Last Week - Finally got rid of dimers

Hi this is Harry Zhang, and I just finished my 5-week lab work with Dr. Ballatore at UPenn. I participated in Dr. Ballatore's current project of making and categorizing carboxylic acid isosteres.

Methylation and LDA alkylation both failed to get rid of the CPD dimer, so Dr. Ballatore suggested another possible factor: the concentration of the starting material. If the starting CPD is too concentrated, it would be easier for it to attach to itself and form dimers. Using the rest of the new batch of CPD, I ran the same alkylation, again with LDA, but this time added more solvent so the starting CPD was ten times more dilute. Since this was the last week, I really hoped that this would work because we have been tackling this problem for almost the full duration of my stay and I would not have really contributed to the making of model compounds if this method did not work. Thankfully, after a work up and column, the LC-MS graph showed only our desired product's mass, and no dimers. That basically concluded my work with Dr. Ballatore and he said that he would send me more data once he finishes the whole project.

Although I have really only made one new model compound (the unmethylated CPD isostere was already made before and I only did it as a practice), I got a taste of what it is like to work with a scientific problem in real labs. By working at the issue in different angles, accompanied with constant testing, we were able to solve the persistent dimerization problem.

Overall, I think the 5 weeks at UPenn showed me what it is like to work in the field of scientific research. It consists of both logical and creative thinking, a lot of cool technology, but also a lot of repetitive work that sometimes even drives Dr. Ballatore crazy.

Week 4 - In the process of solving the dimerization issue

Hi this is Harry Zhang, I'm working at the Ballatore lab at UPenn and this is the 4th week that I've been working with Dr.Ballatore on making carboxylic isosteres (model compounds).

Towards the end of last week, I made another batch of protected CPD for the new LDA alkylation. Making the LDA was also a tedious process, and that was why Dr. Ballatore preferred using KHMDS. The making of LDA was mostly done by him and I was responsible for the alkylation. Since I've already done at least three alkylations, the process was familiar and I didn't make any mistakes so the color of the product was more "right", according to Dr. Ballatore. After working up the product and doing an HPLC, we still found the existence of the dimer. Dr. Ballatore collected samples for NMR to further analyze it while I made more methylated CPD.

I think after three weeks of practice, this week's lab work felt a lot more manageable and I actually had confidence that I wouldn't make a mistake like spilling dichloryl methane or making a cloud of silica powder. I felt that I was able to get a lot more done without the help of either Dr. Ballatore or the post-doc Bryant.

This was also the last week of the stay at Peddie. Over the weekend I moved to a hotel 2 blocks away from the lab to make life a lot easier (no more 3 hour commutes every day).

More Dimers - Week 3

Hi I'm Harry and I'm working in the Ballatore lab at UPenn, I'm joining in the lab's project to create a set of carboxylic acid isosteres.

After finding the dimerization issue, we tried two different things: 1. Do the same alkylation with the left-over CPD but with a 1:1:1 CPD base and benzyl bromide ratio, we did this because the dimerization might have been caused by excess CPD and not enough base and benzyl bromide to react with. 2. Do the same alkylation but this time with a methylated version of CPD. My PI predicts that the methyl group should prevent the formation of CPD dimers. The LC-MS of the product of both methods showed significantly greater yield of the product we want but unfortunately we still see the dimers. We deprotected the products and using high-performance liquid chromatography (HPLC), which accurately separates our desired product and the impurities, we collected our products.

To deal with the persisting dimerization issue, my PI suggested we change the base from KHMDS to LDA, a stronger base because he had done an akylation with LDA without encountering any dimers. To do this I had to make more protected CPD. Meanwhile we set up a separate reaction with a completely different starting material. However, the reaction did not produce any of our desired product and was considered a failed reaction.

This is the general outline of the week's lab work but also a lot of time was used in regular column chromatography, extraction, and the most annoying part: waiting for the extracted product to dry. I'm starting to repeat the same procedures for each reaction: reaction setup, monitoring using TLC and LC-MS, then quenching/extracting with water and ethyl acetate, chromatography (HPLC or regular chromatography), and finally an NMR analysis. It's great that I'm finally starting to do these steps by myself without my PI or post-doc.

Product Characterization - Week 2

Hi I'm Harry and I'm currently working with Dr. Ballatore in a chemistry lab at UPenn focusing on chemical synthesis of isosteres.

