Photoswitchable luminescent lanthanide complexes controlled and interrogated by four orthogonal wavelengths of light.

Optical information storage requires careful control of excitation and emission wavelengths in a reversible and orthogonal manner to enable efficient reading, writing, and erasing of information. Photochromic systems, in which a photoswitch is typcially coupled to an emissive organic fluorophore, have much promise in this regard. However, these suffer from considerable spectral overlap between the switch and fluorophore, such that their emissive and photoswitchable properties are not orthogonal. Here, we overcome this limitation by coupling visible/NIR emissive lanthanide complexes with molecular photoswitches, enabling reversible and orthogonal photoswitching with visible light. Crucially, photoswitching does not lead to sensitised emission from the lanthanide, while excitation of the lanthanide does not induce photoswitching, enabling the state of the system to be probed without perturbation of the switch. This opens up the possibility of developing multi-colour read-write methods for information storage using emissive photoswitches.

Isolation of the elusive [Ru(bipy)3]+: a key intermediate in photoredox catalysis.

Photoredox catalysis has flourished in recent years, but due to its widespread utility applications have grown faster than mechanistic understanding. In this report we help to address this deficit by isolating and characterising one of the intermediates of the iconic photocatalyst [Ru(bipy)3]2+, and testing its initial photoreactivity towards common substrates.

Understanding catalytic synergy in dinuclear polymerization catalysts for sustainable polymers.

Understanding the chemistry underpinning intermetallic synergy and the discovery of generally applicable structure-performances relationships are major challenges in catalysis. Additionally, high-performance catalysts using earth-abundant, non-toxic and inexpensive elements must be prioritised. Here, a series of heterodinuclear catalysts of the form Co(III)M(I/II), where M(I/II) = Na(I), K(I), Ca(II), Sr(II), Ba(II) are evaluated for three different polymerizations, by assessment of rate constants, turn over frequencies, polymer selectivity and control. This allows for comparisons of performances both within and between catalysts containing Group I and II metals for CO2/propene oxide ring-opening copolymerization (ROCOP), propene oxide/phthalic anhydride ROCOP and lactide ring-opening polymerization (ROP). The data reveal new structure-performance correlations that apply across all the different polymerizations: catalysts featuring s-block metals of lower Lewis acidity show higher rates and selectivity. The epoxide/heterocumulene ROCOPs both show exponential activity increases (vs. Lewis acidity, measured by the pKa of [M(OH2)m]n+), whilst the lactide ROP activity and CO2/epoxide selectivity show linear increases. Such clear structure-activity/selectivity correlations are very unusual, yet are fully rationalised by the polymerization mechanisms and the chemistry of the catalytic intermediates. The general applicability across three different polymerizations is significant for future exploitation of catalytic synergy and provides a framework to improve other catalysts.

Heterometallic lanthanide complexes with site-specific binding that enable simultaneous visible and NIR-emission.

Macrocyclic lanthanide complexes have become widely developed due to their distinctive luminescence characteristics and wide range of applications in biological imaging. However, systems with sufficient brightness and metal selectivity can be difficult to produce on a molecular scale. Presented herein is the stepwise introduction of differing lanthanide ions in a bis-DO3A/DTPA scaffold to afford three trinuclear bimetallic [Ln2Ln'] lanthanide complexes with site-specific, controlled binding [(Yb2Tb), (Eu2Tb), (Yb2Eu)]. The complexes display simultaneous emission from all LnIII centers across the visible (TbIII, EuIII) and near infra-red (YbIII) spectrum when excited via phenyl ligand sensitization at a wide range of temperatures and are consequently of interest for exploiting imaging in the near infra-red II biological window. Analysis of lifetime data over a range of excitation regimes reveals intermetallic communication between TbIII and EuIII centers and further develops the understanding of multimetallic lanthanide complexes.

Chelating chloride using binuclear lanthanide complexes in water.

