Development of low-cost, compact chiroptical imaging systems.

Circular dichroism spectroscopy is a key probe of the structural and optical properties of chiral materials, however, commercial circular dichroism spectrometers are large, prohibitively expensive and rarely offer environmental control of the sample under test. Using Fresnel rhombs as inexpensive broadband quarter-wave plates, we demonstrate two novel, low-cost (<£2000) and portable imaging systems controlled by our own bespoke open-source control software which are capable of spatially mapping the circular dichroism of chiral solid state films. By coupling these imaging systems with a temperature controlled stage, we show that we can rapidly identify the thermal processing conditions required to maximise circular dichroism in chiral solid state films by measuring circular dichroism in situ during thermal annealing of a sample under test. The accuracy and spatial resolution of these circular dichroism imagers are cross-compared against our previous studies using an existing circular dichroism imaging system at the Diamond Light Source and are shown to be in good agreement, with a sensitivity down to 250 mdeg and a spatial resolution of 100 µm.

Enhancing Circularly Polarized Electroluminescence through Energy Transfer within a Chiral Polymer Host.

Organic light-emitting diodes (OLEDs) that are able to emit high levels of circularly polarized (CP) light hold significant promise in numerous future technologies. Such devices require chiral emissive materials to enable CP electroluminescence. However, the vast majority of current OLED emitter classes, including the state-of-the-art triplet-harvesting Thermally Activated Delayed Fluorescence (TADF) materials, produce very low levels of CP electroluminescence. Here we showcase a host-guest strategy that allows for energy transfer between a chiral polymer host and a representative chiral TADF emitter. Such a mechanism results in large amplification of the circular polarization of the emitter. As such, this study presents a promising avenue to further boost the performance of CP-OLED devices, enabling their further development and eventual commercialization. This article is protected by copyright. All rights reserved.

Rational Design of New Conjugated Polymers with Main Chain Chirality for Efficient Optoelectronic Devices: Carbo[6]Helicene and Indacenodithiophene Copolymers as Model Compounds.

The unique properties of conjugated polymers (CPs) in various optoelectronic applications are mainly attributed to their different self-assembly processes and superstructures. Various methods are utilized to tune and control CP structure and properties with less attention paid to the use of chirality. CPs with main chain chirality are rare and their microscopic and macroscopic properties are still unknown. In this work, the first experimental results are provided along these lines by synthesizing a series of racemic and enantiopure CPs containing statistical and alternating carbo[6]helicene and indacenodithiophene moieties and evaluating their microscopic (optical, energy levels) and macroscopic properties (hole mobilities, photovoltaic performance). It is demonstrated that a small statistical insertion of either the racemic or enantiopure helicene into the polymer backbone finely tunes the microscopic and macroscopic properties as a function of the statistical content. The microscopic properties of the enantiopure versus the racemic polymers with the same helicene loading remain similar. On the contrary, the macroscopic properties, and more interestingly those between the two enantiomeric forms, are altered as a function of the statistical content. Once incorporated into a solar cell device, these chiral CPs display better performance in their enantiopure versus racemic forms.

Tuning dipolar and multipolar resonances of chiral silicon nanostructures for control of near field superchirality.

Chiral materials display a property called optical activity, which is the capability to interact differentially with left and right circularly polarised light. This leads to the ability to manipulate the polarisation state of light, which has a broad range of applications spanning from energy efficient displays to quantum technologies. Both synthesised and engineered chiral nanomaterials are exploited in such devices. The design strategy for optimising the optical activity of a chiral material is typically based on maximising a single parameter, the electric dipole-magnetic dipole response. Here we demonstrate an alternative approach of controlling optical activity by manipulating both the dipole and multipolar response of a nanomaterial. This provides an additional parameter for material design, affording greater flexibility. The exemplar systems used to illustrate the strategy are nanofabricated chiral silicon structures. The multipolar response of the structures, and hence their optical activity, can be controlled simply by varying their height. This phenomenon allows optical activity and the creation of so called superchiral fields, with enhanced asymmetries, to be controlled over a broader wavelength range, than is achievable with just the electric dipole-magnetic dipole response. This work adds to the material design toolbox providing a route to novel nanomaterials for optoelectronics and sensing applications.

Circularly Polarized Microscopy of Thin Films of Chiral Organic Dyes.

