Bioorthogonal Chemistry, From Basic Science to Clinical Translation — Carolyn Bertozzi

The speaker playfully addresses the academic rivalry between Stanford and Berkeley, expressing disappointment at the lack of enthusiasm from the Stanford attendees. They introduce the topic of Bioorthogonal Chemistry, a field that has its roots in Berkeley between 1996 and 2010. The speaker's research at Berkeley focused on the basic science aspect, while their work at Stanford has been more translational. The backstory of Bioorthogonal Chemistry is tied to the speaker's PhD and postdoctoral training, with a focus on glycoscience—the study of sugar molecules and their role in cell interactions and diseases. The speaker reminisces about their time as a graduate student in organic synthesis and the foundational tool of organic chemists, the round bottom flask, which allows for precise control over chemical reactions. This control is essential for the chemist to perform reactions that have been developed over a century. The speaker's interest in conducting chemistry in less controllable biological settings, such as living cells or animals, led to the concept of Bioorthogonal Chemistry.

The speaker delves into the concept of Bio-orthogonal Chemistry, which involves chemical reactions that do not interfere with biological systems. They describe the universe of all possible chemical reactions and identify a 'galaxy' within it known as the biological reactivity space, which includes all the chemicals found in the human body. The speaker's lab aimed to discover a bio-orthogonal reactivity space, which would consist of chemical functional groups not found in nature and would not crosstalk with biological chemicals. This idea was driven by the speaker's interest in glycoscience and the challenges of imaging sugars in living systems. The speaker also discusses their experience as a postdoctoral fellow at UCSF, studying changes in cell surface glycans associated with diseases like cancer, and the lack of technology for imaging sugars at the time.

The speaker recounts the development of Bioorthogonal Chemistry, beginning with their idea for imaging sugars in living systems during their postdoctoral years. They describe attending a conference where they learned about the biosynthesis of cylic acid and the potential for chemically modified metabolic precursors to be incorporated into cell surface glycans. Inspired by this, the speaker proposed a two-step process for imaging sugars in living systems, which involved feeding cells a chemically modified simple sugar and then using a complementary chemical group to react with the sugar and form a bond with an imaging probe. This approach required the development of bio-orthogonal chemical groups that would selectively react with each other without interfering with biological processes. The speaker admits they did not know what these functional groups could be at the time, but this idea became the foundation for their future research.

The speaker introduces the Staudinger Ligation as the first bioorthogonal reaction developed in their lab. This reaction is a modification of the classic Staudinger reduction, which involves the reaction between triphenylphosphine and an azide to form an intermediate called an Aza illid. The speaker explains how they adapted this reaction to create a stable product by introducing a methyl ester on one of the benzene rings, allowing the intermediate to cyclize and form an amide bond, resulting in a stable cyclic amide. This stable product enabled the reaction to be used for imaging experiments in living systems, such as attaching imaging probes to sugars. The speaker also discusses the process of incorporating azides into various sugars and using the Staudinger Ligation to conjugate them with phosphine reagents for imaging purposes in living cells and animals.

The speaker discusses the limitations of the Staudinger Ligation in terms of reaction kinetics, which was too slow for many imaging applications. They needed a reaction that was at least two orders of magnitude faster. Around the early 2000s, there was an interest in the reactivity of azides, particularly their ability to act as one-three dipoles in dipolar cyclo additions with alkynes, a chemistry known as click chemistry. However, the classic Huisgen cyclo addition was too slow and required heating, which is not viable for living systems. The speaker's lab was exploring ways to accelerate the azide-alkyne cyclo addition without using a toxic metal catalyst. They were inspired by the concept of ring strain in organic chemistry and considered the reactivity of strained alkynes with azides, which led to the discovery of a faster bio-orthogonal reaction.

The speaker describes the discovery of a copper-free click chemistry, which was a significant advancement in bioorthogonal chemistry. This new reaction was based on the use of ring-strained alkynes and azides, which was inspired by the teachings of organic chemistry regarding the reactivity of strained rings. The speaker's team found that a cyclooctyne with a phenylazide reacted explosively, indicating a highly reactive and fast reaction. This discovery was a promising alternative to the copper-catalyzed click chemistry, which was not suitable for biological systems due to the toxicity of copper. The team's work on this copper-free click chemistry opened up new possibilities for fast and efficient bioorthogonal reactions in living systems.

The speaker shares how the developed bioorthogonal reactions were applied to study glycosylation changes in zebrafish, a model organism for vertebrate development. The zebrafish's translucent body allows for easy optical imaging, and its rapid development from a single cell to a larval fish in just five days provides a unique opportunity to observe the entire developmental process. The speaker's team used metabolic and chemical labeling techniques to integrate azidosugars into cell surface glycans during development. By adding a bioorthogonal reactive group, such as difo linked to a fluorescent dye, they could label and image the sugars incorporated into cell surface glycans at different time points. This approach led to the discovery of the role of sugars in cell division and tissue development.