Halide recognition by supramolecular receptors and coordination complexes in water is a long-standing challenge. In this work, we report chloride binding in water and in competing media by pre-organised binuclear kinetically inert lanthanide complexes, bridged by flexible -(CH2)2- and -(CH2)3- spacers, forming [Ln2(DO3A)2C-2] and [Ln2(DO3A)2C-3], respectively. These hydrophilic, neutral lanthanide coordination complexes are shown to bind chloride with apparent association constants of up to 105 M-1 in water and in buffered systems. Hydroxide bridging was observed in these complexes at basic pH, which was proven to be overcome by chloride. Thus, these lanthanide complexes show promise towards chloride recognition in biology and beyond. The results described here have clearly identified a new area of anion coordination chemistry that is ripe for detailed exploration.

Two Color Imaging of Different Hypoxia Levels in Cancer Cells.

Hypoxia (low oxygen levels) occurs in a range of biological contexts, including plants, bacterial biofilms, and solid tumors; it elicits responses from these biological systems that impact their survival. For example, conditions of low oxygen make treating tumors more difficult and have a negative impact on patient prognosis. Therefore, chemical probes that enable the study of biological hypoxia are valuable tools to increase the understanding of disease-related conditions that involve low oxygen levels, ultimately leading to improved diagnosis and treatment. While small-molecule hypoxia-sensing probes exist, the majority of these image only very severe hypoxia (<1% O2) and therefore do not give a full picture of heterogeneous biological hypoxia. Commonly used antibody-based imaging tools for hypoxia are less convenient than small molecules, as secondary detection steps involving immunostaining are required. Here, we report the synthesis, electrochemical properties, photophysical analysis, and biological validation of a range of indolequinone-based bioreductive fluorescent probes. We show that these compounds image different levels of hypoxia in 2D and 3D cell cultures. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Me-Tokyo Green-based probe 4 was only activated in severe hypoxia─0.5% O2 and less. Simultaneous application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Compounds 2 and 4 are therefore useful tools to study hypoxia in a cellular setting and represent convenient alternatives to antibody-based imaging approaches.

Boronic ester functionalised 1,8-diboryl-naphthalene scaffolds: fluoride versus oxide chelation.

1,8-Bis(boronic ester) derivatives of naphthalene, 1,8-C10H6{B(OR)2}2, present an attractive target as receptors for the fluoride ion via B-F-B chelation, but are synthetically challenging to access due to the competing formation of a very stable anhydride containing a B-O-B motif. By contrast, unsymmetrical systems of the type 1,8-C10H6{B(OR)2}(BR'2) can be synthesized for (OR)2 = 1,2-O2C6H4 (i.e. Cat) and R' = Mes. This system is shown to be competent for the uptake of F-, making use of a chelating mode of action and the formation of a bridging B-F-B motif between the two boron centres. However, both experimental and quantum chemical studies indicate that the µ2-F adduct is the kinetic product of fluoride uptake, with an alternative structural motif featuring a terminal B-F bond and a B-O-B bridge using one of the catechol oxygens being (marginally) more favourable thermodynamically.

Cell-permeable lanthanide-platinum(IV) anti-cancer prodrugs.

Platinum compounds are a vital part of our anti-cancer arsenal, and determining the location and speciation of platinum compounds is crucial. We have synthesised a lanthanide complex bearing a salicylic group (Ln = Gd, Eu) which demonstrates excellent cellular accumulation and minimal cytotoxicity. Derivatisation enabled access to bimetallic lanthanide-platinum(ii) and lanthanide-platinum(iv) complexes. Luminescence from the europium-platinum(iv) system was quenched, and reduction to platinum(ii) with ascorbic acid resulted in a "switch-on" luminescence enhancement. We used diffusion-based 1H NMR spectroscopic methods to quantify cellular accumulation. The gadolinium-platinum(ii) and gadolinium-platinum(iv) complexes demonstrated appreciable cytotoxicity. A longer delay following incubation before cytotoxicity was observed for the gadolinium-platinum(iv) compared to the gadolinium-platinum(ii) complex. Functionalisation with octanoate ligands resulted in enhanced cellular accumulation and an even greater latency in cytotoxicity.

A Model System to Explore the Detection Limits of Antibody-Based Immuno-SPECT Imaging of Exclusively Intranuclear Epitopes.

