Update!

I hope everyone has been “enjoying” this lovely rainy weather, I’m starting to forget what sunshine is…

Since coming back to school I’ve been quite busy in lab. Unfortunately, I wish my progress could reflect the work I’ve put in, but that’s lab work for yah. For the Azobenzene project, I haven’t been able to find a synthetase to house my amino acid. I decided to try the synthesis one more time, to see if I could make the UAA more pure. Surprisingly, the synthesis worked extremely well. For the TMS and Boc removals, I used a very high solvent system (30:1 DCM:MeOH) which really separated my products. After deprotecting the Boc group, dissolving the UAA into a 100 mM solution of base and water proved to be quite difficult. It was suggested to me that I used a 10% solution of DMSO, which worked quite well! The UAA dissolved into a nice brown homogeneous solution (yum). I then tried seven different synthetases using a 96-well plate, testing for fluorescence. Unfortunately there didn’t seem to be any difference between the plus and the minus wells. I am now transforming a new synthetase while simultaneously cutting down on the amount of UAA I use, just in case it’s cytotoxic. In our lab, we have a new method of producing protein (thanks to J$), an hopefully that, coupled with the new synthetase, will help my azobenzene find a home.

The microwave click project is going better. I pretty much redid all the reactions with different alkynes and azides. For one set of alkynes, I have had great yields, with beautiful spectra. I attempted using an UAA azide, but that had little success, same with a more polar azide. This may be evident that the reaction only works with certain types of molecules (i.e. nonpolars, aliphatics, etc.). I am continuing exploring the possibilities of this reaction by synthesizing more azides. My chart of click reactions is up to fifteen now.

The selenocysteine project is going decently. We successfully completed the first round of the selection, but the second round is proving more difficult. We have taken a hiatus from the selection, and instead, we have been using the azobenzene synthetase assay as an excuse to test more synthetases with our selenocysteine UAA. It seems that a 100 mM solution of the UAA is cytotoxic, so we needed to bump the concentration down by about a tenth. Similar to azobenzene, we weren’t able to find a difference between the plus and minus wells, but we are continuing to test more synthetases.

There are more results to come, thanks for reading!