Ab initio-based determination of lanthanoid-radical exchange as visualised by inelastic neutron scattering.

Magnetic exchange coupling can modulate the slow magnetic relaxation in single-molecule magnets. Despite this, elucidation of exchange coupling remains a significant challenge for the lanthanoid(iii) ions, both experimentally and computationally. In this work, the crystal field splitting and 4f-π exchange coupling in the erbium-semiquinonate complex [ErTp2dbsq] (Er-dbsq; Tp- = hydro-tris(1-pyrazolyl)borate, dbsqH2 = 3,5-di-tert-butyl-1,2-semiquinone) have been determined by inelastic neutron scattering (INS), magnetometry, and CASSCF-SO ab initio calculations. A related complex with a diamagnetic ligand, [ErTp2trop] (Er-trop; tropH = tropolone), has been used as a model for the crystal field splitting in the absence of coupling. Magnetic and INS data indicate antiferromagnetic exchange for Er-dbsq with a coupling constant of Jex = -0.23 meV (-1.8 cm-1) (-2Jex formalism) and good agreement is found between theory and experiment, with the low energy magnetic and spectroscopic properties well modelled. Most notable is the ability of the ab initio modelling to reproduce the signature of interference between localised 4f states and delocalised π-radical states that is evident in the Q-dependence of the exchange excitation. This work highlights the power of combining INS with EPR and magnetometry for determination of ground state properties, as well as the enhanced capability of CASSCF-SO ab initio calculations and purposely developed ab initio-based theoretical models. We deliver an unprecedentedly detailed representation of the entangled character of 4f-π exchange states, which is obtained via an accurate image of the spin-orbital transition density between the 4f-π exchange coupled wavefunctions.

Deuteration for biological SANS: Case studies, success and challenges in chemistry and biology.

Small angle neutron scattering is a powerful complementary technique in structural biology. It generally requires, or benefits from, deuteration to achieve its unique potentials. Molecular deuteration has become a mature expertise, with deuteration facilities located worldwide to support access to the technique for a wide breadth of structural biology and life sciences. The sorts of problems well answered by small angle scattering and deuteration involve large (>10Å) scale flexible movements, and this approach is best used where high-resolution methods (crystallography, NMR, cryo-EM) leave questions unanswered. This chapter introduces deuteration, reviewing biological deuteration of proteins, lipids and sterols, and then steps through the ever-expanding range of deuterated molecules being produced by chemical synthesis and enabling sophisticated experiments using physiologically relevant lipids. Case studies of recent successful use of deuteration may provide illustrative examples for strategies for future experiments. We discuss issues of nomenclature for synthesised molecules of novel labeling and make recommendations for their naming. We reflect on our experiences, with cost associated with achieving an arbitrary deuteration level, and on the benefits of experimental co-design by user scientist, deuteration scientist, and neutron scattering scientist working together. Although methods for biological and chemical deuteration are published in the public domain, we recommend that the best method to deuterate is to engage with a deuteration facility.

Controlling phase and rheological behaviours of hexagonal lyotropic liquid crystalline templates for nanostructural administration and retention.

Introducing polymerizable monomers into a binary hexagonal lyotropic liquid crystalline (LLC) template is a straightforward way for retaining the nanostructure but will decrease attractive intra- and inter- aggregate interactions. It is therefore crucial to understand the interfacial interactions at nanoscale after introducing the monomers but prior to polymerization. Herein, active species, poly (ethylene glycol) diacrylate (PEGDA) and 2-hydroxyethyl methacrylate (HEMA), were introduced into hexagonal LLC of dodecyl trimethylammonium bromide and water to explore the structural variables, dimensional stability, and dynamic property. At a proper volume ratio of PEGDA/HEMA (1/4), the system presents excellent homogeneity with a higher dimensional stability and lower dynamic property from rheological assessments, thereby achieving robust, free-standing, and transparent membranes after photo-polymerization. The unique property of the system also lies in the much lower order-disorder transition temperature (45 °C) that facilitates the reorientation of mesochannels. They are in contrast inaccessible for the ternary system only with PEGDA, though the nanostructure for both systems could be retained. An insight into subtle variations in these parameters allows us to prepare a polymerizable template possessing higher dimensional stability and suitable flexibility via molecular design, thereby enabling simultaneous structural alignment and retention for the development of functional nanomaterials.

Diffusion in Organic Film Stacks Containing Solution-Processed Phosphorescent Poly(dendrimer) Dopants.

