The atomic surface site interaction point description of fluid phase non-covalent interactions was implemented in a temperature-dependent version of SSIMPLE to predict vapour–liquid equilibria for 171 pure compounds and for 196 binary mixtures.

Organic compounds based on 1,3,5-triazine scaffolds are utilised in practical applications in agriculture, as well as in the pharmaceutical and plastic industries. In fundamental research, 1,3,5-triazines are used as building blocks for the construction of oligomers that are relevant to the areas of supramolecular chemistry, chemical biology and polymer science. Here, we review the molecular recognition and self-assembly properties of monodisperse linear and branched oligomers, macrocycles, and dendrimers of 1,3,5-triazine. We focus mainly on experimental studies conducted in solution, describing the key interactions and structural features of these systems.

Cooperativity between interactions in H-bonded networks can increase the strengths of H-bonds involving hydroxyl groups by up to 50%. The effect of changing the geometry of an intramolecular hydroxyl-hydroxyl H-bond on cooperativity with an intermolecular hydroxyl·quinuclidine H-bond was quantified by comparing the H-bonding properties of a series of hydroxycresols with the corresponding series of bisphenols. In the hydroxycresols, the intramolecular H-bond forms a 6-membered ring, and X-ray crystallography showed that the H-bond is distorted away from the ideal linear O-H⋯O geometry by up to 35°. In the bisphenols, the intramolecular H-bond forms an 8-membered ring, and the geometry is close to ideal, with the OH bond of the donor pointing directly at the lone pair of the acceptor. The presence of the intramolecular H-bonding interactions in solution was confirmed using 1H NMR spectroscopy, and NMR titrations were used to measure the association constants for formation 1 : 1 complexes with quinuclidine in n-octane. Compared with the non-cooperative H-bond formed by benzyl alcohol with quinuclidine, the strength of the intermolecular H-bond formed by the hydroxycresols increased by between -8 kJ mol-1 and -14 kJ mol-1, depending on the substituent para to the phenol OH donor. Electron-withdrawing substituents make the phenol a better H-bond donor, and the increase in the strength of the intramolecular H-bond leads to an increase in the strength of the intermolecular H-bond with quinuclidine. For the bisphenols, the cooperative effects were very similar: the substituent effects were practically identical, and the presence of the intramolecular H-bond increased the strength of the intermolecular interaction by between -10 kJ mol-1 and -16 kJ mol-1. The results show that cooperativity in H-bonded networks depends strongly on the polarity of the interacting groups but is relatively insensitive to the precise geometric arrangement.

The free energy contribution due to cooperativity between an n–π* interaction and a H-bond was found to be within the experimental error, suggesting that associated contributions to the stability of folded proteins are small.

Protein aggregates are promising biomarkers for early diagnosis of neurodegenerative disorders. Single-Molecule Array (SiMoA) is a powerful method to detect these aggregates at ultra-low concentrations in biofluids. Herein, we report a next-generation SiMoA assay using chemically synthesized small molecules, rather than antibodies, to capture alpha-synuclein aggregates, a protein hallmark in Parkinson's Disease and other synucleinopathies. These small molecule-based capturing agents contain aggregate-binding head groups, and a backbone functionalized with a primary amine for bead conjugation in the SiMoA assay. The most promising molecule, BF-79-2, captured recombinant alpha-synuclein aggregates, specifically excluding monomers, at picomolar concentrations. BF-79-2 also captured alpha-synuclein aggregates in human blood. Replacing antibodies with small molecules as capturing agents on the SiMoA platform enhances the assay versatility, since small molecules can be screened in silico and synthesized without laborious molecular biology techniques. The application of small molecules as capturing agents broadens the capabilities of the SiMoA platform, rendering it more adaptable for biomarker discovery and disease diagnostics.

