The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 7

The analogous 7-aminocoumarins such as 11 - 13 are also important
fluorophores. Compound 11 shares similar spectral properties to
hydroxycoumarin 10 (l max /l em ¼ 351/430 nm, e ¼ 1.8 10 4 M 1 cm 1 ,
F ¼ 0.75), but the fluorescence of this dye is insensitive to pH levels above

pH 5. 2,3 Moreover, alkylation of the nitrogen substituent can yield
significant bathochromic shifts, allowing access to molecules with longer
wavelengths such as 7-(diethylamino)-4-methylcoumarin 12 ( l max /

l ¼ 380/456 nm) and julolidine derivative 13 ( l max / l ¼ 396/489 nm, em em

0.66). 25
The fluorescence of coumarins can be modulated by structural modifi-
cations using several strategies. For hydroxycoumarins, attachment of acyl or
alkyl substituents on the oxygen elicits a hypsochromic shift and lowers fluo-
rescence quantum yield. 26 Likewise, attachment of electron-withdrawing
acyl groups on the nitrogen of aminocoumarins decreases fluorescence. 27
As detailed below, this property has been exploited in the design of numer-
ous useful probes. In addition, chemical reactions that construct or modulate
the coumarin core can be used to create fluorogenic compounds.

F ¼

4.2. Fluorogenic enzyme substrates
The strategies detailed above have been used to generate numerous fluo-
rogenic enzyme substrates based on the flexible coumarin core. Attachment
of acyl groups on 4-MU can yield useful substrates for esterases and lipases
such as acetate 14 . 28,29 The phosphate ester 15 and sulfate ester 16 also
serve as probes for phosphatases 26 and sulfatases, 30 respectively. Simple
alkoxy derivatives, such as ethoxycoumarin 17 , are valuable substrates
for cytochrome P450 enzymes where oxidative dealkylation releases
4-MU. 31 Alkylation with more exotic moieties can enable measurement
of other enzyme activities. Hydrolysis of the b -lactam amide bond in
ether 18 by b -lactamase yields an intermediate that decomposes to release
fluorescent 10 . 32 Substrates for the widely used b -galactosidase can also
be built from coumarins. Compound 19 is a galactosidase substrate that
bears two fluorine substituents, which lower the p K a and increase the
fluorescence of the resulting coumarin dye at physiological pH. 26 Other
glycosylated coumarins have been used as enzyme substrates. 33 Alkylated
hydroxycoumarins can be employed as esterase substrates by appending
acyloxymethyl ethers to the phenolic oxygen to yield compounds such as
20 . 34 These highly stable esterase substrates can enable careful screening
of esterase activity 35 and delivery of labels inside living cells. 36