Periodic Reporting for period 1 - MuST ArtEM (Multicomponent Supramolecular Structures as Artificial Enzyme Mimics)
Período documentado: 2024-06-03 hasta 2026-06-02
Resumen del contexto y de los objetivos generales del proyecto
Nature has evolved highly efficient catalytic systems that operate with unmatched selectivity, adaptability, and spatial organization. Enzymes achieve these properties through complex and dynamic architectures that position catalytic units with nanometre precision, creating local microenvironments that enhance reaction rates and regulate molecular recognition. Replicating such sophisticated functions using synthetic materials remains a major challenge in chemistry and materials science, yet it is essential for the development of more sustainable and efficient catalytic technologies.
The MuST ArtEM project addressed this challenge by exploring supramolecular polymers as dynamic and modular platforms for catalysis. Unlike conventional covalent polymers, supramolecular polymers are assembled through reversible non-covalent interactions, enabling adaptive behaviour, self-healing properties, and straightforward incorporation of functional components. These characteristics make them promising candidates for the construction of enzyme-inspired materials capable of combining structural precision with functional complexity.
The project was situated within a broader scientific and strategic context that seeks to develop new approaches for sustainable chemical processes, advanced functional materials, and bioinspired technologies. Developing self-assembled catalytic materials that can dynamically reorganise and concentrate reactants could contribute to more efficient chemical transformations while advancing fundamental understanding of how function emerges from molecular organisation.
To address these challenges, MuST ArtEM pursued three interconnected objectives. First, it aimed to develop functional supramolecular polymers containing catalytic units and investigate how their molecular composition and structural organisation influence catalytic performance. Second, it sought to understand how multiple molecular components can be integrated through non-covalent synthesis to create more complex and multifunctional architectures. Third, it explored liquid-liquid phase separation (LLPS) as a strategy for generating compartmentalised catalytic microreactors that mimic the membraneless organelles increasingly recognised as important functional compartments in living cells.
The project pathway was built around the design and investigation of complementary supramolecular systems based on benzene-1,3,5-tricarboxamide (BTA) and ureidopyrimidinone (UPy) motifs. A key achievement was the synthesis of a new zinc-containing BTA-based monomer and its incorporation into supramolecular polymers. These studies demonstrated how functional units can be integrated into dynamic assemblies and revealed that increasing the density of catalytic sites within the supramolecular structures enhances oligonucleotide cleavage activity. This finding provided evidence that supramolecular organisation can generate cooperative catalytic effects through substrate clustering, offering valuable insight into how enzyme-like behaviour can emerge from synthetic systems.
A second major research direction investigated whether phase-separated supramolecular assemblies could create catalytic environments analogous to biological condensates. Although phase separation could not be achieved with the initial BTA-based system, the project successfully established LLPS of UPy-based supramolecular polymers functionalised with catalytic amino acid residues into tactoid-like structures. While only limited catalytic enhancement was observed under the investigated conditions, the work established an experimental platform for studying the relationship between compartmentalisation, molecular organisation, and catalytic function. These results provide important knowledge for future efforts aimed at engineering synthetic condensates with improved reactivity and selectivity.
In addition, MuST ArtEM expanded into the emerging area of photoactive supramolecular materials through a collaboration with the group of Professor Luka Đorđević. By combining aminonaphthalene imide (ANI)-based photoresponsive molecules with established BTA and UPy systems, the project demonstrated that photocatalytic functionality can be introduced into supramolecular assemblies through minimal incorporation of photoactive components while preserving the structural characteristics of the host polymers. Furthermore, coupling these systems with LLPS generated tactoid-like condensates, opening new opportunities for the development of light-responsive catalytic compartments. This interdisciplinary direction broadened the scientific scope and potential impact of the project by linking supramolecular chemistry, photocatalysis, and biomolecular-inspired compartmentalisation.
The expected impact of MuST ArtEM extends beyond the immediate scientific results. At the fundamental level, the project contributes to a deeper understanding of how molecular architecture, dynamic self-assembly, and compartmentalisation influence catalytic performance. Such knowledge is essential for the rational design of next-generation adaptive materials that emulate key aspects of biological systems. At the technological level, the developed concepts may ultimately support the creation of more efficient catalytic platforms for chemical synthesis, biotechnology, and environmental applications.