Efficient organic light-emitting diodes (OLEDs) consist of an emissive layer comprising a blend of a light-emitting and host material in contact with one or more charge transporting layers. The distribution of the active material in the guest-host emissive layer blend and the changes that may occur upon thermal annealing are two important factors in determining the stability and efficiency of OLEDs. We have combined neutron reflectometry and photoluminescence measurements to investigate the structures of films comprising an emissive layer containing a phosphorescent poly(dendrimer) material blended with 4,4'-N,N'-di(carbazolyl)biphenyl. This combination has been shown to give rise to highly efficient OLEDs. Here, we show that the emissive poly(dendrimer) material was not uniformly distributed in the host, but formed a concentration gradient within the emissive layer. Upon heating, the adjacent electron transport layer was found to intermix with the emissive layer, accompanied by changes in the material distribution in the emissive layer. The intermixing of the materials led to a decrease in the photoluminescence from the poly(dendrimer) within the film. The decrease in the photoluminescence was ascribed to an increase in interchromophore interactions that could arise from a conformational change of the poly(dendrimer) or phase separation leading to aggregation. The results indicate that, while uniform mixing of the guest and host is not essential for efficiency, the thermal stabilities of both host and charge transport materials are important for device durability.

Membrane Protein Structures in Lipid Bilayers; Small-Angle Neutron Scattering With Contrast-Matched Bicontinuous Cubic Phases.

This perspective describes advances in determining membrane protein structures in lipid bilayers using small-angle neutron scattering (SANS). Differentially labeled detergents with a homogeneous scattering length density facilitate contrast matching of detergent micelles; this has previously been used successfully to obtain the structures of membrane proteins. However, detergent micelles do not mimic the lipid bilayer environment of the cell membrane in vivo. Deuterated vesicles can be used to obtain the radius of gyration of membrane proteins, but protein-protein interference effects within the vesicles severely limits this method such that the protein structure cannot be modeled. We show herein that different membrane protein conformations can be distinguished within the lipid bilayer of the bicontinuous cubic phase using contrast-matching. Time-resolved studies performed using SANS illustrate the complex phase behavior in lyotropic liquid crystalline systems and emphasize the importance of this development. We believe that studying membrane protein structures and phase behavior in contrast-matched lipid bilayers will advance both biological and pharmaceutical applications of membrane-associated proteins, biosensors and food science.

Evolution of structural dimensions in mesoporous template precursor from hexagonal lyotropic liquid crystals.

Producing nanopores from hexagonal lyotropic liquid crystals (LLCs) templates requires not only retaining phase morphology of the templates but also precisely controlling structural dimensions of unit cells. In this study, SAXS and 2H NMR are used to investigate dimensional evolutions of ternary systems consisting of polymerizable species, (ethylene glycol) diacrylate (PEGDA) and/or 2-hydroxyethyl methacrylate (HEMA), in a LLCs template of hexagonally packed cylinders formed from dodecyl trimethylammonium bromide (DTAB) and water. With the addition of those polymerizable species, the system rearranges into a new hexagonal system with a smaller aggregation number, smaller pores and a thicker pore wall thickness. The hexagonal system will coexist with an aqueous-rich phase containing isotropically distributed DTAB if sufficient PEGDA is applied but the single hexagonal system could be restored by partially replacing the PEGDA with HEMA. The mobility of DTAB molecules within the aggregates varies depending on monomer compositions. The changes in structural dimensions of the unit cells and phase behaviors after adding polymerizable monomers allow dimensional control of mesochannels and potentially enable the control of selectivity and robustness of polymerized nanomaterials via molecular design.

Protein-Eye View of the in Meso Crystallization Mechanism.

For evolving biological and biomedical applications of hybrid protein?lipid materials, understanding the behavior of the protein within the lipid mesophase is crucial. After more than two decades since the invention of the in meso crystallization method, a protein-eye view of its mechanism is still lacking. Numerous structural studies have suggested that integral membrane proteins preferentially partition at localized flat points on the bilayer surface of the cubic phase with crystal growth occurring from a local fluid lamellar L? phase conduit. However, studies to date have, by necessity, focused on structural transitions occurring in the lipid mesophase. Here, we demonstrate using small-angle neutron scattering that the lipid bilayer of monoolein (the most commonly used lipid for in meso crystallization) can be contrast-matched using deuteration, allowing us to isolate scattering from encapsulated peptides during the crystal growth process for the first time. During in meso crystallization, a clear decrease in form factor scattering intensity of the peptides was observed and directly correlated with crystal growth. A transient fluid lamellar L? phase was observed, providing direct evidence for the proposed mechanism for this technique. This suggests that the peptide passes through a transition from the cubic QII phase, via an L? phase to the lamellar crystalline Lc phase with similar layered spacing. When high protein loading was possible, the lamellar crystalline Lc phase of the peptide in the single crystals was observed. These findings show the mechanism of in meso crystallization for the first time from the perspective of integral membrane proteins.

