Wen Chyan

Reaction-based fluorescent sensor for investigating mobile Zn2+ in mitochondria of healthy versus cancerous prostate cells.

Significance Mobile zinc plays important roles in mammalian physiology. Understanding the action of mobile zinc requires tools to follow it within and between live cells. Zinc-selective fluorescent probes offer a facile means for detecting such mobile zinc, but small molecules constructed to perform this task typically have an unpredictable cellular distribution. Subtle changes in chemical structure can radically alter subcellular localization. To overcome this challenge, we installed a chemical unit to direct the sensor specifically to mitochondria and discovered that tumorigenic cells lose their ability to accumulate mobile zinc within these organelles. To carry out this work, we devised a reaction-based sensor that undergoes zinc-mediated chemistry, converting a nonfluorescent molecule into one that emits brightly and avoiding undesired sequestration in endo/lysosome. Chelatable, mobile forms of divalent zinc, Zn(II), play essential signaling roles in mammalian biology. A complex network of zinc import and transport proteins has evolved to control zinc concentration and distribution on a subcellular level. Understanding the action of mobile zinc requires tools that can detect changes in Zn(II) concentrations at discrete cellular locales. We present here a zinc-responsive, reaction-based, targetable probe based on the diacetyled form of Zinpyr-1. The compound, (6-amidoethyl)triphenylphosphonium Zinpyr-1 diacetate (DA-ZP1-TPP), is essentially nonfluorescent in the metal-free state; however, exposure to Zn(II) triggers metal-mediated hydrolysis of the acetyl groups to afford a large, rapid, and zinc-induced fluorescence response. DA-ZP1-TPP is insensitive to intracellular esterases over a 2-h period and is impervious to proton-induced turn-on. A TPP unit is appended for targeting mitochondria, as demonstrated by live cell fluorescence imaging studies. The practical utility of DA-ZP1-TPP is demonstrated by experiments revealing that, in contrast to healthy epithelial prostate cells, tumorigenic cells are unable to accumulate mobile zinc within their mitochondria.

Peptide-based Targeting of Fluorescent Zinc Sensors to the Plasma Membrane of Live Cells

Combining fluorescent zinc sensors with the facile syntheses and biological targeting capabilities of peptides, we created green- and blue-emitting probes that, (i) are readily prepared on the solid-phase, (ii) retain the photophysical and zinc-binding properties of the parent sensor, and (iii) can be directed to the extracellular side of plasma membranes in live cells for detection of mobile zinc.

Cytosolic Uptake of Large Monofunctionalized Dextrans

Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. Here, we report on a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos.

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging

Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chemical biology. Those probes that respond to enzymatic catalysis illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, we review recent advances in enzyme-activated fluorogenic probes for biological imaging. We organize our survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymatic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside therapeutic, diagnostic, and theranostic opportunities.