Supramolecular Optical Chemosensors and Assays for Sensing of Bioactive Analytes in Water and Biofluids

The recognition and detection of biologically important analytes, especially small biomolecules, is of prime relevance and has become an upsurging area of research in chemistry and biology. Consequently, the development of robust chemical molecular sensors (“chemosensors”) based on artificial recognition elements with the potential to detect molecules with high sensitivity and selectivity and coupled with a sensitive signal transduction strategy continues to attract considerable attention. Optical methods based on fluorescence are highly desirable for signal transduction because of their versatility, high sensitivity, low cost with readily available instrumentation, and potential for real-time analysis. Thus, optical/fluorescent chemosensors, in combination with innovative assay protocols, find broad application potential in many disciplines, such as biochemistry and clinical and medical diagnostics. They offer a cost efficient alternative to conventional instrumental analytical methods, such as HPLC-MS, GC-MS, and NMR, and are superior to biosensors in terms of stability, equilibration time, price, and scope for small molecule detection. However, developing chemosensors that fully meet the requirements for practical applications is still challenging. The low binding affinity or selectivity of chemosensors for most biomolecules or their metabolites in biofluids, as well as the low stability of the chemosensor\u27s guest-host ensemble (e.g., upon dilution), are main reasons why the practical application potential of artificial chemosensors has not yet been fully realized. In this work, artificial chemosensors based on supramolecular host guest chemistry coupled with optical signal transduction are utilized to realize both detection and chirality sensing of biologically relevant analytes in aqueous media and complex biofluids. In addition, the various aspects of realizing their practical diagnostic applications are addressed. The first research project involves the development of electronic circular dichroism (ECD) based chemosensors for the detection and chirality sensing of diverse chiral organic analytes in water. Chemosensors that can detect molecular chirality are crucial due to the significance of chiral bio-relevant molecules and the influence of chirality on their related biological activity, e.g., in drug production. However, only a few chirality-based chemosensors are available to date for the detection of compounds in aqueous media. My thesis utilized achiral chromophoric hosts, i.e., acyclic cucurbit[n]urils and molecular tweezers as recognition elements in the chemosensor. The achiral chromophoric hosts were found to respond with information-rich induced ECD signals to the presence of micromolar concentrations of chiral small molecule guests, such as chiral hydrocarbons, terpenes, amino acids and their derivatives, steroids, and drugs in water. In favorable cases, this also allowed for analyte identification and reaction monitoring. In the second research project, fluorescence-detected circular dichroism (FDCD) spectroscopy is applied for the first time for the chiroptical analysis of supramolecular host guest and host protein systems and compared to the widely utilized electronic circular dichroism (ECD). The main goal was to explore the utility of FDCD to improve the sensitivity and selectivity of chiroptical supramolecular assays. The comprehensive investigations demonstrate that FDCD is an excellent choice for common supramolecular applications, e.g., the detection and chirality sensing of chiral organic analytes and label free reaction monitoring. FDCD can be conducted in favorable circumstances at much lower concentrations than ECD measurements, even in chromophoric and auto-emissive biofluids such as blood serum, overcoming the sensitivity limitation of absorbance-based chiroptical spectroscopy. Furthermore, the combined use of FDCD and ECD provided additional valuable information about the system, e.g., the chemical identity of an analyte or hidden aggregation phenomena. The third research project addresses the importance of thermodynamic and kinetic investigations to properly analyze the association and dissociation processes of supramolecular host-guest recognition interactions, which are crucial to designing host guest systems with improved properties and advancing their practical applications. However, kinetic descriptions of supramolecular systems are scarce in the literature, mainly due to the lack of suitable experimental protocols. Thus, three novel fluorescence-based time resolved approaches are introduced that allowed the convenient determination of kinetic rate constants of spectroscopically silent and even insoluble guests with the macrocyclic cucurbit[n]uril and human serum albumin as representative hosts. Furthermore, a new kinetic method is adopted to achieve selective analyte sensing even in situations of poor thermodynamic selectivity due to the host’s often observed similar binding affinities for structurally similar analytes. The method allowed a selective identification and quantification of analytes without the need to modify the parent host synthetically. The fourth research project involves the development of a novel fluorescent chemosensor for the detection of biogenic polyamines, which serve as health indicators in the human body. The fluorescent chemosensor self-assembled from sulfonated pillar[n]arene host in combination with suitable dicationic indicator dyes responds instantly with a fluorescence “turn on” signal to the presence of biogenic polyamines. The photophysical and binding properties of the new fluorescent chemosensor explored in detail in both saline buffers and biologically relevant media display their excellent functionality for polyamine sensing with no salt interferences on the sensing assay. Moreover, the chemosensor allowed the detection of biogenic polyamines down to the low micromolar concentration range in biofluids, such as urine and saliva, with good selectivity even in the presence of potential interferents present in the media. Thus, because of its simplicity, cost-effectiveness, and fast detection capabilities, the newly developed fluorescent chemosensor for polyamines will assist the future development of rapid diagnostic tests for home-use and point-of-care applications. In summary, this doctoral thesis highlights the different strategies for developing supramolecular optical chemosensors for sensitive and selective analyte detection, which are also applicable in biologically relevant media. Future research and development of sensors with improved practical applicability will contribute significantly to the advancement of analytical chemistry and biochemical/medical research