We introduce an optical microscopy technique, circularly polarized microscopy or CPM, able to afford spatially resolved electronic circular dichroism (ECD) of thin films of chiral organic semiconductors through a commercial microscope equipped with a camera and inexpensive optics. Provided the dichroic ratio is sufficiently large, the spatial resolution is on the order of the µm and is only limited by the magnification optics integrated in the microscope. We apply CPM to thin films of small chiral π-conjugated molecules, which gave rise to ordered aggregates in the thin layer. Primarily, conventional ECD can reveal and characterize chiral supramolecular structures and possible interferences between anisotropic properties of solid samples; however, it cannot generally account for the spatial distribution of such properties. CPM offers a characterization of supramolecular chirality and of commingling polarization anisotropies of the material, describing their local distribution. To validate CPM, we demonstrated that it can be adopted to quantify the local ECD of samples characterized by intense signals, virtually on any standard optical microscope.

Interaction of Blenoxane and Congeners Bleomycins A2 and B2 with Human Plasma Proteins Using Circular Dichroism Spectroscopy.

Bleomycin is a glycopeptide congeners' family of antitumor antibiotics employed for the treatment of several types of tumors such as squamous cell carcinomas and malignant lymphomas. The general chemical structure is constituted by three main portions: (i) a metal binding domain that is recognized to be responsible for the DNA cleavage activity; (ii) a DNA binding domain via the 1-4' bithiazole moiety; and (iii) a carbohydrate domain thought to be responsible for the accumulation of bleomycin in some cancer cells. To date, a limited number of protein interactions with bleomycin have been studied, but the plasma binding has not yet been determined. Here, we explore this aspect of the protein binding capacity of bleomycin to the two most abundant plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AGP), which are known to bind and to be carriers of many drug molecules using spectroscopic techniques, such as circular dichroism, UV-vis absorbance, and fluorescence. The results showed that bleomycin binds to plasma proteins with an order-of-magnitude higher affinity for AGP than HSA. This is particularly important as AGP is an acute phase protein and is overexpressed in cancer patients. This should be taken into consideration as it could affect the therapeutic effect of the bleomycin dosage.

Insights into the origins of inverted circular dichroism in thin films of a chiral side chain polyfluorene.

We synthesized a fluorene-bithiophene co-polymer with chiral side chains (cPFT2) and investigated its chiroptical properties via synchotronradiation circular dichroism. We observed that thin films of the polymer display an intense circular dichroism (CD) upon annealing, which is of opposite handedness to the CD reported for similar polyfluorenes bearing the same enantiomeric chiral side chain. We then contrast the properties of this polymer with chiral side chain fluorene homopolymer (cPF) and observe large differences in their thin film morphology. Using photoluminescence spectroscopy, we uncover evidence of polymer chain bending in cPFT2, which is further supported by theoretical calculations, and propose an explanation for the observed inverted optical activity.

Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.

Mueller Matrix Polarimetry on Cyanine Dye J-Aggregates.

Cyanine dyes are known to form H- and J-aggregates in aqueous solutions. Here we show that the cyanine dye, S0271, assembles in water into vortex induced chiral J-aggregates. The chirality of the J-aggregates depends on the directionality of the vortex. This study utilised both conventional benchtop CD spectropolarimeters and Mueller matrix polarimetry. It was found that J-aggregates have real chirality alongside linear dichroism and linear and circular birefringence. We identify the factors that are key to the formation of metastable chiral J-aggregates and propose a mechanism for their assembly.

Self-assembly of chiral diketopyrrolopyrrole chromophores giving supramolecular chains in monolayers and twisted microtapes.

Chiral diketopyrrolopyrroles appended with enantiomeric ethyl lactate functions through an ether linkage to the aryl backbone of the chromophore were synthesized via the Mitsunobu reaction. The molecules have good solubility and excellent optical properties, high molar absorption coefficients, and fluorescence quantum yields. Helical aggregates with circular dichroism arising from the supramolecular arrangement are seen in both solution and thin films, and the aggregates also display circularly polarized luminescence (glum  ≈ ±0.1). The molecules assemble to give monolayers on graphite and precipitate from solution forming supramolecular twisted tapes hundreds of microns long.

Modulating the poly-L-lysine structure through the control of the protonation-deprotonation state of L-lysine.