Overall, MuST ArtEM established new methodologies, molecular building blocks, and design principles for the construction of functional supramolecular catalytic systems. Although several challenges remain before fully enzyme-like synthetic microreactors can be realised, the project generated significant foundational knowledge and opened new research avenues at the interface of catalysis, self-assembly, phase separation, and photocatalysis. These advances provide an important basis for future developments aimed at creating adaptive and highly efficient catalytic materials inspired by the organisational principles of living systems.
The MuST ArtEM project addressed this challenge by exploring supramolecular polymers as dynamic and modular platforms for catalysis. Unlike conventional covalent polymers, supramolecular polymers are assembled through reversible non-covalent interactions, enabling adaptive behaviour, self-healing properties, and straightforward incorporation of functional components. These characteristics make them promising candidates for the construction of enzyme-inspired materials capable of combining structural precision with functional complexity.
The project was situated within a broader scientific and strategic context that seeks to develop new approaches for sustainable chemical processes, advanced functional materials, and bioinspired technologies. Developing self-assembled catalytic materials that can dynamically reorganise and concentrate reactants could contribute to more efficient chemical transformations while advancing fundamental understanding of how function emerges from molecular organisation.
To address these challenges, MuST ArtEM pursued three interconnected objectives. First, it aimed to develop functional supramolecular polymers containing catalytic units and investigate how their molecular composition and structural organisation influence catalytic performance. Second, it sought to understand how multiple molecular components can be integrated through non-covalent synthesis to create more complex and multifunctional architectures. Third, it explored liquid-liquid phase separation (LLPS) as a strategy for generating compartmentalised catalytic microreactors that mimic the membraneless organelles increasingly recognised as important functional compartments in living cells.
The project pathway was built around the design and investigation of complementary supramolecular systems based on benzene-1,3,5-tricarboxamide (BTA) and ureidopyrimidinone (UPy) motifs. A key achievement was the synthesis of a new zinc-containing BTA-based monomer and its incorporation into supramolecular polymers. These studies demonstrated how functional units can be integrated into dynamic assemblies and revealed that increasing the density of catalytic sites within the supramolecular structures enhances oligonucleotide cleavage activity. This finding provided evidence that supramolecular organisation can generate cooperative catalytic effects through substrate clustering, offering valuable insight into how enzyme-like behaviour can emerge from synthetic systems.
A second major research direction investigated whether phase-separated supramolecular assemblies could create catalytic environments analogous to biological condensates. Although phase separation could not be achieved with the initial BTA-based system, the project successfully established LLPS of UPy-based supramolecular polymers functionalised with catalytic amino acid residues into tactoid-like structures. While only limited catalytic enhancement was observed under the investigated conditions, the work established an experimental platform for studying the relationship between compartmentalisation, molecular organisation, and catalytic function. These results provide important knowledge for future efforts aimed at engineering synthetic condensates with improved reactivity and selectivity.
In addition, MuST ArtEM expanded into the emerging area of photoactive supramolecular materials through a collaboration with the group of Professor Luka Đorđević. By combining aminonaphthalene imide (ANI)-based photoresponsive molecules with established BTA and UPy systems, the project demonstrated that photocatalytic functionality can be introduced into supramolecular assemblies through minimal incorporation of photoactive components while preserving the structural characteristics of the host polymers. Furthermore, coupling these systems with LLPS generated tactoid-like condensates, opening new opportunities for the development of light-responsive catalytic compartments. This interdisciplinary direction broadened the scientific scope and potential impact of the project by linking supramolecular chemistry, photocatalysis, and biomolecular-inspired compartmentalisation.
The expected impact of MuST ArtEM extends beyond the immediate scientific results. At the fundamental level, the project contributes to a deeper understanding of how molecular architecture, dynamic self-assembly, and compartmentalisation influence catalytic performance. Such knowledge is essential for the rational design of next-generation adaptive materials that emulate key aspects of biological systems. At the technological level, the developed concepts may ultimately support the creation of more efficient catalytic platforms for chemical synthesis, biotechnology, and environmental applications.
Overall, MuST ArtEM established new methodologies, molecular building blocks, and design principles for the construction of functional supramolecular catalytic systems. Although several challenges remain before fully enzyme-like synthetic microreactors can be realised, the project generated significant foundational knowledge and opened new research avenues at the interface of catalysis, self-assembly, phase separation, and photocatalysis. These advances provide an important basis for future developments aimed at creating adaptive and highly efficient catalytic materials inspired by the organisational principles of living systems.
Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha
The functional BTAZn monomer was obtained from previously synthetized BTA, which was firstly tosylated and then reacted with di-Boc protected 1,4,7-triazacyclononane. Subsequent deprotection and Zn metalation afforded the desired compound, as confirmed by NMR spectroscopy and mass spectrometry. The co-assembly of BTAZn with BTA stacks was investigated using UV–Vis spectroscopy, cryo-TEM, and scattering techniques.
The binding of the assemblies to oligonucleotides of different lengths was assessed by fluorescence measurements using dye-labelled DNA and BTA, with Förster resonance energy transfer (FRET) serving as an indicator of the proximity and dynamic association between the oligonucleotides and BTA stacks. The results showed stronger binding for longer oligonucleotide strands, suggesting that the interaction is driven by multivalent binding between the polyanionic oligonucleotides and the positively charged BTA assemblies.
The catalytic activity of the assemblies towards oligonucleotide cleavage was investigated by capillary electrophoresis, enabling the degradation of the oligonucleotides to be monitored over time. At a constant concentration of functional BTAZn, higher catalytic efficiency was observed for assemblies containing a higher density of BTAZn units, suggesting a cooperative effect between neighbouring Zn catalytic sites during phosphate hydrolysis.
The resulting assemblies were able to cleave both RNA and DNA, which hindered their integration with DNA/RNA hybrid systems aimed at enabling the subsequent self-assembly of DNA nanotubes. Moreover, the BTAZn co-assemblies proved sensitive to the reaction environment, leading to poor reproducibility in the presence of more complex reaction media. Consequently, this objective could not be achieved within the course of the action.
In response, an alternative research direction was pursued through an unplanned collaboration with the group of Professor Luka Đorđević at the University of Padova. This collaboration aimed to combine the amphiphilic photocatalytic systems developed by their group with the supramolecular polymers developed in our laboratory, with the broader objective of introducing photocatalytic functionality into supramolecular assemblies through non-covalent incorporation.
The system investigated consisted of an amphiphilic surfactant based on an aminonaphthalene imide (ANI) core, which was co-assembled with BTA. We found that incorporation of ANI within the BTA supramolecular stacks induced a structural transition from double to single helices, as suggested by UV–Vis and fluorescence spectroscopy, eventually leading to dissolution of the assemblies above a critical ANI concentration. The resulting assemblies were characterized using NMR spectroscopy, confocal microscopy, cryo-TEM, and AFM. Further image analysis is required to conclusively establish the morphological changes associated with the ANI-induced structural transition.
Having established the influence of ANI incorporation on the structure of the BTA assemblies, we subsequently investigated their photocatalytic properties. In the presence of salts, ANI self-assembles into nanoribbons and is capable of catalysing the oxygen reduction reaction (ORR) under 450 nm irradiation, resulting in the formation of H2O2. In contrast, no significant catalytic activity was observed for ANI at low concentrations or in the absence of salts. Remarkably, incorporation of a small amount of ANI as a dopant within the BTA stacks was sufficient to activate the photocatalytic properties, demonstrating that supramolecular co-assembly can be used to introduce photocatalytic functionality into otherwise non-photocatalytic BTA assemblies.
Finally, the role of supramolecular structure in determining catalytic activity was investigated by incorporating a glycerol-derived chiral surfactant known to induce the double-to-single helix transition in BTA assemblies. This enabled a direct comparison of the photocatalytic activity of the single and double helical structures. The single helical assemblies displayed higher catalytic activity than their double helical counterparts, providing evidence that the supramolecular organization of the catalytic units plays a key role in determining their reactivity and highlighting the importance of controlling higher-order structure to tune functionality.
Attempts to achieve LLPS with the BTAZn system were not successful. Instead, use of UPy functionalized with aminoacidic residues, such as histidine and lysine, resulted in phase separation into tactoid-like structures catalysing esters hydrolysis and hydrazone formation, respectively. the use fluorescent substrates allowed to monitor the progress of the hydrazone formation reaction inside the tactoids by means of confocal microscopy. However, under the explored conditions, substrates confinement did not result in enhancement of reaction rate, which was the same as the one observed not in the phase separation conditions. This is most likely due to the high dynamicity of the obtained tactoids, thus further research is currently ongoing to find a way to rigidify such macromolecular condensates.
A similar approach was employed with the ANI system. Due to its fluorescence, it was possible to visualize ANI within the tactoids formed by UPy supramolecular polymers with confocal microscopy, confirming the incorporation of the photoactive amphiphile in the stacks without hampering their ability to phase separate. The optimization of the reaction conditions to perform photocatalysis is currently ongoing.
The binding of the assemblies to oligonucleotides of different lengths was assessed by fluorescence measurements using dye-labelled DNA and BTA, with Förster resonance energy transfer (FRET) serving as an indicator of the proximity and dynamic association between the oligonucleotides and BTA stacks. The results showed stronger binding for longer oligonucleotide strands, suggesting that the interaction is driven by multivalent binding between the polyanionic oligonucleotides and the positively charged BTA assemblies.
The catalytic activity of the assemblies towards oligonucleotide cleavage was investigated by capillary electrophoresis, enabling the degradation of the oligonucleotides to be monitored over time. At a constant concentration of functional BTAZn, higher catalytic efficiency was observed for assemblies containing a higher density of BTAZn units, suggesting a cooperative effect between neighbouring Zn catalytic sites during phosphate hydrolysis.
The resulting assemblies were able to cleave both RNA and DNA, which hindered their integration with DNA/RNA hybrid systems aimed at enabling the subsequent self-assembly of DNA nanotubes. Moreover, the BTAZn co-assemblies proved sensitive to the reaction environment, leading to poor reproducibility in the presence of more complex reaction media. Consequently, this objective could not be achieved within the course of the action.
In response, an alternative research direction was pursued through an unplanned collaboration with the group of Professor Luka Đorđević at the University of Padova. This collaboration aimed to combine the amphiphilic photocatalytic systems developed by their group with the supramolecular polymers developed in our laboratory, with the broader objective of introducing photocatalytic functionality into supramolecular assemblies through non-covalent incorporation.
The system investigated consisted of an amphiphilic surfactant based on an aminonaphthalene imide (ANI) core, which was co-assembled with BTA. We found that incorporation of ANI within the BTA supramolecular stacks induced a structural transition from double to single helices, as suggested by UV–Vis and fluorescence spectroscopy, eventually leading to dissolution of the assemblies above a critical ANI concentration. The resulting assemblies were characterized using NMR spectroscopy, confocal microscopy, cryo-TEM, and AFM. Further image analysis is required to conclusively establish the morphological changes associated with the ANI-induced structural transition.
Having established the influence of ANI incorporation on the structure of the BTA assemblies, we subsequently investigated their photocatalytic properties. In the presence of salts, ANI self-assembles into nanoribbons and is capable of catalysing the oxygen reduction reaction (ORR) under 450 nm irradiation, resulting in the formation of H2O2. In contrast, no significant catalytic activity was observed for ANI at low concentrations or in the absence of salts. Remarkably, incorporation of a small amount of ANI as a dopant within the BTA stacks was sufficient to activate the photocatalytic properties, demonstrating that supramolecular co-assembly can be used to introduce photocatalytic functionality into otherwise non-photocatalytic BTA assemblies.
Finally, the role of supramolecular structure in determining catalytic activity was investigated by incorporating a glycerol-derived chiral surfactant known to induce the double-to-single helix transition in BTA assemblies. This enabled a direct comparison of the photocatalytic activity of the single and double helical structures. The single helical assemblies displayed higher catalytic activity than their double helical counterparts, providing evidence that the supramolecular organization of the catalytic units plays a key role in determining their reactivity and highlighting the importance of controlling higher-order structure to tune functionality.
Attempts to achieve LLPS with the BTAZn system were not successful. Instead, use of UPy functionalized with aminoacidic residues, such as histidine and lysine, resulted in phase separation into tactoid-like structures catalysing esters hydrolysis and hydrazone formation, respectively. the use fluorescent substrates allowed to monitor the progress of the hydrazone formation reaction inside the tactoids by means of confocal microscopy. However, under the explored conditions, substrates confinement did not result in enhancement of reaction rate, which was the same as the one observed not in the phase separation conditions. This is most likely due to the high dynamicity of the obtained tactoids, thus further research is currently ongoing to find a way to rigidify such macromolecular condensates.
A similar approach was employed with the ANI system. Due to its fluorescence, it was possible to visualize ANI within the tactoids formed by UPy supramolecular polymers with confocal microscopy, confirming the incorporation of the photoactive amphiphile in the stacks without hampering their ability to phase separate. The optimization of the reaction conditions to perform photocatalysis is currently ongoing.
Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)
The MuST ArtEM project delivered new molecular tools and fundamental insights for the development of bioinspired supramolecular catalytic systems. The project successfully synthesized and characterized a novel zinc-functionalized benzene-1,3,5-tricarboxamide monomer (BTAZn) and demonstrated its incorporation into supramolecular polymers. The resulting assemblies enabled the study of the relationship between catalyst density, molecular organization, and catalytic activity. A key finding was that increasing the density of catalytic units within the assemblies enhanced oligonucleotide cleavage, providing evidence that supramolecular organization can generate cooperative catalytic effects.
The project also expanded current knowledge on the use of liquid-liquid phase separation (LLPS) in functional supramolecular materials. Although phase separation could not be achieved in the BTA-based catalytic system, LLPS was successfully established in amino-acid-functionalized UPy supramolecular polymers. The results showed that compartmentalization alone is insufficient to guarantee improved catalytic performance, highlighting the importance of understanding how molecular exchange, reactant partitioning, and catalyst organization influence reactivity within condensates.
A further significant result was the demonstration that photocatalytic functionality can be introduced into BTA supramolecular polymers through the incorporation of small amounts of aminonaphthalene imide (ANI)-based monomers. Additionally, combining ANI-containing assemblies with UPy systems enabled the formation of phase-separated tactoid-like condensates, opening new opportunities to investigate photocatalysis within biomimetic compartmentalized environments.
Collectively, these results provide new design principles for constructing functional supramolecular materials in which catalytic activity emerges from the dynamic assembly of multiple molecular components. The project has strengthened the knowledge base at the interface of supramolecular chemistry, catalysis, systems chemistry, and soft matter science.
The main impact of the project is scientific and methodological. The generated knowledge contributes to the long-term development of synthetic systems that mimic key features of enzymes and biological condensates, including dynamicity, compartmentalization, and cooperative function. Such systems could ultimately contribute to more efficient catalytic processes, responsive materials, and sustainable chemical technologies.
To maximize the impact of the project, further research is required to improve catalytic performance under phase-separated conditions and to establish clear structure-function relationships governing activity in supramolecular condensates. In particular, the photocatalytic properties of the newly developed ANI-containing phase-separated assemblies should be investigated, as these systems represent a promising platform for light-driven catalysis. Future work should also explore a broader range of catalytic reactions and assess the robustness and scalability of the developed supramolecular architectures.
The project has therefore delivered both tangible research outputs and new research directions. While the developed systems are currently at an early-stage, fundamental-research level, they provide a foundation for future collaborative projects aimed at developing adaptive catalytic materials inspired by living systems.
The project also expanded current knowledge on the use of liquid-liquid phase separation (LLPS) in functional supramolecular materials. Although phase separation could not be achieved in the BTA-based catalytic system, LLPS was successfully established in amino-acid-functionalized UPy supramolecular polymers. The results showed that compartmentalization alone is insufficient to guarantee improved catalytic performance, highlighting the importance of understanding how molecular exchange, reactant partitioning, and catalyst organization influence reactivity within condensates.
A further significant result was the demonstration that photocatalytic functionality can be introduced into BTA supramolecular polymers through the incorporation of small amounts of aminonaphthalene imide (ANI)-based monomers. Additionally, combining ANI-containing assemblies with UPy systems enabled the formation of phase-separated tactoid-like condensates, opening new opportunities to investigate photocatalysis within biomimetic compartmentalized environments.
Collectively, these results provide new design principles for constructing functional supramolecular materials in which catalytic activity emerges from the dynamic assembly of multiple molecular components. The project has strengthened the knowledge base at the interface of supramolecular chemistry, catalysis, systems chemistry, and soft matter science.
The main impact of the project is scientific and methodological. The generated knowledge contributes to the long-term development of synthetic systems that mimic key features of enzymes and biological condensates, including dynamicity, compartmentalization, and cooperative function. Such systems could ultimately contribute to more efficient catalytic processes, responsive materials, and sustainable chemical technologies.
To maximize the impact of the project, further research is required to improve catalytic performance under phase-separated conditions and to establish clear structure-function relationships governing activity in supramolecular condensates. In particular, the photocatalytic properties of the newly developed ANI-containing phase-separated assemblies should be investigated, as these systems represent a promising platform for light-driven catalysis. Future work should also explore a broader range of catalytic reactions and assess the robustness and scalability of the developed supramolecular architectures.
The project has therefore delivered both tangible research outputs and new research directions. While the developed systems are currently at an early-stage, fundamental-research level, they provide a foundation for future collaborative projects aimed at developing adaptive catalytic materials inspired by living systems.