Characterization of Azobenzene-based Photo-switches and their Evaluation for In Vivo Applications

• Main Author: Beharry, Andrew
• Other Authors: Woolley, Andrew
• Language: en_ca
• Published: 2012
• Subjects: Biological Chemistry; 0490
• Online Access: http://hdl.handle.net/1807/32665

Photoisomerization of azobenzene can be used to reversibly photo-control peptide and protein structure, thereby offering the potential to probe peptide and protein function directly in living systems. Most azobenzene photo-switches to date however require the use of UV light, which suffers from poor tissue penetration and can lead to cell damage. In addition, the unknown stability of azobenzene photo-switches within the reducing intracellular environment has limited these switches to extracellular applications. The ability to red-shift the photo-switching wavelength and tune the thermal cis-to-trans rate independently from one another has been a central challenge. Incorporating a piperazine-like moiety at the 4,4′-para positions red-shifts the π-π* band of an azobenzene-based cross-linker such that trans-to-cis photoisomerization can be triggered with blue light. Accompanying this red-shift was a faster cis-to-trans thermal rate, which allowed for fast secondary structural changes of the attached peptides (τ½ ~2s). Piperazine-like moieties at the 2,2′-ortho positions of an azobenzene-based cross-linker resulted in a similar red-shift in the π-π* band but a longer-lived cis isomer relative to its para-counterpart (τ½ ~minutes). These results suggest that the thermal rate could be tuned independently from the photo-switching wavelength by appropriate para or ortho substitution. The effect of 2,2′,6,6′-ortho-tetramethoxysubstitution provided an alternative approach to red-shifting the photo-switching wavelength. These groups caused an unconventional red-shift in the n-π* band of the trans isomer allowing for trans-to-cis and cis-to-trans photoisomerization to occur with green and blue light, respectively. In this case, the half-life of the cis isomer was not shortened, but rather extended relative to its parent compound (τ½ ~days versus minutes). These results provide progress in tuning the photo-switching wavelength independently from the thermal rate. A fluorescent reporter was developed to determine the stability of a commonly employed 4,4′-diamido derivative in vivo. Photoisomerization was found to cause time-dependent changes in fluorescein emission intensity. The reporter was microinjected in zebrafish embryos and photo-switching could be imaged for at least two days. This work provides the first direct evidence of azobenzene photo-switching in vivo, and indicates that it will be possible, in general, to photo-control peptide and protein function in living systems.