Imaging of intranuclear epitopes using antibodies tagged to cell-penetrating peptides has great potential given its versatility, specificity, and sensitivity. However, this process is technically challenging because of the location of the target. Previous research has demonstrated a variety of intranuclear epitopes that can be targeted with antibody-based radioimmunoconjugates. Here, we developed a controlled-expression model of nucleus-localized green fluorescent protein (GFP) to interrogate the technical limitations of intranuclear SPECT using radioimmunoconjugates, notably the lower target-abundance detection threshold. Methods: We stably transfected the lung adenocarcinoma cell line H1299 with an enhanced GFP (EGFP)-tagged histone 2B (H2B) and generated 4 cell lines expressing increasing levels of GFP. EGFP levels were quantified using Western blot, flow cytometry, and enzyme-linked immunosorbent assay. An anti-GFP antibody (GFP-G1) was modified using dibenzocyclooctyne-N3-based strain-promoted azide-alkyne cycloaddition with the cell-penetrating peptide TAT (GRKKRRQRRRPPQGYG), which also includes a nuclear localization sequence, and the metal ion chelator N3-Bn-diethylenetriamine pentaacetate (DTPA) to allow radiolabeling with 111In. Cell uptake of 111In-GFP-G1-TAT was evaluated across 5 cell clones expressing different levels of H2B-EGFP in vitro. Tumor uptake in xenograft-bearing mice was quantified to determine the smallest amount of target epitope that could be detected using 111In-GFP-G1-TAT. Results: We generated 4 H1299 cell clones expressing different levels of H2B-EGFP (0-1 million copies per cell, including wild-type H1299 cells). GFP-G1 monoclonal antibody was produced and purified in house, and selective binding to H2B-EGFP was confirmed. The affinity (dissociation constant) of GFP-G1 was determined as 9.1 ± 3.0 nM. GFP-G1 was conjugated to TAT and DTPA. 111In-GFP-G1-TAT uptake in H2B-EGFP-expressing cell clones correlated linearly with H2B-EGFP expression (P < 0.001). In vivo xenograft studies demonstrated that 111In-GFP-G1-TAT uptake in tumor tissue correlated linearly with expression of H2B-EGFP (P = 0.004) and suggested a lower target-abundance detection threshold of approximately 240,000 copies per cell. Conclusion: Here, we present a proof-of-concept demonstration that antibody-based imaging of intranuclear targets is capable both of detecting the presence of an epitope of interest with a copy number above 240,000 copies per cell and of determining differences in expression level above this threshold.

Enhancing 31P NMR relaxation rates with a kinetically inert gadolinium complex.

The kinetically stable heptadentate gadolinium complex Gd.pDO3A (1.Gd) demonstrates significant 31P nuclear magnetic resonance (NMR) relaxation enhancement of biologically relevant phosphate species; adenosine triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate. Gd.pDO3A (1.Gd) binds these species in fast exchange, enabling the relaxation of the bulk phosphate species in solution. This gives rise to 31P relaxation enhancements up to 250-fold higher than those observed for 31P relaxation enhancements with the commercial MRI contrast agent Gd.DOTA (DOTAREM), 2. Gd.pDO3A-like complexes may have potential applications as 31P magnetic resonance contrast agents, since shortening the T1 relaxation time of phosphate species would reduce the time needed to acquire 31P-MR spectra.

INDIANA: An in-cell diffusion method to characterize the size, abundance and permeability of cells.

NMR and MRI diffusion experiments contain information describing the shape, size, abundance, and membrane permeability of cells although extracting this information can be challenging. Here we present the INDIANA (IN-cell DIffusion ANAlysis) method to simultaneously and non-invasively measure cell abundance, effective radius, permeability and intrinsic relaxation rates and diffusion coefficients within the inter- and intra-cellular populations. The method couples an experimental dataset comprising stimulated-echo diffusion measurements, varying both the gradient strength and the diffusion delay, together with software to fit a model based on the Kärger equations to robustly extract the relevant parameters. A detailed error analysis is presented by comparing the results from fitting simulated data from Monte Carlo simulations, establishing its effectiveness. We note that for parameters typical of mammalian cells the approach is particularly effective, and the shape of the underlying cells does not unduly affect the results. Finally, we demonstrate the performance of the experiment on systems of suspended yeast and mammalian cells. The extracted parameters describing cell abundance, size, permeability and relaxation are independently validated.

