Probing metalloenzyme dynamics in living systems: Contemporary advances in fluorescence imaging tools and applications.

Metalloenzymes are essential to cellular function, and their overexpression or enhanced activation are potential therapeutic targets. However, the study of metalloenzymes in vitro presents various challenges, leading many to develop tools to study them in their native cellular environment. Small-molecule fluorescence probes are commonly used to monitor metalloenzyme function, activity, and distribution in situ. These include probes that are activity-based (fluorescence is mediated by enzyme activity) or binding-based (fluorescence is mediated by interactions with the enzyme upon binding its metal cofactor). We discuss recent innovations that overcome key design challenges, such as the rapid diffusion of activity-based probes, the difficulty of probing redox-active enzymes, the selectivity of binding-based probes, and the poor penetration depth of fluorescence, and describe novel applications of these tools.

Dual Nanoparticle Conjugates for Highly Sensitive and Versatile Sensing Using 19 F Magnetic Resonance Imaging.

Fluorine magnetic resonance imaging (19 F MRI) has emerged as an attractive alternative to conventional 1 H MRI due to enhanced specificity deriving from negligible background signal in this modality. We report a dual nanoparticle conjugate (DNC) platform as an aptamer-based sensor for use in 19 F MRI. DNC consists of core-shell nanoparticles with a liquid perfluorocarbon core and a mesoporous silica shell (19 F-MSNs), which give a robust 19 F MR signal, and superparamagnetic iron oxide nanoparticles (SPIONs) as magnetic quenchers. Due to the strong magnetic quenching effects of SPIONs, this platform is uniquely sensitive and functions with a low concentration of SPIONs (4 equivalents) relative to 19 F-MSNs. The probe functions as a "turn-on" sensor using target-induced dissociation of DNA aptamers. The thrombin binding aptamer was incorporated as a proof-of-concept (DNCThr ), and we demonstrate a significant increase in 19 F MR signal intensity when DNCThr is incubated with human α-thrombin. This proof-of-concept probe is highly versatile and can be adapted to sense ATP and kanamycin as well. Importantly, DNCThr generates a robust 19 F MRI "hot-spot" signal in response to thrombin in live mice, establishing this platform as a practical, versatile, and biologically relevant molecular imaging probe.

An Fe complex for 19F magnetic resonance-based reversible redox sensing and multicolor imaging.

We report a first-in-class responsive, pentafluorosulfanyl (-SF5)-tagged 19F MRI agent capable of reversibly detecting reducing environments via an FeII/III redox couple. In the FeIII form, the agent displays no 19F MR signal due to paramagnetic relaxation enhancement-induced signal broadening; however, upon rapid reduction to FeII with one equivalent of cysteine, the agent displays a robust 19F signal. Successive oxidation and reduction studies validate the reversibility of the agent. The -SF5 tag in this agent enables 'multicolor imaging' in conjunction with sensors containing alternative fluorinated tags and this was demonstrated via simultaneous monitoring of the 19F MR signal of this -SF5 agent and a hypoxia-responsive agent containing a -CF3 group.

Structural insights into the design of reversible fluorescent probes for metallo-ß-lactamases NDM-1, VIM-2, and IMP-1.

Metallo-ß-lactamases (MBLs) are enzymes that are capable of hydrolyzing most ß-lactam antibiotics and all clinically relevant carbapenems. We developed a library of reversible fluorescent turn-on probes that are designed to directly bind to the dizinc active site of these enzymes and can be used to study their dynamic metalation state and enzyme-inhibitor interactions. Structure-function relationships with regards to inhibitory strength and fluorescence turn-on response were evaluated for three representative MBLs.

Visualizing the Dynamic Metalation State of New Delhi Metallo-ß-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe.

New Delhi metallo-ß-lactamase (NDM) grants resistance to a broad spectrum of ß-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metalation state of NDM within Escherichia coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.

Copper(II) Pyridyl Aminophenolates: Hypoxia-Selective, Nucleus-Targeting Cytotoxins, and Magnetic Resonance Probes.

