The development of fluorescent tools for selective labeling of biomolecules [electronic resource]
Keywords: Chemistry.

The ability to label biomolecules with fluorophores has greatly aided in the understanding of many of life's diverse processes. Technologies that allow for the rapid, specific, and covalent attachment of small molecule fluorophores to targets of interest in the complex cellular milieu are highly desirable. From the design of novel ligands for the HaloTag enzyme and its variants to the development of dialdehyde precursors for oxime and hydrazone formation, the work described herein aims to augment and improve upon the tools available for fluorescent labeling in biological systems. Chapter 1 documents our effort to develop a HaloTag-derived enzyme/ligand pair that displays orthogonal reactivity to the original HaloTag system. Self-labeling enzymatic protein tags have allowed for experiments not previously feasible with conventional fluorescent protein tags. While a number of such tags are available, the fast kinetics, low background signal, and simple substrates make the HaloTag system stand out among them. To follow more than one protein of interest simultaneously, an orthogonal enzyme with properties similar to HaloTag would be beneficial. To develop such a system, we first altered the structure of a HaloTag substrate to inhibit reaction with the original HaloTag enzyme. We then used this alternative substrate to select for HaloTag mutants that it effectively labels. An orthogonal enzyme that functions in E. coli was developed. In Chapter 2, a novel strategy for the development of fluorogenic HaloTag substrates is explored. For intracellular fluorescent labeling of enzymatic protein tags, the need to wash away excess unreacted label can add 30 minutes or more to an experiment before making cellular measurements. This extra time prevents the measurement of processes on shorter timescales. A number of fluorogenic labels for these self-labeling tags have been reported, with the goal of near-instantaneous no-wash imaging. The previous best fluorogenic HaloTag ligand displayed a 12-fold increase in fluorescence intensity upon reaction with the enzyme, but washing steps were still performed in cellular imaging experiments. We introduce a series of dimethylaminostilbazolium-based HaloTag substrates that light up upon reaction with HaloTag due to their twisted internal charge transfer structure. The best-performing ligand becomes 27-fold brighter after reacting with HaloTag, and is used in the no-wash imaging of subcellular structures in E. coli after only five minutes of labeling. This work has been adapted in part with permission from: Clark, S. A.; Singh, V.; Vega Mendoza, D.; Margolin, W.; Kool, E. T. Bioconjug. Chem. 2016, 27 (12), 2839. Copyright 2016 American Chemical Society. Chapter 3 details our strategy for incorporating stable dialdehyde precursors into fluorescent labels for hydrazone and oxime formation. The condensation between alpha nucleophiles and carbonyl compounds is one of the classic bioconjugation reactions. The small size of the linkage and simplicity of the reactive substrates are both attractive features; however, the rates of these reactions are typically much slower than more recently developed bioorthogonal reactions. Recent studies have reported that dialdehyde-containing substrates can react with aminooxy compounds with rates competitive with the fastest bioorthogonal reactions, but strategies for introducing this functionality into a variety of probes is currently lacking. Additionally, aldehydes are often subject to air oxidation, complicating storage of such reagents. We demonstrate that pyrrolidinediol can transform NHS-esters and isothiocyanates into stable cyclic diols. These diols are readily oxidized to dialdehydes under mild conditions in situ and subsequently rapidly react with a range of alpha nucleophiles at physiological pH, including with a DNA-oxyamine oligomer.