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ConspectusAs the remit of chemistry expands beyond molecules to systems, new synthetic targets appear on the horizon. Among these, life represents perhaps the ultimate synthetic challenge. Building on an increasingly detailed understanding of the inner workings of living systems and advances in organic synthesis and supramolecular chemistry, the de novo synthesis of life (i.e., the construction of a new form of life based on completely synthetic components) is coming within reach. This Account presents our first steps in the journey toward this long-term goal. The synthesis of life requires the functional integration of different subsystems that harbor the different characteristics that are deemed essential to life. The most important of these are self-replication, metabolism, and compartmentalization. Integrating these features into a single system, maintaining this system out of equilibrium, and allowing it to undergo Darwinian evolution should ideally result in the emergence of life. Our journey toward de novo life started with the serendipitous discovery of a new mechanism of self-replication. We found that self-assembly in a mixture of interconverting oligomers is a general way of achieving self-replication, where the assembly process drives the synthesis of the very molecules that assemble. Mechanically induced breakage of the growing replicating assemblies resulted in their exponential growth, which is an important enabler for achieving Darwinian evolution. Through this mechanism, the self-replication of compounds containing peptides, nucleobases, and fully synthetic molecules was achieved. Several examples of evolutionary dynamics have been observed in these systems, including the spontaneous diversification of replicators allowing them to specialize on different food sets, history dependence of replicator composition, and the spontaneous emergence of parasitic behavior. Peptide-based replicator assemblies were found to organize their peptide units in space in a manner that, inadvertently, gives rise to microenvironments that are capable of catalysis of chemical reactions or binding-induced activation of cofactors. Among the reactions that can be catalyzed by the replicators are ones that produce the precursors from which these replicators grow, amounting to the first examples of the assimilation of a proto-metabolism. Operating these replicators in a chemically fueled out-of-equilibrium replication-destruction regime was found to promote an increase in their molecular complexity. Fueling counteracts the inherent tendency of replicators to evolve toward lower complexity (caused by the fact that smaller replicators tend to replicate faster). Among the remaining steps on the road to de novo life are now to assimilate compartmentalization and achieve open-ended evolution of the resulting system. Success in the synthesis of de novo life, once obtained, will have far-reaching implications for our understanding of what life is, for the search for extraterrestrial life, for how life may have originated on earth, and for every-day life by opening up new vistas in the form living technology and materials.

Central to the origin of life is the question how a chemical system transitioned from interacting molecules to an entity with the capacity for self-replication, diversification and adaptive evolution. Here, we study a chemical system that is comprised of macrocycles that have been shown to spontaneously give rise to self-replicating entities. By combining experimental and theoretical approaches, we strive to understand the evolutionary potential of this system. In particular, we apply eco-evolutionary reasoning to investigate whether and when this system of chemical replicators can diversify. Here, we report first results of a simplified stochastic chemical reaction model that is parameterized on the basis of experimental data. The model considers the competition of two replicators that do not interact directly but need similar building blocks for their growth and reproduction. Interestingly, the replicator that emerges first is being overtaken by the later one. By means of stochastic simulations, we will explore how the competitive ability of a replicator is determined by its chemical characteristics, and under which conditions replicators can coexist. The results will subsequently inform the design of future experiments.

The behaviour of aqueous dynamic combinatorial libraries (DCLs) containing either electron-rich donor building blocks based on dioxynaphthalene (DN), or electron-deficient acceptor building blocks based on naphthalenediimide (NDI) are described. The influence of concentration and ionic strength on library distribution and diversity, together with the responses to electronically-complementary templates have been explored in detail, establishing the principles to be employed in more complex libraries leading to a new generation of catenanes.

This thesis presents a few contributions to the field of systems chemistry, ranging from dynamic combinatorial chemistry to photoizomerization. In the preface and the first chapter the reader is introduced to the general concepts and recent developments in systems chemistry. In Chapter 2 the concept of antiparallel dynamic covalent chemistry is introduced as a new kind of interaction between different combinatorial chemistries, and also as a tool to control the behaviour of dynamic combinatorial libraries. In Chapter 3 the interface between dynamic combinatorial chemistries is again explored, as well as a new kind of interaction between different combinatorial chemistries – in particular the catalysis of E/Z-isomerization of hydrazones by thiols. In Chapter 4 a long standing problem of slow hydrazone exchange is tackled. In particular, using UV irradiation as a means of introducing energy into system enables faster exchange due to the presence of reactants richer in energy. In Chapter 5 UV irradiation is used to establish photostationary states between two isomers of a hydrazone distributed between two immiscible solvents. Such a system is very easy to prepare, and yet shows complex emergent behaviour. In the final chapter all results are summarized and put in the perspective of possible future developments.

