UC Berkeley

Imaging represents a powerful method for advancing our understanding of biology. In particular, it has been used as a tool for the diagnosis and monitoring of diseases in vivo. Bioluminescence imaging (BLI) represents one of the molecular imaging modalities and has been applied to the study of numerous processes in cells and in animals. However, there is a need for the design of new bioluminescence imaging probes for the study of several key metabolic processes. Activatable bioluminescence imaging probes represent an attractive approach to this problem and are the subject of this dissertation. Activatable bioluminescence imaging probes have several desirable characteristics, including high sensitivity and the ability to be triggered in response to various stimuli. In particular, we describe the design, synthesis and biological evaluation of activatable bioluminescence imaging probes for the study of two metabolic processes: glycosylation and fatty acid uptake. Specific emphasis is placed on the development of probes that have optimal properties for imaging of these processes in mice.

Chapter 1 introduces the basics of bioluminescence imaging with an overview of the different luciferins and luciferases. The advantages of bioluminescence imaging are discussed. Additionally, the uses of bioluminescence imaging are reviewed. An emphasis is placed on activatable bioluminescence imaging probes, with two main types of stimuli discussed: enzymatic and small molecule. The potential of bioluminescence imaging for the study of two new biological processes, glycosylation and fatty acid uptake, is presented along with an overview of these two fields.

In Chapter 2, the synthesis and evaluation of a bioluminescence imaging probe for the study of glycosylation is discussed. A phosphine–luciferin probe was designed to undergo Staudinger ligation with metabolically incorporated azidosugars. The probe was synthesized and evaluated for its ability to label cell–surface glycans in a luciferase–expressing prostate cancer cell line. The probe was able to successfully image these glycans and demonstrated higher sensitivity than other previously published fluorescent phosphine probes.

Chapter 3 describes the design, synthesis, and evaluation of a bioluminescence imaging probe for the study of fatty acid uptake. The probe is activated inside of cells after uptake by fatty acid transport proteins (FATPs). The fatty–acid luciferin probe was synthesized and evaluated for physiological uptake in vitro using luciferase–expressing adipocytes. The probe was next tested in vivo using several modes of administration. Imaging of intestinal fatty acid uptake and uptake by brown adipose tissue (BAT) was demonstrated.

In the next two chapters, we switch our focus and describe a different project involving the synthesis and use of peptoids. In Chapter 4, the design of novel architectures of peptoids is discussed. The synthesis of dendritic and cyclic peptoid architectures is presented using various diamine monomers. Several synthetic parameters were changed in order to gain an understanding and control of which architecture is ultimately obtained. Optimized conditions were found for synthesis of dendritic and cyclic architectures. Dendrimers composed of aromatic diamines were subsequently used in the synthesis of two classes of amphiphilic molecules.

Chapter 5 discusses the synthesis and sequencing of combinatorial peptoid libraries. A number of amines were optimized for incorporation in peptoid libraries and three combinatorial libraries were synthesized. In addition, we report a rapid, high–throughput, sensitive, and inexpensive sequencing method for the identification of peptoids on a single bead. This method is based on partial Edman degradation/mass spectrometry (PED/MS) and should help facilitate the screening of large libraries of peptoids.