Abstract: Unnatural Amino Acid Azobenzene Derivatives


My name is Marshall Padilla and I am soon to be a rising Senior Chemistry Major. I am back again this summer to continue my azobenzene work through an Honors Fellowship. I would first like to thank the Chemistry Department here at William & Mary for granting me the Llanso-Sherman scholarship, which will fund my Honors research this summer. Last summer I synthesized azobenzene, an unnatural amino acid (UAA) that has an azobenzene R group. I inserted it into Green Fluorescent Protein (GFP) at several different spots and demonstrated that by utilizing azobenzene’s photoisomerability, I could alter the fluorescence of GFP reversibly- meaning I could shift the fluorescence of GFP and reconvert it back. This was shown both in pure protein extract and in vivo, as bacteria were grown that were made able to synthesize of GFP, and demonstrate fluorescence reversibility due to azobenzene.

This summer, I plan on extending my research on azobenzene by creating azobenzene derivatives. I will be derivatizing these UAAs by adding an alkyne, an olefin, and an amine group to the 4′ carbon of the terminal benzene group of azobenzene. With these three different derivatives, I hope to prove fluorescence reversibility, but alter fluorescence in a non-toxic manner. Normal photoisomerability happens at wavelengths under 400 nm, which in cells, can cause severe problems such as DNA mutations. By adding these molecular groups onto azobenzene, I hope to be able to photoisomerize at non-phototoxic wavelengths. This would create a more practical application for my UAA. Therefore, I will be making fluorescence measurements at various wavelengths in order to find a reasonable and effective wavelength in which to demonstrate this idea.

In addition to altering fluorescence of GFP, I hope to incorporate the alkyne azobenzene UAA into GFP, and undergo a Glaser-Hay reaction. Glaser-Hay reactions are reactions between two alkyne groups to form alkyne bridges. More specifically, I will perform a Glaser-Hay reaction with an alkyne fluorophore, or a type of molecular marker. This fluorophore will allow easy verification of the relative success of the Glaser-Hay reaction.

With these projects in mind, my research over the summer will primarily be twofold: synthesis and protein expression. I have developed a series of reactions involving azo-coupling to develop the azobenzene derivatives. Once they have been synthesized and purified, I will insert them into transformed bacteria, in order to utilize bacterial machinery to create protein with my UAAs.

I will hopefully posting frequently, in order to let y’all know how the research is fairing.

Thanks for reading!


  1. Congratulations on your recent publication. There is a lot more to azobenzenes. I’ll be happy to discuss them with you if you like.