Caged fluorescent probes

Figure 11.14 Photochemical un-caging using two-photon excitation. Inactive ( caged ) fluorescent probes within the focal volume of the laser (a) are first activated by two-photon-induced photochemical un-caging (b) diffusion of the probes from the focal volume is then monitored by subsequent two-photon-induced fluorescence...

Prochazka et al. [31] studied solvent relaxatitm in the shells of polystyrene-WocA -poly(ethylene oxide) (PS-PEO), polystyrene-Woc)t-poly(2-vinylpyridine) (PS-PVP), and polystyrene-Woc)t-poly(2-vinylpyridine)-Woc)t-poly(ethylene oxide) (PS-PVP-PEO) in aqueous solutions, using Patman as a fluorescent probe. While in the case of the PEO shell of the PS-PEO micelle, a slow relaxation was observed because the mobility of water molecules in the shell was hindered by the strong hydrogen bonding to PEO chains, the solvent cage of the probe in the shell of PS-PVP micelles appeared to relax much faster. Interestingly, water in PS-PVP-PEO terpolymer micelles exhibited a slow relaxation as in the case of the PS-PEO diblock, but the relaxation time became pH responsive due to the presence of the weak polyelectrolyte PVP block. 

An overview of the synthesis, structure, photophysical properties, and applications of squaraine rotaxanes as fluorescent imaging probes and chemosensors is provided in a recent review [67]. Although a variety of squaraine dyes form rotaxanes with the molecular cage 25 or with a tetralactam macrocyclic system introduced by Leigh and co-workers [16, 17], there is no evidence in the literature that conventional cyanine dyes can be embedded in these macrocycles. 

Recently, a Ser-caged, photoactivatable fluorescent peptide probe that monitors protein kinase C (PKC) activity was described [75]. As expected, the Ser-caged peptide failed to serve as an effective PKC substrate in vitro, but upon light-induced deprotection (300-400nm, A.max360nm, 90s), the serine became phosphorylated and enzyme activity was recorded as a convincing change in the fluorescent properties of the probe. Photoconversion was estimated to occur with 60% yield and a quantum yield of 0.06. 

With this probe, the investigators also studied the light-induced sampling of PKC activity in HeLa cells in vivo. Exposure of cells to phorbol ester (TPA) normally induces PKC activity. HeLa cells microinjected with the caged probe at an estimated concentration of 20 pM failed to display a fluorescent response to TPA, while a robust response was recorded as a result of a concomitant TPA treatment and UV irradiation (365 nm at 1J cm 2). 

Vazquez, M. E. Nitz, M. Stehn, J. Yaffe, M. B. Imperiali, B. Fluorescent caged phospho-serine peptides as probes to investigate phosphorylation-dependent protein associations. 

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