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Protonation States of Novel Therapeutics for the Resurrection of Organophosphorus-Aged Acetylcholinesterase

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2020, Master of Science, Ohio State University, Chemistry.
Acetylcholinesterase (AChE) is an enzyme which catalyzes the degradation of acetylcholine to acetic acid and choline within neuromuscular junctions. Upon inhibition of AChE by some toxicant, acetylcholine begins to build up at the neuromuscular junctions and results in a cholinergic crisis, resulting in a variety of symptoms which can be summarized by the SLUDGE acronym: salivation, lacrimation, urination, defecation, gastrointestinal distress, and emesis. Left untreated and if toxic exposure is sufficient, a cholinergic crisis will result in death. While a non-lethal cholinergic crisis can result from a variety of triggers, organophosphorus (OP) compounds pose a global threat to human populations. Responsible for approximately 200,000 deaths annually, OP compounds inhibit AChE via phosphylation of a serine residue situated within the active site of AChE. Current medical treatment for inhibited AChE involves the use of nucleophilic oximes, which displace the phosphorus species. Individuals exposed to OPs are also given a variety of other medicines to treat the SLUDGE symptoms described above. Although the use of a nucleophilic oxime can displace the phosphorus moiety, OP-inhibited AChE can also undergo a process known as aging. Aging occurs when the phosphorus moiety undergoes spontaneous O-dealkylation. Once AChE has aged, the phosphorus center is less electrophilic which limits the use of a nucleophilic treatment such as pyridinium oximes. Currently, there is no approved therapeutic which can restore aged AChE to the native state. Our group has recently shown that quinone methide precursors (QMPs) can both resurrect OP-aged AChE and reactive OP-inhibited AChE back to the native state of the enzyme. However, the ability of a QMP to resurrect OP-aged AChE and reactive OP-inhibited AChE has been shown to be dependent on the amine leaving group of the QMP. More recently, computational efforts by Joseph Fernandez, a graduate student on our research team, have indicated that the protonation state of our QMPs may also impact the ability of a QMP to resurrect OP-aged AChE and reactive OP-inhibited AChE. Ola Nosseir, another member of our research group, has used EPIK to generate preliminary protonation state assignments for our QMPs at physiological pH. The work in this thesis describes the use of an FT-IR and 1H NMR spectroscopic methods in order to provide experimental data so as to confirm the assignments made by the EPIK program. Initial protonation state assignments were simply based on simplistic pKa assignments generated from 1H NMR data. These pKa assignments were generated by plotting the chemical shift of various hydrogens of our QMPs versus the pH of an aqueous solution. As the pH of a solution is increased, the deprotonation of a titratable proton causes an upfield shift for other protons in the molecule. When these chemical shifts are graphed, these deprotonation events are indicated by a sigmoidal curve. These sigmoidal curves are known to represent the pKa values for the various titratable protons found in the molecule. However, while appropriate for initial protonation state assignments, this method is quite crude as the assignments are only based on chemical intuition and knowledge of various pKa values. Thus, we sought additional experimental data, which could be corroborated computationally, to confirm our protonation state assignments. First, we report that the 4JH-H meta coupling constants on the aromatic ring can also be used to assign the protonation state of a molecule. Like the chemical shift data described above, the 4JH-H meta coupling constants can be plotted versus the pH of an aqueous solution to generate sigmoidal curves. These sigmoidal curves were found to occur at the same pH as the sigmoidal curves described earlier and were consequently associated with deprotonation events of titratable protons. We initially chose to probe the 4JH-H meta coupling constants found in two of our core frameworks. Calculated 4JH-H meta coupling constants for all protonation states for these frameworks were generated at the B3LYP/aug-cc-pvtz level of theory. Notably, at both low and high pH, the calculated 4JH-H meta coupling constants for the fully protonated and deprotonated forms our frameworks were in agreeance with the experimental 4JH-H meta coupling constants obtained at an appropriate pH. The initial success in using the 4JH-H meta coupling constants to assign protonation states for our core frameworks, prompted us to investigate the couplings of a model QMP. Thus, the coupling constants for the various protonation states of a model QMP were calculated and compared to experimental data. Like the core frameworks, at both low and high pH, the calculated 4JH-H meta coupling constants for the fully protonated and deprotonated forms of this model QMP agreed with the experimental coupling constants. We then hypothesized that the 4JH-H meta coupling constants of this model QMP near physiological pH might agree well with the calculated coupling constants of one of the protonation states. The comparison of the 4JH-H meta coupling constant of this model QMP near physiological pH was found to agree well with the calculated 4JH-H meta coupling constants obtained from the QMP in a zwitter-ionic protonation state. While 1H NMR provided preliminary protonation state assignments, we also report that experimental IR spectroscopy can be used to corroborate our protonation state assignment. To make this assignment, Boltzmann distributions for each possible protonation state of the same model QMP from above were computed at the B3LYP/6-311+G(d,p) level of theory. These different Boltzmann distributions were then compared to an experimental IR spectrum obtained near physiological pH. After considering the effects of intramolecular hydrogen bonding, the Boltzmann distribution of two distinct zwitter-ionic forms was found to represent the experimental IR (taken near physiological pH) of our model QMP.
Christopher Hadad (Advisor)
Jon Parquette (Committee Member)
90 p.

Recommended Citations

Citations

  • Hopf, R. G. (2020). Protonation States of Novel Therapeutics for the Resurrection of Organophosphorus-Aged Acetylcholinesterase [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595331575488179

    APA Style (7th edition)

  • Hopf, Ryan. Protonation States of Novel Therapeutics for the Resurrection of Organophosphorus-Aged Acetylcholinesterase. 2020. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1595331575488179.

    MLA Style (8th edition)

  • Hopf, Ryan. "Protonation States of Novel Therapeutics for the Resurrection of Organophosphorus-Aged Acetylcholinesterase." Master's thesis, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595331575488179

    Chicago Manual of Style (17th edition)