Effect of deuteration on the phase behaviour and structure of lamellar phases of phosphatidylcholines - Deuterated lipids as proxies for the physical properties of native bilayers.

Deuteration of phospholipids is a common practice to elucidate membrane structure, dynamics and function, by providing selective visualisation in neutron scattering, nuclear magnetic resonance and vibrational spectroscopy. It is generally assumed that the properties of the deuterated lipids are identical to those of the protiated lipids, and while a number of papers have compared the properties of different forms, to date this has been no systematic study of the effects over a range of conditions. Here we present a study of the effects of deuteration on the organisation and phase behaviour of four common phospholipids (DSPC, DPPC, DMPC, DOPC), observing the effect of chain deuteration and headgroup deuteration on lipid structure and phase behaviour. For saturated lipids in excess water the gel-fluid phase transition temperature is 4.3 ± 0.1 °C lower for lipids with deuterated chains compared to protiated chains, consistent with previous work. Despite this significant change, well away from the transition structural changes as measured by powder small angle X-ray scattering are small and within errors. To investigate this further, measurements were carried out on oriented multilamellar stacks of DOPC in the fluid phase at reduced hydration. Neutrons are used in conjunction with contrast variation to elucidate the role of the deuteration explicitly. It is found that deuterated chains cause a reduction in the lamellar repeat spacing and bilayer thickness, but deuterated headgroups cause an increase. Consequences for the interpretation of Neutron Scattering data with deuterated lipids are discussed.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Quantitative 3D determination of self-assembled structures on nanoparticles using small angle neutron scattering.

The ligand shell (LS) determines a number of nanoparticles' properties. Nanoparticles' cores can be accurately characterized; yet the structure of the LS, when composed of mixture of molecules, can be described only qualitatively (e.g., patchy, Janus, and random). Here we show that quantitative description of the LS' morphology of monodisperse nanoparticles can be obtained using small-angle neutron scattering (SANS), measured at multiple contrasts, achieved by either ligand or solvent deuteration. Three-dimensional models of the nanoparticles' core and LS are generated using an ab initio reconstruction method. Characteristic length scales extracted from the models are compared with simulations. We also characterize the evolution of the LS upon thermal annealing, and investigate the LS morphology of mixed-ligand copper and silver nanoparticles as well as gold nanoparticles coated with ternary mixtures. Our results suggest that SANS combined with multiphase modeling is a versatile approach for the characterization of nanoparticles' LS.

Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22.

Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1-7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1-7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.

A Fluorine-18 Radiolabeling Method Enabled by Rhenium(I) Complexation Circumvents the Requirement of Anhydrous Conditions.

Azeotropic distillation is typically required to achieve fluorine-18 radiolabeling during the production of positron emission tomography (PET) imaging agents. However, this time-consuming process also limits fluorine-18 incorporation, due to radioactive decay of the isotope and its adsorption to the drying vessel. In addressing these limitations, the fluorine-18 radiolabeling of one model rhenium(I) complex is reported here, which is significantly improved under conditions that do not require azeotropic drying. This work could open a route towards the investigation of a simplified metal-mediated late-stage radiofluorination method, which would expand upon the accessibility of new PET and PET-optical probes.

Synthesis of Perdeuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D82 ]POPC) and Characterisation of Its Lipid Bilayer Membrane Structure by Neutron Reflectometry.

