Third Post!

While my time in the Young Lab has come to a close, I still have much to discuss. In the last few weeks I started on an actual experiment for the AzoBen UAA. I took agar plates, which are plates that bacteria grow on, and created an environment where bacteria can express the photoswitchable nature of GFP with my UAA. I took four plates, and to three of them I added 10 ml of agar, 10  μl of IPTG, 10  μl of Arabinose, 10  μl of chloramphenicol, 10 μl of Ampicillin, and 100 μl of AzoBen. On the fourth plate, I repeated the same procedure, except I did not add AzoBen. I then plated bacteria that could inherently grow GFP. The purpose of this setup, was to test whether I could detect changes in fluorescence when I irradiated the bacteria. Since three of the plates contained GFP with AzoBen, they theoretically should be able to change the fluorescence. I took one plate, irradiated half of it for 10 minutes, at 365nm. I then left the plate overnight in an incubator. When I came back and checked the fluorescence, I noticed that both sides were the same. This meant that either the experiment did not work, or that the plate did change, but changed back. Hoping that the result meant the latter, I took one plate and irradiated half of it for 10 minutes at 365nm, and checked it after an hour. To my joy, one side had a different fluorescence reading than the other. This meant that the experiment had work; but now I needed to check if the bacteria reverted back. After a few hours of hopeful checking, I saw that the irradiated bacteria were changing back to their original fluorescence! This was fantastic news, as I now have supporting evidence that my UAA is able to alter the function of GFP, but is also able to undo these changes.

My journey to synthesize ProTyro has been long, but I finally say I have successfully produced it! My third attempt produced what I thought was the UAA, but I wasn’t positive. Before my fourth attempt, I analyzed all of my reactions and tried altering some of the conditions. To my surprise, on the first reaction, I achieved an 83% yield! After careful synthesis, I ended up with a product, that when I NMR’d, verified that my results had been a success. Throughout my attempts, the last step (microwave deprotection) was always garnering low yields. I realized that I didn’t need to microwave with power. To check this, I divided my sample into two separate vials, and microwaved one with power and one without power. After NMRing both products, I concluded that I got a higher yield when microwaving without power.

Since it is towards the end of the summer, I am not able to do too much with this new UAA, but during the school year, I main priority will be working with ProTyro.
I hope everyone is doing well!
-Marshall