Designing the architecture of L-lysine-based polymeric structures is a highly challenging task that requires careful control of the amino acid reactive groups. Conventional processes to obtain branched polylysine need several steps and the addition of specific catalysts. In the present work, to gain a better understanding and control of the formation of L-lysine-based polymers, we have investigated the correlation between the protonation state of L-lysine and the corresponding hydrothermally grown structures. The samples have been characterized by combining optical spectroscopies, such as UV-Vis, fluorescence, and synchrotron radiation circular dichroism with structural analysis by Nuclear Magnetic Resonance, Fourier Transform Infrared spectroscopy, and dynamic light scattering. We have observed that aqueous precursor solutions with alkaline pHs promote the formation of branched structures. In contrast, high pHs favour the reactivity of the ε-amino groups leading to linear structures, as shown by circular dichroism analyses. On the other hand, acidic conditions trigger the branching of the amino acid. Interestingly, the polymeric forms of L-lysine emit in the blue because the increasing number of intermolecular hydrogen bonds promote the intermolecular charge transfer responsible for the emission. Understanding the correlation between the L-lysine charged states and the polymeric structures that could form controlling the protonation-deprotonation states of the amino acid opens the route to a refined design of polypeptide systems based on L-lysine.

A conformational switch controlling the toxicity of the prion protein.

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and ß2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.

Effect of Trehalose and Ceftriaxone on the Stability of Aggregating-Prone Tau Peptide Containing PHF6* Sequence: An SRCD Study.

The tau protein, a soluble protein associated with microtubules, which is involved in the assembly and stabilization of cytoskeletal elements, was found to form neurofibrillary tangles in different neurodegenerative diseases. Insoluble tau aggregates were observed to be organized in paired helical filaments (PHFs) and straight filaments (SFs). Recently, two small sequences (306-311 and 275-280) in the microtubule-binding region (MTBR), named PHF6 and PHF6*, respectively, were found to be essential for tau aggregation. Since a possible therapeutic approach consists of impairing amyloid formation either by stabilizing the native proteins or reducing the level of amyloid precursors, here we use synchrotron radiation circular dichroism (SRCD) at Diamond B23 beamline to evaluate the inhibitory effects of two small molecules, trehalose and ceftriaxone, against the aggregation of a small peptide containing the PHF6* sequence. Our results indicate that both these molecules, ceftriaxone and trehalose, increased the stability of the peptide toward aggregation, in particular that induced by heparin. With trehalose being present in many fruits, vegetables, algae and processed foods, these results support the need to investigate whether a diet richer in trehalose might exert a protective effect toward pathologies linked to protein misfolding.

Crystal structure and metal binding properties of the periplasmic iron component EfeM from Pseudomonas syringae EfeUOB/M iron-transport system.

EfeUOB/M has been characterised in Pseudomonas syringae pathovar. syringae as a novel type of ferrous-iron transporter, consisting of an inner-membrane protein (EfeUPsy) and three periplasmic proteins (EfeOPsy, EfeMPsy and EfeBPsy). The role of an iron permease and peroxidase function has been identified for the EfeU and EfeB proteins, respectively, but the role of EfeO/M remains unclear. EfeMPsy is an 'M75-only' EfeO-like protein with a C-terminal peptidase-M75 domain (EfeOII/EfeM family). Herein, we report the 1.6 Å resolution crystal structure of EfeMPsy, the first structural report for an EfeM component of P. syringae pv. syringae. The structure possesses the bi-lobate architecture found in other bacterial periplasmic substrate/solute binding proteins. Metal binding studies, using SRCD and ICP-OES, reveal a preference of EfeMPsy for copper, iron and zinc. This work provides detailed knowledge of the structural scaffold, the metal site geometry, and the divalent metal binding potential of EfeM. This work provides crucial underpinning for a more detailed understanding of the role of EfeM/EfeO proteins and the peptidase-M75 domains in EfeUOB/M iron uptake systems in bacteria.

Imaging deposition-dependent supramolecular chiral organisation.

Thin films of a chiral diketopyrrolopyrrole derivative were imaged with spatially-defined Mueller Matrix Polarimetry, focussing on the Circular Dichroism signal, giving unique insight into the impact that deposition techniques and thermal annealing can have on chiral supramolecular structures in the solid state, where homogeneity was observed for spun-coated films while drop-coating afforded chiroptical diversity in the material, a feature invisible to absorption spectroscopy or optical microscopy.

Self-assembly of Gold Nanoparticles into an Adjustable Plasmonic 3D Lattice using Janus-type Forked Mesogenic Ligands.

