Chemical transformations that join two molecular components together rapidly while remaining highly efficient and selective are valued for their elegant simplicity and effectiveness in a myriad of applications. By applying the principles of ‘click’ chemistry to biology, information about molecular interactions in vivo can therefore be gained from minimally perturbing bioorthogonal coupling reactions. Developing bioorthogonal ‘click’ reactions – reactions that do not cross-react with biological components – provides new ways to accurately study biological systems at the molecular level. This thesis describes the development of such tools. Strain-promoted alkyne-nitrone cycloadditions (SPANC) represent rapid, efficient, selective, and tunable conjugation strategies that are applicable to biomolecular labelling experiments. Herein, SPANC reactions with bicyclo[6.1.0]nonyne are examined using physical organic methods to determine the stereoelectronic factors governing SPANC reactivity. Second-order rate constants (k2) of up to 1.49 M-1s-1 were measured and the resulting cycloadditions are applied to the design and synthesis of nitrone-based molecular probes. The first example of SPANC-mediated metabolic labelling in live-cell bacteria is reported, establishing SPANC as an efficient and bioorthogonal metabolic labelling strategy for cellular labelling.

Les isoprénoïdes, présents dans tous les organismes vivants, sont synthétisés selon deux processus: la voie du Mevalonate et la voie Méthylérythritol phosphate (MEP). Cette dernière, absente chez l’humain, est très étudiée car elle représente une cible pour le développement de nouveaux antimicrobiens. Le ME-N3, un analogue du méthylérythritol portant un azoture, a été synthétisé et exploité dans des expériences de marquage métabolique de la voie MEP en utilisant un couplage bioorthogonale suivi d’une analyse par LC/MS. De façon intéressante, nous avons découvert que le MEP-N3, un analogue du MEP, inhibe l'enzyme IspD d’ E. coli (3ème enzyme de la voie MEP). Les études cinétiques ont révélé que le MEP-N3 possède la meilleure activité inhibitrice sur IspD d’ E.coli en comparaison avec les inhibiteurs connus, et que le mécanisme d'inhibition est de type mixte. Une étude détaillée du mécanisme de la réaction catalysée par IspD a été réalisée pour la première fois, en utilisant une analyse cinétique à deux substrats. / Isoprenoids, present in all living organisms, are synthesised according to two routes: the Mevalonate and the Methylerythritol phosphate (MEP) pathways. The MEP pathway, absent in humans, is extensively investigated as it is a target for the development of new antimicrobials. ME-N3 an azide tagged analogue of methylerythritol was synthesised and utilised for metabolic labelling studies of the MEP pathway using bioorthogonal ligation followed by LC-MS analysis. Interestingly, we found that MEP-N3, an analogue of MEP, inhibits E.coli IspD (3rd enzyme of the MEP pathway). Further inhibition kinetic studies revealed that MEP-N3 possesses the highest inhibitory activity on E.coli ispD when compared to known inhibitors. In addition, the mechanism of inhibition of E.coli ispD by MEP-N3 was found to be best described using a mixed type model. Moreover, determination of the IspD reaction mechanism has been carried out for the first time, by virtue of a bisubstrate steady state kinetic analysis.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Inositolpolyphosphate (InsPs) bilden eine ubiquitäre Gruppe an hochphosphorylierten, intrazellulären Signalmolekülen in eukaryotischen Zellen. Trotz deren Beteiligung an unzähligen biologischen Prozessen bleibt die Detektion von InsPs (insb. einzelner Enantiomere) eine Herausforderung, da die momentan verfügbaren Analysemethoden immer noch limitiert sind. In der vorliegenden Arbeit wird die stabile Isotopenmarkierung von myo-Inositol (Ins) und InsPs in Kombination mit Kernspinresonanzspektroskopie (engl. Nuclear Magnetic Resonance spectroscopy, NMR) erkundet, um diese Lücke zu schließen. Die Abhängigkeit von NMR-Daten und chemischer Struktur erlaubte die Analyse komplexer Mixturen aus InsPs aus in vitro-Experimenten und biologischen Proben. Durch stereospezifische 13C-Markierung konnten sogar Enantiomere voneinander unterschieden werden. Mit Hilfe dieser Methode wurden mehrere InsP-Stoffwechselwege untersucht. Als Erstes wurde das menschliche, Phytase-artige Enzym MINPP1 (engl. Multiple Inositol Polyphosphate Phosphatase 1) detailliert in vitro und in lebenden Zellen charakterisiert. Dabei wurde ein bisher unbeschriebener InsP-Stoffwechselweg in menschlichen Zellen erstmals beschrieben. Als Zweites wurden InsP verdauende Bakterien aus der menschlichen Darmflora untersucht, sodass der Abbauweg von Inositolhexakisphosphat beleuchtet werden konnte. Als Drittes wurden DUSP-Enzyme (engl. Dual-Specificity Phosphatases) identifiziert und in vitro charakterisiert, die in der Lage sind, die Phosphoanhydrid-Bindung von Inositolpyrophosphaten (PP-InsPs) zu spalten. Die vorliegende Arbeit demonstriert, dass 13C-Markierung in Verbindung mit NMR ein mächtiges Werkzeug darstellt, um InsP-Stoffwechselvorgänge zu untersuchen. / Inositol polyphosphates (InsPs) comprise a ubiquitous group of densely phosphorylated intracellular messengers in eukaryotic cells. Despite their contributions to a myriad of biological processes the detection of InsPs remains challenging to this day, especially with regards to differentiating enantiomers, as the available analytical toolset is still limited. In this thesis the use of stable isotope labelling of myo-inositol (Ins) and InsPs is explored to address this shortcoming. Combining 13C-labelling and nuclear magnetic resonance spectroscopy (NMR) provides both enhanced sensitivity and makes use of NMR’s strong structure-data dependency. This enabled the deconvolution of complex mixtures of InsPs from in vitro experiments or biological samples. With stereo-specific 13C-labels InsP mixtures could be resolved to individual enantiomers. Using this technique several InsP metabolic pathways were examined. Firstly, the human phytase-like enzyme Multiple Inositol Polyphosphate Phosphatase (MINPP1) was characterized in depth in vitro and in living cells, establishing a hitherto undescribed inositol polyphosphate metabolic path in humans. Secondly, inositol phosphate digesting bacteria isolated from the human gut microbiome were investigated, shedding light on the metabolic fate of inositol hexakisphosphate in the digestive track. Thirdly, a set of Dual-Specificity Phosphatases (DUSPs) were identified to be able to hydrolyze the phosphoanhydride bond of inositol pyrophosphates (PP-InsPs) and characterized in vitro. The 13C-labelling approach of InsPs in junction with NMR represents a powerful tool for the study of inositol polyphosphate metabolism. In the thesis at hand, this method has facilitated our understanding of inositol polyphosphate pathways and it will be continuing doing so in the future in several biological contexts.