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Thesis 11-25-15 Torkabadi.pdf (3.7 MB)

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Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.

Heidari Torkabadi, Hossein
http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373

Abstract Details

2016, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Raman spectroscopy utilizing a Raman microscope can be used to study various biological samples. Based on approaches using difference spectroscopy, rapid mix -rapid freeze, and freeze drying of enzyme-inhibitor mixtures and bacterial cells, we developed several novel protocols. These enabled us to identify the enzymatic reaction intermediates and inhibition pathways in aqueous solutions and inside cells. The recent results complement the reactions of various ß-lactamases with different inhibitors in single crystals, elucidated in the Carey laboratory for more than a decade. Taken together, the in vitro results allow us to characterize the populations and reactions of drug molecules inside bacterial cells. A Raman microscope was used with a combination of rapid mix - rapid freeze, and freeze drying to study the reaction of SHV-1 ß-lactamase with tazobactam, and trap early intermediates in a dilute aqueous solution milliseconds after mixing. To extend the comparison of solution and crystal reactions, we used another clinically important ß-lactamase enzyme, CTX-M-9 reacting with clavulanic acid, sulbactam, or tazobactam. We identified an infrequent path of inhibition for this enzyme when reacted with each of the three inhibitors. In solution and in single crystals, each formed an irreversible ß-alkoxyacrylate species. Using Raman microscopy to characterize freeze-dried bacterial cells, we studied the populations and reactions of two different classes of inhibitors inside the cells. With clavulanic acid and tazobactam, which are known inhibitors of SHV-1 ß-lactamase, we identified the formation of enamine intermediates inside cells expressing SHV-1. The population of free clavulanic acid was measured inside each bacterial cell when the cells were not expressing the target enzyme. For the second class of compounds we measured the population of dihyofolate reductase (DHFR) inhibitors, (the propargyl-based UCP 1038 and UCP 1089) inside Gram-negative and Gram-positive bacteria after different times of exposure to the inhibitors. Single crystal studies indicate that the protonation of the diaminopyrimidine ring of the inhibitors occurs upon binding to DHFR. Identical spectral changes occur in bacterial cells, suggesting that the protonation is occurring for inhibitors upon binding to the intra-cellular targets.
Paul Carey (Advisor)
Mary Barkley (Committee Chair)
Robert Bonomo (Committee Member)
Thomas Gerken (Committee Member)
Witold Surewicz (Committee Member)
Focco Van den Akker (Committee Member)
140 p.
Biochemistry; Chemistry; Pharmaceuticals

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Citations

  • Heidari Torkabadi, H. (2016). Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells. [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373

    APA Style (7th edition)

  • Heidari Torkabadi, Hossein . Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells. 2016. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373.

    MLA Style (8th edition)

  • Heidari Torkabadi, Hossein . "Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells." Doctoral dissertation, Case Western Reserve University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373

    Chicago Manual of Style (17th edition)

case1448904373
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© 2016, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.