Celebrating the Contributions of Carolyn Bertozzi to Bioorthogonal Chemistry and its Application to Glycoscience

Abstract

This issue celebrates the Wolf Prize given to Carolyn Bertozzi for her transformative contributions to the creation and development of bioorthogonal chemistry and its applications for understanding the role of carbohydrates in biology. Chemistry is a discipline fundamentally focused on the properties of molecular bonds. Understanding these properties has allowed chemists to establish “rules” of reactivity that provide the foundation for chemical processes that underly almost all of the aspects of our modern world, from agriculture and materials to pharmaceuticals. Chemical reactions have made significant contributions to the field of biology for many decades. For example, organic chemistry provided the means to generate complex natural products and other probes to selectively perturb enzymes and cellular pathways. Likewise, the development of the reactions for solid-phase DNA synthesis helped push the molecular biology revolution forward. Despite their clear power, however, many chemical reactions have selectivity limitations that require them to be performed under controlled conditions (e. g., inert solvents, limited number of reactants, etc.). This means that the direct application of chemical reactions to biological systems in water and the milieu of reactive groups on proteins, nucleic acids, etc. is extremely challenging. However, Carolyn saw the notable utility of being able to perform “bioorthogonal chemistry,” where two reactive partners are so well paired that they prefer to react selectively with each other over all of the other possibilities in biology. Through her combination of incredible insight, determination, and inspiration, Carolyn created the Staudinger ligation, and perhaps more importantly the concept of the azide as a powerful functional group for bioorthogonal chemistry. The subsequent ∼20 years has seen the continued development and application of bioorthogonal chemistries by the Bertozzi lab and others to the point where these transformations make up a cornerstone of chemical biology. As three of Carolyn's previous trainees, we had a front row seat for the creation, development, and application of bioorthogonal chemistry, and all three of us routinely use these reactions in our own work. Matthew Pratt was present during the earlier days of the Bertozzi lab as a member of her 5th PhD class in 1999. During this time, it was clear that the development of bioorthogonal chemistry was inextricably linked to Carolyn's passion to glycoscience. The intellectual spark that resulted in the development of the Staudinger ligation came from the desire to do chemistry on the surface of living cells to study glycans. Building upon the work of Werner Reutter, the Bertozzi lab had shown that monosaccharide-derivatives containing ketones could be metabolized by cells and displayed in native glycans on cells. These ketones could be detected with aminooxy reagents through the selective formation of oximes, achieving the original goal. However, Carolyn was not satisfied with the pioneering result, as the interior of cells is full of metabolites that contain ketones and aldehydes that can undergo this same reaction. Instead she considered other reactions that can occur in water, leading to the insight that an intermediate in the Staudinger reaction between azides and phosphines can be trapped to form a covalent bond, giving the Staudinger ligation. The lab fairly quickly demonstrated that the azide can be incorporated into multiple monosaccharides and other metabolites for detection of different biomolecules. It is no accident that the boundaries of bioorthogonal chemistry have been and continue to be pushed in the pursuit of understanding glycans. In comparison to the other major biomolecules proteins and nucleic acids that benefitted from the revolution of genomics in the last decades, there simply was no corresponding solution for glycans. Innovative chemical approaches enabled by Carolyn are a testament to the fact that necessity is, after all, the mother of invention. Following the introduction of the azide as a convenient bioorthogonal tag, the potential impact for proteomic characterization of glycoproteins, glycan engineering, protein labelling, etc. began to emerge, but the first generations of chemical probes were often too immature for these applications. However, Carolyn was undaunted and showed incredible determination to contribute to pushing bioorthogonal chemistry further, even if the applications were years or sometimes decades away from materialization. One example was the idea to render chemically tagged monosaccharides specific to the glycosyltransferase enzyme that introduced them. While conventional bioorthogonal labelling tools are fairly promiscuous for the glycans they tag, being able to trace glycans back to their biosynthetic origin promised an understanding of the disease