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Reversible detection of bio-reduction by 19F magnetic resonance using an FeIII complex with a unique –SF5 tag for multiplexed sensing.

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.

Extraction of metal ions into perfluorocarbon solvents using fluorinated ligands has shown promise in a number of applications ranging from catalysis to biomedical imaging. However, the speciation of the extracted metal complexes is poorly understood hindering the design and implementation of new fluorous extraction systems. The extraction of s-, p-, d-, and f-block metal ions from the aqueous phase to the fluorous phase (perfluoro-15-crown-5 ether) with a fluorinated acetylacetonate pro-ligand was investigated and the speciation of the extracted metal complexes characterized, including coordinatively saturated mononuclear and polynuclear species and coordinatively unsaturated mononuclear species. The leaching of these species back into aqueous media was studied along with the effects of synergistic extraction using a combination of fluorinated acac and nonfluorinated neutral ligands. Our findings indicate that formation of coordinatively saturated mononuclear metal complexes in the fluorous phase is essential for retaining extracted species and suggest an important role for synergistic extraction for metal ions that do not form these types of metal complexes with acac ligands alone.

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 β-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.

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.

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.

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.

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.

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.

Metal complexes containing ligands that can be cleaved using biological reductants are possible alternatives to traditional photocaged metal ions. This strategy has been demonstrated in this proof-of-concept study with a disulfide containing Fe3+ complex, FeL1Cl. The loss of tight Fe3+ complexation due to disulfide reduction is observed through a decrease in a ligand to metal charge transfer band in its UV–vis spectrum. The mechanism of this reaction was investigated using mass spectrometry and stopped flow kinetics. A coumarin fluorescence assay was used to determine the ability of this complex to catalyze Fenton chemistry and produce hydroxyl radical. Hydroxyl radical production by FeL1Cl was low but in the presence of the reductant glutathione, hydroxyl radical production is increased, suggesting that reduction of the disulfide bond by glutathione uncages the reactivity of the Fe center in this complex.

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., 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.

Three thermodynamically stable nano-sized complexes containing 42 Ln3+ ions in a single molecule were synthesized via a self-assembly process. The compounds have torus or wheel-like architectures and have the general formula Ln42L14(OAc)82(OH)30 (Ln = Eu, Tb, Gd, L = deprotonated ortho-vanillin, OAc = acetate). The Eu and Tb complexes represent the first reported visibly luminescent lanthanide nanotoroids. The single crystal structures of all three complexes and their photophysical properties are reported.

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.

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.

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.

A fluorinated, thulium(iii) complex (Tm-PFZ-1) serves as an off-on

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 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.

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 i...

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. 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. 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. 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.

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. The reactivity with cysteine was studied by monitoring the appearance of a robust diamagnetic 19F signal following addition of cysteine in conjunction with UV-vis and EPR spectroscopies. Importantly, complexes with –CH2CF3 tags display good water solubility. Studies with this complex in HeLa cells demonstrate the applicability of these probes to detect cysteine in complex biological environments.

19F 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 19F magnetic resonance agents for sensing cellular hypoxia. The synthesized complexes feature a hypoxia-targeting Cu2+ coordination core, nine equivalent fluorine atoms connected via a variable-length poly(ethylene glycol) linker. Introduction of the fluorine moiety maintains the planar coordination geometry of the Cu2+ center, while the linker length modulates the Cu2+/+ reduction potential, 19F NMR relaxation properties, and lipophilicity. In particular, the 19F NMR relaxation properties were quantitatively evaluated by the Solomon–Bloembergen model, revealing a regular pattern of relaxation enhancement tuned by the distance between Cu2+ and F atoms. Finally, the potential utility of these complexes for sensing reductive environments was demonstrated using both 19F MR phantom i...

Previous work has shown that fluctuations in zinc content and subcellular localization plays 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.

We report the first dual-responsive (19)F MRI and fluorescence imaging probe for cellular hypoxia. The Cu(2+)-based probe exhibits no (19)F 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.