Recognition-encoded melamine oligomers (REMO) are synthetic polymers composed of repeating triazine-piperazine units and equipped with phenol and phosphine oxide side-chains. Short oligomers have previously been shown to form length- and sequence-selective H-bonded duplexes in non-polar solvents. Here, automated solid phase synthesis was used to prepare homo-sequence REMO with either twelve phenol recognition units or twelve phosphine oxide recognition units. The ends of the oligomers were functionalised with an azide and an alkyne group to allow investigation of duplex formation by covalent trapping with copper-catalysed azide-alkyne cycloaddition (CuAAC) reactions. The oligomers were also functionalised with a dansyl fluorophore or a dabcyl quencher dye to allow investigation of duplex formation by Förster resonance energy transfer (FRET). Covalent trapping showed that the duplex is the major species present in a 1 : 1 mixture of the phenol 12-mer and phosphine oxide 12-mer at micromolar concentrations in dichloromethane. FRET titration experiments showed that the association constant for duplex formation is greater than 108 M-1 in chloroform, and DMSO denaturation experiments showed that duplex formation is highly cooperative. The Hill coefficient for denaturation of the 12-mer duplex was 4.6, which is significantly higher than the value measured for the corresponding 6-mer duplex (1.9). This behaviour mirrors that observed for nucleic acid duplexes, where denaturation becomes increasingly cooperative as more base-pairs are added to the duplex.

Recognition-encoded melamine oligomers (REMO) are composed of an alternating piperazine-triazine backbone and side-chains equipped with either a H-bond donor (phenol, D) or a H-bond acceptor (phosphine oxide, A). Complementary homo-oligomers form stable duplexes in organic solvents, due to intermolecular base-pairing interactions between the phenol and phosphine oxide side-chains. For mixed-sequence oligomers, the major pathway that competes with duplex formation is folding due to intramolecular base-pairing interactions. Automated solid phase synthesis was used to prepare the self-complementary REMO DADA, and this oligomer was used to investigate the competition between intermolecular and intramolecular H-bonding interactions. Isothermal titration calorimetry in chloroform showed that DADA forms a dimeric complex, but with reduced stability compared with the duplexes formed by shorter oligomers. The results indicate that a folded state with intramolecular interactions between the two terminal recognition units is significantly populated. The dimeric complex formed at higher concentrations could involve the interaction of two folded oligomers in a kissing stem-loops structure, or the oligomer could unfold to give the duplex with four intermolecular base-pairs. One end of the oligomer was equipped with an azide and the other with an alkyne, so that the dimeric complex could be covalently trapped using copper-catalysed azide-alkyne cycloaddition reactions. The major product was the macrocyclic duplex with small amounts of the macrocyclic single-strand, which shows that the DADA·DADA duplex dominates at millimolar concentrations. Understanding the propensity of the REMO architecture to fold will help guide the future design principles for synthesis of more complex functional assemblies.

The templated ligation of DNA oligomers allows quantitative coupling reactions at very low concentrations and with very high selectivity. Recognition-encoded melamine oligomers (REMO) also form sequence-selective duplexes, and it should therefore be possible to ligate them in the same way. REMO duplex formation is based on H-bonding interactions between phosphine oxide (A) and 4-nitrophenol (D) recognition units, so two DDD oligomers could be ligated using an AAAOAAA template (where O represents a blank recognition unit). Two different types of chemistry were investigated: SNAr coupling of a piperazine with a dichlorotriazine, and CuAAC coupling of an azide and an alkyne. Quantitative templated ligation was observed in the presence of competing reagents, whereas statistical mixtures of different products were obtained in the absence of template. The effective molarities for the intramolecular reaction between the two substrates bound to the template are 5 mM for the SNAr coupling and 3 mM for the CuAAC coupling, which means that template-directed REMO ligations at micromolar concentrations give quantitative yields with very high selectivity in the presence of large amounts of competing reactants.

Replication of molecular information in nature is based on the synthesis of the backbone of the copy strand by polymerization of monomers bound to a template. An alternative strategy is to use a preassembled polymer backbone devoid of sequence information as the copy strand and to attach side chains in a sequence determined by binding to a template, i.e., base-filling. Base-filling strategies were investigated for template-directed synthesis of recognition-encoded melamine oligomers (REMO) using H-bond base-pairing interactions between 4-nitrophenol and phosphine oxide side chains. A template with three 4-nitrophenol H-bond donor recognition units was used with a blank copy strand equipped with three aldehyde groups for the reversible attachment of amine recognition units via dynamic imine chemistry. Equilibration of the template and blank strands in dichloromethane in the presence of benzylamine and a phosphine oxide recognition unit equipped with an amine resulted in selective incorporation (79%) of the phosphine oxide recognition unit into the resulting copy strand. Covalent attachment of the blank strand to the template with a diester linker increased the selectivity of the base-filling process to 85%, and carrying out the experiment in toluene further increased the selectivity to 92%. The imines in the copy strand were trapped by reduction, and cleavage of the ester linkages allowed recovery of the template strand along with the kinetically stable tris-phosphine oxide copy. Fidelity of templating is determined by the concentration of the template strand, the association constant for the base-pairing interaction, and the effective molarities of the intramolecular interactions in the duplex.

