Smaller research institutions often lack the enormous budgets of academic heavyweights, and this places limitations on the systems they can acquire and, ultimately, the research they can conduct. However, as technology continues to advance novel solutions are being developed which allow smaller institutions to access the same analytical capabilities. This means new research can be undertaken and students can be taught to the same standard as the larger institutions.
One of these solutions is the Spinsolve from Magritek, a low-maintenance, benchtop NMR system. AZoM spoke to Dr. Irosha Nawarathne, of Lyon College, on how she uses the Spinsolve in her research into the next generation of tuberculosis drugs, how she incorporates the Spinsolve in her teaching, and why she chose benchtop NMR over a cryogenic system.
Tell us about the research on tuberculosis medication you have been carrying out at Lyon College?
We have been developing new types of tuberculosis (TB) drugs based on rifampin (rifampicin), a drug currently in use. While rifampin is already an effective TB treatment, there is a continuous emerging of multidrug resistant TB bacterial (Mycobacterium tuberculosis - MTB) strains. We are in the process of developing novel drugs to treat patients with rifampin resistant TB. Rifampin works by binding to the RNA polymerase (RNAP) in TB bacterium, and this binding prevents the bacteria from replicating.
However, the TB bacterium is always mutating, hence evolving. Some of these mutations change the chemistry at the rifampin binding site in RNAP, which prevents rifampin from binding. Thus TB bacterium survives in the presence of rifampin drug. We work with rifampin-like drugs, and more specifically with a family of antibiotics called rifamycins, which we modify in response to the changes at the binding site of the TB bacteria. Our goal is to develop analogues of rifamycins that could bind to the mutated RNAPs in rifampin resistant TB bacteria.
How are you modifying Rifampin?
Our modifications are always in response to the prevalent RNAP mutations in rifampin resistant strains. Rifampin binds to RNAP majorly due to several hydrogen bonding interactions. These interactions are disrupted by some of the mutations occur at the RNAP binding site, which increase the survival of mutated MTB strains in the presence of the TB drug, rifampin. Say, for example, serine residue is being swapped for leucine residue through a mutation. That prevents rifampin from binding to the mutated MTB RNAP, as leucine has no hydrogen acceptor or a donor in the side chain. Also its bulkier size prevents rifampin from reaching a strong bonding distance.
We look at the changes that occur to the binding site and modify rifamycin core in a way that means it can still bind to the modified MTB RNAP. So if the site goes from polar to non-polar, we modify rifamycin so it is also non-polar, or if the amino acid residue becomes bulkier we make sure that the groups on the modified rifamycin is a little less bulky so it can still fit in the binding cavity.
Serine to leucine mutation is present in around 42% of resistant TB strains so we’ve focused on finding new drugs for this specific modification. We’ve successfully synthesized a range of rifampin-like molecules, rifamycin analogues, which we are going to test on mutated RNAP. If this happens to be a success, we will have produced a new drug for the treatment of resistant MTB strains, as the rifamycin core has proven drug characteristics.
How have you been using the Spinsolve as part of this research? Can the SpinSolve still be used for large molecules such as Rifampin?
The Spinsolve is designed to study smaller molecules. The resolution is not as great as that of high frequency cryogenic NMR. I used to use a 400-600 MHz NMR systems to study rifamycins and that was still difficult – they are very sophisticated molecules, with a number of chiral centres and polar (thus variable) regions.
However, we’ve managed to work around these resolution issues of the Spinsolve and the rifamycins by conducting our reactions using smaller molecules that are structurally similar to rifamycins. For example, when we attempt to develop sensitive reactions at the hydroxyquinone moiety of rifamycin, we carry out the reaction on a smaller hydroxyquinone that can be analysed by the Spinsolve.
Consequently, we determine the method that works the best and then use that method on rifamycins. Once the process is complete, we send our modified rifamycin to an external lab for a complete high frequency NMR and LC-MS analyses.
We have also installed the fluorine protocol for the Spinsolve which is especially useful as we do a lot of work involving fluorinations on both rifamycin and Paclitaxel (Taxol®) molecules. Fluorinations are difficult reactions to run even on simple molecules, never mind on sensitive and highly functionalized molecules such as rifamycin and paclitaxel, so we spend a lot of time developing our synthetic methodologies (A side note: we work on paclitaxel (antimitotic agent) to understand its biosynthetic pathway in detail; hence we can develop highly efficacious analogues through biocatalysis).
Rifamycin S and Paclitaxel's (Taxol®) complex structure makes it difficult to resolve using low frequency NMR
How are you planning on using the Spinsolve's fluorine modes in your research?
The fluorine protocol will allow us to see if fluorine has been incorporated into our product molecules. Previously, we had to send the samples away and get the results from an external lab for LC-MS data to confirm if fluorine has been incorporated, yet we were not able to tell at what position. Now we can use the Spinsolve in 19F mode and determine the success of fluorination reactions right away. For Taxol, we will now be able to use the Spinsolve to do everything including determining where in the molecular framework fluorine is on various molecules.
For rifamycins, which are more complicated, it will still be difficult to determine where exactly the fluorine is. However, the fluorine protocol will be very useful for our purifications. Usually, when we run a preparative TLC or a column chromatography after a reaction we have three or four different products, and we have no way of knowing which one is the correct product except for guessing based on the polarity. With the Spinsolve's fluorine mode, we will be able to almost immediately know which spot contains our product.