We reported a set of water-soluble transition metal complexes that can serve as both (19)F and PARACEST magnetic resonance imaging agents. The high number of equivalent fluorine atoms and the paramagnetic effect of metals offer these complexes high (19)F sensitivity as demonstrated by in vitro (19)F MRI experiments. The complexes contain carboxyamide groups appended onto a cyclen macrocycle, which provide (1)H 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 (19)F MR and PARACEST as complementary imaging techniques was demontrated with the Fe(II) complex.

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 forCrystal 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 quadratureNiLThe significant difference in the chemical shift and relaxation times between NiL

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.

We report a pair of fluorinated, redox-active copper complexes for potential use as (19)F MRI contrast agents for detecting cellular hypoxia. Trifluorinated Cu(II) ATSM-F3 displays the appropriate redox potential for selective accumulation in hypoxic cells and a completely quenched (19)F NMR signal that is "turned on" following reduction to Cu(I). Incubation of cancer cells with CuATSM-F3 resulted in a selective detection of (19)F signal in cells grown under hypoxic conditions.

Two series of Cd-Ln and Ni-Ln clusters [Ln8Cd24L12(OAc)44(48)Cl4(0)] and [Ln8Ni6L6(OAc)24(EtOH)6(H2O)2] were constructed using a flexible ligand. The Cd-Ln clusters exhibit interesting nano-drum-like structures which allows direct visualization by TEM. Luminex MicroPlex Microspheres loaded with the Cd-Sm cluster were visualized using epifluorescence microscopy. Cytotoxicity studies on A549 and AGS cancer cell lines showed that the materials have mild to moderate cytotoxicity.

A fluorinated, air-stable cobalt(II) complex serves as a turn-on 19F magnetic resonance imaging (MRI) tracer for reactive oxygen species including H2O2. Upon oxidation with H2O2, the complex converts from paramagnetic high spin CoII to diamagnetic low spin CoIII resulting in a chemical shift change and enhancement in 19F NMR signal. Further, the oxidation can be reversed in the presence of reductant Na2S2O4. The turn-on response is demonstrated by 19F MRI, characterized by a ∼2–3 fold enhancement in signal.

Background: Zinc is the most abundant transition metal in the mammalian oocyte, and dynamic fluxes in intracellular concentration are essential for regulating both meiotic progression and fertilization. Whether the defined pathways of zinc utilization in female meiosis directly translate to mitotic cells, including the mammalian preimplantation embryo, has not been studied previously. Results: We determined that zinc is the most abundant transition metal in the preimplantation embryo, with levels an order of magnitude higher than those of iron or copper. Using a zinc-specific fluorescent probe, we demonstrated that labile zinc is distributed in vesicle-like structures in the cortex of cells at all stages of preimplantation embryo development. To test the importance of zinc during this period, we induced zinc insufficiency using the heavy metal chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). Incubation of embryos in media containing TPEN resulted in a developmental arrest that was specific to zinc chelation and associated with compromised mitotic parameters. The developmental arrest due to zinc insufficiency was associated with altered chromatin structure in the blastomere nuclei and decreased global transcription. Conclusions: These results demonstrate for the first time that the preimplantation embryo requires tight zinc regulation and homeostasis for the initial mitotic divisions of life. Developmental Dynamics 244:935–947, 2015. © 2015 Wiley Periodicals, Inc.

Rapid cellular zinc influx regulates early mammalian development during the oocyte-to-egg transition through modulation of the meiotic cell cycle. Despite the physiological necessity of this zinc influx, the molecular mechanisms that govern such accumulation are unknown. Here we show that the fully grown mammalian oocyte does not employ a transcriptionally based mechanism of zinc regulation involving metal response element-binding transcription factor-1 (MTF-1), as demonstrated by a lack of MTF-1 responsiveness to environmental zinc manipulation. Instead, the mammalian oocyte controls zinc uptake through two maternally derived and cortically distributed zinc transporters, ZIP6 and ZIP10. Targeted disruption of these transporters using several approaches during meiotic maturation perturbs the intracellular zinc quota and results in a cell cycle arrest at a telophase I-like state. This arrest phenocopies established models of zinc insufficiency during the oocyte-to-egg transition, indicating the essential function of these maternally expressed transporters. Labile zinc localizes to punctate cytoplasmic structures in the human oocyte, and ZIP6 and ZIP10 are enriched in the cortex. Altogether, we demonstrate a mechanism of metal regulation required for female gamete development that may be evolutionarily conserved.

