Date of Award

12-2024

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry/Organic Chemistry

First Advisor

Béla Török

Second Advisor

Marianna Török

Third Advisor

Jason Evans, Guodong Zhang

Abstract

High-resolution mass spectrometry (HRMS), as its name suggests, possesses high resolving power that enables the separation of ions with very close mass values. This is particularly beneficial for analyzing complex samples where numerous compounds may have similar masses, as it helps prevent signal overlaps and ensures an accurate spectral analysis. The current work explores the diverse applications of high-resolution mass spectrometry in oligonucleotide research, organic synthesis and medicinal chemistry. Oligonucleotide research: Chapters 2-5 present the research results from studies that employed ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) to understand the pharmacokinetics of RNA interference (RNAi) therapeutics. The investigations, which used REVERSIR, givosiran, and 2’-deoxy-2’-fluoro nucleotides as examples, showcased the application of HRMS in studying the ADME (absorption, distribution, metabolism, and excretion) properties of oligonucleotides in silencing RNA. The REVERSIR study involved a single-stranded GalNAc-siRNA-mediated oligonucleotide and was carried out using samples from rat and monkey plasma, liver, and kidney. A novel single-stranded deaminated metabolite from REVERSIR was identified in monkey liver, characterized by the deamination of the terminal 2′-O-methyladenosine nucleotide to 2′-O-methylinosine, and its structure was confirmed with HRMS. Quantification methods for both the parent compound and the identified metabolites were developed and validated for various concentration ranges in rat and monkey plasma, liver, and kidney, showing high intra- and inter-day accuracy and precision. Givosiran, an RNAi therapeutic, was characterized using HRMS to elucidate its distribution and metabolism in nonclinical studies, providing essential insights into the drug's behavior within biological systems. The key findings include comprehensive analyses of the drug's metabolic pathways and its distribution in different tissues, which contribute to a better understanding of its efficacy and safety profile. A non-naturally occurring 2′-deoxy-2′-fluoro-modified nucleotide unit is present in GalNAc-siRNA conjugates. HRMS was used to identify, confirm, and qualify modified nucleotides within siRNA therapeutics. The in vitro study assesses the integration of these modifications into RNA strands, their metabolites, and metabolic stability. The results indicated that the modified nucleotides are neither inhibitors nor preferred substrates for human polymerases, and no obligation or non-obligate chain termination was observed. Therefore, they can be safely applied in the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity, allowing for low dose and infrequent dosing. Organic synthesis and medicinal chemistry: HRMS has emerged as a pivotal tool in organic synthesis for characterizing complex molecular structures and reaction intermediates, ensuring the purity and specificity of the synthesized compounds. Chapter 6 describes the HRMS applications in the development of new β-carboline derivatives targeting the pathology of Alzheimer’s disease. HRMS was used to analyze and confirm the interaction of these compounds with Aβ by observing inhibitor-Aβ complex formation thereby gaining insights into the mode of action of the compounds and affirming their potential in therapeutic applications.

Comments

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