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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2017

Month Degree Awarded

May

First Advisor

Igor A. Kaltashov

Second Advisor

Richard W. Vachet

Third Advisor

Stephen J. Eyles

Fourth Advisor

James J. Chambers

Subject Categories

Analytical Chemistry | Medicinal-Pharmaceutical Chemistry | Organic Chemistry

Abstract

Acquired immunodeficiency syndrome (AIDS) remains one of the most serious public health challenges and a significant cause of mortality for certain populations. Despite the large number of antiretrovirals (mostly nucleotide and nucleoside analogs) developed in the past two decades, the inability of small molecule therapeutics to target HIV reservoirs directly creates a significant obstacle to their effective utilization. Indeed, achieving the desired therapeutic effect in the absence of the effective targeted delivery must rely on dosage escalation, which frequently causes severe toxicity. This problem may be solved by conjugation of antiretroviral agents to endogenous proteins (e.g., hemoglobin haptoglobin complex) that are specifically recognized by HIV reservoirs (such as macrophages) for internalization and catabolism. However, conjugation of a large class of antiretroviral agents (acyclic nucleoside phosphonates, such as adefovir) to a protein is challenging due to the rapid decay (including hydrolysis and dimerization) of the activated form of the drug (adefovir phosphonoimidazolide) during transition (ether precipitation) from organic phase to aqueous phase. This work introduces a novel synthetic strategy which overcomes the instability of the activated form of adefovir by emulating the first step of its metabolic pathway (phosphorylation), making it highly reactive towards primary amine groups of proteins. The effective conjugation of phosphorylated form of adefovir to protein via an imidazolide based intermediate was demonstrated using lysozyme as a model carrier protein. Mass spectrometry (MS) based analytical methods were used to support design and optimization of all those conjugations. Further optimization of adefovir’s conjugation with hemoglobin (Hb), a drug carrier which targets macrophage via haptoglobin (Hp)-CD163 mediated heme scavenging system, was pursued using another novel linker, phosphonoacetate, which allows reactions to be performed at neutral pH with a satisfactory yield. Successful loading of adefovir to Hp, the obligatory partner of Hb for targeted drug delivery, via Hb·Hp binding was demonstrated by MS. Lastly, a new strategy was developed for detecting and quantitating exogenous Hp·Hb complex with high sensitivity in complex biological samples using gallium as a tracer of this protein and inductively coupled plasma mass spectrometry (ICP MS) as a method of detection.

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