H-bonding interactions in networks are stabilised by cooperativity, but the relationship between the chemical structures of the interacting functional groups and the thermodynamic consequences is not well-understood. We have used compounds with an intramolecular H-bond between a pyridine H-bond acceptor and an amide NH group to quantify cooperative effects on the H-bond acceptor properties of the amide carbonyl group. 1H NMR experiments in n-octane confirm the presence of the intramolecular H-bond and show that this interaction is intact in the 1 : 1 complex formed with perfluoro-tert-butanol (PFTB). UV-vis absorption titrations were used to measure the relationship between the association constant for formation of this complex and the H-bond acceptor properties of the pyridine involved in the intramolecular H-bond. Electron-donating substituents on the pyridine increase the strength of the intermolecular H-bond between PFTB and the amide. There is a linear relationship between the H-bond acceptor parameter β measured for the amide carbonyl group and the H-bond acceptor parameter for the pyridine. The cooperativity parameter κ determined from this relationship is 0.2, i.e. β for an amide carbonyl group is increased by one fifth of the value of β of an acceptor that interacts with the NH group. This result is reproduced by DFT calculations of H-bond parameters for the individual molecules in the gas phase, which implies that the observed cooperativity can be understood as polarisation of the electron density in the amide π-system in response to formation of a H-bond. The cooperativity parameter κ measured for the secondary amide H-bond donor and H-bond acceptor is identical, which implies that polarisation of an amide mediates the interaction between an external donor or acceptor in a reciprocal manner.

Musketeer is a powerful open-source software tool for the analysis of titration data, featuring a simple cross-platform graphical interface for importing data directly from UV-vis, fluorescence and NMR spectrometers, or from spreadsheets. The fast data analysis algorithm can be used to obtain equilibrium constants for simple binding isotherms, as well as for more complicated systems with multiple competing equilibria. Applications of Musketeer for the analysis of a range of different supramolecular and biomolecular systems are illustrated, including titrations with multiple spectroscopically active species, competitive binding assays, denaturation experiments, optimisation of concentrations as variables. The software also includes a number of tools that can be used to select the binding isotherm that represents the best model to describe a dataset.

Molecular electrostatic potential surfaces (MEPS) calculated using density functional theory have been used to develop a simplified description of the non-covalent interaction properties of organic molecules. The Atomic Interaction Point (AIP) model introduced here represents an evolution of the Surface Site Interaction Point (SSIP) model described previously, in which a molecule is represented by a discrete set of interaction points that define sites of interaction with other molecules. The interaction sites are described by interaction parameters that are equivalent to the experimentally determined H-bond donor and acceptor parameters α and β. By using high electron density MEPS that lie inside the van der Waals surface, it is possible to obtain accurate interaction parameters and locations for polar sites (s-holes, H-bond donors and acceptors), which are identified as local maxima and minima on the MEPS. For non-polar sites that represent π-systems and halogens, an approach based on molecular orbitals was used to assign the locations of the AIPs, and the interaction parameters were obtained using a lower electron density MEPS that lies close to the van der Waals surface. The AIP descriptions can be implemented directly in the Surface Site Interaction Point Model for Liquids at Equilibrium (SSIMPLE) to calculate solvation free energies, and the free energy of transfer of 1504 compounds from n-hexadecane to water was predicted with a root mean square error of 5 kJ mol-1. AIPs also provide a useful tool for mapping non-covalent interactions in intermolecular complexes, and examples are provided showing how X-ray crystal structures can be converted into AIP interaction maps that allow quantification of the free energy contributions of both polar and non-polar interactions to the stabilities of complexes in solution.

