Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / Nature has developed a generous number of peptides carrying out various essential functions in all living organisms. Human body produces peptides as signaling molecules, such as hormones, to transmit messages from cell to cell and regulate metabolic homeostasis. Microbes synthesize peptides as antibiotics to inhibit the growth of other microorganisms. These peptides display an exceeding diversity of amino acid composition, peptide sequence, secondary structure and post-translational modification. Inspired by nature, researchers have developed peptides as a unique modality of therapeutics, combining the best attributes of small-molecule drugs and protein-based biopharmaceuticals. This work has sought to explore the potential of peptides as synthetic receptors, binders of protein and probes for bacteria detection. The research started from a foldable cyclic peptide scaffold, prolinomycin, a proline-rich analogue of valinomycin. The peptide can chelate a potassium ion folding into a drum like structure, which provides a platform to display and preoganize functional side chains for target binding. We first investigated its folding behavior under physiological conditions. We demonstrate that the metal-assisted folding of the prolinomycin scaffold tolerates various side chain mutations. The stability of the structure can be improved by introducing crosslinking moieties. Based on this scaffold, we rationally designed synthetic receptors of various amines by utilizing iminoboronate chemistry with acetylphenyl boronic acid (APBA). Furthermore, I pursued phage display, a powerful technique to develop high affinity peptide binders of protein targets. Proteins are the most appealing targets for drug development and disease biomarkers discovery. We chose sortase A (SrtA) as a model target protein to screen for potent peptide binders. A peptide inhibitor of sortase A with single-digit micromolar affinity was identified from a cyclic peptide library displayed by phage. In addition, from the chemically modified phage display peptide library presenting APBA motifs, peptide binders with specificity and micromolar affinity towards SrtA were discovered. Instead of binding to the active site, the peptide could recognize the surface of the protein. Additionally, to further expand the chemical space of phage display, I constructed a phage display peptide library presenting N-terminal cysteine (NCys) which can undergo site-specific chemical modifications. Two pieces of chemistry were applied, including thiazolidino boronate (Tzb) mediated acylation reaction of NCys and 2-cyanobenzothiazole (CBT)-NCys condensation. The site-specific dual modifications on NCys and internal Cys of phage-encoded peptides were achieved. Furthermore, a strategy to N, S-doubly label NCys via an alternative pathway of CBT condensation was reported, which presents a significant addition to the toolbox for site-specific protein modifications. Finally, by functionalizing graphene field effect transistors (G-FET) with peptide probes, we developed the first selective, electrical detection of the pathogenic bacterial species Staphylococcus aureus and antibiotic resistant Acinetobacter baumannii on a single platform. Overall, peptides provide enormous opportunities for therapeutics development. Research herein demonstrated principles of peptide design for specific molecular recognition. Novel chemistry strategies have been developed to expand the molecular diversity of peptide libraries. We believed that the advances in peptide design and screening would promote peptide-based drug discovery. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Signal integration and transduction by cell-surface receptors is a complex, multi-layered process resulting in tight regulation of downstream mediators, which in turn elicit pre-defined native cellular responses. The modular architecture of transmembrane receptors provides a unique opportunity for engineering de novo sensor/effector circuits, enabling the development of custom cellular functions for research and therapeutic applications. The signal transduction module of most existing chimeric receptors consists of either native intracellular domains or effectors domains fused to non-programmable DNA binding proteins. Therefore, these receptors can only engage in natural signalling pathways or drive the expression of artificial, pre-integrated transgenes. By harnessing the programmability of a nuclease deficient CRISPR/Cas9 (dCas9) signal transduction module and leveraging the evolutionarily optimised ligand-sensing capacity of native receptors, I have created a novel class of dCas9-based synthetic receptors (dCas9-synR). I demonstrate that an optimised split dCas9-based core architecture and custom protease-based signal release mechanism can be standardised across multiple classes of extracellular domains to engineer receptor tyrosine kinase (RTK)-based and G-protein-coupled receptor (GPCR)-based chimeric receptors. dCas9-synRTK and dCas9-synGPCR integrate a broad variety of input signals (peptides, proteins, lipids, sugars) with highly specific and robust activation of any custom output transcriptional programme in an agonist dose-dependent manner. Finally, to showcase the therapeutic potential of dCas9-synRs, I used them to convert a pro-angiogenic signal into an anti-angiogenic response, deploy a chemokine/cytokine programme in response to tumour-enriched biomolecules, and induce insulin expression following glucose stimulation. The performance of dCas9-synRs and their unique versatility in redirecting the information flow makes them ideally suited to engineer designer cells capable of sensing specific disease markers and in turn drive various therapeutic programmes.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.

Chimie dynamique: reconnaissance de nucléobases par des récepteurs synthétiques et isomérie cis-trans d'hydrazones acylées.Ce travail traite du développement des systèmes moléculaires qui peuvent s'adapter à l'addition de substances qui agissent comme un gabarit. Cette approche permet d'isoler une espèce majeure à partir d'un mélange de composés par le biais de la chimie combinatoire dynamique (CCD). La première partie de ma thèse de doctorat inclus l'utilisation d'un ADN simple brin (ADNsb) comme un gabarit pour le transfert d'information par auto-assemblage de récepteurs sans avoir besoin d'enzyme. De nouveaux récepteurs de l'adénine et de la guanine (pinces A et G) solubles dans l'eau ont été conçues dans ce but. Une approche utilisant la résonance magnétique nucléaire (RMN) a été utilisée pour déterminer l'affinité de liaison comme preuve d'une reconnaissance spécifique et efficace. Une évaluation dans l'eau par dichroïsme circulaire (CD) et mesure de la température de fusion par UV (Tm) a été réalisée. Cela a permis de tester respectivement la capacité d'auto-assemblage entre les pinces et un modèle ADNsb, et la force du processus de coopérativité. La deuxième partie de ce travail est axée sur le tri spontanné de motifs pyridine acylhydrazone et sur les configurations intéressantes qu'ils adoptent. Nous avons étudié la synthèse d'une série de motifs pyridine acylhydrazone: dimère, trimères et pentamères. Des études RMN ont permis d'évaluer les changements dans l'équilibre configurationnel cis / trans de ces systèmes dynamiques. Les études ont montré que l'équilibre attendu est biaise la cis acylhydrazone pyridine isomère a été observée par diffraction des rayons X. / Dynamic chemistry: nucleobase recognition by synthetic receptors and cis-trans acylhydrazone isomerism. This work deals with the development of molecular systems which can adapt upon the addition of substances that act as templates. This approach enables one major species to be identified from a mixture of compounds through the use of dynamic combinatorial chemistry (DCC). The first part of my PhD included the use of a single stranded DNA (ssDNA) as a template for information transfer via the self-assembly of receptors without the need for enzymes. New water soluble adenine and guanine receptors (A and G clamps) were designed and synthesised for this purpose. Nuclear magnetic resonance (NMR) titration studies were carried out to calculate the binding affinity and as a proof of specific and efficient recognition. An assessment in water via circular dichroism (CD) and UV temperature melting (Tm) studies was carried out. This tested the ability for self-assembly between the clamps and a ssDNA template and the strength of the cooperative process respectively. The second part of my PhD focused on the self-sorting of acylhydrazone pyridine motifs and the interesting configurations they adopt. The feasibility to synthesise these acylhydrazone pyridine motifs (dimer, trimers and pentamers) was investigated. X-ray and NMR studies showed that the equilibrium was found to be biased in an unusual way, and the cis acylhydrazone pyridine isomer was observed.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.