the pi net framework. In spite of the stability, both bis(carbenes) are readily trapped by molecular oxygen to afford bis(ketones). Presumably, the reaction of the upper-lying localized quintet states with oxygen is much faster than that for lower-lying states.