DEAR LAB NOTEBOOK …
By: Sarah Anderson
As a graduate student getting my PhD in chemistry, I’m expected to keep a laboratory notebook. It’s where I document what I do each day: the aim of the experiment, the conditions I use and the outcome. My lab notebook serves as a memory aid, allowing me to access the details of my previous attempts to inform my future work. As with any daily account of one’s progress toward meeting a certain goal, whether it’s achieving a scientific result or asking your crush out on a date, my entries tend to take on an emotional undertone. I’ve exploited my lab notebook as an experimental record/personal diary hybrid, so it provides an accurate picture of life as a graduate-level researcher. Below are a few excerpts from a period of my grad school career I feel is representative of the research process: my first and only experience with the infamous Organic Synthesis.
Organic synthesis entails converting one molecule — a collection of atoms bonded together in a specific arrangement — into a different molecule. The initial molecule is reacted with chemicals that are known to attach certain groups of atoms to or remove them from the original structure, yielding the desired product. In theory, the concept can be compared to remodeling a house. Let’s say you want to add a room. You start with a house, and then you “react” it with wood, metal, and concrete to produce a bigger house. In practice, organic synthesis looks less like a construction site and more like the stereotypical visual that pops into your head when you think “chemistry:” an array of multicolored liquids and concoctions simmering in beakers. All of this remodeling happens on a tiny atomic scale imperceptible to the eye, which is partly why it’s so difficult. But I was up for the challenge, as I needed to make a molecule that could get into the cell and help stimulate the production of another molecule I was studying.
Notebook 1, Page 234 01/09/2018 TIC synthesis attempt #1
“NMR looks exactly the same as spectrum for isocitrate — the esterification did not work. Isocitrate was insoluble in methanol + benzene and yellow color persisted long after 30 min; reaction did not go to completion.”
Here, I reacted isocitrate (the starting house) with a chemical called trimethylsilyldiazomethane (the raw building materials) to attach three carbon atoms (the additional rooms) at specific sites to form a new molecule called trimethylisocitrate, or TIC (the finished, larger house). The general procedure I used indicated that the yellow color of the reaction should disappear as the new molecule formed, but my solution remained suspiciously yellow. I used a technique called nuclear magnetic resonance, or NMR, which displays a peak for each carbon atom, to analyze the molecule. I didn’t see any peaks corresponding to the new carbon atoms, which confirmed that the reaction did not occur. I wasn’t all that surprised because I had noticed that the isocitrate didn’t dissolve very well in the solvent. This insolubility was likely the problem, as molecules and chemicals need to be together in the solution phase to be close enough to interact with one another. It was like I was throwing concrete and metal and wood at the house from a mile away.
Notebook 1, Page 249 02/02/2018 isocitrate potassium saltà carboxylic acid
“Sonnication, heating, and using HUGE volumes of solvent has not significantly improved solubility. Alison recommended taking the salt to the carboxylic acid before proceeding with synthesis. Simple acidification with HCl. The verdict? Completely soluble. GOD BLESS ALISON”
I was really struggling to get the starting compound to dissolve, so I talked to my colleague, who is an organic chemistry whiz. She suggested converting the initial isocitrate molecule into isocitric acid, a slightly different molecule whose chemical properties make it easier to dissolve in the solvent. It worked like a charm.
Notebook 1, Page 254 02/08/2018 TIC synthesis attempt #8
“Ran the synthesis with the carboxylic acid. NMR confirmed esterification, but saw two sets of peaks. It freaking formed diastereomers. Cried in Chick-fil-A.”
This was a rough day. While changing the chemical properties of the initial molecule helped it to dissolve and react, it also messed up its spatial orientation. Molecules are three-dimensional structures, and the atoms that comprise them occupy fixed positions relative to one another. The chemicals involved in the preliminary conversion step caused these positions to shift out of place. And when it comes to making molecules, having the right atoms in the wrong place is as problematic as having a basement on the top floor of a house. I realized I needed to start with the original molecule.
Notebook 1, Page 266 03/19/18 TIC synthesis attempt #11
“Testing new synthetic route with trimethylsilylchloride in MeOH and isocitrate salt. Result: I GOT IT!!! AHHHHHHH!!!”
… And this was a great day. Just as there are many types of building materials one can employ to add a room onto a house, there are several chemicals that can bring about the same modifications to a molecule. I discovered a new chemical that could attach the carbon atoms in the right spots through a different reaction mechanism, and its accompanying solvent was much more compatible with my original starting material. The isocitrate dissolved, its spatial orientation was preserved and the TIC molecule was successfully formed.
Notebook 1, Page 267: 03/22/18 TIC + cells test #1
“Added to media at 5mM, incubated for 48 hours, pelleted, lysed. No AKG peak in SAMDI spectrum L Increase concentration/incubation period?”
Oh, you thought we were done? Keep in mind, the whole reason I was making the TIC molecule was so that I could use it to promote the intracellular production and enhance the detection of another molecule called alpha-ketoglutarate (AKG). AKG levels can signal certain genetic adaptations exploited by brain cancer cells, and measuring AKG can help to evaluate the efficacy of cancer treatments that target those changes. To determine if the TIC I made had its intended effect on the cell, I analyzed cells I had treated with TIC using a technique developed in my lab that can capture specific molecules and quantify them by their mass. Unfortunately, I didn’t observe any increase in AKG in the cells I had treated compared to cells that were left alone. And with that, I was met with an entirely new set of obstacles to overcome.
So, there you have it: a snapshot of scientific research. It’s optimization. It’s problem solving. It’s troubleshooting. It’s asking for help from people who know more than you do. It’s starting over with a new approach. Sometimes, it’s having a mental breakdown in a fast food restaurant. If you’re lucky, it’s receiving a text consisting solely of prayer hands emojis from your mom on the day you’re trying the mental breakdown-inducing experiment again. It’s clinging to each success, however short-lived, as motivation to sustain you through the inevitable failures. It’s fighting for every slide you present, every sentence you write, every graph you publish. Perhaps scientists aren’t always transparent about this process because they feel the impact of their work is somehow cheapened if it’s apparent that they didn’t figure it out on the first try. Personally, I find nothing more impressive than persistence. I’ll try to keep this in mind as I continue to fill the pages of my lab notebook. They will surely include more enthusiastic declarations in all caps, more frowny faces, more thinly-veiled curse words. But, as the great Theodore Roosevelt once said, “Nothing in the world is worth having or doing unless it means effort, pain and difficulty.” Hmmm. Currently Googling if Teddy has a PhD …
Sarah Anderson is a fourth year PhD candidate in the chemistry department. She works on developing tools to accelerate processes related to drug discovery, molecular sensing and fundamental biological questions in Professor Milan Mrksich’s lab. In the future, Sarah hopes to pursue a career in science writing to help make science accessible to everyone it affects. Follow her on Twitter at @seanderson63