Document Type


Date of Degree

Spring 2016

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Wiemer, David F.

First Committee Member

Pigge, F. Christopher

Second Committee Member

Quinn, Daniel M.

Third Committee Member

Stone, Elizabeth A.

Fourth Committee Member

Wiemer, Andrew J.


Phosphorus is an element that is essential for life, and is used in the synthesis of many proteins, carbohydrates and deoxyribonucleic acids. Phosphorus often exists in the form of phosphate when found in the biological systems. Clinical development of possible pharmaceutical agents have used phosphorus in the form of phosphonates to increase the metabolic stability of the potential drug. Some of these phosphonates target the isoprenoid biosynthetic pathway (IBP). The IBP plays an important role in the synthesis of cholesterol and in other aspects of cellular metabolism. The enzymes of the IBP have been the target of possible therapeutic agents for treatment of multiple diseases, including cancer. Often these phosphonic acids are masked by an enzymatically cleavable group in order to increase their bioavailability and activity.

Phosphoantigens are small organo-phosphorus molecules that stimulate the expansion of Vγ9Vδ2 T-cells which detect and eliminate infected cells. Both natural and non-natural phosphoantigens have exhibited a wide range of effective concentrations (EC50) for γδ T-cells. The most potent phosphoantigen is E-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP), which is an intermediate in the bacterial IBP. Nanomolar concentration of this compound stimulate T-cell proliferation. While HMBPP is highly potent, it undergoes rapid decomposition when injected into the blood stream. Synthesis of more stable phosphonate analogues can show better activity for expansion of the γδ T-cell population. Increased activity was observed in T-cell assays after masking the phosphonic acids to increase the bioavailability of the active phosphoantigen.

Because some phosphoantigen showed strong activity with masked phosphonic acids, families of phosphonate analogues now have been prepared. Most use selenium dioxide mediated oxidation to incorporate the terminal alcohol and ester exchange to provide prodrugs to study the structure-activity-relationships. The biological activity of these compounds has been investigated and new phosphoantigens were shown to be strong activators of γδ T-cells. Furthermore, the phosphoantigens have been shown to bind to the protein butyrophilin 3A1 (BTN3A1) at an intracellular domain. A second family of phosphoantigen derivatives, masked by a new pH fluorescent cell-cleavable ester, were prepared and tested by our collaborators to explore the compounds’ activity and to investigate the mechanism of action.

Finally, a new class of phosphorus compounds alkyl 1, 1, 1-trisphosphonates has been studied to obtain salts that might be biologically active. Trisphosphonates contain a unique arrangement of phosphonate groups on a single carbon and could provide charge states unseen in the more traditional bisphosphonates. A general route to asymmetric trisphosphonates through a step-wise phosphorylation of each phosphonate has been developed. Selective phosphonate ester cleavage would allow for the ability to obtain a multitude of charge states and possible biological activity.

Public Abstract

Phosphorus is an element that is essential for life, and is found in proteins, carbohydrates and nucleic acids (DNA). The metabolically more stable phosphonates may represent a more attractive template for pharmaceutical agents, and phosphonates which target the isoprenoid biosynthetic pathway (IBP) are known. This pathway has been the target of clinical drugs used to treat multiple diseases including cancer. Often these phosphonic acids are masked by cell-cleavable groups in order to increase their bioavailability and activity.

My research has focused on the synthesis of phosphonates that stimulate immune cells to detect and eliminate infections. Synthesis of phosphonate analogues of natural phosphoantigens determined that they show biological activity, specifically the ability to expand immune cell populations. Families of phosphonate analogues now have been prepared to study how different structures affect their biological activity. One family of phosphonate derivatives, masked by a new pH-sensitive, fluorescent, cell-cleavage ester, was prepared and tested as a biological probe.

A relatively new class of phosphorus compounds that has yet to be biologically explored would be the trisphosphonates. Trisphosphonates contain three phosphorus groups on a single carbon. Selective preparation of various trisphosphonate salts may lead to biological activity. The preparation of these molecules will be examined in detail.


Fluorescent, Phosphoanitgens, Phosphonates, Prodrug, Trisphosphonates


xviii, 190 pages


Includes bibliographical references (pages 186-190).


Copyright © 2016 Rebekah Ruth Shippy

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