Summer Research 2015: First Update!


The first few weeks of research have gone by fairly quickly, but I’m happy to report great progress! Since I work full time, I have been assigned several projects that I work on throughout the week, let me quickly go over them.

My Honors Project is synthesizing and incorporating azobenzene derivatives into proteins. I have three derivatives in mind: an alkyne, an alkene, and a dimethylamine. A few weeks ago we finally got the materials necessary to construct the first derivative, the alkyne. Part of that reason is that we needed to develop a synthesis that was effective and had high yields. Much of the problem with developing the synthesis was the Boc protecting group, which protects the N-terminus of the amino acid. At low pH, the Boc group is removed, therefore it is pertinent that we avoid using strong acids. We deciding on purchasing an alkyne aniline (protected with a TMS group), and reducing the amine to a nitroso via Oxone, a peroxide. This created a nitrosoarene which we used to create an azo bond with an amino acid precursor with the wonderful name Boc-Phe-4′-NH2. My first attempt at this was a success, but I tried to knock off both the TMS and Boc protecting groups by concentrated HCl and dioxanes; however, this seemed to muck up the product. My second attempt is ongoing, I am having trouble separating the azo formation reaction, but I have cleaved the TMS group with TBAF. Now all I need to do is run another column to separate the products and cleave the Boc group.

My second project has been utilizing a microwave to perform click reactions (which are just azide and alkyne cycloadditions) with small molecules and then proteins and resin. On the first try we were able to react benzyl azide and phenylacetylene in the microwave with TCEP and copper sulfate. The protein proved to be more difficult, as we seemed to have denature the protein in the strong microwave conditions. Perhaps we could use high power and low microwave time? The biggest surprise came when we decided to utilized a control for the small molecule clicks by performing the reaction without the copper catalyst. To our surprise, the reaction still worked! And even more interestingly, the regioisomer concentration seemed to be about 1:1, where with the catalyst it is about 1:8. Instead of focusing with protein, we have been recently trying to optimize the copper-free microwave reaction to have more regioselectivity, therefore completely avoiding the need for copper. At several attempts at increasing the temperature and time, we increased the power to 300 watts, which only reacted for about a minute, but produced almost identical results as the click with copper! We just need to acquire yields and good NMRs in order to understand the reaction more.

I end with my final project, the Protyro UAA and Glaser-Hay. For about a year I have been synthesizing and trying to react an unnatural amino acid nicknamed protyro (which is a fancy progaryl tyrosine). Last summer I underwent the six step synthesis and produced a great abundance, and over the school year I found a synthetase that I could utilize, named ambryx. Recently I have been trying to undergo a Glaser-Hay reaction (which is the formation of an alkyne-alkyne bond) with the UAA in GFP reacted with an alkyne-fluorophore, using TMEDA and CuI. The reaction was producing limited success until I was able to optimize it, and got it to work! The next step was cleaving  the protein from the flurophore using light. I ran at least one gel a day for three weeks, doing a Glaser-Hay almost everyday. I made a super-concentrated batch of Protryo-GFP and reacted it with the fluorophore for four hours. Then I irradiated the protein at 254 nm for thirty minutes, and it worked! I got a fantastic gel and almost cried. All I need to do now is collect all of my spectra and results, and type up the paper for publication.

Thanks for reading, hopefully I can begin the Selenocysteine project and continue with the other active projects.



  1. esbrown17 says:

    I am amazed at how much work you are getting done over the summer. I look forward to seeing the final products of all of your projects.


  2. I am excited about this! I really want to see the final project!