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), an unsaturated acyl chain containing lipid, is often the predominant lipid in eukaryotic cell membranes in which it is crucial for the fluidity of membranes under physiological conditions. Commercially available, partially deuterated [D31 ]1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D31 ]POPC) does not provide sufficient isotopic contrast for detailed structural studies of multicomponent membranes through neutron techniques. Herein, a relatively straightforward and generic chemical deuteration method is discussed for the asymmetric synthesis of perdeuterated [D31 ]1-palmitoyl-[D33 ]2-oleoyl-sn-[D5 ]glycero-[D13 ]3-phosphocholine ([D82 ]POPC) that also allows selective deuteration of any of its constituent groups. Neutron reflectivity of a [D82 ]POPC-supported bilayer was used to experimentally determine the neutron scattering length density profile of the lipid. The acyl chains of [D82 ]POPC are closely contrast-matched to heavy water, whereas the very high scattering length density of the deuterated glycerophosphocholine head groups provides good contrast to membrane-binding agents in both deuterated and non-deuterated solvent environments.

In Vivo Evaluation of Radiofluorinated Caspase-3/7 Inhibitors as Radiotracers for Apoptosis Imaging and Comparison with [18F]ML-10 in a Stroke Model in the Rat.

PURPOSE: The first biological evaluation of two potent fluorine-18 radiolabelled inhibitors of caspase-3/7 was achieved in a cerebral stroke rat model to visualize apoptosis. PROCEDURES: In vivo characteristics of isatins [(18)F]-2 and [(18)F]-3 were studied and compared by µPET to previously described 1-[4-(2-[(18)F]fluoroethyl)benzyl]-5-(2-methoxymethylpyrrolidin-1-ylsulfonyl)isatin ([(18)F]-1) and to 2-(5-[(18)F]fluoropentyl)-2-methyl-malonic acid ([(18)F]ML-10) used as a reference radiotracer in a rat stroke model. RESULTS: [(18)F]-2 and [(18)F]-3 were radiolabelled with high radiochemical purity and high specific radioactivity. Radioactivity uptakes in ischemic and contralateral brain regions were weak for the three radiolabelled isatins and lower for [(18)F]ML-10. In µPET, time activity curves showed significant uptake differences between both regions of interest for [(18)F]-1 after 45 min. No differences were observed for [(18)F]ML-10. CONCLUSIONS: Radiolabelled isatins are more promising radiotracers to image apoptosis than [(18)F]ML-10 in this stroke animal model without craniectomy. In particular, [(18)F]-1 presented significant uptake in apoptotic area 45 min after administration.

Novel Fluorinated 8-Hydroxyquinoline Based Metal Ionophores for Exploring the Metal Hypothesis of Alzheimer's Disease.

Zinc, copper, and iron ions are involved in amyloid-beta (Aß) deposition and stabilization in Alzheimer's disease (AD). Consequently, metal binding agents that prevent metal-Aß interaction and lead to the dissolution of Aß deposits have become well sought therapeutic and diagnostic targets. However, direct intervention between diseases and metal abnormalities has been challenging and is partially attributed to the lack of a suitable agent to determine and modify metal concentration and distribution in vivo. In the search of metal ionophores, we have identified several promising chemical entities by strategic fluorination of 8-hydroxyquinoline drugs, clioquinol, and PBT2. Compounds 15-17 and 28-30 showed exceptional metal ionophore ability (6-40-fold increase of copper uptake and >2-fold increase of zinc uptake) and inhibition of zinc induced Aß oligomerization (EC50s < ∼5 µM). These compounds are suitable for further development as drug candidates and/or positron emission tomography (PET) biomarkers if radiolabeled with (18)F.

Synthesis and in Vivo Evaluation of [123I]Melanin-Targeted Agents.

This study reports the synthesis, [(123)I]radiolabeling, and biological profile of a new series of iodinated compounds for potential translation to the corresponding [(131)I]radiolabeled compounds for radionuclide therapy of melanoma. Radiolabeling was achieved via standard electrophilic iododestannylation in 60-90% radiochemical yield. Preliminary SPECT imaging demonstrated high and distinct tumor uptake of all compounds, as well as high tumor-to-background ratios compared to the literature compound [(123)I]4 (ICF01012). The most favorable compounds ([(123)I]20, [(123)I]23, [(123)I]41, and [(123)I]53) were selected for further biological investigation. Biodistribution studies indicated that all four compounds bound to melanin containing tissue with low in vivo deiodination; [(123)I]20 and [(123)I]53 in particular displayed high and prolonged tumor uptake (13% ID/g at 48 h). [(123)I]53 had the most favorable overall profile of the cumulative uptake over time of radiosensitive organs. Metabolite analysis of the four radiotracers found [(123)I]41 and [(123)I]53 to be the most favorable, displaying high and prolonged amounts of intact tracer in melanin containing tissues, suggesting melanin specific binding. Results herein suggest that compound [(123)I]53 displays favorable in vivo pharmacokinetics and stability and hence is an ideal candidate to proceed with further preclinical [(131)I] therapeutic evaluation.