Targeting the low-oxygen (hypoxic) environments found in many tumours by using redox-active metal complexes is a strategy that can enhance efficacy and reduce the side effects of chemotherapies. We have developed a series of CuII complexes with tridentate pyridine aminophenolate-based ligands for preferential activation in the reduction window provided by hypoxic tissues. Furthermore, ligand functionalization with a pendant CF3 group provides a 19 F spectroscopic handle for magnetic-resonance studies of redox processes at the metal centre and behaviour in cellular environments. The phenol group in the ligand backbone was substituted at the para position with H, Cl, and NO2 to modulate the reduction potential of the CuII centre, giving a range of values below the window expected for hypoxic tissues. The NO2 -substituted complex, which has the highest reduction potential, showed enhanced cytotoxic selectivity towards HeLa cells grown under hypoxic conditions. Cell death occurs by apoptosis, as determined by analysis of the cell morphology. A combination of 19 F NMR and ICP-OES indicates localization of the NO2 complex in HeLa cell nuclei and increased cellular accumulation under hypoxia. This correlates with DNA nuclease activity being the likely origin of cytotoxic activity, as demonstrated by cleavage of DNA plasmids in the presence of the CuII nitro complex and a reducing agent. Selective detection of the paramagnetic CuII complexes and their diamagnetic ligands by 19 F MRI suggests hypoxia-targeting theranostic applications by redox activation.

Versatile Nickel(II) Scaffolds as Coordination-Induced Spin-State Switches for 19 F Magnetic Resonance-Based Detection.

19 F magnetic resonance (MR) based detection coupled with well-designed inorganic systems shows promise in biological investigations. Two proof-of-concept inorganic probes that exploit a novel mechanism for 19 F MR sensing based on converting from low-spin (S=0) to high-spin (S=1) Ni2+ are reported. Activation of diamagnetic NiL1 and NiL2 by light or ß-galactosidase, respectively, converts them into paramagnetic NiL0 , which displays a single 19 F NMR peak shifted by >35 ppm with accelerated relaxation rates. This spin-state switch is effective for sensing light or enzyme expression in live cells using 19 F MR spectroscopy and imaging that differentiate signals based on chemical shift and relaxation times. This general inorganic scaffold has potential for developing agents that can sense analytes ranging from ions to enzymes, opening up diverse possibilities for 19 F MR based biosensing.

Responsive fluorinated nanoemulsions for 19F magnetic resonance detection of cellular hypoxia.

We report two highly fluorinated Cu-based imaging agents, CuL1 and CuL2, for detecting cellular hypoxia as nanoemulsion formulations. Both complexes retained their initial quenched 19F MR signals due to paramagnetic Cu2+; however, both complexes displayed a large signal increase when the complex was reduced. DLS studies showed that the CuL1 nanoemulsion (NECuL1) had a hydrodiameter of approximately 100 nm and that it was stable for four weeks post-preparation. Hypoxic cells incubated with NECuL1 showed that 40% of the Cu2+ taken up was reduced in low oxygen environments.

Visible Light Mediated Bidirectional Control over Carbonic Anhydrase Activity in Cells and in Vivo Using Azobenzenesulfonamides.

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation. Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.

Harnessing chemical exchange: 19F magnetic resonance OFF/ON zinc sensing with a Tm(iii) complex.

A fluorinated, thulium(iii) complex (Tm-PFZ-1) serves as an off-on 19F magnetic resonance probe for Zn(ii). Rapid exchange among different conformations combined with paramagnetic relaxation and chemical shift effects of Tm(iii) effectively eliminate the 19F NMR/MRI signal in Tm-PFZ-1. Chelation of Zn(ii) induces increased structural rigidity and reduces exchange rate, affording a robust 19F NMR/MRI signal. Tm-PFZ-1 represents a first-in-class paramagnetic 19F MR agent that exploits a novel sensing mechanism for Zn(ii) and is the first 19F MR-based scaffold to provide an "off-on" response to Zn(ii) in aqueous solution.

Reversible Solid-State Isomerism of Azobenzene-Loaded Large-Pore Isoreticular Mg-CUK-1.

A large-pore version of Mg-CUK-1, a water-stable metal-organic framework (MOF) with 1-D channels, was synthesized in basic water. Mg-CUK-1L has a BET surface area of 2896 m2 g-1 and shows stark selectivity for CO2 sorption over N2, O2, H2, and CH4. It displays reversible, multistep gated sorption of CO2 below 0.33 atm. The dehydrated single-crystal structure of Mg-CUK-1L confirms retention of the open-channel structure. The MOF can be loaded with organic molecules by immersion in hot melts, providing single crystals suitable for X-ray diffraction. trans-Azobenzene fills the channels in a 2 × 2 arrangement. Solid-state UV-vis spectroscopy reveals that azobenzene molecules undergo reversible trans-cis isomerization, despite being close-packed; this surprising result is confirmed by DFT-simulated UV-vis spectra.

19F Magnetic Resonance Activity-Based Sensing Using Paramagnetic Metals.