This dissertation is focused on two major investigations. The first one is about the mechanism with which a known self-replicator can create copies of itself. This study led to the first real-time visualization of a molecule capable of self-replication. The second is about the introduction of additional functionalities within self-replicating systems of molecules. Here, we were able to show emergent behavior: catalytic activity that would only start when the self-replication process takes place.

Inspired by nature, chemists have envisioned that it is possible to obtain organized structures (and functions) using complex networks of interacting molecules in a synthetic setting. Thus far, functional molecular systems have been recognized as fruitful platforms, suitable to challenge and complement prevalent strategies for innovation in areas ranging from biology and medicine to energy sustainability. To this end, this thesis describes studies on self-assembling systems and their emergent properties and functions. The research presented teeters at the interface of fundamental and applied research as we address conceptually very different topics from a unique starting point. The work in Chapters 3 and 4 utilizes reversibility of covalent and non-covalent interactions to explore the modes of organization in peptide-based systems. In particular, we investigated the tendency of self-assemblies towards post-modification, self-replication and folding. In Chapters 5 and 6 we explored an alternative synthetic mimic of the extracellular matrix (ECM). The basic scaffold was designed in such a way to facilitate chemical modifications on demand (easily and quickly). In this approach, only minimal synthetic efforts are required to readily diversify a polymeric material using various bioactive entities (e.g., cell adhesion ligands, RGD or LDV).In the future, functional systems that make use of dynamic bonds will continue to address highly compelling and challenging questions. By answering those, we aim to make advancements in fundamental topics such as the origin of life, as well as health and technology.

In this thesis, we employed synthetic self-replicating systems attempting to mimic and understand life’s central characteristics including diversity, out-of-equilibrium states, complexity and homochirality.

In the quest for greener chemistry to substitute the often toxic, waste-producing and polluting processes of the chemical industry, nature is frequently taken as inspiration. Enzymes are the best chemists on earth, they have been evolved to become extremely efficient and selective for a specific activity. Artificial (metallo)enzymes (ArMs) are hybrid bio-chemical systems aiming to combine the broad reaction scope of organic chemistry and the high efficiencies and selectivities of enzymes, while working under environmentally friendly conditions.This thesis describes the design and creation of several ArMs based on non-catalytic Multidrug Resistance Regulator proteins. Here, we show how the multispecificity of these proteins allows the incorporation of several metal complexes and substrates into their scaffolds to create a range of ArMs for different reactivities using different assembly methodologies.

For over 60 years computers have been used to simulate biological systems in molecular detail using classical mechanics, using so-called molecular dynamics simulations. To perform a molecular dynamics simulation the system of interest has to be described using simulation input files, ensuring that the system is described using an appropriate model which reproduces the physical behaviour of the real system. Preparing these input files has, until now, only been possible for common, linear, polymers, such as proteins and DNA due to a historical focus on these systems. In chapter 2 we present software that is capable of setting up simulations for more complex systems such as branched or cyclic polymers using models at several resolutions. However, to assemble the required parameters for these polymers, building blocks are required. For atomistic molecular dynamics models, where every atom is represented as a separate particle, so-called automatic topology builders are commonplace. These tools can generate input parameters for arbitrary molecules automatically, without human intervention. For coarse-grained molecular dynamics models, where multiple atoms are grouped together to form a single, coarse, interaction site, these tools are not commonplace however. In chapter 3 we present the initial work to reformulate the problem of automatically creating coarse-grained input files as a clustering problem, which we aim to solve using machine learning techniques.Finally, in chapter 4, we present combined experimental and simulation work where we study an artificial peptide system, which is capable of self-replication, in an attempt to elucidate the self-replication mechanism.

Making artificial (“de-novo”) life from completely synthetic molecules is a challenging endeavour that could help us understand life, detect it, and gain some insight on its origins. The aim of this thesis is to combine in a single system several properties that are fundamental to living systems – specifically self-replication, compartmentalization, and metabolism.This work is based on self-replicating fibres that emerge from a dynamic combinatorial library. Through the thesis, we study their self-replication mechanism using HS-AFM, use complex coacervates to compartmentalize them, and combine them with photocatalytic cofactors to obtain the first synthetic example of a light-driven protometabolism. Finally, we combine this protometabolic feedback loop with out-of-equilibrium conditions to design a synthetic oscillator.The results shown here are first steps towards the synthesis of de-novo life, and can lead in the future to more complex processes such as ecological relationships or Darwinian evolution.

The work carried out in this thesis provides examples on how variations in building block design affect self-assembly and self-replication behavior in dynamic combinatorial libraries. Chapter 1 provides an introduction in which possible definitions of life are discussed, together with the main characteristics of living systems (like self-replication and metabolism) and an overview of recent developments on self-replicators based on peptides and nucleobases. In Chapter 2 we report two peptide-based self-replicators that were utilized to synthesize supramolecular polymers with controllable size and composition. Our observations indicated that the nature of the polymers was strongly affected by the morphology of the sheared seeds. In Chapter 3 we show how self-replication can be tuned by the length of the spacer in DCLs made from two structurally different building blocks. In Chapter 4 we discovered parasitic behavior between self-replicators formed from building blocks that differ by a single methylene unit. We observed that a 6-ring replicator can only emerge when assisted by a pre-existing 8-ring self-replicator. Aiming for diversification of self-replicators through a cross-catalytic pathway under out-from-equilibrium conditions, we used continuous flow-set-ups in Chapter 5. First, we set up a system with two building blocks and optimized the experimental conditions in terms of flow rate, time and starting replicator composition. We also report attempts to create a cross-catalytic cycle by introducing a third building block. Finally, in Chapter 6, our studies are placed in a broader perspective and future prospects are discussed.

This thesis intends to capture the processes of self-replication and folding individually or combined by using dynamic combinatorial chemistry. This thesis is composed of 6 chapters, each of them dealing with a different aspect of folding and replication.In Chapter 1, we briefly introduced the basic concepts of systems chemistry and the origin of life. In order to mimic some of life’s important features and to achieve life-like behavior, it is particularly important to construct complex systems that combine the processes of self-replication and folding. In Chapter 2, we described how a dynamic combinatorial selection approach allows access to a foldamer of remarkable complexity constituted by 15 identical peptide-nucleobase building blocks. In Chapter 3, we reported the discovery of foldamers that form autonomously in aqueous media based on simple dipeptides using a systems chemistry approach. In Chapter 4, we described the emergence of self-replicating molecules that contain both amino acids and nucleobase from DCLs. In Chapter 5 we described for the first time the self-sorting between self-replicating and folded molecules in a DCL composed of two building blocks. Furthermore, we developed a transient self-replicating system using the same building blocks. Conclusions are drawn in Chapter 6.

This thesis focuses on the emergence of novel peptide self-replicators from dynamic combinatorial libraries. Thiol functionalised peptide building blocks, first reported by our group in 2010, form the basis of all chapters. The work performed here has two main objectives: First, to deepen the insights obtainable from self-replicating systems and second, to use peptide building blocks to mimic life-like features such as replication. Alongside this, we developed novel strategies to access novel replicators and to dictate their properties.

Complex (supra)molecular systems are ubiquitous in living organisms as well as in synthetic context but understanding them in depth is still out of reach, mainly because we do not have much insight into the gradual formation of complex behavior from more simple system elements. The motivation of this thesis is to show how complex chemical systems can be constructed and as a result, how the emergence of novel systems phenomena can be understood with the aid of the applied bottom-up approach.