Supporting Methods Supporting Figures 1-11 Supporting Tables 1-3 Supporting Figure 1. Synthesis route for the smart, targeted hydrogen peroxide probe. Reagents and conditions: (a) 4-Bromo-1-butyne, NaI, MeCN, reflux. (b) POCl3, DMF. (c) Aniline, EtOH. (d) NaOAc, EtOH, reflux. (e) Piperazine, Cs2CO3, MeCN. (f) 1-Azido-1-deoxy-β-D-glucopyranoside, CuSO4, Na-ascorbate, t-BuOH, H2O. (g) 4-Nitrophenylchloroformate, Pyridine, DCM. (h) 4, DIPEA, DMF. (i) 1-Azido-1-deoxy-β-D-glucopyranoside, Cu(CH3CN)4, TBTA, ACN. Supporting Figure 2. Calibration of the LC-MS/MS study. This study was performed in multiple reaction monitoring mode (MRM-MS). (A) Chemical structure of JW35mono which is used as internal standard (IS) for MRM-MS analysis. Standard calibration curves of JW41 and JW35 prepared in tumor or liver matrix were extracted for ex vivo quantification of JW35 and JW41total by MRM-MS. The signal ratio of the peak area of JW35 to the peak area of the IS versus the JW35 concentration (ï­M) is plotted in (B); the peak area of JW41total versus its concentration (ï­M) in (C). For each study a new calibration curve was acquired before and after running the samples. To validate the calibration line and the status of the analytical system, six quality control (QC) samples at three known concentrations were run before and after the biological samples. One representative calibration curve together with the QC samples are shown for JW35 and JW41total in (B) and (C) respectively. The colored symbols represent values obtained from the calibration samples, the crosses represent values obtained from the QC samples run before and after the biological samples, validating the calibration curve. The boronate groups of JW41 are partially hydrolyzed to give JW41hydro by the aqueous and acidic conditions required for the LC-MS/MS analysis. The linearity curves shown in (D) suggest that the loss of the concentration of JW41 is compensated by the gain in concentration of JW41hydro. Therefore, the peak area of the MRM transitions of JW41 and JW41hydro were added together to JW41total, which was used for the analysis. (E) Calculated total concentration of JW41total and JW35 24 h after injection of JW41 / JW41:JW35. The results of 4 tumors where the mice received an injection of JW41 alone and 3 tumors where mice received an injection of 2:1 JW41:JW35 solution are shown. Note these values cannot be interpreted as absolute due to the aforementioned hydrolysis challenges. Supporting Figure 3. LC-MS/MS chromatograms of JW41 (75 nM), JW35 (38 nM) and the internal standard IS (JW35Mono, 190 nM). Representative ion chromatogram of JW41, JW41hydro, JW35 and the IS are depicted in (A). Fragmentation of the parent compounds JW41 (m/z=1379.67), JW41hydro (m/z=1297.61), JW35 (m/z=560.30) and IS (m/z=914.48), are depicted in (B), (D), (F) and (H), respectively. Multiple reaction monitoring MRM-MS peaks for JW41 (monitoring m/z=1379.67 transition to m/z=913.48), JW41hydro (monitoring m/z=1297.60 transition to m/z=831.39), JW35 (monitoring m/z=560.30 transition to m/z=208.10) and IS (monitoring m/z=914.48 transition to m/z=161.11) are shown in (C), (E), (G) and (I), respectively. Supporting Figure 4. Linker evaluation. (A) Synthesis of the different dyes for linker evaluation studies (JW7/8/11/13/14/16/17). Reagents and conditions: (a) III, N-(4-hydroxyphenyl)acetamide, K2CO3, MeCN(anh). (b) tert-Butyl(4-hydroxyphenyl)carbamate, prepared from 4-aminophenol, di-tert-butyl-dicarbonate, THF(anh). (c) III, TEA, MeCN(anh) (d) JW11, TFA, DCM(anh). (e) III, piperazine, TEA, MeCN(anh). (f) JW8, imidazole, acetyl chloride, THF(anh). (g) JW8, di-tert-butyl dicarbonate, TEA, THF(anh). (h) JW8, TEA, trimethylacteyl chloride, THF(anh). To find an efficient linker unit able to elicit a shift in the optical properties of the dye upon reaction with H2O2 we explored benzene-1,4-diamine (B) and piperazine (C) as potential linker structures with different steric and electric nitrogen modifications. Representative, normalized absorbance spectra of the different dye products (solvent 15 μM in MeOH) are shown. The results indicate that 4-aminophenol is not suitable as a linker for our purpose since no significant absorption shift is observed by derivatizing the nitrogen, whereas the piperazine-derivatives features promising characteristics with absorption spectra sensitive to sterically and electrical modifications on the nitrogen. Supporting Figure 5. Theoretical investigation of the effect of the different linker units towards the dye backbone. The minimum-energy conformations of JW13 (A), JW11 (B), JW35 (C) and JW41 (D) were obtained through geometry optimization in the gas phase using density functional theory. To