Recognition- and Reactivity-Based Fluorescent Probes for Studying Transition Metal Signaling in Living Systems

Abstract

ConspectusMetals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed “recognition” and “reactivity”. Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent examples from our laboratory and collaborations in which applications of chemical probes reveal that labile copper contributes to various physiologies. The first example shows that copper is an endogenous regulator of neuronal activity, the second illustrates cellular prioritization of mitochondrial copper homeostasis, and the third identifies the “cuprosome” as a new copper storage compartment in Chlamydomonas reinhardtii green algae. Indeed, recognition- and reactivity-based fluorescent probes have helped to uncover new biological roles for labile transition metals, and the further development of fluorescent probes, including ones with varied Kd values and new reaction triggers and recognition receptors, will continue to reveal exciting and new biological roles for labile transition metals.

Exploiting 1,2,3-Triazolium Ionic Liquids for Synthesis of Tryptanthrin and Chemoselective Extraction of Copper(II) Ions and Histidine-Containing Peptides

“…Here, we chose copper(II) as our initial target ion so that its chemoselective extraction based on molecular recognition could be facilitated using an affinity ionic liquid 3 (AIL 3). The copper ion is abundant in human serum as well as the brain [34], and plays an important role as an endogenous regulator of neuronal activity in living systems [35,36] and is closely correlated with the pathogenesis of Alzheimer's disease [37]. Moreover, exposure to high copper concentrations can cause gastrointestinal disturbances, liver or kidney damage.…”

In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease

“…This same redox activity also poses a potential danger, requiring highly orchestrated regulation of copper pools to prevent oxidative stress and free radical damage events that are detrimental to health (12)(13)(14)(15)(16)(17)(18). Indeed, genetic disorders that disrupt copper homeostasis lead to severe and lethal conditions such as Menkes and Wilson's diseases (13,19,20), and imbalances in physiological copper levels and tissue miscompartmentalization arising from genetic and/or dietary factors are correlated with cancer, neurodegenerative diseases, and metabolic disorders such as obesity, diabetes, and nonalcoholic fatty liver disease (NAFLD) (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32).…”

“…This central importance of copper in biology motivates the development of technologies to enable monitoring of labile, loosely bound copper pools in living systems to disentangle contributions of global (whole animal) and/or local (tissue specific) copper dynamics in healthy and disease states (9,16,33). However, the vast majority of these tools have been limited to dissociated cell culture and related thin specimens (e.g., zebrafish), and current examples of in vivo copper imaging in mammalian models remain rare owing to the challenges of coupling a selective and sensitive response to copper with modalities that offer tissue penetration at appropriate depths (16,(34)(35)(36)(37)(38)(39).…”

“…However, the vast majority of these tools have been limited to dissociated cell culture and related thin specimens (e.g., zebrafish), and current examples of in vivo copper imaging in mammalian models remain rare owing to the challenges of coupling a selective and sensitive response to copper with modalities that offer tissue penetration at appropriate depths (16,(34)(35)(36)(37)(38)(39). Moreover, none of these technologies allow for in vivo copper imaging in a targeted cell-or tissue-specific manner.…”

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

“…In this regard, detection of iron with both metal and oxidation state specificity is of central importance, because while iron is stored primarily in the ferric oxidation state, a ferrous iron pool loosely bound to cellular ligands, defined as the labile iron pool (LIP), exists at the center of highly regulated networks that control iron acquisition, trafficking, and excretion. Indeed, as a weak binder on the Irving-Williams stability series (13), Fe 2+ provides a challenge for detection by traditional recognition-based approaches (14), and as such we (15)(16)(17) and others (18)(19)(20) have pursued activity-based sensing approaches to detect labile Fe 2+ stores in cells (21)(22)(23)(24)(25). These tools have already provided insights into iron biology, as illustrated by the direct identification of elevations in LIPs during ferroptosis (26,27), an emerging form of cell death, using the ratiometric iron indicator FIP-1 (15).…”