A fluorinated, air-stable cobalt(ii) complex serves as a turn-on

The brain is a singular organ of unique biological complexity that serves as the command center for cognitive and motor function. As such, this specialized system also possesses a unique chemical composition and reactivity at the molecular level. In this regard, two vital distinguishing features of the brain are its requirements for the highest concentrations of metal ions in the body and the highest per-weight consumption of body oxygen. In humans, the brain accounts for only 2% of total body mass but consumes 20% of the oxygen that is taken in through respiration. As a consequence of high oxygen demand and cell complexity, distinctly high metal levels pervade all regions of the brain and central nervous system. Structural roles for metal ions in the brain and the body include the stabilization of biomolecules in static (e.g., Mg2+ for nucleic acid folds, Zn2+ in zinc-finger transcription factors) or dynamic (e.g., Na+ and K+ in ion channels, Ca2+ in neuronal cell signaling) modes, and catalytic roles for brain metal ions are also numerous and often of special demand.

Upon activation, mammalian eggs release billions of zinc ions in an exocytotic event termed the “zinc spark.” The zinc spark is dependent on and occurs coordinately with intracellular calcium transients, which are tightly associated with embryonic development. Thus, we hypothesized that the zinc spark represents an early extracellular physicochemical marker of the developmental potential of the zygote. To test this hypothesis, we monitored zinc exocytosis in individual mouse eggs following parthenogenetic activation or in vitro fertilization (IVF) and tracked their development. Retrospective analysis of zinc spark profiles revealed that parthenotes and zygotes that developed into blastocysts released more zinc than those that failed to develop. Prospective selection of embryos based on their zinc spark profile significantly improved developmental outcomes and more than doubled the percentage of embryos that reached the blastocyst stage. Moreover, the zinc spark profile was also associated with embryo quality as the total cell number in the resulting morulae and blastocysts positively correlated with the zinc spark amplitude (R = 0.9209). Zinc sparks can thus serve as an early biomarker of zygote quality in mouse model.

A new synthetic clay, iC4-LMS, which is a lamellar mesostructured silicate with isobutyl groups cova- lently attached to silicate sheets, was synthesized with the goal to increase the compatibility of the inorganic sheets with polypropylene (PP) in a melt-blending process. The lamellar morphology of iC4-LMS was confirmed using X- ray diffraction and transmission electron microscopy. Based on 29 Si and 13 C{ 1 H} CP-MAS NMR spectra, isobutyl functional groups were attached to at least 10 mol % of sili- cate tetrahedral sites in the inorganic layers. These surface groups mimic the subunits in the PP chains. Samples of iC4-LMS were mixed with several organic solvents and sonicated. The solvent most like PP, tetramethylpentade- cane, had the highest viscosity, forming a gel which indi- cates very good dispersion of the clay. However, when iC4- LMS was melt-blended with PP, it did not show significant increase in rheology. This modest effect on rheology may arise from fracture of iC4-LMS layers as a result of the shear stresses during melt blending produced by the viscous PP. 2007 Wiley Periodicals, Inc. J Appl Polym Sci 105: 1456- 1465, 2007

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent “zinc spark.” The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.

