Fluorogenic substrate probes

Internally quenched fluorogenic substrate probes have been shown to be useful in a variety of contexts, from conventional enzymology and inhibitor evaluation, to searches for important new therapeutic protease targets. The development of automated methods for synthesis of these substrate probes has enabled access to a wide range of substrate sequences, and facilitated detailed studies of enzyme-substrate sub-site interactions. The selection of the Edans-Dabcyl fLuorophore-quencher pair has led to the synthesis of efficiently quenched peptide substrates containing more than 30 amino add residues. The overall versat bty of these substrate probes ensures widespread future application to studies of many new and existing proteases of interest. 

The introduction of FRET pairs with improved properties allows the generation of probes suitable for the measurement of spatio-temporal activities in living cells and in vivo. One example is the imaging of caspase-3 activation in HeLa cells after inducing apoptosis. Previous fluorogenic substrates, such as DEVD-MCA, could not be used in living cells because the autofluorescence interfered with the low emission wavelength of the coumarin dye MCA [123], With the incorporation of the FRET pair 6-carboxydichlorofluorescein and 5-carboxytetramethylrhodamine into another peptide sequence cleavable by caspase-3, Mizukami et al. measured caspase-3-like activity in HeLa cells stimulated by an inducer of apoptosis [124],... 

Fluorescence-based assays employ reagents to characterize a wide range of cellular components, including nucleic acids, proteins, biogenic amines, carbohydrates, and lipids. These assays are often based on affinity probes, such as oligonucleotides, antibodies, aptamers, and lectins, which are used to characterize a specific component within the cell. In addition, fluorogenic substrates are used to characterize enzyme activity in single cells. 

If the intrinsic fluorescence of the extract is a serious problem limiting sensitivity, one can either extract the fluorescent compounds prior to analysis (see above) or use one of the other fluorogenic substrates synthesized by Molecular Probes, Inc. In particular, resorufin glucuronide yields resorufin, which has an extraordinarily high extinction coefficient and quantum efficiency, and its excitation (570 nm) and emission (590 nm) are conveniently in a range where plant tissue does not heavily absorb or fluoresce. In addition, it fluoresces maximally at neutral pH, making it unnecessary to stop the reaction. Because of the tendency of resorufin to be reduced to a non-fluorescent form, omit DTT or beta-mercaptoethanol from the reaction mix. 

Thus, because the test can be carried out on 96-well microtiter plates, high throughput is possible. Of course, the inherent disadvantage noted above for some of the colorimetric tests also applies here, namely, the fact that the optimization of a potential catalyst is focused on a specific substrate 11 modified by incorporation of a probe, in this case the fluorogenic moiety 14. However, one can expect the test to be useful in directed evolution projects in which proof of principle is the goal. Moreover, this kind of approach can be used in very practical applications, namely as a pre-test for the activity of enzymes. 

Scope and Limitations of the TK Fluorogenic Assay The designed fluorogenic probe 1 was used as a potential donor substrate of yeast TK. Indeed, TK was able to cleave the C2-C3 bond of 1 to generate the aldehyde 4. A fluorescence signal appeared because 4 proved rather unstable and, in the presence of BSA, underwent a p-elimination to release umbeUiferone, a highly fluorescent compound (Scheme 15.6). 

In this way, compound 1 is a substrate for TK, yielding a-hydroxyl and P-coumarinyl substituted aldehyde. The slow release of fluorescence compared with the results obtained with transaldolase from an appropriate fluorogenic probe is... 

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