Adaptieve moleculaire systemen waarin op spiropyran gebaseerde moleculaire schakelaars een cruciale rol spelen staan centraal in dit proefschrift. Zulke systemen kunnen reageren op prikkels (zoals licht, pH, elektriciteit, etc.) met een verandering in moleculaire, en daarbij materiële, eigenschappen zoals kleur, zuurgraad, of geleiding, onder andere. Hoewel spiropyrans en gerelateerde moleculen al 100 jaar lang bekend zijn, is het nog steeds een veelvoorkomend gebruiksmiddel in de moderne wetenschappelijke literatuur en, steeds meer, in commerciële toepassingen (bijv. kleurveranderende brilglazen). De grote uitdaging is het vertalen van de verandering in een enkel molecuul naar de macroscopische wereld zodat wij op onze schaal de verandering ook kunnen ervaren en benutten. In dit proefschrift wordt allereerst bewijs van principe geleverd door synthese en karakterisatie van modelstoffen die in staat zijn effectief polymeren te genereren, waarin het daaropvolgende schakelen tussen eigenschappen wordt hersteld. Vervolgens wordt deze kennis gebruikt om functionele polymeren te maken van spiropyran, waarbij tevens nieuwe functies werden blootgelegd. Een dezer functies is inherent aan de spiropyran en is schijnbaar over het hoofd gezien al deze tijd, en is daarom onderzocht in een apart hoofdstuk. Ten slotte wordt een nieuwe variatie van spiropyran onderzocht door de structuur van twee zulke moleculen te verenigen, wat tot nieuwe, en bovenal interessante, eigenschappen leidt. In zijn geheel draagt dit proefschrift bij aan het vooruitstreven van dit enerverend onderzoeksgebied van spiropyran moleculaire schakelaars, terwijl tegelijkertijd enkele belangwekkende fundamentele kwesties opgelost zijn aangaande de kleurveranderingen van spiropyrans. Maar natuurlijk zijn in dit proces ook weer vele nieuwe kwesties opgegraven.

We weten dat het huidige leven het resultaat is van miljoenen jaren van evolutie. Maar wat ging er aan dit proces vooraf? Hoe zijn de allereerste levensvormen ontstaan? We weten nog maar erg weinig van hoe primitief leven is gevormd uit de prebiotische “soep”. Dit proefschrift richt zich op deze vragen en beschrijft hoe een aantal fundamentele karakteristieken van het leven spontaan kunnen ontstaan uit mengsels van in elkaar overgaande moleculen die interacties met elkaar aangaan. Dit heeft geleid tot de ontdekking van moleculen die, afhankelijk van de omstandigheden, zichzelf kunnen reproduceren (kopieën van zichzelf kunnen maken) of membraanstructuren kunnen vormen die lijken op een lege cel. Zelf-replicatie en de vorming van cel-achtige compartimenten zijn belangrijke stappen in het ontstaan van het level en in het maken van synthetisch leven.

Self-replicating molecules play a fundamental role in origins of life studies. In Chapter 2, evidence is provided that specific self-replicating molecules are capable of growing exponentially. This is a very important breakthrough: exponentially growing replicators are better prone to survive during Darwinian evolution. Almost all replicators discovered up to the latest years were not capable of exponential growth. Primordial life likely resulted from repeated attempts of replication in a destructive environment. This replication and destruction process is at the core of Darwinian evolution. In Chapter 3, such a replication/destruction system is studied experimentally and computationally. Based on these studies, it is possible to set up an experimental system where the replicators are constantly broken down into building blocks, and where the latter replicate again. Computer simulations allow the determination of optimal energy coupling conditions for such systems. In Chapter 4, information exchange pathways in peptide fibres were studied. The results show that the exchange of building blocks from one fibre to another one takes place at the extremities thereof, while building blocks in the fibre cores are hardly used in the replication of other fibres. These are therefore protected from replication and the information that they carry can hardly be exchanged, unless the fibres continuously break into smaller pieces. While in our experiments Darwinian evolution does not yet take place, we could observe significant processes for enabling Darwinian evolution: exponential growth of fibres, destruction of replicators and selective exchange of building blocks.

This thesis presents another approach for direct cytosolic delivery via membrane fusion. This approach is based on a complementary pair of coiled-coil forming peptides, K (KIAALKE)4 and E (EIAALEK)4 and is mimicking the action of the SNARE-complex. The SNARE-complex is responsible for fusion between vesicles and membranes of cells in the synapse and is also composed of coiled-coil forming peptides. In our system, the peptides are conjugated to a cholesterol anchor via a polyethylene glycol (PEG) spacer, yielding lipopeptides CP4K4 and CP4E4. Membrane fusion between liposomes and the plasma membrane of the cells is triggered by these two lipopeptides when each embedded within the lipid bilayer of the liposomes or the plasma membrane of the cell.

The interactions of a Dynamic Combinatorial Library (DCL) of molecules with specific targets leads to composition changes of the library which can reveal potential guests and / or catalysts. In this thesis some chemical systems have been proposed to achieve a certain level of molecular complexity and interactions within molecular networks by using DCC. These systems were implemented for the discovery of catalysts and the construction of molecular communication networks.