Motivated by a need for synthetic clays that can be dispersed and exfoliated in polymer melts without added compatibilizers, lamellar mesostructured silicates and aluminosilicates with covalently attached hexadecyl functional groups (C 16 -LMS and C 16 -LMAS, respectively) were prepared by sol–gel syntheses and their structures were characterized. Based on XRD and TEM data, lamellar products with layer spacings of 4.8–4.9 nm were obtained between room temperature and 60 °C (C 16 -LMS) or 70 °C (C 16 -LMAS). The degree of condensation of the aluminosilicate layers increased at the higher synthesis temperatures. Attachment of organic groups to the inorganic sheets was confirmed by 29 Si solid state MAS NMR and IR spectroscopy. The sheets of C 16 -LMS consisted of single or double layers of tetrahedral silicate groups, each attached to a hexadecyl chain. C 16 -LMAS was composed of pyrophyllite-like layers (Si:Al=2) with an octahedral aluminum layer sandwiched between two tetrahedral silicate layers and hexadecyl surface groups. Tetrahedral aluminum sites were also present. The clay layer spacing could be increased to 5.2 nm by addition of tetraethoxysilanes during the synthesis (C 16 -SiO 2 -LMAS). C 16 -SiO 2 -LMAS was structurally similar to C 16 -LMAS; however, the presence of additional silicate groups in this structure increased the inorganic layer thickness and introduced further structural disorder.

Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.

Mammalian oogenesis is a discontinuous process that begins during fetal development, arrests at birth, and then resumes on a cyclical basis from puberty to menopause to produce fully mature oocytes that are competent for fertilization . Females are born with a fixed number of oocytes arrested at the prophase I stage of meiosis . This arrest is maintained until oocytes are selected to resume growth by gonadotropins, which are released from the pituitary upon entering puberty. At the time of ovulation, a fully grown oocyte completes maturation and arrests again at metaphase of meiosis II until fertilization occurs. Sperm binding triggers egg activation and release from metaphase II arrest. This entire process is tightly controlled, as it has a significant impact on egg quality and the developmental potential of the resulting embryo . The underlying mechanisms regulating oocyte meiotic entry, arrest, and exit—which can occur over a span of decades—have always been of great interest in the reproductive biology field, and many groundbreaking discoveries have revealed the existence of a dynamic network of hormones, receptors, kinases, and second messengers that control this process. Recent work has expanded this regulatory network to include the transition metal zinc , which adds a new level of complexity and fine-tuning to the regulation of the oocyte meiotic cell cycle . Dynamic accrual, sequestration, and exocytosis of zinc through controlled pathways are crucial for appropriate timing of the meiotic cell cycle as oocytes progress through maturation and activation. This work is important to our general understanding of oocytes and will have implications for reproductive interventions in the future.

X-ray fluorescence nanotomography provides unprecedented sensitivity for studies of trace metal distributions in whole biological cells. Dose fractionation, in which one acquires very low dose individual projections and then obtains high statistics reconstructions as signal from a voxel is brought together (Hegerl & Hoppe, 1976), requires accurate alignment of these individual projections so as to correct for rotation stage runout. It is shown here that differential phase contrast at 10.2 keV beam energy offers the potential for accurate cross-correlation alignment of successive projections, by demonstrating that successive low dose, 3 ms per pixel, images acquired at the same specimen position and rotation angle have a narrower and smoother cross-correlation function (1.5 pixels FWHM at 300 nm pixel size) than that obtained from zinc fluorescence images (25 pixels FWHM). The differential phase contrast alignment resolution is thus well below the 700 nm × 500 nm beam spot size used in this demonstration, so that dose fractionation should be possible for reduced-dose, more rapidly acquired, fluorescence nanotomography experiments.

This tutorial review highlights progress in the development of responsive magnetic resonance imaging (MRI) contrast agents for detecting and sensing biologically relevant metal ions. Molecular imaging with bioactivatable MRI indicators offers a potentially powerful methodology for studying the physiology and pathology of metals by capturing dynamic three-dimensional images of living systems for research and clinical applications. This emerging area at the interface of inorganic chemistry and the life sciences offers a broad palette of opportunities for researchers with interests ranging from coordination chemistry and spectroscopy to supramolecular chemistry and molecular recognition to metals in biology and medicine.

