Second Post!

Hello Friends!
I hope everyone’s summer has been going well. Here in the ISC I have been productive in synthesizing my new unnatural amino acid protyro. This UAA has the ability to “click” onto other molecules, meaning it has an alkyne group that is able to link to other alkynes, azides, etc. Creating protyro has been an adventure by itself. The actual synthesis is six steps, that involve a variety of organic reactions such as brominations, reductions, nitrations, and deprotections. I have attempted the synthesis twice now. The first time the reactions were going well until the second to last reaction, which was a malonic ester synthesis done via a microwave. When doing a microwave reaction, one needs to add a stir bar to allow a thorough microwaving, but I had forgotten about this fact. After finishing the microwaving, my professor informed me that I had forgot the stir bar, so I added the stir bar and re-microwaved. It is tough to know whether this is what caused the reaction to fail, but after I ran a column, I generated very little product. I decided to start over with precursor that I saved. This time I successfully completed (or thought I had successfully completed) all six steps. I tried to take a H NMR of the final product, and even though they say twelfth time is the charm, all twelve of my attempts were to no avail. This may be due to the fact that I had created very little product, or that the UAA was just difficult in general to read. I tried to NMR in various solvents: CDCL3, Methanol-D4, and even D2O. I decided to go ahead and insert protyro into GFP Waldo, a dimer GFP hybrid, where the UAA was inserted into the 151st residue. Despite having an “iffy” product, my confidence in this insertion lowered once our lab realized our comp cells were tainted. Comp cells are cells whose plasmids are easily edited, and this insertion and selection is done via inserting  antibiotic resistance, namely Ampicillin and Chloramphenicol. Without going into too much detail, we found antibiotics inherent in the bacteria, which means many of our bacteria colonies could be contaminated. Over the course of the next few days, my goal is to successfully synthesize protyro, which means having high yields for each of the synthesis steps.
My time with Florence the fluorimeter has been intriguing, yet tiresome. I have done over a hundred scans of different proteins with my AzoBenzene compound, many of these with varying solutions such as PBS buffer and 1X tris buffer. I have seen many interesting traits from the fluorimeter, but handling the data can be overwhelming. Each scan produces several hundred data points, all of which have to be transferred onto a separate excel sheet and compiled. While this is a very necessary part of my research, I have become weary of the constant scans, and I’m worried I have become inundated with graphs on lines.
I have much more information and results to come in the next few days so stay posted, and thanks for reading!

Comments

  1. jkvilla says:

    Hello Marshall!

    A six step synthesis sounds challenging! Not to seem pessimistic but there appears to be a lot of steps that could go wrong! I find it ironic that each of us had errors with adding stir bars with the microwave: you forgot to add and I accidentally added a stir bar. Have you added your protyro to the new comp cells, or are you just going to roll with the synthesis again? What exactly is the purpose of the protyro if you don’t mind my asking?

    And after my own experiences with Florence, I begin to see your disagreements with him. It truly is graphs on giraffes on graphs!

    I wish you the best of luck with the new synthesis and analysis!! 🙂

  2. aekelley says:

    It is always interesting to read about chemical research which varies so greatly from the physical chemistry that goes on in my lab. I find the idea of being able to essentially hand design the features a protein will have to be fascinating. I look forward to seeing if your results are successful and the implications that brings for designing proteins in the future. As far as the data analysis grind goes, that is something I can completely empathize with as much of my research involves analyzing data points and interpreting what properties they translate to in the physical world. Perhaps to expedite the process, you might consider writing a simple MATLAB script which will produce graphs for you, cutting down on the time and tedium of sorting through the data yourself? Just a thought. Good luck with your synthesis!