Light-mediated liberation of enzymatic activity: "small molecule" caged protein equivalents

Abstract

Light-activatable ("caged") proteins have been used to correlate, with exquisite temporal and spatial control, intracellular biochemical action with global cellular behavior. However, the chemical or genetic construction of caged proteins is nontrivial, with subsequent laborious introduction into living cells, potentially problematic competition with natural endogenous counterparts, and challenging intracellular incorporation at levels equivalent to the natural enzymes. We describe the design, synthesis, and characterization of small molecular equivalents of a caged Src kinase. These compounds are easy to prepare and function by inhibiting the action of the natural unmodified enzyme.

Figure 1

A Photocleavable Caged Protein Equivalent.

Figure 2

Enzymatic activity (ΔF vs time) as a function of irradiation time of compound 6 (1.9 μM). Assays were performed as previously described. Experimental conditions: A (0 min hv), B (2 min hv), C (15 min hv), D (120 min hv), and E (0 min hv + 1.9 μM compound 2).

Figure 3

Presence of Src kinase on (A) nonirradiated and (B) irradiated UltraLink^® beads modified with bivalent inhibitor 11 as assessed by exposure to an Alexa Fluo488-labeled monoclonal Src antibody.