Principles of Fluorescence Spectroscopy

80 FLUOROPHORES
Figure 3.29. Fluorogenic reagents for amines.
of amino acids was selected to be specific for a protease
found in HIV. Cleavage of the peptide results in a greater
distance between the donor and acceptor and increased
donor intensity. This concept of a decrease in energy transfer
upon cleavage has also been applied to lipases that
hydrolyze phospholipids.
3.6.2. Structural Analogues of Biomolecules
One approach to designing fluorophores is to make the
shape similar to the parent biomolecule. This approach was
used to make fluorescent analogues of steroids. Two examples
are shown in Figure 3.31. Cholesterol is an essential
component of cell membranes, and estradiol is important
for the expression of female sexual characteristics. Both
molecules are nonfluorescent, but structurally similar molecules
have been synthesized which display useful fluorescence.
Dehydroergosterol is a fluorescent analogue of cholesterol
that displays absorption and emission maxima near
325 and 390 nm, respectively. Dehydroergosterol has been
used as a probe for the interactions of steroids with membranes.
103–106 Ligand analogues have also been reported for
the estrogen receptor. 107–108
3.6.3. Viscosity Probes
Fluorescent quantum yields are often dependent on viscosity,
but there are relatively few fluorophores characterized
Figure 3.30. Fluorogenic probes for HIV protease. The fluorescence signal is generated when HIV protease releases the fluorophore (F) from the
quenching effects of the nearby acceptor chromophore (Q).