We report the formation of a 3D body-centred self-assembled superlattice of gold nanoparticles whose interparticle gap, and hence its plasmonic properties, are adjustable exclusively in the xy-plane. Thus, even though the particles are spherical, their anisotropic packing generates tailorable plasmonic dichroism. The gold nanoparticles are coated with forked ligands containing two mesogens: either two cholesterols ("twin"), one cholesterol and one azobenzene ("Janus"), or a mixture of the two. Beside the body-centered arrangement of gold nanoparticles, the structure also contains unusual two-dimensionally x-y dual undulated (eggbox-like) smectic-like layers of mesogens. Moreover, the presence of azobenzene mesogens allows the superlattice to be melted through UV-induced photo-isomerization; the process is reversible displaying low fatigue on repeated cycling.

Modelling the adsorption of proteins to nanoparticles at the solid-liquid interface.

HYPOTHESIS: We developed a geometrical model to determine the theoretical maximum number of proteins that can pack as a monolayer surrounding a spherical nanoparticle. We applied our new model to study the adsorption of receptor binding domain (RBD) of the SARS-CoV-2 spike protein to silica nanoparticles. Due to its abundance and extensive use in manufacturing, silica represents a reservoir where the virus can accumulate. It is therefore important to study the adsorption and the persistence of viral components on inanimate surfaces. EXPERIMENTS: We used previously published datasets of nanoparticle-adsorbed proteins to validate the new model. We then used integrated experimental methods and Molecular Dynamics (MD) simulations to characterise binding of the RBD to silica nanoparticles and the effect of such binding on RBD structure. FINDINGS: The new model showed excellent fit with existing datasets and, combined to new RBD-silica nanoparticles binding data, revealed a surface occupancy of 32% with respect to the maximum RBD packing theoretically achievable. Up to 25% of RBD's secondary structures undergo conformational changes as a consequence of adsorption onto silica nanoparticles. Our findings will help developing a better understanding of the principles governing interaction of proteins with surfaces and can contribute to control the spread of SARS-CoV-2 through contaminated objects.

Electronic Circular Dichroism Imaging (ECDi) Casts a New Light on the Origin of Solid-State Chiroptical Properties.

Solid-state ECD (ss-ECD) spectra of a model microcrystalline solid, finasteride, dispersed into a KCl pellet were recorded by using the synchrotron radiation source at the Diamond B23 beamline. Scanning a surface of 36 mm2 with a step of 0.5 mm, we measured a set of ECD imaging (ECDi) spectra very different from each other and from the ss-ECD recorded with a bench-top instrument (1 cm2 area). This is due to the anisotropic part of the ECD (ACD), which averages to zero in solution or on a large number of randomly oriented crystallites, but can otherwise be extremely large. Two-way singular value decomposition (SVD) analysis, through experimental and simulated TDDFT spectra, disclosed that the measured and theoretical principal components are in line with each other. This finding demonstrates that the observed isotropic ss-ECD spectrum is governed by the anisotropy of locally oriented crystals. It also introduces a new quality for ss-ECD measurements and opens a new future for probing and mapping chiral materials in the solid state such as active pharmaceutical ingredients (APIs).

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution.

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Interaction of a Short Peptide with G-Quadruplex-Forming Sequences: An SRCD and CD Study.

G-quadruplex (G4) forming DNA sequences were recently found to play a crucial role in the regulation of genomic processes such as replication, transcription and translation, also related to serious diseases. Therefore, systems capable of controlling DNA and RNA G-quadruplex structures would be useful for the modulation of various cellular events. In particular, peptides represent good candidates for targeting G-quadruplex structures, since they are easily tailored to enhance their functionality. In this work, we analyzed, by circular dichroism and synchrotron radiation circular dichroism spectroscopies, the interaction of a 25-residue peptide deriving from RHAU helicases (Rhau25) with three G-quadruplex-forming oligonucleotide sequences, in both sodium- and potassium-containing buffers, the most relevant monovalent cations in physiological conditions. The peptide displayed greater affinity for the G4 sequences adopting a parallel structure. However, it showed the ability to also interact with antiparallel or hybrid G-quadruplex structures, inducing a conformation conversion to the parallel structure. The stability of the oligonucleotide structure alone or in presence of the Rhau25 peptide was studied by temperature melting and UV denaturation experiments, and the data showed that the interaction with the peptide stabilized the conformation of oligonucleotide sequences when subjected to stress conditions.