Imaging of protease activity in live cells using activity based probes
Methods and materials for the imaging of cells containing active proteases such as cathepsin are disclosed. The present materials include activity based probes that bind to an enzyme and are subsequently cleaved. Cleavage results in a fluorescent signal due to removal of a quenching group which, when present on the probe causes altered or no fluorescence. The probes employ an acyloxymethyl ketone reactive group, one or more amino acids for determining specificity, a fluorophore and a quencher. The probes are cell permeable and may use, for example, a QSY7 (diarylyrhodamine) quencher and a BODIPY (bora-diaza-indecene) dye.
1. An activity based probe for labeling an active protease, having the formula
where
(a) AA 1 is a phenylalanine side chain;
(b) n is four;
(c) R 2 is a QSY® quencher, wherein the quencher is linked through a lower alkyl, aryl or lower alkyl-aryl linker;
(d) R 3 is a cyanine dye and a lower alkyl; and
(e) R 4 is a capping group selected from the group consisting of aliphatic ester, aromatic ester or heterocyclic ester.
2. A compound having the formula:
where R10 has the formula below where * indicates a point of attachment:
3. A compound having the formula:
where R 10 has the formula:
in which * indicates a point of attachment.