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Reactivity and Assembly of Multifunctional, Stimuli-responsive Encapsulation Structures

Periodic Reporting for period 3 - RAMSES (Reactivity and Assembly of Multifunctional, Stimuli-responsive Encapsulation Structures)

Reporting period: 2019-12-01 to 2021-05-31

All matter surrounding us, including earth and wind, materials we use, food we eat as well as our own bodies consist of molecules, which themselves are composed of different atoms from the periodic table of elements. Molecules can be small or large, of simple shape or structurally complex, flat or voluminous, dense or hollow, soluble or solid. Whereas minerals are usually rather simple in composition and internal structure, biologically derived molecules, such as proteins, DNA and vitamins are more complex, both in der 3-dimensional structure as well as specific function. For example, enzymes are able to manufacture („catalyze“) the formation of other complex molecules and they possess control switches and input sites for chemical fuels to regulate their activity.
Over billion years of evolution, nature has brought the production and function of its building blocks to perfection. They are capable of specific recognition, information storage, signal transduction, self-repair and replication. Acting together, they can function as molecular machines with the ability to maneuver atoms and molecules on the nanometer scale (as Richard Feynman already recognized in the 50th) and produce new molecules, thus acting as microscopic factories. Structural biologists and chemists have elucidated breathtaking details about the shape and motion of the molecules forming the living world.
In the last decades, driven by an inherent curiosity, researchers started to rebuilt, mimic or in some cases even surpass natural paradigms by synthetic laboratory work. They systematically created man-made molecular structures from scratch and tested their ability to reach the structural and functional complexity of what we know from the biological world. After all, they used the same chemical elements also available to nature and built on the same physical laws.
Here is where synthetic supramolecular chemistry comes into play, a dedicated branch of chemical research which aims at creating molecular assemblies with ever increasing complexity. After 1987s Nobel Prize for Host-Guest Chemistry kicked off a flourishing width of research activities, the 2016 Nobel Prize for Molecular Machines showed the impressive progress in the meantime. Nevertheless, compared with biological machines and networks, the synthetic systems available to date are still in their infancy.
Within the ERC project RAMSES (Reactivity and Assembly of Multifunctional, Stimuli-responsive Encapsulation Structures) the Clever Lab has specialized on the synthesis of hollow molecules ("cages") that are able to include smaller molecules in their interior. We use "self-assembly", a process comparable to a self-solving puzzle game where building blocks, organic molecules and metal ions that fit perfectly to each others, form a larger structure by just shaking them together long enough for the final result to emerge. We further use a special class of DNA structures, called G-quadruplexes, with the ability to bind metal ions, to create bio-hybrid nano-structures. In both compound classes, we integrate functions such as switches (triggered by light), catalytic sites (to promote chemical reactions) and many other chemical groups.
Our mission is to gather fundamental knowledge about non-covalent assembly processes, develop new routes towards structural and functional complexity on the nanoscale and turn this into practical application, highly relevant for sustainable industrial processes, new medical and diagnostic developments and smart materials used in future devices.
RAMSES addresses a fundamental discrepancy between naturally derived structures with nanoscale cavities and most artificial self-assembled nano-confinements: while the biological structures, e.g. enzymes, are usually low symmetric (as are their substrates) and densely equipped with multiple functionalities, man-made mimics are of rather high symmetry and low diversity regarding the number of functions implemented into one structural motif. The reason is the following: nature employs a multi-step strategy starting with the synthesis of a covalently joined block polymer containing about 20 different functional units in a predetermined (genetically encoded) sequence. This polypeptide chain then folds in a non-covalent fashion (often assisted by dynamic-covalent cross-linking steps) into a single type of 3-dimensional object, containing internal pockets and surface features that are lined with functionalities, acting together to bestow the folded protein with its evolved function. Self-assembly in the laboratory, however, lives from simplifying the employed components, usually giving them symmetric shapes and directed connectivities to produce highly symmetric objects such as squares, cubes, tetrahedra, octahedra or Archimedean spheres following fundamental geometric principles. While the spontaneous emergence of the latter structures is of unquestionable elegance, it brings the problem, that the resulting structures are structurally far from the complexity of biological compounds.

