UC Berkeley

The ability to chemically modify biomolecules has facilitated our ability to detect, manipulate, and study them both in vitro and in vivo, as well as to prepare and tailor the properties of biologics and other pharmaceuticals based on natural products. This thesis describes several projects united around the theme of new methods to chemically modify biomolecules. The bulk of the thesis describes a new method to prepare protein bioconjugates relevant to diagnosis and treatment of human disease, while the last chapter describes the development of a new research tool to image protein glycosylation in vivo.

Chapter 1 describes the state of the art in protein modification methods by examining them through the lens of site-specific antibody–drug conjugates. Many methods for site-specific protein modification are now known in the literature, but only the subset that have met stringent requirements with respect to reagent and conjugate stability, minimal side reactivity, fast reaction kinetics, and amenability to structure-activity relationship studies have been seriously considered for use on a commercial scale. Thus, rather than cataloging every known method for site-specific protein modification, this chapter harnesses the collective wisdom of the field in detailing the origins and practical uses of the most popular and well-validated methods for site-specific protein modification.

Chapters 2 and 3 describe my contributions to the field resulting in a reaction for protein modification known as the Pictet–Spengler ligation. Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. The Pictet–Spengler ligation addresses this problem by providing a means to generate a stable linkage to protein aldehydes and ketones.

Aside from their hydrolytic instability, another drawback of oxime linkages is that the optimal conditions for their formation are acidic (pH 4.5), preventing their use with acid-sensitive proteins and post-translational modifications. The work in Chapter 3 describes a variant of the Pictet–Spengler ligation, the hydrazino-Pictet–Spengler ligation, that proceeds quickly near neutral pH. This work was carried out at Redwood Bioscience (now part of Catalent Pharma Solutions), a biotechnology company based in Emeryville, CA that uses aldehyde-functionalized proteins to prepare site-specifically modified antibody-drug conjugates.

Chapter 4 transitions from protein modification to glycan modification, describing a new method that combines the Bertozzi lab’s longstanding interest in metabolic glycoengineering with recent advances in fluorogenic bioorthogonal reactions to image internal cell-surface glycans in live zebrafish. Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. We have previously used the chemical reporter strategy in conjunction with bioorthogonal chemistry to image glycans on the enveloping layer of live zebrafish embryos; however, the ability to image glycans systemically inside a live organism has remained elusive. This chapter describes a method that combines metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a fluorogenic tetrazine ligation reaction, allowing us to image sialylated glycoconjugates within in live zebrafish embryos.