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

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

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

Teaching indicators to unravel the kinetic features of host-guest inclusion complexes

Both thermodynamic and kinetic insights are needed for a proper analysis of association and dissociation processes of host–guest interactions. However, kinetic descriptions of supramolecular systems are scarce in the literature because suitable experimental protocols are lacking. We introduce here three time-resolved methods that allow for convenient determination of kinetic rate constants of spectroscopically silent or even insoluble guests with the macrocyclic cucurbit[n]uril family and human serum albumin (HSA) protein as representative hosts

Chirality Sensing of Terpenes, Steroids, Amino Acids, Peptides and Drugs with Acyclic Cucurbit[n]urils and Molecular Tweezers

Achiral chromophoric hosts, i.e. acyclic cucurbit[ n ]urils and molecular tweezers, were found to respond with characteristic Circular Dichroism (CD) spectra to the presence of micromolar concentrations of chiral hydrocarbons, terpenes, steroids, amino acids and their derivates, and drugs in water. In favourable cases, this allows for analyte identification or for reaction monitoring

Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes

Fluorescence-detected circular dichroism (FDCD) spectroscopy is applied for the first time to supramolecular host–guest and host–protein systems and compared to the more known electronic circular dichroism (ECD). We find that FDCD can be an excellent choice for common supramolecular applications, e.g. for the detection and chirality sensing of chiral organic analytes, as well as for reaction monitoring. Our comprehensive investigations demonstrate that FDCD can be conducted in favorable circumstances at much lower concentrations than ECD measurements, even in chromophoric and auto-emissive biofluids such as blood serum, overcoming the sensitivity limitation of absorbance-based chiroptical spectroscopy. Besides, the combined use of FDCD and ECD can provide additional valuable information about the system, e.g. the chemical identity of an analyte or hidden aggregation phenomena. We believe that simultaneous FDCD- and ECD-based chiroptical characterization of emissive supramolecular systems will be of general benefit for characterizing fluorescent, chiral supramolecular systems due to the higher information content obtained by their combined use

Chirality sensing of terpenes, steroids, amino acids, peptides and drugs with acyclic cucurbit[n]urils and molecular tweezers

Achiral chromophoric hosts, i.e. acyclic cucurbit[ n ]urils and molecular tweezers, were found to respond with characteristic Circular Dichroism (CD) spectra to the presence of micromolar concentrations of chiral hydrocarbons, terpenes, steroids, amino acids and their derivates, and drugs in water. In favourable cases, this allows for analyte identification or for reaction monitoring