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!

First Post!

I realize that I am late to the “first post” party, but I guarantee you, I have been busy at work! Along with my current project, I have undertaken another, more grande assignment, so trying to balance the syntheses and biological applications of both projects have been tedious, yet fulfilling. For those who don’t know, my research is in the synthesis and application of unnatural amino acids (UAAs). My current project L-phenylalanine-4′-azobenzene (AzoBen) has been going somewhat smoothly. Since I have already created this UAA, I have been testing its abilities. The majority of my time spent testing AzoBen has been spent with my new friend Florence the Fluorimeter. A fluorimeter measures excitation and emission spectra, and since my UAA is photosensitive, I can understand how different wavelengths of light change the functionality of proteins with AzoBen. My protein of choice is Green Fluorescent Protein (GFP) which derives from the jellyfish  Aequorea victoria, and naturally glows. I have been comparing the wild type, or normal GFP, with GFP with AzoBen inserted into the 151 residue, and also with AzoBen inserted into the 66 residue. Through the data given by Florence, I was able to note several interesting changes in emission spectra. When GFP was irradiated with light (365nm) the emission graphs would both shift up and shift down, and well as shift left at certain excitation wavelengths. This indeed means that AzoBen’s photoisomerable ability is affecting this protein, so this validates my project! While this is exciting, much more fluorimetry data needs to had before I can fully comprehend the specific effects.

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Synthesis and Application of L-phenylalanine-4′-azobenzene

Hello! My name is Marshall Padilla, and I am a chemistry major and sophomore here at the College. I will be working with Dr. Young in the Chemistry Department to develop an unnatural amino acid. Amino acids are the building blocks of proteins, which in turn, are the workhorses of the cell. There exists twenty natural amino acids; but recently, researchers have began synthesizing unnatural amino acids. My amino acid (let’s call it AzoPhe for short) has a very peculiar property: when AzoPhe comes into contact with a specific wavelength of light, part of the molecule actually shifts, a feature called photoisomerability. This feature is useful, because when AzoPhe is inserted into a protein, and a specific wave length of light is shined on it, the amino acid will deactivate the protein. AzoPhe could also be utilized to block ion channels, which are important proteins that regulate ion concentration. After creating this amino acid, I will (hopefully) insert it into a protein called GFP, which is a fluorescent protein. If I successfully insert the amino acid into specific locations (residues) of the protein, I will be able to activate and deactivate the protein’s ability to fluoresce via AzoPhe’s photoisomerability. The ability to control a protein offers a powerful medical tool, as the misregulation and misexpression of proteins is often the root of health conditions.

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