Multimetallic Lanthanide Complexes: Using Kinetic Control To Define Complex Multimetallic Arrays.

Kinetically inert lanthanide complexes are proving to be highly effective building blocks for the preparation of complex heterometallic architectures, allowing complete control of metal ion domains, which cannot be achieved under thermodynamic control. Kinetic stability may render perceivable labile coordination bonds more durable than several types of covalent interactions. For complexes in clinical use, the significance of kinetic stability cannot be overstated, and this Account treats the topic accordingly. Kinetically inert complexes can be used as building blocks for elaborate synthesis. For instance, it is now possible to prepare heterometallic lanthanide complexes containing two or more different lanthanide ions by linking kinetically robust complexes together. This approach can yield bimetallic (f-f' or d-f) and trimetallic (f-f'-f″) lanthanide complexes. In this Account, we describe our studies exploiting the slow dissociation of lanthanide complexes derived from 1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid (DOTA) related ligands to link complexes together through synthetic manipulation of pendent groups on the ligand skeleton or through coordination of bridging donor groups to a d-block metal center. In the course of this work, we have developed a variety of such methods, ranging from peptide coupling and diazotization to Ugi and click chemistry and have also explored the use of alternative strategies that combine orthogonal protecting group chemistry with sequential complexation of different lanthanide ions or that use self-assembly to deliver well-defined multimetallic systems. These well-defined bimetallic systems also have considerable scope for exploitation. Since the earliest studies, it has been clear that there is potential for application in the burgeoning field of molecular imaging. Heterometallic lanthanide complexes can be used as single-molecule bimodal imaging agents through incorporation of MRI active and luminescent components. Alternatively, conventional luminescence methods can be exploited in conjunction with lanthanide luminescence. In the simplest cases, a single lanthanide can be used to achieve a switchable response in combination with a transition metal complex. Bimetallic f-f' complexes allow the full potential of the approach to be realized in systems in which one lanthanide responds to changes in the concentration of an analyte, while a second lanthanide center can be used to define the concentration of the probe itself. This offers a new solution to the old dichotomy of ratiometric imaging that can potentially be applied widely.

Shining light on the antenna chromophore in lanthanide based dyes.

Lanthanide based dyes and assays exploit the antenna effect, where a sensitiser-chromophore is used as a light harvesting antenna and subsequent excited state energy transfer populates the emitting lanthanide centred excited state. A rudimentary understanding of the design criteria for designing efficient dyes and assays based on the antenna effect is in place. By preparing kinetically inert lanthanide complexes based on the DO3A scaffold, we are able to study the excited state energy transfer from a 7-methoxy-coumarin antenna chromophore to europium(iii) and terbium(iii) centred excited states. By contrasting the photophysical properties of complexes of metal centres with and without accessible excited states, we are able to separate the contributions from the heavy atom effect, photoinduced electron transfer quenching, excited state energy transfer and molecular conformations. Furthermore, by studying the photophysical properties of the antenna chromophore, we can directly monitor the solution structure and are able to conclude that excited state energy transfer from the chromophore singlet state to the lanthanide centre does occur.

Brook trout distributional response to unconventional oil and gas development: Landscape context matters.

We conducted a large-scale assessment of unconventional oil and gas (UOG) development effects on brook trout (Salvelinus fontinalis) distribution. We compiled 2231 brook trout collection records from the Upper Susquehanna River Watershed, USA. We used boosted regression tree (BRT) analysis to predict occurrence probability at the 1:24,000 stream-segment scale as a function of natural and anthropogenic landscape and climatic attributes. We then evaluated the importance of landscape context (i.e., pre-existing natural habitat quality and anthropogenic degradation) in modulating the effects of UOG on brook trout distribution under UOG development scenarios. BRT made use of 5 anthropogenic (28% relative influence) and 7 natural (72% relative influence) variables to model occurrence with a high degree of accuracy [Area Under the Receiver Operating Curve (AUC)=0.85 and cross-validated AUC=0.81]. UOG development impacted 11% (n=2784) of streams and resulted in a loss of predicted occurrence in 126 (4%). Most streams impacted by UOG had unsuitable underlying natural habitat quality (n=1220; 44%). Brook trout were predicted to be absent from an additional 26% (n=733) of streams due to pre-existing non-UOG land uses (i.e., agriculture, residential and commercial development, or historic mining). Streams with a predicted and observed (via existing pre- and post-disturbance fish sampling records) loss of occurrence due to UOG tended to have intermediate natural habitat quality and/or intermediate levels of non-UOG stress. Simulated development of permitted but undeveloped UOG wells (n=943) resulted in a loss of predicted occurrence in 27 additional streams. Loss of occurrence was strongly dependent upon landscape context, suggesting effects of current and future UOG development are likely most relevant in streams near the probability threshold due to pre-existing habitat degradation.