Fluorine magnetic resonance imaging (19F MRI) is a promising bioimaging technique due to the favorable magnetic resonance properties of the 19F nucleus and the lack of detectable biological background signal. A range of imaging agents have been developed for this imaging modality including small molecule perfluorocarbons, fluorine-rich macromolecules and nanoparticles, and paramagnetic metal-containing agents. Incorporation of paramagnetic metals into fluorinated agents provides a unique opportunity to manipulate relaxation and chemical shift properties of 19F nuclei. Paramagnetic centers will enhance relaxation rates of nearby 19F nuclei through paramagnetic relaxation enhancement (PRE). Further, metals with anisotropic unpaired electrons can induce changes in 19F chemical shift through pseudocontact shift (PCS) effects. PRE and PCS are dependent on the nature of the metal center itself, the molecular scaffold surrounding it, and the position of the 19F nucleus relative to the metal center. One intriguing prospect in 19F magnetic resonance molecular imaging is to design responsive agents that can serve to provide a read out biological activity, including the activity of enzymes, redox activity, the activity of ions, etc. Paramagnetic agents are well suited for this activity-based sensing as metal complexes can be designed to respond to specific biological activities and give a corresponding 19F response that results from changes in the metal complex structure and subsequently PRE/PCS. Broadly speaking, when designing paramagnetic 19F MR biosensors, one can envision that in response to changes in analyte activity, the number of unpaired electrons of the metal changes or the ligand conformation/chemical composition changes. This Account highlights activity-based probes from the Que lab that harness paramagnetic metals to modulate 19F signal. We discuss probes that use conversion from Cu2+ to Cu+ in response to reducing environments to dequench the 19F MR signal. Probes in which oxidants convert Co2+ to Co3+, resulting in chemical shift responses, are also described. Finally, we explore our foray into using Ni2+ coordination switching to furnish probes with different 19F signals when they are converted between 4-coordinate square planar and higher coordination numbers. A major barrier for 19F MR molecular imaging is in vivo application, as signal sensitivity is relatively low, requiring long imaging times to detect imaging agents. Nanoparticle and macromolecular agents show promise due to their higher fluorine density and longer circulation times; however, their analyte scope is limited to analytes that induce cleavage events. A grand challenge for researchers in this area is adapting lessons learned from small molecule paramagnetic probes with promising in vitro activities for the development of probes with enhanced in vivo utility for basic biological and clinical applications.

Interrogating Intracellular Zinc Chemistry with a Long Stokes Shift Zinc Probe ZincBY-4.

Previous work has shown that fluctuations in zinc content and subcellular localization play key roles in regulating cell cycle progression; however, a deep mechanistic understanding requires the determination of when, where, and how labile zinc pools are concentrated into or released from stores. Labile zinc ions can be difficult to detect with probes that require hydrolysis of toxic protecting groups or application at high concentrations that negatively impact cell function. We previously reported a BODIPY-based zinc probe, ZincBY-1, that can be used at working concentrations that are 20-200-fold lower than concentrations employed with other probes. To better understand how zinc pools can be visualized at such low probe concentrations, we modulated the photophysical properties via changes at the 5-position of the BODIPY core. One of these, ZincBY-4, exhibits an order of magnitude higher affinity for zinc, an 8-fold increase in brightness in response to zinc, and a 100 nm Stokes shift within cells. The larger Stokes shift of ZincBY-4 presents a unique opportunity for simultaneous imaging with GFP or fluorescein sensors upon single excitation. Finally, by creating a proxy for the cellular environment in spectrometer experiments, we show that the ZincBY series are highly effective at 50 nM because they can pass membranes and accumulate in regions of high zinc concentration within a cell. These features of the ZincBY probe class have widespread applications in imaging and for understanding the regulatory roles of zinc fluxes in live cells.

Highly fluorinated metal complexes as dual 19F and PARACEST imaging agents.

We reported a set of water-soluble transition metal complexes that can serve as both 19F and PARACEST magnetic resonance imaging agents. The high number of equivalent fluorine atoms and the paramagnetic effect of metals offer these complexes high 19F sensitivity as demonstrated by in vitro19F MRI experiments. The complexes contain carboxamide groups appended onto a cyclen macrocycle, which provide 1H CEST peaks well differentiated from bulk water. The Co(ii) agent displays two CEST peaks that can be utilized for ratiometric pH determination and the concept of combining 19F MR and PARACEST as complementary imaging techniques was demonstrated with the Fe(ii) complex.

A dual-responsive probe for detecting cellular hypoxia using 19F magnetic resonance and fluorescence.