What aspects of chemistry are you currently teaching? How does your research tie into the teaching that you're doing at Lyon College?
I am an organic chemist, so I primarily teach organic chemistry and instrumental analysis. I have also taught advanced inorganic chemistry at Lyon College. I always try to use the examples from my research in teaching.
My organic and instrumental classes really easily tie into my research as the concepts align directly with my research interests and practices – Class can easily focus on building a hypothesis, synthesis, purification, and analysis of products. Inorganic chemistry is further away from my expertise and biomedical research. But I frequently utilize organometallic catalysts in my research. I bring up those applications in the inorganic chemistry class.
What experiments have your students been using the Spinsolve for? How do you think using it has enhanced their understanding?
My students adore it. They are amused by the idea that they get hands-on experience on a state-of-the-art instrument in biomedical research. We end up using the Spinsolve in almost every experiment that we run in the teaching labs. My students are regularly synthesizing small molecules, like aspirin, or are carrying out syntheses to further understand the reaction mechanisms such as dehydrations or Grignard reactions, as part of their lab projects.
The NMR spectra generated using the Spinsolve allow the students to determine if their reaction has been successful. It also gives them insight into the mechanics of the reaction itself, which promotes their deep learning. The NMR spectra can be used to determine the purity of a product or whether there is any residual starting material or intermediates. They enjoy determining the molecular framework of the product using the instrument. The Spinsolve makes it easier to teach important concepts in synthetic and analytical chemistry.
Dr. Nawarathne uses the SpinSolve to demonstrate NMR to her students
What are the advantages of using benchtop NMR over the other conventional benchtop techniques, such as FT-IR and UV-Vis spectroscopy?
We also have FT-IR and UV-Vis at Lyon College. For an example, before we acquired the Spinsolve, FT-IR was our instrument of choice for every experiment we did in the organic chemistry labs. This meant we were limited in what we could demonstrate to our students.
For example, if the students had carried out a dehydration reaction, using FT-IR, we could see the disappearance of the hydroxyl absorption of the starting material as the reaction progress, but we couldn’t draw further conclusions. Now with the Spinsolve, we can show the change in functional group and exactly where the modifications occur on the molecular framework.
The Spinsolve also eliminates the problem of interference from impurities during sample analysis. For example, during the dehydration experiment, if the product is not distilled properly there will be some residual water left in the final product. Water would show a hydroxyl absorption in an FT-IR spectrum, making it looks like the starting material, which also has a hydroxyl group. This isn’t a huge problem with NMR as it provides clear and detailed information of the molecules that are being analysed. As NMR does not just focus on the functional group, the analysis from the Spinsolve is more detailed and meaningful yet it is a quick analysis. After all, NMR is the instrument of choice of an organic chemist.
Why did you choose the Spinsolve over a cryogenic NMR system?
It is simply because of the easy maintenance. We have had a cryogenic system at Lyon College several years ago and there have been a lot of maintenance issues. There were not enough staff members to take care of the system and the school didn’t have the financial support to sustain it, thus the system eventually failed.
It was important to bring back the NMR system as it is a necessity for our research and teaching programs. Also NMR is widely used in biomedical field in which my students are fond of. Hence I wanted them to become experts in NMR so that they can be leaders in their fields in the future. However, I knew that we couldn’t maintain a cryogenic system. We just don’t have the time to maintain it ourselves and can’t justify the cost of a technician. So for that reason, we chose to acquire the Spinsolve, which uses a permanent magnet with minimal/no maintenance requirement.
The SpinSolve 43 from Magritek
Where can our readers find out more about your research, Lyon College, and the SpinSolve?
You can find further information on the Spinsolve at the Magritek website. Any inquiries regarding my research program or the use of the Spinsolve in teaching at Lyon College need to be directed to me at [email protected], unless you can find the answers on my faculty webpage.
About Dr. Irosha Nawarathne
Irosha Nawarathne is an assistant professor at Lyon College in Arkansas. She earned her Bachelor’s degree (First Class Honors) in chemistry from University of Colombo in Sri Lanka and her PhD in bioorganic chemistry from Michigan State University in East Lansing. For her dissertation research, she successfully utilized Taxus acyltransferases in biocatalysis in order to modify biologically important natural product scaffolds such as paclitaxel (Taxol®).
Upon graduation in 2011, she joined the College of Pharmacy at University of Michigan in Ann Arbor as a research fellow, where she gained medicinal chemistry skills by conducting research on antibiotic development. Her current research lab at Lyon College focuses on the development of efficacious therapeutics from large natural product scaffolds, with proven biomedical applications, through targeted synthesis.
Currently, her research team full of undergraduates is working on the development of novel rifamycins to combat multi-drug resistant Mycobacterium tuberculosis (MTB) strains by gaining a deeper understanding of molecular interactions between rifamycins and MTB RNA polymerase. Her laboratory also attempts to modify precursors of paclitaxel to further understand the biosynthetic pathway of paclitaxel.
Dr. Nawarathne is funded through National Institutes of Health and General Medical Sciences (P20 GM103429). She is passionate in creating opportunities for undergraduate students to explore the process of drug discovery and challenging them with intriguing questions, while contributing to the advancement of human health through drug discovery.
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