relevance of individual glycosyltransferases. But how to achieve enzyme specificity, given the general lack of variability in the way glycosyltransferases recognize the activated nucleotide-sugars? Shortly after the first bioorthogonal reactions in complex model organisms were applied, the Bertozzi group engaged in interactions with the Shokat lab that pioneered the concept of applying bump-and-hole engineering to achieve allele-specific kinase inhibition. An idea was born to apply the tactic to glycosyltransferases, too: engineering a transferase to contain a “hole” that would specifically accommodate a “bumped”, chemically modified nucleotide-sugar substrate and, prima facie, act as a specific reporter system for the activity of that transferase. It is not hard to convey how much ahead of its time this idea was: there was a lack of crystal structures of glycosyltransferases to inform protein engineering; methods of mass spectrometry were not sensitive enough; chemoenzymatic procedures for the synthesis of nucleotide-sugars were yet to be established; there was no CRISPR for facile genome manipulation. It would take twenty years for these roadblocks to be overcome. Finally, a team of trainees that included Ben Schumann brought the tactic, conceived by Carolyn with an entirely different generation of Bertozzians, into fruition. The approaches described above allow for the labeling, visualization, and identification of glycans in living systems. However, they do not necessarily allow one to ask another key question: what biology is controlled by one glycan structure and how does that biology change upon structural alterations to that glycan? This is a hard question to answer using traditional biological tools, as the extracellular glycans that make up the cellular glycocalyx are endogenously heterogeneous and enzymatically synthesized in a non-templated fashion. A major strength for chemists has always been the synthesis of defined structures. Carolyn and her trainees have exploited bioorthogonal and other chemistries to develop techniques to chemically synthesize high molecular weight, highly glycosylated molecules of the glycocalyx and clever methods to attach those molecules to the cell surface. Her approaches have been used to probe phenomena essential for human health such as infection, immunity, and cancer. Knowledge gleaned from her lab's work is now opening the door to entirely new therapeutic classes reliant on specific glycosylation. For example, modifications to glycans in the glycocalyx could improve outcomes in difficult-to-treat cancers. Her lab has also designed synthetic glycopeptides that target associated cargo for degradation, which could be used to eliminate disease causing proteins. Carolyn's trainees in this area, including Jessica Kramer, continue to use these approaches to make advancements in our understanding of epithelial biology. We would also be remiss if we didn't mention Carolyn's skill set outside of science. Matthew Pratt remembers fondly the tireless effort Carolyn put into all areas of trainee mentorship, particularly the exposure she provided students and postdocs to other scientific leaders. She took opportunities to put her trainees front and center, allowing them to build professional relationships that pay dividends for their entire careers. Jessica Kramer was a lab member during the transition from UC Berkeley to Stanford and recalls how Carolyn carefully considered the effects of the move on each individual she mentored. We were even invited to participate in the design of the new lab space to ensure it fit our needs. Carolyn is also a true renaissance woman who excels at essentially everything she takes on, including karaoke and softball – and she is a beast on the dancefloor! Ben Schumann was one of the first lab members who started after the move to Stanford. Joining a big lab at a major U.S. university with a 2-months-old baby can be nerve-wracking, but Carolyn set the perfect example of a leader who walks the walk of supporting individual circumstances. You would often see her pick up her kids after work, or bump into her taking them for ice cream on weekends. Her lab is run based on the trust that people achieve their best when they are enthusiastic about their work, which is easy with Carolyn radiating enthusiasm in every second of the day. We thank Carolyn for her mentorship and inspiration and congratulate her on the prestigious Wolf Prize and also the subsequent awarding of the Nobel Prize in Chemistry for similar contributions to bioorthogonal chemistry. We would not be the scientists we are today without the training she provided and her continued guidance. We also extend our sincere thanks to the authors of each of the articles. The various review and research articles speak to the broad reach and impact of Carolyn's work. We hope that this collection serves as a catalyst for readers interested in chemical biology, bioorthogonal chemistry, and their applications to glycoscience.