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.

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

Fluorine magnetic resonance imaging ( F MRI) has emerged as an attractive alternative to conventional 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 F MRI. DNC consists of core-shell nanoparticles with a liquid perfluorocarbon core and a mesoporous silica shell ( F-MSNs), which give a robust F MR signal, and superparamagnetic iron oxide nanoparticles (SPIONs) as magnetic quenchers.

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

We report a first-in-class responsive, pentafluorosulfanyl (-SF)-tagged F MRI agent capable of reversibly detecting reducing environments an Fe redox couple. In the Fe form, the agent displays no F MR signal due to paramagnetic relaxation enhancement-induced signal broadening; however, upon rapid reduction to Fe with one equivalent of cysteine, the agent displays a robust F signal. Successive oxidation and reduction studies validate the reversibility of the agent.

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

Metallo-β-lactamases (MBLs) are enzymes that are capable of hydrolyzing most β-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-β-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe.

New Delhi metallo-β-lactamase (NDM) grants resistance to a broad spectrum of β-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.

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 Cu complexes with tridentate pyridine aminophenolate-based ligands for preferential activation in the reduction window provided by hypoxic tissues. Furthermore, ligand functionalization with a pendant CF group provides a F spectroscopic handle for magnetic-resonance studies of redox processes at the metal centre and behaviour in cellular environments.

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

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 F MR sensing based on converting from low-spin (S=0) to high-spin (S=1) Ni are reported. Activation of diamagnetic NiL and NiL by light or β-galactosidase, respectively, converts them into paramagnetic NiL , which displays a single F NMR peak shifted by >35 ppm with accelerated relaxation rates.

Responsive fluorinated nanoemulsions for F 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.

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

Two azobenzenesulfonamide molecules with thermally stable configurations resulting from fluorination of positions to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the and configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the 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.

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

A fluorinated, thulium(iii) complex (Tm-PFZ-1) serves as an off-on F 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 F NMR/MRI signal in Tm-PFZ-1. Chelation of Zn(ii) induces increased structural rigidity and reduces exchange rate, affording a robust F NMR/MRI signal.

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 m g and shows stark selectivity for CO sorption over N, O, H, and CH. It displays reversible, multistep gated sorption of CO below 0.

F Magnetic Resonance Activity-Based Sensing Using Paramagnetic Metals.

Fluorine magnetic resonance imaging (F MRI) is a promising bioimaging technique due to the favorable magnetic resonance properties of the F 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 F nuclei.

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.

A dual-responsive probe for detecting cellular hypoxia using F 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.

Highly fluorinated metal complexes as dual F 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.

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, Zn -containing protein.

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 Zn, thereby inhibiting enzyme activity. Following irradiation with either 365 or 410 nm light, the CAP probes isomerize to their cis form.

Copper(ii) complexes for cysteine detection using F magnetic resonance.

Cysteine plays an essential role in maintaining cellular redox homeostasis and perturbations in cysteine concentration are associated with cardiovascular disease, liver disease, and cancer. 19F MRI is a promising modality for detecting cysteine in biology due to its high tissue penetration and negligible biological background signal. Herein we report fluorinated macrocyclic copper complexes that display a 19F NMR/MRI turn-on response following reduction of the Cu(ii) complexes by cysteine.

Towards Ni(II) complexes with spin switches for F 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 F 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.

F PARASHIFT Probes for Magnetic Resonance Detection of HO and Peroxidase Activity.

Elevated levels of reactive oxygen species and peroxidase expression are often associated with inflammation and inflammatory diseases. We developed two novel Co(II) complexes that can be used to detect oxidative activity associated with inflammation using F magnetic resonance imaging (MRI). These agents display a large change in F chemical shift upon oxidation from Co(II) to Co(III), facilitating selective visualization of both species using chemical shift selective pulse sequences.

A new probe for detecting zinc-bound carbonic anhydrase in cell lysates and cells.

We report the synthesis and application of a small molecule probe for carbonic anhydrase (CA) to track holo-CA in cell lysates and live-cell models of zinc dyshomeostasis. The probe displays a 12-fold increase in fluorescence upon binding to bovine CA and also responds to human CA isoforms.

Hypoxia-Responsive F MRI Probes with Improved Redox Properties and Biocompatibility.

F magnetic resonance imaging (MRI), an emerging modality in biomedical imaging, has shown promise for in vitro and in vivo preclinical studies. Here we present a series of fluorinated Cu(II)ATSM derivatives for potential use as F magnetic resonance agents for sensing cellular hypoxia. The synthesized complexes feature a hypoxia-targeting Cu coordination core, nine equivalent fluorine atoms connected via a variable-length poly(ethylene glycol) linker.

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy.

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as 'zinc sparks.' The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as 'hardening', which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy.