A dual encapsulation strategy to generate anion-responsive luminescent lanthanide hydrogels.

We report a new method to generate ion-responsive luminescent hydrogels, involving encapsulation of a luminescent lanthanide probe within crosslinked amphiphilic polymer particles and subsequent entrapment within a hydrogel. The resulting hydrogels are capable of reversible bicarbonate sensing, exhibit no leaching, and can be tuned for a range of sensing applications.

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange.

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the 10 functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter et al., 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.

Sterically demanding macrocyclic Eu(iii) complexes for selective recognition of phosphate and real-time monitoring of enzymatically generated adenosine monophosphate.

The design of molecular receptors that bind and sense anions in biologically relevant aqueous solutions is a key challenge in supramolecular chemistry. The recognition of inorganic phosphate is particularly challenging because of its high hydration energy and pH dependent speciation. Adenosine monophosphate (AMP) represents a valuable but elusive target for supramolecular detection because of its structural similarity to the more negatively charged anions, ATP and ADP. We report two new macrocyclic Eu(iii) receptors capable of selectively sensing inorganic phosphate and AMP in water. The receptors contain a sterically demanding 8-(benzyloxy)quinoline pendant arm that coordinates to the metal centre, creating a binding pocket suitable for phosphate and AMP, whilst excluding potentially interfering chelating anions, in particular ATP, bicarbonate and lactate. The sensing selectivity of our Eu(iii) receptors follows the order AMP > ADP > ATP, which represents a reversal of the order of selectivity observed for most reported nucleoside phosphate receptors. We have exploited the unique host-guest induced changes in emission intensity and lifetime for the detection of inorganic phosphate in human serum samples, and for monitoring the enzymatic production of AMP in real-time.

Localising individual atoms of tryptophan side chains in the metallo-ß-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

The metallo-ß-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time-Resolved Luminescence Studies.

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

Critical Evaluation of the Interaction of Reactive Oxygen and Nitrogen Species with Blood to Inform the Clinical Translation of Nonthermal Plasma Therapy.

Non-thermal plasma (NTP), an ionized gas generated at ambient pressure and temperature, has been an emerging technology for medical applications. Through controlled delivery of reactive oxygen and nitrogen species (ROS/RNS), NTP can elicit hormetic cellular responses, thus stimulating broad therapeutic effects. To enable clinical translation of the promising preclinical research into NTP therapy, a deeper understanding of NTP interactions with clinical substrates is profoundly needed. Since NTP-generated ROS/RNS will inevitably interact with blood in several clinical contexts, understanding their stability in this system is crucial. In this study, two medically relevant NTP delivery modalities were used to assess the stability of NTP-generated ROS/RNS in three aqueous solutions with increasing organic complexities: phosphate-buffered saline (PBS), blood plasma (BP), and processed whole blood. NTP-generated RNS collectively (NO2 -, ONOO-), H2O2, and ONOO- exclusively were analyzed over time. We demonstrated that NTP-generated RNS and H2O2 were stable in PBS but scavenged by different components of the blood. While RNS remained stable in BP after initial scavenging effects, it was completely reduced in processed whole blood. On the other hand, H2O2 was completely scavenged in both liquids over time. Our previously developed luminescent probe europium(III) was used for precision measurement of ONOO- concentration. NTP-generated ONOO- was detected in all three liquids for up to at least 30 seconds, thus highlighting its therapeutic potential. Based on our results, we discussed the necessary considerations to choose the most optimal NTP modality for delivery of ROS/RNS to and via blood in the clinical context.

Time-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe.

Peroxynitrite (ONOO-) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO- in cell biology, improved methods for the selective detection and real-time analysis of ONOO- are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO- in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO- in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment.