Regioselective magnesiation of N-heterocyclic molecules: securing insecure cyclic anions by a β-diketiminate-magnesium clamp

Using a specially designed magnesium metallating manifold, combining kinetically activated TMP amide base with a sterically amplified β-diketiminate ligand, this study has established a new regioselective strategy for magnesiation of challenging N-heterocyclic molecules. The broad scope of the approach is illustrated through reactions of pyrazine, triazoles and substituted pyridines through isolation and structural elucidation of their magnesiated intermediates

Synthetic Fermentation as a Platform for Library Synthesis, Drug Discovery, and Chemical Outreach

The generation and biological screening of chemical libraries has long been a valuable tool for the discovery of new drugs. Synthetic fermentation is a set of techniques for chemical library synthesis under conditions compatible with direct biological assessment of the resulting products. To make this possible, certain criteria must be met with regard to the chemical reactions employed. The chemistry must be carried out in aqueous media, without the need for catalysts, enzymes or reagents, and any byproducts generated must be non-toxic. Building from our initial disclosure of a first generation synthetic fermentation, this thesis discusses various advances in our application of this concept, detailing the use of synthetic fermentation in phenotypic screening, high throughput synthesis and scientific outreach. Chapter 2 of this thesis discusses the use of synthetic fermentation for phenotypic screening, leading to the discovery of a family of antibacterial peptides. A large collection of building blocks was synthesized and used in an initially random screening process. Active mixtures were identified, and simple deconvolution led to the identification of a micromolar inhibitor of Bacillus subtilis growth. Structure-activity relationship studies led us to a peptide with increased antimicrobial activity and selectivity over HEK293 cells. Variants of this lead compound bearing photoaffinity probes and dyes were synthesized by synthetic fermentation and utilized for mechanism of action studies, leading to the discovery that our peptide had a nanomolar affinity for pencillin-binding protein 4, a known target for beta-lactam antibiotics. Chapter 3 details the development of a new synthetic fermentation reaction capable of the high-throughput synthesis of drug-like small molecule libraries. A three-component coupling reaction between potassium acyltrifluoroborate initiators, isoxazolidine monomers and aminothiol terminators was optimised, and a selection of new complexity-generating building blocks were synthesised. A library of 2184 compounds was generated through the combinatorial application of 39 building blocks, and analysed in an automated manner by LC/MS coupled to a coding script capable of detecting both the desired product of the reaction and known side-products. A number of trends and discoveries were observed from this initial data set, which enables the incorporation of automation, machine learning and prediction into the synthetic fermentation workflow. Chapter 4 of this thesis describes the development of “Make a Molecule”, a scientific outreach program based on synthetic fermentation chemistry. Based on our phenotypic screening of antibiotics, Make a Molecule aims to provide high school students with insight and hands-on experience of the drug discovery process through the opportunity to synthesise and biologically assess compounds for antimicrobial activity. After the optimisation of scientific protocols and development of an reaction guide web app, our Make a Molecule workshop was introduced to the classrooms of Kantonschule Zürich Nord, allowing two classes of students to experience the thrill of discovering biologically active molecules

Synthetic fermentation of β-peptide macrocycles by thiadiazole-forming ring-closing reactions

Macrocyclic β-peptides were efficiently prepared using a thiadiazole-forming cyclization reaction between an α-ketoacid and a thiohydrazide. The linear β-peptide precursors were assembled from isoxazolidine monomers by α-ketoacid-hydroxylamine (KAHA) ligations with a bifunctional initiator – a process we have termed ‘synthetic fermentation’ due to the analogy of producing natural product-like molecules from simpler building blocks. The linear synthetic fermentation products underwent Boc-deprotection/thiadiazole-forming macrocyclization under aqueous, acidic conditions to provide the cyclic products in a one-pot process. This reaction sequence proceeds from easily accessed initiator and monomer building blocks without the need for additional catalysts or reagents, enabling facile production of macrocyclic β-peptides, a relatively underexplored structural class.ISSN:2041-6520ISSN:2041-653

Antibiotic Discovery with Synthetic Fermentation: Library Assembly, Phenotypic Screening, and Mechanism of Action of Beta-Peptides Targeting Penicillin-Binding Proteins

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing for the resulting cultures to be directly screened for biological activity. In this work, a novel beta-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing for simple identification of active cultures. Subsequent deconvolution, focused screening and structure-activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human Hek293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant (Kd) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds

Antibiotic Discovery with Synthetic Fermentation: Library Assembly,Phenotypic Screening, and Mechanism of Action of β‑PeptidesTargeting Penicillin-Binding Proteins

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing the resulting cultures to be directly screened for biological activity. In this work, a novel β-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing simple identification of active cultures. Subsequent deconvolution, focused screening, and structure–activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human HEK293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant (Kd) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds.ISSN:1554-8929ISSN:1554-893

Make a Molecule: A Synthetic Organic and Medicinal Chemistry Workshop for High School Students

This manuscript describes an chemical outreach program developed to provide high school students the opportunity to make new organic molecules in a safe and user friendly fashion and test them for antibacterial activity

Make a Molecule: A Synthetic Organic and Medicinal Chemistry Workshop Program for High School Students

Despite the importance of organic chemistry to human health and quality of life, very few outreach programs have been successful in communicating this message. This contributes to negative public perceptions of organic chemistry and obscures the enormous advances in quality of life engendered by organic synthesis. New chemical outreach programs that provide insight and hands-on experience of small molecule chemistry are vital to improving public engagement with the science. Make a Molecule is an organic chemistry workshop program that provides high school students with the opportunity to synthesize and biologically assess short peptides in a safe and easy manner, allowing them to experience the thrill of making and testing new bioactive molecules in a classroom environment. This manuscript details the development of the Make a Molecule project from its origins in our “synthetic fermentation” chemistry to its initial application in high schools. Scientific protocols were optimized to suit a classroom setting with the help of high school students. An accompanying website was constructed to serve as an experimental guide, aid with the workshop execution, and provide an educational resource for students and teachers. With everything in hand, the first Make a Molecule workshops were conducted in a local high school in Zurich, Switzerland, during which students were able to successfully synthesize and test novel bioactive molecules.ISSN:0021-9584ISSN:1938-132

Palladium-Catalyzed Ring Expansion of Spirocyclopropanes to Form Caprolactams and Azepanes

A palladium(0)-catalyzed rearrangement of piperidones and piperidines bearing a spirocyclopropane ring was developed. The ring expansion reaction led to a variety of functionalized caprolactam and azepane products in good to excellent yields. Experimental and computational mechanistic studies revealed an initial oxidative addition of the distal carbon–carbon bond of a cyclopropane ring to the palladium(0) catalyst and the relief of ring strain as a driving force for product formation