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

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

Bibliographic Details
Main Author: Heidari Torkabadi, Hossein
Language:English
Published: Case Western Reserve University School of Graduate Studies / OhioLINK 2016
Subjects:
Biochemistry
Chemistry
Pharmaceuticals
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-case14489043732021-08-03T06:34:09Z Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells. Heidari Torkabadi, Hossein Biochemistry Chemistry Pharmaceuticals 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. 2016-01-27 English text Case Western Reserve University School of Graduate Studies / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373 http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection NDLTD
language English
sources NDLTD
topic Biochemistry
Chemistry
Pharmaceuticals
spellingShingle Biochemistry
Chemistry
Pharmaceuticals
Heidari Torkabadi, Hossein
Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.
author Heidari Torkabadi, Hossein
author_facet Heidari Torkabadi, Hossein
author_sort Heidari Torkabadi, Hossein
title Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.
title_short Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.
title_full Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.
title_fullStr Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.
title_full_unstemmed Raman Microscopic Studies of Antimicrobial Reactions in Solution, Crystals, and Bacterial cells.
title_sort raman microscopic studies of antimicrobial reactions in solution, crystals, and bacterial cells.
publisher Case Western Reserve University School of Graduate Studies / OhioLINK
publishDate 2016
url http://rave.ohiolink.edu/etdc/view?acc_num=case1448904373
work_keys_str_mv AT heidaritorkabadihossein ramanmicroscopicstudiesofantimicrobialreactionsinsolutioncrystalsandbacterialcells
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