quantify the relative orientation of the two terminal polycyclic moieties, a dihedral angle was defined as shown in the picture (defined with the main axis across the central aliphatic chain and terminating in the two distal benzene rings) and measured on the optimized structures. Supporting Figure 5. Theoretical investigation of the effect of the different linker units towards the dye backbone. The minimum-energy conformations of JW13 (A), JW11 (B), JW35 (C) and JW41 (D) were obtained through geometry optimization in the gas phase using density functional theory. To quantify the relative orientation of the two terminal polycyclic moieties, a dihedral angle was defined as shown in the picture (defined with the main axis across the central aliphatic chain and terminating in the two distal benzene rings) and measured on the optimized structures. Supporting Figure 7. TPA assay to investigate GLUT specific uptake. Pharmacological activation of glucose transport in (A) MDA-MB-231 and (B) MCF7 cells was induced with TPA (13 μM) 1 h prior dye addition and led to increased uptake of JW41 and the positive control IR800CW-2DG but not of the negative control IR800CW. Statistical significance was assessed by unpaired 2 tailed t-test; *p<0.05, **p<0.01,****p<0.0001 Supporting Figure 8. In vivo characterization of 2:1 JW41:JW35 mixture in subcutaneous MDA-MB-231 tumors in nude mice. A) Representative PAI slice through MDA-MB-231 tumors (top row) and kidney, spleen and liver (bottom row) before and 15-20 min after injection of the 2:1 JW41-JW35 mixture. The regions of interest (ROIs) are indicated with white borders (LT = left tumor, RT = right tumor, L = liver, S = spleen, K = kidney, C = control region). The signal of JW41 is shown in yellow, the signal of JW35 in magenta. (B) Changes in the PA spectra and an increase in the PA signal between 700 and 810 nm were detected in the ROIs upon injection of the mixture. Representative graphs for tumor (top row, n = 4) and liver (bottom row, n = 2) are presented. (C) The time course of the total probe signal at the isosbestic point of the capped and uncapped probe up to 24 h after injection. (D) Fluorescence images before, 1 h and 24 h post injection of the JW41-JW35 mixture. Fluorescence in vivo imaging results support the PA findings indicating selective in vivo accumulation of the new probe in subcutaneous MDA-MB-231 tumors and the liver. (E) The specific increase in radiant efficiency in the left tumor and liver upon injection of the dye mixture (values from the control region, C, were used for background correction). Only the signal of the left tumor is considered since the position of the right tumor was hard accessible for the fluorescence imaging and the signal was mixed with the liver signal. (F) PA signal change at 765nm over time in the control regions confirming that the PA signal kinetic data (illustrated in C) are specific to the organs of interest. Supporting Figure 9. In vivo PA signal from control regions following injection of JW41 alone. ROIs illustrated in main text Figure 5. Supporting Figure 10. Time course of the in vivo PA signals of JW41 and JW35 derived using spectral unmixing. (A) JW41 PA signal time course following injection of JW41 alone. (B) JW41 PA signal time course following injection of the 2:1 mixture of JW41:JW35; (C) JW35 PA signal time course following injection of JW41 alone. (D) JW35 PA signal time course following injection of the 2:1 mixture of JW41:JW35. Background subtraction was performed in both cases by averaging the values obtained before injection (first 3 time points) and subtracting the average. Signals for the injection of JW41 alone are from ROIs drawn in main text Figure 5; signals for the injection of the mixture of JW41:JW35 are from ROIs drawn in Supplementary Figure 8. Supporting Figure 11. Ex vivo characterization of in vivo studies with JW35 alone and 2:1 JW41-JW35 mixture. (A) Representative H&E stained section of frozen MDA-MB-231 tumor from mouse treated with JW35 alone and (B) 2:1 JW41-JW35 mixture. The necrotic area is indicated with the white line. Representative widefield fluorescence image of a consecutive tumor section after formaldehyde fixation and staining with DAPI (blue) are shown to the right in both cases. Magnification of the widefield image allowing the localization of the dye signal into the cytosols of the cells. Supporting Table 1. Table of photophysical properties of JW41 and JW35. These were obtained from bulk UV-vis absorption and fluorescence measurements of the dyes. Supporting Table 2. Spectral unmixing studies of JW41 and JW35 in tissue-mimicking phantoms. Supporting Table 3. Concentrations of standard and QC samples for LC-MS/MS analysis.

