Teaching old indicators even more tricks: binding affinity measurements with the guest-displacement assay (GDA)

A simple change has important consequences: the guest-displacement assay (GDA) is introduced which allows for binding affinity determinations of supramolecular complexes with spectroscopically silent hosts and guests. GDA is complementary to indicator-displacement assay for affinity measurements with soluble components, but is superior for insoluble or for weakly binding guests

Analyte sensing with unselectively binding synthetic receptors: virtues of time-resolved supramolecular assays

The cross-reactivity to many analytes is one major limitation of most synthetic receptors (SRs) known so far. Herein, we show that through time-resolved competitive binding assays, even unselectively binding SRs can be utilized for analyte distinction and quantification. Furthermore, our methodology has also been applied to analyte mixtures and can be used in a microplate format

Rituximab-Containing Treatment Regimens May Imply a Long-Term Risk for Difficult-To-Treat Chronic Hepatitis E

Hepatitis E virus (HEV) infection is an emerging disease in industrialized countries which is usually characterized by a self-limited course. However, there is an increased risk of HEV persistence in immunocompromised risk populations, comprising patients following solid organ transplantation or hematological malignancies. Recently, chronic HEV infection following rituximab-containing treatment regimens has been described. Here we report five patients with chronic hepatitis E after prior rituximab therapy for various indications. We determined the immunological characteristics of these patients and analyzed the development of ribavirin (RBV) treatment failure-associated mutations in the HEV genome. One patient became chronically HEV-infected 110 months after administration of rituximab (RTX). Immunological characterization revealed that all patients exhibited significant hypogammaglobulinemia and CD4+ T cell lymphopenia. One patient permanently cleared HEV following weight-based ribavirin treatment while three patients failed to reach a sustained virological response. In depth mutational analysis confirmed the presence of specific mutations associated with RBV treatment failure in these patients. Our cases indicate that rituximab-containing treatment regimens might imply a relevant risk for persistent HEV infection even years after the last rituximab application. Moreover, we provide further evidence to prior observations suggesting that chronically HEV infected patients following RTX-containing treatment regimens might be difficult to treat

Kinetics and Mechanism of Cation‐Induced Guest Release from Cucurbit[7]uril

Abstract: The release of two organic guests from cucurbit[7]uril (CB7) was selectively monitored by the stoppedflow method in aqueous solutions of inorganic salts to reveal the mechanistic picture in detail. Two contrasting mechanisms were identified: The symmetric dicationic 2,7- dimethyldiazapyrenium shows a cation-independent complex dissociation mechanism coupled to deceleration of the ingression in the presence of alkali and alkaline earth cations (M$^{+n}$) due to competitive formation of CB7–M$^{+n}$ complexes. A much richer, unprecedented kinetic behaviour was observed for the ingression and egression of the monocationic and non-symmetric berberine (B$^{+}$). The formation of ternary complex B+–CB7–M$^{+n}$ was unambiguously revealed. A difference of more than two orders of magnitude was found in the equilibrium constants of Mn+ binding to B$^{+}$–CB7 inclusion complex. Large cations, such as K$^{+}$ and Ba$^{2+}$, also promoted B$^{+}$ expulsion from the ternary complex in a bimolecular process. This study reveals a previously hidden mechanistic picture and motivates systematic kinetic investigations of other host–guest systems

Kinetics and Mechanism of Cation-Induced Guest Release from Cucurbit[7]uril

The release of two organic guests from cucurbit[7]uril (CB7) was selectively monitored by the stopped-flow method in aqueous solutions of inorganic salts to reveal the mechanistic picture in detail. Two contrasting mechanisms were identified: The symmetric dicationic 2,7-dimethyldiazapyrenium shows a cation-independent complex dissociation mechanism coupled to deceleration of the ingression in the presence of alkali and alkaline earth cations (Mn+) due to competitive formation of CB7-Mn+ complexes. A much richer, unprecedented kinetic behaviour was observed for the ingression and egression of the monocationic and non-symmetric berberine (B+). The formation of ternary complex B+-CB7-Mn+ was unambiguously revealed. A difference of more than two orders of magnitude was found in the equilibrium constants of Mn+ binding to B+-CB7 inclusion complex. Large cations, such as K+ and Ba2+, also promoted B+ expulsion from the ternary complex in a bimolecular process. This study reveals a previously hidden mechanistic picture and motivates systematic kinetic investigations of other host-guest systems