A detailed risk assessment of shale gas development on headwater streams in the Pennsylvania portion of the Upper Susquehanna River Basin, U.S.A.

The development of unconventional oil and gas (UOG) involves infrastructure development (well pads, roads and pipelines), well drilling and stimulation (hydraulic fracturing), and production; all of which have the potential to affect stream ecosystems. Here, we developed a fine-scaled (1:24,000) catchment-level disturbance intensity index (DII) that included 17 measures of UOG capturing all steps in the development process (infrastructure, water withdrawals, probabilistic spills) that could affect headwater streams (<200km2 in upstream catchment) in the Upper Susquehanna River Basin in Pennsylvania, U.S.A. The DII ranged from 0 (no UOG disturbance) to 100 (the catchment with the highest UOG disturbance in the study area) and it was most sensitive to removal of pipeline cover, road cover and well pad cover metrics. We related this DII to three measures of high quality streams: Pennsylvania State Exceptional Value (EV) streams, Class A brook trout streams and Eastern Brook Trout Joint Venture brook trout patches. Overall only 3.8% of all catchments and 2.7% of EV stream length, 1.9% of Class A streams and 1.2% of patches were classified as having medium to high level DII scores (>50). Well density, often used as a proxy for development, only correlated strongly with well pad coverage and produced materials, and therefore may miss potential effects associated with roads and pipelines, water withdrawals and spills. When analyzed with a future development scenario, 91.1% of EV stream length, 68.7% of Class A streams and 80.0% of patches were in catchments with a moderate to high probability of development. Our method incorporated the cumulative effects of UOG on streams and can be used to identify catchments and reaches at risk to existing stressors or future development.

In Vivo Pretargeted Imaging of HER2 and TAG-72 Expression Using the HaloTag Enzyme.

A novel pretargeted SPECT imaging strategy based on the HaloTag enzyme has been evaluated for the first time in a living system. To determine the efficacy of this approach, two clinically relevant cancer biomarkers, HER2 and TAG-72, were selected to represent models of internalizing and noninternalizing antigens, respectively. In MDA-MB-231/H2N (HER2-expressing) and LS174T (TAG-72-expressing) xenograft tumors in mice, pretargeting experiments were performed in which HaloTag-conjugated derivatives of the antibodies trastuzumab (anti-HER2) or CC49 (anti-TAG-72) were utilized as primary agents, and the small molecule HaloTag ligands 111In-HTL-1, -2, and -3 were evaluated as secondary agents. While this approach was not sufficiently sensitive to detect the internalizing HER2 antigen, pretargeting experiments involving the most optimal secondary agent, 111In-HTL-3, were successful in detecting the noninternalizing antigen TAG-72 and provided high-contrast SPECT images at 4 and 24 h postinjection.

Lanthanide Complexes that Respond to Changes in Cyanide Concentration in Water.

Cyanide ions are shown to interact with lanthanide complexes of phenacylDO3A derivatives in aqueous solution, giving rise to changes in the luminescence and NMR spectra. These changes are the consequence of cyanohydrin formation, which is favored by the coordination of the phenacyl carbonyl group to the lanthanide center. These complexes display minimal affinity for fluoride and can detect cyanide at concentrations less than 1 µm. By contrast, lanthanide complexes with DOTAM derivatives display no affinity for cyanide in water, but respond to changes in fluoride concentration.