The Design, Synthesis, and Biological Incorporation of Azobenzenes Towards Spatiotemporal Control of Proteins
John, Alford Antoine
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The azo group of azobenzene can undergo isomerization using visible and/or UV light to interconvert between the trans and cis isomers. The change in properties associated with isomerization allows the application of in different areas of chemistry. Azobenzenes have been synthesized using many different techniques with varying degrees of success. However, very few methods exist for the synthesis of symmetric azobenzenes from both mono-substituted and tri-substituted anilines. In Chapter 1, N -haloimide and DBU was used to rapidly generate the di-substituted symmetric azobenzenes in good to excellent yields. The robust method for the synthesis of di-substituted symmetric azobenzenes have been applied to the synthesis of ortho-substituted red-shifted azobenzenes. The latter represents a class of azobenzenes that has gained notable recognition in the scientific community because of ability to induce reversible isomerization using less damaging visible light. In Chapter 2, a second method was developed for the synthesis of unsymmetrical azobenzene-alanines in good to high yield. In addition, azobenzenes bearing clickable functional groups were synthesized uneventfully. Next, the photophysical properties of azobenzenes with two fluorine or chlorine atoms in both ortho positions were crucial to give rise to azobenzene-alanines that can undergo reversible isomerization using visible light. Finally, the genetic incorporation of the azobenzene-alanine was performed to give rise to ten superfolder green fluorescent protein bearing azobenzene in the S2 position. We perceived that spatiotemporal control of proteins could be accessed by controlling peptide conformation using visible light. To that end, we genetically encoded unnatural azobenzene-alanines on the NoxaBH3 domain of the Noxa-Ubiquitin fusion protein using amber codon suppression in Chapter 3. The successful incorporation of red-shifted azobenzene-alanine represent a bolder step towards protein control using visible light.