Bioorthogonal is defined as not interfering or interacting with biology. Chemical reactions that are bioorthogonal have recently become valuable tools to visualize biomolecules in their native environments, particularly those that are not amenable to traditional genetic modification. The field of bioorthogonal chemistry is rather young, with the first published account of the term bioorthogonal in 2003, yet it is expanding at a rapid rate. The roots of this unique subset of chemistry are in classic protein modification and subsequent bioconjugation efforts to obtain uniformly and site-specifically functionalized proteins. These studies are highlighted in Chapter 1. Chapter 2 opens with a summary of the bioorthogonal chemical reporter strategy, a two-step approach where a bioorthogonal functional group is installed into a biomolecule of interest, most often using endogenous metabolic machinery, and detected through a secondary covalent reaction with an appropriately functionalized chemical partner. It is this chemical reporter strategy that empowers bioorthogonal chemistry and allows for a wide variety of biological species to be assayed.
Chapter 2 proceeds to outline the discovery of the Staudinger ligation, the first chemical reaction developed for use in the bioorthogonal chemical reporter strategy. The Staudinger ligation employed the azide as the chemical reporter group and, since its debut in 2000, many laboratories have capitalized on the exquisite qualities of the azide (small, abiotic, kinetically stable) that make it a versatile chemical reporter group. The success of the azide prompted the development of other bioorthogonal chemistries for this functional group. One of these chemistries, Cu-free click chemistry, is the 1,3-dipolar cycloaddition between cyclooctynes and azides. The cycloaddition is promoted at physiological conditions by the ~18 kcal/mol of ring strain contained within cyclooctyne, and further modifications to the cyclooctyne reagents have lead to increased reactivity through augmentation of the ring strain or optimization of orbital overlap. When I began my graduate work, a difluorinated cyclooctyne (DIFO), which was 60-fold more reactive than other existing bioorthogonal chemistries, had just been synthesized and employed for labeling azides on live cells and within living mice. DIFO performed very well on cultured cells, but it was outperformed by the slower Staudinger ligation in the more complex environment of the mouse. We hypothesized that DIFO was too hydrophobic to be effective in mice and designed a more hydrophilic cyclooctyne reagent, a dimethoxyazacyclooctyne (DIMAC). DIMAC was synthesized in nine steps in a 10% overall yield (Chapter 3). As predicted, DIMAC displayed reaction kinetics similar to early generation cyclooctynes, but exhibited improved water-solubility. Consequently, DIMAC labeled cell-surface azides with comparable efficiencies to the early generation cyclooctynes but greater signal-to-noise ratios were achieved due to minimal background staining. Encouraged by these results, we assayed the ability for DIMAC to label azides in living mice and found that DIMAC was able to modify azides in vivo with moderate signal over background. However, the Staudinger ligation was still the superior bioorthogonal reaction for labeling azides in vivo. Our results collectively indicated that both hydrophilicity and reactivity are important qualities when optimizing the cyclooctynes for in vivo reaction with azides (Chapter 4).
We were also interested in modifying DIMAC so that it would become fluorescent upon reaction with an azide. Previous work in the lab had established that fluorogenic reagents could be easily created if a functional group was cleaved from the molecule upon reaction with an azide. We envisioned a leaving group could be engineered into the azacyclooctyne scaffold by strategically positioning a labile functional group across the ring from a nitrogen atom. The cyclooctyne structure should be stable, as it is rigid and intramolecular reactions are not favorable. However, upon reaction with an azide, a significant amount of strain is liberated and the intramolecular reaction should readily occur. Efforts toward the synthesis of this modified DIMAC reagent are chronicled in Chapter 5.
Chapter 6 is a very short account of our early work to use DIFO-based reagents for proteomics. The results contained in this chapter are preliminary and further endeavors towards this goal are underway by others within the group.
Chapters 7, 8 and 9 are devoted to strategies to increase the second-order rate constant of Cu-free click chemistry. In Chapter 7, various routes toward a tetrafluorinated cyclooctyne are outlined, although none of them successfully yielded this putatively highly reactive cyclooctyne. Chapter 8 describes the synthesis of a difluorobenzocyclooctyne (DIFBO), which is more reactive than DIFO, but unstable due to its propensity to form trimer products. However, DIFBO can be kinetically stabilized by encapsulation in beta-cyclodextrin. Only beta-cyclodextrin and not the smaller (alpha) or larger (gamma) cyclodextrins were able to protect DIFBO. We did observe an intriguing result when complexation with the larger gamma-cyclodextrin was attempted. It appears as though two DIFBO molecules can fit inside the gamma-cyclodextrin and dimeric products, which were not apparent in the absence of gamma-cyclodextrin, were observed. We hypothesized that all oligomer products of DIFBO were derived from a common cyclobutadiene intermediate. While DIFBO was chemically interesting, it was not a useful reagent for labeling azides in biological settings. Thus, Chapter 9 is devoted to the modification of DIFBO, with the aim of identifying a reactive yet stable cyclooctyne. The data from Chapter 9 suggest we are rapidly approaching the reactivity/stability limit for cyclooctyne reagents.
The results contained within Chapters 7-9 indicated that it was time to explore other bioorthogonal chemistries. When embarking on the development of a new bioorthogonal chemical reaction, we aimed to explore unrepresented reactivity space, such that the new reaction would be orthogonal to existing bioorthogonal chemistries. We became attracted to the highly strained hydrocarbon quadricyclane and performed a screen to find a suitable reactive partner for this potential chemical reporter group (Chapter 10). Through this analysis, we discovered that quadricyclane cleanly reacts with Ni bis(dithiolene) reagents and this transformation appeared to be a good prototype for a new bioorthogonal chemical reaction. After a thorough mechanistic investigation and many rounds of modification to the Ni bis(dithiolene) species, a nickel complex with suitable reaction kinetics, water-solubility, and stability was obtained (Chapter 11). Gratifyingly, this Ni bis(dithiolene) reagent selectively modified quadricyclane-labeled bovine serum albumin, even in the presence of cell lysate (Chapter 12). Other results in Chapter 12 highlight that this new bioorthogonal ligation reaction is indeed orthogonal to Cu-free click chemistry as well as oxime ligation chemistry. Additionally, quadricyclane-dependent labeling is observed on live cells, although further optimization is necessary.
The final chapter of this dissertation outlines the current state of the field. There are now many methods to modify biomolecules including several new, although relatively untested, bioorthogonal chemistries. The rapid pace of this field makes it an exciting time to be pursuing bioorthogonal chemistry.