The Chemistry of Small-Molecule Fluorogenic Probes - Progress in Molecular Biology and Translational Science - page 5

A second approach to modulate fluorescence is to use F¨ rster resonance
energy transfer (FRET). 14,15 FRET entails energy transfer from the excited
state of one “donor” dye to another “acceptor” dye. In most cases,
FRET occurs between two structurally distinct dyes, where the emission
spectrum from the donor fluorophore overlaps the absorption spectrum
of the acceptor dye. 15 In dyes with small Stokes shifts, however, FRET
between two dyes with the same structure (i.e., homo-FRET) is
favorable. A useful application of this energy transfer uses boron
dipyrromethene (BODIPY) dyes ( Section 5 ), which are environmentally
insensitive and show small Stokes shifts of < 20 nm. 3 An example of a
fluorogenic compound exploiting homo-FRET between two BODIPY
dyes is phospholipase substrate 3 ( Fig. 1.1B ), which is relatively
nonfluorescent because of energy transfer between the two fluorophore
moieties incorporated into the lipid chains. Enzyme-catalyzed hydrolysis
of the ester bond yields fatty acid 4 and phospholipid 5 , both of which
are highly fluorescent ( 10 4 M 1 cm 1 ,

l max / l em ¼ 505/514 nm, e ¼ 9.1

0.94). 2,3,16
A third method to modulate the fluorescence of fluorophores is to mod-
ify the core structure of the dye using chemistry. An example of this strategy
is the trans -cinnamic acid derivative 6 shown in Fig. 1.1C . Illumination of
such molecules with UV light causes trans

and F ¼

cis isomerization. The cis form
undergoes rapid lactonization with cleavage of the ester bond, generating a
fluorescent coumarin 7 (see also Section 4 ). 17,18 Changes in environment
such as polarity can also elicit increases in fluorescence, resulting in
fluorogenic dyes. One example of this phenomenon is the phenoxazine
dye Nile Red ( 8 ; Fig. 1.1D ; Section 8 ). Compound 8 absorbs at 591 nm,
emits at 657 nm, and is relatively nonfluorescent in aqueous solution. In
nonpolar media, such as xylene, Nile Red undergoes a dramatic
hypsochromic shift (

!

523/565 nm) and an increase in quantum
yield. 19 This allows fluorescent staining of hydrophobic regions, such as
lipid droplets, in living cells. 20
Finally, changes in the electronic structure of the dye or its appendages
can cause changes in fluorescence. 1 An example of a probe type utilizing this
process are the fluorescent Ca 2 þ indicators 21 such as Fluo-4 ( 9 ; Fig. 1.1E ).
In the apo state, the lone pairs of electrons on the aniline moieties of the
1,2-bis( o -aminophenoxy)ethane- N , N , N 0 , N 0 -tetraacetic acid (BAPTA) che-
lation motif 22 quench fluorescence because of photoinduced electron trans-
fer (PeT). Ca 2 þ chelation changes the energy of these lone pairs of electrons,
making PeT less efficient and leading to a large increase in fluorescence. 21,23

l max / l ¼ em