Over the past 70 years fluorescence has evolved into an indispensable tool for examining biological phenomena. Today fluorescent molecules are used extensively as labels for biomolecules, substrates for enzymes, indicators for ions, and stains for subcellular domains. Small organic molecules constitute an important subclass of helpful fluorophores, and countless small-molecule probes are available either commercially or through de novo design and synthesis. Attachment of different reactive groups, substrate moieties, chelating components, and other chemical functionalities to a fairly small set of dye scaffolds gives rise to the large collection of fluorescent probes now available. A panel of 30 common fluorescent dyes spanning the ultraviolet, visible, and near-infrared spectra regions are shown in the figure (available soon).
Our laboratory uses organic chemistry in the design and synthesis of new fluorescent probes from these foundational fluorescent molecules. In particular, we are interested in developing probes in which the chemical and photophysical properties can be masked by assorted molecular functionalities and then unmasked by a user-designated process such as light, enzymatic activity, or other environmental changes. This chemical masking can suppress unwanted fluorescence signals in various applications, thereby functioning as a filter for bioimaging and other experiments. By combining this strategy with advances in instrumentation, protein engineering, and genetic techniques, we hope to devise sophisticated ways to illuminate biological systems.
Photoactivation of Fluorophores
Light can provide exquisite spatiotemporal control over chemical structure. Photochemical uncaging of small-molecule agonists, fluorescent dyes, and fluorescent proteins can be used in sophisticated experiments to deliver biologically active molecules or to facilitate complex imaging. An exciting technology that uses photoactivatable fluorophores (PAFs) is photoactivated localization microscopy (PALM), developed by Eric Betzig (HHMI, JFRC) and Harold Hess (HHMI, JFRC). This technique involves iterative activation and measurement of caged fluorophores within a sample, allowing construction of an ultrahigh-resolution image. The current palette of protein-based PAFs facilitates two-color imaging; we seek to complement the proteinous caged probes with small-molecule fluorophores, thereby increasing the range of spectral properties accessible with this technique.
We are also exploring strategies for the attachment of small-molecule dyes to biomolecules in a specific fashion. Traditional strategies use biological nucleophiles, but recent advances allow labeling of small molecules inside living cells through genetic incorporation of proteinic reactive tags that react in an orthogonal fashion with small-molecule ligands. In collaboration with Loren Looger (HHMI, JFRC) and colleagues in the industrial sector, we hope to use and expand the chemistries involved in these ligation strategies.
Enzymatic Activation of Fluorophores
In addition to light, enzymatic activity can be used to manage fluorophore properties. For example, endogenous enzymes facilitate the delivery of various fluorescent molecules to biological systems. In a common example, polar moieties on the dye are cloaked as simple esters to allow efficient passage of the molecule through lipid bilayers. Once inside the cell, nonspecific esterases catalyze the hydrolysis of the masking ester bonds, releasing the fluorescent molecule. This method can be used to deliver simple fluorophores or fluorescent ion indicators to live cells. In previous work, sponsored by grants from the National Institutes of Health, we exploited esterase-mediated unmasking of fluorophores by developing fluorogenic labels to examine biomolecule trafficking.
Although this esterase strategy can work well for cultured cells, the use of fluorescent molecules in vivo presents a difficult problem. Together with Karel Svoda (HHMI, JFRC) and Loren Looger, we are pursuing techniques to develop further the masking techniques of small-molecule sensors. Improvements in the chemical stability of masking groups and ultimate subcellular localization of fluorescent indicators could advance the utility of these compounds in vivo. Another useful technique goes beyond the simple advancement of global cellular delivery of such molecules. Orthogonal enzyme-substrate pairs could be used to target indicators to genetically defined cellular populations and subcellular locales.
Expanding Dye Synthesis
As toolmakers, our overall goal is developing novel molecules to advance bioresearch. Along the way, however, we seek innovative paths to the synthesis of fluorescent dyes and their derivatives. Our projects take advantage of the ability of synthetic organic chemistry to tune small-molecule fluorophores to a specific function. Increasing the repertoire of chemical reactions that can construct and modify dye structures is therefore important to any effort involving the synthesis of new probes. Rather than be satisfied with the current portfolio of chemical transformations used in dye research (some reactions date back to the 19th century) we aim to explore new synthesis techniques and apply them to the creation of novel dye derivatives. This effort may lead to facile synthesis of fluorophore libraries, allowing the screening of dye structures to find compounds with nuanced properties for challenging biological experiments.
Our close interactions with both engineers and biologists at Janelia Farm Research Campus will provide a fertile environment for discovering new applications for organic chemistry. The unique power of the chemical discipline is the capacity to construct molecules of definite structure. As chemists at Janelia, we have the opportunity to ply our craft in concert with scientists from other disciplines to shed light on complex problems in neurobiology and beyond.
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