At the end of the previous week we were trying to figure out the content of a product that was not what we expected. To further characterize the compound, in addition to LC-MS, the post-doc Brian took me down to the nuclear magnetic resonance (NMR) machine. Apparently the magnetic field around the machine is so strong that if you had a credit card in your pocket it would be deactivated. Brian tried to explain to me how the NMR works in simple terms because the actual theory requires chemistry and physics knowledge that are well beyond my level. So basically the NMR creates a magnetic field that can change the direction an electron spins (it can only spin in two possible directions), and the machine can measure the amount of energy required to make that change. The energy required is presented on the resulting graph as a chemical shift, which is essentially the position of a peak on the graph. In a proton NMR, the integration of each peak shows the number of protons at that location; in a carbon NMR, each peak represents one carbon atom. Because the energy required to make the change in spin direction is related to the chemical environment that the atom is in (which simply means the other atoms around it in the chemical structure), each proton or carbon in a specific location will have a specific range of chemical shift. So if you know that a carbon atom in a carbonyl group will always show up on the left end of the graph, a peak on the left end will indicate that you have a carbonyl carbon. The NMR essentially gives us more information on the structure of the compound that we have made. 

The NMR result certainly did not represent the compound we wanted, so while Dr. Ballatore and Brian were trying to figure out what was going on with the NMR (I can't interpret a NMR graph), he had me set up another reaction to deprotect the compound. The isobutane that was attached in the first step of the reaction was a "protection" of the reactive carbonyl oxygen. Once deprotected, we ran another LC-MS to see what was in the compound. As expected, the largest peak showed the same molecular weight as before minus an isobutane. After some guessing around, Dr. Ballatore figured it out. What happened was that the reaction produced a large amount of dimers, compounds that had two cyclopentanediones. It was pretty amazing how he just guessed the right one.

While all this was happening, we had set up a separate reaction which was a lot simpler than the one we were working on, it only had one step and didn't require reflux or deprotection. The reaction sill uses cyclopentanedione, and it basically adds the benzyl ring on the other side of the structure. Changing the location of the benzyl group can affect the compound's acidity and ability to penetrate membranes.

After figuring out what went wrong, we redid the reaction and this time modified the run-time to minimize dimerization.

Making Isosteres - Week 1

The first week at the lab was really interesting and fun except for the fact that I almost broke a finger (which had nothing to do with the lab but I had to take a day off for it). Overall I was happy that I wasn't burdened with piles of articles but instead got to do a lot of hands-on work. The lab is pretty messy, my PI said that they don't do a very good job making things neat and orderly unless there's an inspection. There are only two other people besides my PI and me in the room, a grad student and an undergrad. They were pretty busy with their own projects (which were similar to what I'm doing) so I mainly worked with my PI directly and a post-doc who's working next door. Unlike some of the other labs, I didn't have to go through hours of training sessions. On the first day, basically as soon as I arrived, my PI started writing chemical structures on a blackboard  and told me that this is the reaction that I'm doing. Then I followed the instructions to weigh out the solids and get the correct volume of liquids and put them in a round-bottom flask. Although I've used all the instruments before (analytical balance, weighing paper, syringes, pipettes) and the task wasn't particularly hard, I was still a little scared because I was handling all these glassware and chemicals on my first day. The reaction I set up was a reflux reaction in which the flask is placed in a hot wax bath with a condenser on top so any solvent that evaporates will go into the condenser instead of falling back into the reaction solution.The purpose of the reaction was to replace a proton with an isobutane, the first step in a three-step process that would result in an isostere of 1,3-cyclopentanedione, the chemical I'm currently working with.

On the second day, I worked with the post-doc the whole time. He demonstrated some of the more complicated (but actually really basic) processes like thin layer chromatography (determines the solvent condition suitable for the compound and checks if there are products made), column chromatography (a tedious process that may take over an hour, separates the product solution into fractions, the fractions are then tested using TLC to see which ones contain the desired product), and purification methods including using water to quench the reaction, using ethyl acetate to separate the organic and inorganic layers, and using sodium sulfate to eliminate any left-over water. These are the standard procedures for almost every reaction that I will do.

Next day morning I got a black fingernail and went to the lab in pain. My PI showed me the Liquid Chromatography-Mass Spectrocoply (LC-MS) machine and tested the solution I collected the day before. The LC-MS basically runs a mini-column and presents a graph on the computer that contains peaks. These peaks represent the compounds in the product solution; the machine gives the molecular weight of every compound and we are trying to find the peak with the molecular weight that corresponds to the product we want. If it is a big peak then that means the reaction is successful and we got the right product. There are other peaks on the graph that corresponds to the left-over reactants and some side-products. Unfortunately we could not find the right peak and even my PI couldn't figure out what happened. After lunch I went to the ER because the finger was starting to bother me a lot. Since I couldn't use my right hand, I stayed in the dorm the next day.

After the weekend, I still couldn't use the bruised finger but my PI said it's fine and he'll help me do anything that required my right hand index finger. I still got to set up the next reaction, this time in a cold bath made of dry ice and acetone. Meanwhile we did more LC-MS tests on the product of the previous reaction and found that our desired product exists but in a very small amount compared to the main peak that had a different molecular weight. So the week ended with an unknown product.