Enzyme/substrate
pairs, such as β-galactosidase with chromogenic
x-gal substrate, are widely used as reporters to monitor biological
events, but there is still a requirement for new reporter systems,
which may be orthogonal to existing systems. Here, we focused on azoreductase
(AzoR). We designed and synthesized a library of azo-rhodamine derivatives
as candidate fluorogenic substrates. These derivatives were nonfluorescent,
probably due to ultrafast conformational change around the NN
bond after photoexcitation. We found that AzoR-mediated reduction
of the azo bond of derivatives bearing an electron-donating group
on the azobenzene moiety was followed by nonenzymatic cleavage to
afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which
was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at
the single cell level.