Periodic Reporting for period 4 - FunCapSys (Functional Systems of Capsules) Reporting period: 2021-07-01 to 2022-12-31 Summary of the context and overall objectives of the project A living organism is composed of myriad chemical subsystems, each consisting of structurally complex biomolecules that interact in many ways; the properties of life emerge from these dense connections. Understanding how these interactions take place will help elucidate the foundations of biology and enable the creation of new chemical networks with targeted functions. Deciphering chemical systems requires new tools to be developed, however.Molecular encapsulation - an area to which the Nitschke group has made significant contributions in recent years - provides a crucial platform for the development of systems chemistry. Binding a network member within a capsule allows it to be hidden and then revealed upon receipt of a release signal, or transported as a cargo between spatially removed parts of a network. Larger capsules may also isolate subsystems from each other in the manner of vesicles. Additionally, capsules may also be designed to respond to specific stimuli to modify their binding pockets to accommodate desired guests.The FunCapSys research programme will develop new means of engineering complex systems of capsules that perform useful functions in response to specific stimuli. Two parallel lines of inquiry will be pursued in a synergistic fashion in order to generate new tools that will feed directly into a third project that aims to develop unprecedented chemical cascades. Firstly, we will investigate capsules that are able to perform useful functions in ionic liquids and that can move between solution phases when subjected to external stimuli. Secondly, we will create adaptable molecular capsules that can bind different guest molecules and modulate their reactivity. Thirdly, we will use the tools developed to build complex chemical networks that are able to carry out complex synthetic tasks and separate valuable products from mixtures. The goals of this research are all based around controlling the behaviour of complex systems and will thus advance the development of systems chemistry.The FunCapSys project will seek to address a wide range of challenges through contributions in fields including coordination chemistry, catalysis, physical organic chemistry, systems chemistry and molecular machines. In the long term, it is anticipated that the groundwork laid in this research programme will provide a toolkit for the design and function of new synthetic chemical systems, paving the way toward new commercial technologies in chemical synthesis and purification. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the first half of the 60 month FunCapSys project we have developed new approaches to enable capsules to sequester guests from an immiscible phase for separations and purifications. We have demonstrated the first example of coordination cages shuttling and transporting molecular cargoes reversibly between immiscible liquid layers and the use of sequential phase transfer of different cage species to separate their respective cargoes. As a proof of concept, this idea constitutes a new approach to molecular separations and may enable the development of lower cost molecular separation methods that would thus enable significant energy saving. We also demonstrated the transfer of a supramolecular cage with controlled directionality between three phases, based on a cage that responds reversibly in two distinct ways to different stimuli. These results will open up opportunities to use such circulatory guest transport as a new mode of chemical purification. Moreover we have programmed information into related phase transfer systems allowing different functional outcomes in response to the distinct stimuli of heat and light. Our research efforts have also shed light on the underlying mechanism of formation of new metal-organic container molecules from the readily available subcomponents. Moreover, we have utilized the host-properties of these systems for a variety of applications such as catalysis and selective sensing of analytes including fluorometric detection of biologically relevant guests in an aqueous environment. We have also developed strategies to rationally modify the binding ability of cages through new post-assembly modification reactions.We have developed a new method for synthesising water soluble and water stable metal-organic container molecules representing a significant step forward in the design of abiological architectures able to trap, transport or transform chemicals in water or physiological media. We hope that the control of the solubility properties and stability of these nanocontainers will allow others and us to build sophisticated systems for sensing low levels of biologically relevant molecules or environmental pollutants present in urine or the bloodstream in the context of clinical diagnosis. Likewise, our structures may be suitable as vehicles to transport dyes for diagnostic imaging, or to deliver drugs across macroscopic distances. Ultimately, we hope that such water-soluble architectures may serve as nanoreactors for the preparation of new molecules of interest, such as pharmaceuticals, or for the degradation of toxic pollutants such as residual medicines and pesticides that can be found in water streams.We also developed a new and useful triangular-prismatic host framework with a remarkable ability to selectively bind a collection of pharmaceutically relevant molecules. Whereas the central binding sites of most previously described capsules are roughly spherical, those of the triangular prisms are prolate. This decreased symmetry promoted the binding of a collection of complex natural products—steroids, opiates, alkaloids, and other drugs bound within the prisms. The flexible nature of the structure induced complex binding interactions involving collections of guests, including cooperative binding events and guest aggregation around the cage, underscoring the utility of our system to generate diverse host–guest dynamics from simple building blocks. The general concept of designing heteroleptic structures with smaller cavities than those of their homoleptic derivatives may provide a new method for optimizing the formation of low-symmetry structures that recognize diverse and targeted sets of prolate guests, including many pharmaceuticals beyond those explored so far. Such structures may serve as the basis of new chemical sensing and purification systems, enabling new liquid extraction methods that select for specific molecules within biological feedstocks. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The scientific results obtained during Project A and Project B to date are such that we can confidently state that we went well beyond the state of the art. We have made great progress during the course of the project in the development of three-dimensional capsules for molecular separations, guest recognition and reactivity modulation. We have also employed state of the art analysis techniques to analyse complex supramolecular systems including slice-selective NMR, Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI‐FTICR‐MS) and Traveling Wave Ion Mobility Spectrometry (TWIMS). Our development of a separation technique based on the addition of anionic stimuli rather than thermal energy is significant in the context of chemical separations, which remain the most energy-intensive processes in industry. We have developed new strategies for the stabilisation of metallo-supramolecular systems in water, greatly expanding their potential applications, and for the synthesis of new low symmetry receptors to target low symmetry different natural products and pharmaceuticals. We will explore the use of these receptors to modify the reactivity of these large and biologically relevant guest molecules in the next part of the project. In the final part of the project will also seek to combine the expertise acquired to date in the project to assemble multi-functional systems capable of separating selected components of mixtures using active transport employ our supramolecular capsules in complex abiological systems.