We present the synthesis and characterization of Copper-Gad-7 (CG7), a new copper-activated magnetic resonance imaging (MRI) contrast agent that possesses a Gd(3+)-DO3A scaffold with an appended thioether-rich receptor for copper recognition. Installation of additional carboxylate groups on the periphery of the CG scaffold affords a practical strategy to increase the absolute relaxivity of these types of copper-responsive MRI sensors as well as reduce their sensitivity to biologically abundant anions. Due in large part to restricted access of inner-sphere water molecules to the paramagnetic Gd(3+) core, in the absence of copper ions, CG7 exhibits a relatively low relaxivity value of r(1) = 2.6 mM(-1) s(-1); addition of Cu(+) triggers a 340% enhancement in relaxivity to r(1) = 11.4 mM(-1) s(-1). For comparison, the relaxivity of the analogous CG2 sensor without peripheral carboxylates increases from r(1) = 1.5 to 6.9 mM(-1) s(-1) upon Cu(+) binding. CG7 features high selectivity for Cu(+) over a range of biologically relevant metal ions, including the cellular abundant alkali and alkaline earth cations and d-block ions Zn(2+) and Cu(2+). Moreover, the Cu(+)-response of the CG7 sensor is not significantly affected by bicarbonate, phosphate, citrate, and lactate anions at cellular levels. (17)O NMR dysprosium-induced shift (DIS) and nuclear magnetic relaxation dispersion (NMRD) experiments suggest that the origin of the improved anion compatibility of CG7 is a reduced q modulation compared to previous members of the CG family, and T(1)-weighted phantom images confirm that CG7 can monitor changes in copper levels by MRI at clinically relevant field strengths.

This tutorial review highlights progress in the development of responsive magnetic resonance imaging (MRI) contrast agents for detecting and sensing biologically relevant metal ions. Molecular imaging with bioactivatable MRI indicators offers a potentially powerful methodology for studying the physiology and pathology of metals by capturing dynamic three-dimensional images of living systems for research and clinical applications. This emerging area at the interface of inorganic chemistry and the life sciences offers a broad palette of opportunities for researchers with interests ranging from coordination chemistry and spectroscopy to supramolecular chemistry and molecular recognition to metals in biology and medicine.

The design, synthesis, and evaluation of the Copper-Gad (CG) family, a new class of copper-activated magnetic resonance imaging (MRI) contrast agents, are presented. These indicators comprise a Gd(3+)-DO3A core coupled to various thioether-rich receptors for copper-induced relaxivity switching. In the absence of copper ions, inner-sphere water binding to the Gd(3+) chelate is restricted, resulting in low longitudinal relaxivity values (r(1) = 1.2-2.2 mM(-1) s(-1) measured at 60 MHz). Addition of Cu(+) to CG2, CG3, CG4, and CG5 and either Cu(+) or Cu(2+) to CG6 triggers marked enhancements in relaxivity (r(1) = 2.3-6.9 mM(-1) s(-1)). CG2 and CG3 exhibit the greatest turn-on responses, going from r(1) = 1.5 mM(-1) s(-1) in the absence of Cu(+) to r(1) = 6.9 mM(-1) s(-1) upon Cu(+) binding (a 360% increase). The CG sensors are highly selective for Cu(+) and/or Cu(2+) over competing metal ions at cellular concentrations, including Zn(2+) at 10-fold higher concentrations. (17)O NMR dysprosium-induced shift and nuclear magnetic relaxation dispersion measurements support a mechanism in which copper-induced changes in the coordination environment of the Gd(3+) core result in increases in q and r(1). T(1)-weighted phantom images establish that the CG sensors are capable of visualizing changes in copper levels by MRI at clinical field strengths.

The brain is a singular organ of unique biological complexity that serves as the command center for cognitive and motor function. As such, this specialized system also possesses a unique chemical composition and reactivity at the molecular level. In this regard, two vital distinguishing features of the brain are its requirements for the highest concentrations of metal ions in the body and the highest per-weight consumption of body oxygen. In humans, the brain accounts for only 2% of total body mass but consumes 20% of the oxygen that is taken in through respiration. As a consequence of high oxygen demand and cell complexity, distinctly high metal levels pervade all regions of the brain and central nervous system. Structural roles for metal ions in the brain and the body include the stabilization of biomolecules in static (e.g., Mg2+ for nucleic acid folds, Zn2+ in zinc-finger transcription factors) or dynamic (e.g., Na+ and K+ in ion channels, Ca2+ in neuronal cell signaling) modes, and catalytic roles for brain metal ions are also numerous and often of special demand.