Simultaneous analyte indicator binding assay (SBA) for the monitoring of reversible host–guest complexation kinetics

Very little information is available on the kinetics of the self-assembly and dissociation of optically silent building blocks despite the importance of such data in the rational design of tailor-made host–guest systems. We introduce here a novel time-resolved method that enables the simultaneous determination of complex formation and complex dissociation rate constants for inclusion-type host–guest complexes. The simultaneous analyte indicator binding assay (SBA) gives also direct access to binding affinities, thus largely simplifying the experimental procedure for a full kinetic and thermodynamic characterisation of host–guest systems

A supramolecular cucurbit[8]uril-based rotaxane chemosensor for the optical tryptophan detection in human serum and urine

Sensing small biomolecules in biofluids remains challenging for many optical chemosensors based on supramolecular host-guest interactions due to adverse interplays with salts, proteins, and other biofluid components. Instead of following the established strategy of developing alternative synthetic binders with improved affinities and selectivity, we report a molecular engineering approach that addresses this biofluid challenge. Here we introduce a cucurbit[8]uril-based rotaxane chemosensor feasible for sensing the health-relevant biomarker tryptophan at physiologically relevant concentrations, even in protein- and lipid-containing human blood serum and urine. Moreover, this chemosensor enables emission-based high-throughput screening in a microwell plate format and can be used for label-free enzymatic reaction monitoring and chirality sensing. Printed sensor chips with surface-immobilized rotaxane-microarrays are used for fluorescence microscopy imaging of tryptophan. Our system overcomes the limitations of current supramolecular host-guest chemosensors and will foster future applications of supramolecular sensors for molecular diagnostics

Cucurbit[n]uril-Immobilized Sensor Arrays for Indicator-Displacement Assays of Small Bioactive Metabolites

The patterned immobilization of chemosensors into nano/microarrays has often boosted utilization in diagnostics and environmental sensing applications. While this is a standard approach for biosensors, e.g., with antibodies, other proteins, and DNA, arraying is not yet adopted widely for supramolecular chemosensors which are still predominantly used in solution systems. Here we introduce the patterned immobilization of cucurbit[n]urils (CBn) into multiplexed microarrays and elucidate their prospects for the advancement of surface-bound indicator-displacement assays to detect small molecule analytes. The microarrays were generated by microchannel cantilever spotting of functionalized CBn and subsequent self-assembly of the corresponding indicator dyes from solution. Enhanced sensitivity of surface-bound microarrays was established in demonstrations with small bioactive metabolites (spermine, amantadine, and cadaverine) compared to bulk assays. Furthermore, the integration of the CBn/indicator microarrays into microfluidic channels provides an efficient route for real-time monitoring of the sensing process, allows easier handling, and reduces need for analyte volume. The concept was further extended to differential sensing of analytes on diplex or multiplex CBn/indicator microarrays, opening up a route for multicomponent sensing of small molecule analytes in complex liquids

Tris(4-azidophenyl)methanol - a novel and multifunctional thiol protecting group

The novel tris(4-azidophenyl)methanol, a multifunctionalisable aryl azide, is reported. The aryl azide can be used as a protecting group for thiols in peptoid synthesis and can be cleaved under mild reaction conditions via a Staudinger reduction. Moreover, the easily accessible aryl azide can be functionalised via copper-catalysed cycloaddition reactions, providing additional opportunities for materials chemistry applications.Peer reviewe

Elucidating dissociation activation energies in host–guest assemblies featuring fast exchange dynamics

The ability to mediate the kinetic properties and dissociation activation energies (E$_{a}$) of bound guests by controlling the characteristics of "supramolecular lids" in host-guest molecular systems is essential for both their design and performance. While the synthesis of such systems is well advanced, the experimental quantification of their kinetic parameters, particularly in systems experiencing fast association and dissociation dynamics, has been very difficult or impossible with the established methods at hand. Here, we demonstrate the utility of the NMR-based guest exchange saturation transfer (GEST) approach for quantifying the dissociation exchange rates (k$_{out}$)) and activation energy (E$_{a, out}$) in host-guest systems featuring fast dissociation dynamics. Our assessment of the effect of different monovalent cations on the extracted E$_{a, out}$ in cucurbit[7]uril:guest systems with very fast k$_{out}$ highlights their role as "supramolecular lids" in mediating a guest\u27s dissociation E$_{a}$. We envision that GEST could be further extended to study kinetic parameters in other supramolecular systems characterized by fast kinetic properties and to design novel switchable host-guest assemblies