Dissipative particle dynamics (DPD) simulations of nonionic surfactants with an added salt show that the Setschenow relationship is reproduced; that is, the critical micelle concentration is log-linearly dependent on the added salt concentration. The simulated Setschenow coefficients depend on the DPD bead-bead repulsion amplitudes, and matching to the experimentally determined values provides a systematic method to parameterize the interactions between salt ion beads and surfactant beads. The optimized ion-specific interaction parameters appear to be transferrable and follow the same trends as the empirical Hofmeister series.

Template-directed synthesis of nucleic acids in the polymerase chain reaction is based on the use of a primer, which is elongated in the replication process. The attachment of a high affinity primer to the end of a template chain has been implemented for templating the synthesis of triazole oligomers. A covalent ester base-pair was used to attach a primer to a mixed sequence template. The resulting primed template has phenol recognition units on the template, which can form noncovalent base-pairs with phosphine oxide monomers via H-bonding, and an alkyne group on the primer, which can react with the azide group on a phosphine oxide monomer. Competition reactions between azides bearing phosphine oxide and phenol recognition groups were used to demonstrate a substantial template effect, due to H-bonding interactions between the phenols on the template and phosphine oxides on the azide. The largest rate acceleration was observed when a phosphine oxide 2-mer was used, because this compound binds to the template with a higher affinity than compounds that can only make one H-bond. The 31P NMR spectrum of the product duplex shows that the H-bonds responsible for the template effect are present in the product, and this result indicates that the covalent ester base-pairs and noncovalent H-bonded base-pairs developed here are geometrically compatible. Following the templated reaction, it is possible to regenerate the template and liberate the copy strand by hydrolysis of the ester base-pair used to attach the primer, thus completing a formal replication cycle.

Formation of a H-bond with an amide carbonyl oxygen atom increases the strength of subsequent H-bonds formed by the amide NH, due to polarisation of the bond. The magnitude of this effect has been quantified by measuring association constants for the formation of 1 : 1 complexes of 2-hydroxylbenzamides with tri-n-butyl phosphine oxide. In 2-hydroxybenzamides, there is an intramolecular H-bond between the phenol OH group and the carbonyl oxygen atom. Comparison of the association constants measured for compounds with and without the 2-hydroxy group allows direct quantification of the effect of the intramolecular H-bond on the H-bond donor properties of the amide NH group. Substituents were used to modulate the strength of the intramolecular and intermolecular H-bonds. The presence of an intramolecular H-bond increases the strength of the intermolecular H-bond by more than one order of magnitude in n-octane solution. The increase in the H-bond donor parameter used to describe the amide NH group is directly proportional to the H-bond donor parameter of the phenol OH group that makes the intramolecular H-bond. These polarisation effects will lead to substantial cooperativity in complex systems that feature networks of non-covalent interactions, and the measurements described here provide a quantitative basis for understanding such phenomena.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The self-assembly of a unique molecular container is reported: a hybrid hydrogen-bonded/metal-coordinated cage where both hydrogen-bonding and metal-coordination form the crucial part of the topology. The hybrid cage was prepared combining hydrogen-bonded rosette motif and palladium(II)/platinum(II) coordination to a pyridine ligand. It was also shown that the hybrid cage could be prepared by integrative self-sorting from simple components. For the first time the genuine dual character of the hybrid cage was manifested as both self-assembling parts responded selectively to different stimuli (such as phosphine and cyanurate), which resulted in the disassembly of the cage.

Thioflavin-T is used to image amyloid aggregates because of the excellent turn-on fluorescence properties, but binding affinities are low. By mounting multiple dye units on the surface of a vesicle, the binding affinity for α-synuclein fibrils is increased by three orders of magnitude, and the optical response is increased. Cooperative interactions of the dye headgroup and lipid with the protein provide a general strategy for the construction of multivalent amyloid probes based on vesicles.

A key process in the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases is the aggregation of proteins to produce fibrillary aggregates with a cross β-sheet structure, amyloid. The development of reagents that can bind these aggregates with high affinity and selectivity has potential for early disease diagnosis. By linking two benzothiazole aniline (BTA) head groups with different length polyethylene glycol (PEG) spacers, fluorescent probes that bind amyloid fibrils with low nanomolar affinity have been obtained. Dissociation constants measured for interaction with Aβ, α-synuclein and tau fibrils show that the length of the linker determines binding affinity and selectivity. These compounds were successfully used to image α-synuclein aggregates in vitro and in the post-mortem brain tissue of patients with Parkinson's disease. The results demonstrate that multivalent ligands offer a powerful approach to obtain high affinity, selective reagents to bind the fibrillary aggregates that form in neurodegenerative disease.