Mild conditions for deuteration of primary and secondary arylamines for the synthesis of deuterated optoelectronic organic molecules.

Deuterated arylamines demonstrate great potential for use in optoelectronic devices, but their widespread utility requires a method for large-scale synthesis. The incorporation of these deuterated materials into optoelectronic devices also provides the opportunity for studies of the functioning device using neutron reflectometry based on the difference in the scattering length density between protonated and deuterated compounds. Here we report mild deuteration conditions utilising standard laboratory glassware for the deuteration of: diphenylamine, N-phenylnaphthylamine, N-phenyl-o-phenylenediamine and 1-naphthylamine (via H/D exchange in D2O at 80 °C, catalysed by Pt/C and Pd/C). These conditions were not successful in the deuteration of triphenylamine or N,N-dimethylaniline, suggesting that these mild conditions are not suitable for the deuteration of tertiary arylamines, but are likely to be applicable for the deuteration of other primary and secondary arylamines. The deuterated arylamines can then be used for synthesis of larger organic molecules or polymers with optoelectronic applications.

Spectroscopic investigations on the interactions of potent platinum(II) anticancer agents with bovine serum albumin.

The interactions of three platinum(II)-based anticancer complexes [(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)](2+), [(5,6-dimethyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)platinum(II)](2+), and [(5,6-dimethyl-1,10-phenanthroline)(1,2-diaminoethane)platinum(II)](2+) (56MEEN) with BSA have been examined by circular dichroism (CD), fluorescence and (1)H pulsed gradient spin-echo (PGSE) diffusion NMR spectroscopy. The number of association constants and sites differed depending upon the spectroscopic method. This may be because each technique monitors different types of interaction/s and/or as a consequence of the different concentration ranges required for each technique. The titration of BSA with the achiral 56MEEN as monitored by CD indicates a reduction in the α-helical nature of the albumin, with the association constant calculated to be ~5 × 10(6) M(-1) for one site. Due to the chiral nature of the other two complexes, their association with albumin was not monitored using CD but was examined using fluorescence and PGSE diffusion NMR. Titration of BSA with any of the three metal complexes resulted in quenching of fluorescence, with the number of association sites calculated to be ~1.1, with an association constant of ~2 × 10(5) M(-1). PGSE diffusion NMR provided insights into interactions occurring with the BSA in its entirety, rather than with individual regions. Metal complex binding sites were estimated (~10 equivalent) from the diffusion data, with the average association constant for all sites ~10(2)-10(3)M(-1). These experiments highlight the information that can be elucidated from complementary spectroscopic techniques and demonstrate the usefulness of PGSE diffusion NMR in monitoring multiple weak binding sites, which is of great importance in studying drug-biomolecule interactions.

Transition metal based anticancer drugs.

With an ageing baby-boomer population in the Western World, cancer is becoming a significant cause of death. The prevalence of cancer and all associated costs, both in human and financial terms, drives the search for new therapeutic drugs and treatments. Platinum anticancer agents, such as cisplatin have been highly successful but there are several disadvantages associated with their use. What is need are new compounds with different mechanisms of action and resistance profiles. What needs to be recognised is that there are many other metal in the periodic table with therapeutic potential. Here we have highlighted metal complexes with activity and have illustrate the different approaches to the design of anticancer complexes.

Advances in platinum chemotherapeutics.

The approved platinum(II)-based anticancer agents cisplatin, carboplatin and oxaliplatin are widely utilised in the clinic, although with numerous disadvantages. With the aim of circumventing unwanted side-effects, a great deal of research is being conducted in the areas of cancer-specific targeting, drug administration and drug delivery. The targeting of platinum complexes to cancerous tissues can be achieved by the attachment of small molecules with biological significance. In addition, the administration of platinum complexes in the form of platinum(IV) allows for intracellular reduction to release the active form of the drug, cisplatin. Drug delivery includes such technologies as liposomes, dendrimers, polymers and nanotubes, with all showing promise for the delivery of platinum compounds. In this paper we highlight some of the recent advances in the field of platinum chemotherapeutics, with a focus on the technologies that attempt to utilise the cytotoxic nature of cisplatin, whilst improving drug targeting to reduce side-effects.