The general topic of this dissertation is the development of synthetic receptors for organic ammonium ions in near physiological conditions using disulfide dynamic combinatorial chemistry (DCC). Chapter 1 explains the importance of this development and the associated difficulties when using the conventional, iterative approach: design, synthesis and testing. Then it introduces the external template effects in disulfide DCC that can be used as an alternative, superior approach to conventional chemistry for obtaining synthetic receptors able to work in near physiological medium. Chapter 2 presents a successful use of this approach to develop a synthetic receptor for nicotine. The receptor features an affinity comparable to those of synthetic receptors reported thus far, and is obtained with relatively better yield. Chapter 3 describes the design of a DCL of which nearly all of its macrocyclic, oligomeric members can be amplified, with size selectivity, after being exposed to 30 organic ammonium-ion templates. Chapter 4 shows that the macrocylic oligomers can be amplified, with length-size selectivity, after being exposed to aliphatic α-ω diamines. In chapter 5, a fitting program (DCLFit) was used to reliably estimated the binding affinities of the amplified DCL members to four chosen templates. The binding constants were in close agreement with the ones obtained by ITC, and found to be the highest reported so far for synthetic receptors. Chapter 6 describes the derivatization of the DCL building blocks with natural amino acids to allow the DCL to acquire additional diversity, chiral centers, recognition property, flexibility or rigidity. Valine side chains played a crucial role in the types of the formed products, resulting DCL distributions and DCL responses to templates. Undesirable side reactions were also observed upon introducing the valine residues.

Dit proefschrift beschrijft de werkzaamheden uitgevoerd op een zelfassemblerende en self-replicating systeem. Het werk is opgedeeld in verschillende studies, waarbij een eerste studie gedaan die een demonstratie en een beschrijving van de exponentiële zelfreplicerende aard van het systeem. Mechanistische studies worden vervolgens blootgesteld, waarbij de hoeveelheid schuifspanning toegepast op het systeem is onderzocht, eerst met een kwalitatieve methode, en vervolgens met behulp van een Couette Cell, een speciaal apparaat dat kwantitatieve metingen toelaat. Tenslotte wordt het laatste deel van het manuscript gericht op de beschrijving van transmissie elektronenmicroscopie gebruikt als instrument voor het karakteriseren van nanodeeltjes van verschillende aard, zoals vezels afkomstig uit het peptide zelf-replicerend systeem, maar ook uit projecten ingeleid samenwerking met andere universiteiten.

In dit proefschrift wordt dynamische combinatoriele chemie (DCC) gebruikt om complexe synthetische systemen te genereren en wordt de zelfassemblage van de moleculen in deze systemen bestudeerd. In hoofdstuk 1 worden de fundamentele principes van DCC geintroduceerd en bespreken we de huidige toestand van DCC. We komen tot de conclusie dat er echter nog genoeg ruimte is voor verdere ontwikkelingen in dit vakgebied. In hoofdstuk 2 worden, door toevoeging van een moleculaire mal in een bibliotheek van disulfide bouwstenen, verschillende catenanen gevormd door de verstrengeling van cyclische structuren van de bouwstenen met ringvormige moleculaire mallen. Een kwantitatieve methode voor het bepalen van de evenwichtsconstantes voor elke individuele stap in de vorming van de catenaan werd ontwikkeld, welke wellicht algemeen toepasbaar is voor catenaan formatie door middel van dynamische covalente bindingen. In hoofdstuk 3 worden complexe systemen gevormd door gelijktijdig gebruik van twee moleculaire mallen in één bibliotheek, met hierin Russische pop-achtige structuren van trimoleculaire complexen ten gevolge van het gelijktijdige “casting” en “molding” proces. Gezien de beperkte hoeveelheid voorbeelden van zelf-replicatie in dynamische combinatoriele bibliotheken (DCBen) worden er in hoofstuk 4 en 5 twee verschillende systemen ontwikkeld waarin aangetoond wordt dat zelfassemblage kan leiden tot zelf-replicatie in de DCBen. In hoofdstuk 6 en 7 worden twee zelf-synthetiserende hydrogels ontwikkeld met behulp van DCC, ten gevolge van zelfassemblage in de DCBen. In hoofdstuk 8 wordt elk aandeel van de hoofstukken in perspectief gebracht en worden de vooruitzichten voor de zelfassemblage in complexe chemische systemen besproken.