We report the first dual-responsive 19F MRI and fluorescence imaging probe for cellular hypoxia. The Cu2+-based probe exhibits no 19F MR signal and reduced fluorescence signal due to paramagnetic quenching; however, the probe turns-on in both modes following reduction to Cu+. This bimodal agent can differentiate hypoxic and normoxic cells in both modalities.

Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation.

Upon fertilization or parthenogenesis, zinc is released into the extracellular space through a series of exocytic events termed zinc sparks, which are tightly coordinated with intracellular calcium transients. The zinc spark reduces the total amount of intracellular zinc, and this reduction is necessary and sufficient to induce egg activation even in the absence of calcium transients. In addition, this zinc release contributes to the block to polyspermy through modification of the zona pellucida. The zinc spark has been documented in all organisms examined to date including the mouse, two species of nonhuman primates, and human. Here we determined whether zinc sparks occur in the bovine, an important model of gamete development in mono-ovulatory mammalian species. We obtained metaphase II-arrested (MII) bovine eggs following in vitro maturation. Total zinc, assessed in single cells using X-Ray Fluorescence Microscopy, was significantly more abundant in the bovine egg compared to iron and copper. Studies with intracellular fluorescent probes revealed that labile zinc pools are localized to discrete cytoplasmic punctae enriched at the cortex. To determine whether zinc undergoes dynamic fluxes during egg activation, we parthenogenetically activated bovine eggs using two approaches: ionomycin or bovine phospholipase C zeta (bPlcζ). Both these methods induced zinc sparks coordinately with intracellular calcium transients. The zinc spark was also observed in bovine eggs following intracytoplasmic sperm injection. These results establish that zinc is the most abundant transition metal in the bovine egg, and zinc flux during egg activation - induced by chemical activation or sperm - is a highly conserved event across mammalian species.

Towards Ni(II) complexes with spin switches for 19F MR-based pH sensing.

OBJECTIVES: Our aim was to demonstrate the potential of exploiting simultaneous changes in coordination geometry and spin state in fluorinated Ni(II) complexes as an avenue for 19F magnetic-resonance (MR)-based pH sensing. MATERIALS AND METHODS: Crystal structures were studied using an Agilent Technologies SuperNova Dual Source diffractometer. Solution magnetic moment was determined using Evan's method. MR images were collected on a 7.0-T MR scanner equipped with a quadrature 19F volume coil. RESULTS: NiL1 and NiL2 were synthesized; crystallographic and spectroscopic data supported NiL1 as being diamagnetic and NiL2 as being paramagnetic. In aqueous solution, ligand dissociation from Ni(II) center was observed for both complexes at around pH 6, precluding their use as reversible pH sensors. The two complexes have distinct 19F nuclear magnetic resonance (NMR) signals in terms of both chemical shift and relaxation times, and selective imaging of the two complexes was achieved with no signal interference using two 19F MRI pulse sequences. CONCLUSION: The significant difference in the chemical shift and relaxation times between NiL1 and NiL2 allowed selective imaging of these species using 19F MRI. While NiL1 and NiL2 were not stable to acidic environments, this report lays the framework for development of improved ligand scaffolds that stably coordinate Ni(II) in acidic aqueous solution and act as agents for ratiometric pH mapping by 19F MRI.

Pull-Down of Metalloproteins in Their Native States by Using Desthiobiotin-Based Probes.

One-third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active-site metals under mild conditions so as to prevent changes in metallation state. The proof-of-concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn2+ -containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull-down of differently metallated CAs was also investigated.

In Situ Photoregulation of Carbonic Anhydrase Activity Using Azobenzenesulfonamides.

We report two small molecule azobenzenesulfonamide probes, CAP1 and CAP2, capable of photomodulating the activity of carbonic anhydrase (CA) on demand. In the trans form, CAP azobenzene probes adopt a linear shape, making them suitable for occupying the CA active site and interacting with Zn2+, thereby inhibiting enzyme activity. Following irradiation with either 365 or 410 nm light, the CAP probes isomerize to their cis form. Because of the change in steric profile, the probe exits the active site, and the activity of the enzyme is restored. The cis isomer can revert back to the trans isomer through thermal relaxation or via photoirradiation with 460 nm light and thereby inhibit protein activity again. This process can be repeated multiple times without any photodegradation and thus can be used to inhibit or activate the protein reversibly. Importantly, we demonstrate our ability to apply CAP azobenzene probes to regulate CA activity both in an isolated protein solution and in live cells, where the two isomers of CAP1 differentially regulate the intracellular cytosolic pH.