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.

Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging.

Fluorogenic probes are invaluable tools for spatiotemporal investigations within live cells. In common fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophore is masked by esterification until entry into a cell, where endogenous esterases catalyze the hydrolysis of the masking groups, generating fluorescence. The susceptibility of masking groups to spontaneous hydrolysis is a major limitation of these probes. Previous attempts to address this problem have incorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. Here, we report on a linker-free strategy that employs adventitious electronic and steric interactions in easy-to-synthesize probes. We find that X···C═O n→π* interactions and acyl group size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stable to spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo, yielding a bright, photostable fluorophore with utility in biomolecular imaging.

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

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.

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.

Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues.

Chelatable, or mobile, forms of zinc play critical signaling roles in numerous biological processes. Elucidating the action of mobile Zn(II) in complex biological environments requires sensitive tools for visualizing, tracking, and manipulating Zn(II) ions. A large toolbox of synthetic photoinduced electron transfer (PET)-based fluorescent Zn(II) sensors are available, but the applicability of many of these probes is limited by poor zinc sensitivity and low dynamic ranges owing to proton interference. We present here a general approach for acetylating PET-based probes containing a variety of fluorophores and zinc-binding units. The new sensors provide substantially improved zinc sensitivity and allow for incubation of live cells and tissue slices with nM probe concentrations, a significant improvement compared to the µM concentrations that are typically required for a measurable fluorescence signal. Acetylation effectively reduces or completely quenches background fluorescence in the metal-free sensor. Binding of Zn(II) selectively and quickly mediates hydrolytic cleavage of the acetyl groups, providing a large fluorescence response. An acetylated blue coumarin-based sensor was used to carry out detailed analyses of metal binding and metal-promoted acetyl hydrolysis. Acetylated benzoresorufin-based red-emitting probes with different zinc-binding sites are effective for sensing Zn(II) ions in live cells when applied at low concentrations (∼50-100 nM). We used green diacetylated Zinpyr1 (DA-ZP1) to image endogenous mobile Zn(II) in the molecular layer of mouse dorsal cochlear nucleus (DCN), confirming that acetylation is a suitable approach for preparing sensors that are highly specific and sensitive to mobile zinc in biological systems.

Peptide Targeting of Fluorescein-Based Sensors to Discrete Intracellular Locales.

Fluorescein-based sensors are the most widely applied class of zinc probes but display adventitious localization in live cells. We present here a peptide-based localization strategy that affords precision in targeting of fluorescein-based zinc sensors. By appending the zinc-selective, reaction-based probe Zinpyr-1 diacetate (DA-ZP1) to the N-terminus of two different targeting peptides we achieve programmable localization and avoid unwanted sequestration within acidic vesicles. Furthermore, this approach can be generalized to other fluorescein-based sensors. When appended to a mitochondrial targeting peptide, the esterase-activated profluorophore 2',7'-dichlorofluorescein diacetate can be used effectively at concentrations four-times lower than previously reported for analogous, non-acetylated derivatives. These results demonstrate on-resin or in-solution esterification of fluorescein to be an effective strategy to facilitate peptide-based targeting in live cells.

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.