This paper from Feringa’s Lab caught our eye. The paper demonstrates the ability to incorporate azobenzene photoswitches onto sites of interest through a bio-orthogonal reaction. The group synthesised two azobenzenes (Figure 1), one with a short PEG motif and one without, evaluated their physical properties when ligated to various targets.
Figure 1: Tool compound with a description of its many business ends and conditions to synthesise some key bonds
Azobenzene can act as photoswitches as they can be isomerised between the cis and trans isomers by irradiation with light. At specific wavelength the cis form can be reached which can either isomerise back to the more stable trans form, either thermally in the dark or by being irradiated with white light. The azobenzene properties (eg isomerisation rate, absorption maximum) can be fine tuned in order for the tool to function for its given task, as described in the paper... “Short-lived cis azobenzenes are required for photomodulating fast biological events, where rapid, reversible changes are desired. In contrast, application of azobenzenes with higher thermal stability is beneficial for studying longer-lasting processes, without the need for constant irradiation.”
The azobenzenes contain a phosphine allowing them to take part in a Staudinger—Bertozzi ligation where they react with azides to form amides. To study the effectiveness of the ligation, the photoswitches were incubated with benzyl azide and the reaction was monitored by 31P NMR. Clean conversions of the compounds was observed over 16 h at room temperature. The long reaction time could be a drawback to the ligation if working with cells or trickier targets. Another drawback is stability of the reagent which can be prone to oxidation of the phosphine, however in this case the reagents were relatively stable with only 15% of the compound oxidised when stored under nitrogen at 4 °C.
“A short polyethylene glycol (PEG) chain was incorporated onto the molecule at the 4-position with the aim of increasing the solubility in water and organic solvents, and exploring additional possibilities offered by the formation of a push-pull-type azobenzene. These additional properties include the red-shift of the UV absorption maximum and the shorter lifetime of cis isomer in polar solvents.” Indeed, a red shift of 31 nm was observed for the PEG version. The PEG version did have a shorter cis isomer lifetime when irradiated by white light or being kept in the dark. As the PEG version has a low thermal stability, the photostationary state (trans to cis ratio after irradiation) could not actually be determined in water, but a 5:95 trans:cis ratio was observed when irradiated in chloroform. The version without the PEG chain had a higher thermal stability in aqueous media. Upon prolonged incubation (41 h) of the photo irradiated sample in the dark at room temperature low conversion to the trans isomer was detected, thus indicating an intriguingly slow thermal reisomerization. After 41 hours the sample was irradiated with white light which resulted in the instantaneous switch back to the trans isomer. Several irradiation cycles were then performed to demonstrate that the molecule fully retained its switchable behaviour upon prolonged incubation in aqueous buffer.
They also demonstrated that the molecule could be attached to functionalised quartz surfaces and a model azide-containing protein. In the case of the quartz surface, the photoswictch properties were observed by monitoring the changes in absorbance of light, after irraditation by light to induce photoisomeriosation. But in the case of modified protein, only the MS results to demonstrate the level of incorporation were included in the paper. It would have been great to have seen evidence of the photoswitch functioning on a biomolecule but I expect we will be seeing this tool compound in action elucidating and creating new biological pathways.
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