We develop new strategies for the self-assembly of functionalized, artificial building blocks that allow for a rational (non-statistical) integration of two or more components into a single architecture. The challenge is to design systems and assembly principles where, for example, components A, B, C and D only assemble into "heteroleptic" product ABCD, without falling into narcissistic self-sorting (A only with A, B only with B…) or a complete statistical mess (here: 55 possible “lantern-shaped”, four-component systems, from AAAA over BCCD to ABCD). Therefore, while interconnectivity between the components has to be secured, entropy – a formidable enemy favoring the formation of convoluted mixtures – has to be overcome by carefully installed molecular "nuts and bolts" or "grooves and tongues". After reliable and robust strategies towards such low-symmetric, multi-component structures have been devised, these will be used to facilitate the implementation of multiple functionalities and study their interplay in the context of photo-redox processes, materials properties and action on internalized substrates.

Within the first half of the RAMSES project, we were able to successfully progress our endeavour on several front lines:

1) We developed three orthogonal self-assembly strategies to obtain new metal-mediated nano-structures by design:
- the "shape-complementary" approach, which allows the combination of different building blocks with a matching overall shape into a single type of structure, see: https://dx.doi.org/10.1039/c7cc03379f and https://dx.doi.org/10.1002/anie.201702573
- the "donor-site-engineering" approach, in which the immediate environment of the metal ions, acting as "glue" to hold the other components together, is carefully designed in a way, that only one solution to the self-assembly puzzle remains possible, see: https://dx.doi.org/10.1002/chem.201802188 and https://dx.doi.org/10.1021/jacs.9b02207
- the "bridged-ring" approach, where metal ions are embedded into a large ring structure and additional organic ligands complement the structure to form 3D cavities (to be published)

2) We established several cutting-edge analytical techniques in the project team to characterize structure and function of the self-assembled materials with high accuracy. For example:
- Trapped Ion Mobility Spectrometry, allowing us to differentiate cages close in size and shape from complex mixtures with unprecedented resolution, see: https://dx.doi.org/10.1039/c9dt01814j
- Isothermal Titration Calorimetry, yielding valuable information about the thermodynamic fundament of self-assembly and host-guest processes, see: https://dx.doi.org/10.1002/cptc.201900038
- Various Electron Microscopic techniques, delivering means of visualizing the formation of higher-order structures, assembled from the developed heteroleptic cages, on a microscopic scale, see: https://dx.doi.org/10.1021/jacs.8b10991 and https://dx.doi.org/10.1002/anie.201800490
- Synchrotron single crystal diffractometry at DESY Hamburg, Petra III, beamline P11 (relevant for all project publications)

3) We drove forward the implementation of functional elements into the self-assembled nano-structures and studied first examples of application:
- Chirality was introduced to generate cages that cannot be superimosed with their mirror images, a geometric property widely found in nature, see:
https://dx.doi.org/10.1002/anie.201812926
- Light-switchable units, allowing to control guest uptake and release. Formerly open questions about the underlying mechanisms could be answered, see:
https://dx.doi.org/10.1021/jacs.8b11872 and https://dx.doi.org/10.1002/cptc.201900038
- Decoration of the newly developed heteroleptic cages with solubility-controlling appendices, leading to anisotropic amphiphiles (a fancy word for soap-like compounds) that were shown to form vesicular structures ("solvent-filled bubbles") that find application as rarely described agents to form so called "oil-in-oil emulsions" with potential use in chemical process technologies, see: https://dx.doi.org/10.1021/jacs.8b10991 and https://dx.doi.org/10.1002/anie.201800490
- "Bowls" instead of cages as new structural motifs were introduced, allowing to bind technologically important fullerenes, select and purify them from mixtures, bring them into a wider range of organic solvents and even control their reactivity, see: https://dx.doi.org/10.1021/jacs.9b02207 and https://www.tu-dortmund.de/storages/tu_website/Referat_1/Presseseite/Archiv_unizet/unizet460_web.pdf

For further news, see our Twitter channel: https://twitter.com/guido_clever and website: www.clever-lab.de
Currently, the RAMSES team is concentrating on the following tasks:

- We further enhance the repertoire of assembly strategies to obtain multifunctional, heteroleptic structures, as we see the contribution of novel assembly principles as a fundamental enabling technique to allow a multitude of future downstream developments and applications. In particular, we are refining the shape-complementarity approach to widen the scope of building blocks and metal cations to be implemented, we obtained exciting, yet unpublished results by combining three different assembly principles in a single one-pot approach and last but not least, we develop DNA G-quadruplex-based structures possessing nanosized cavities carrying multiple functionalities as bio-hybrid alternatives to the fully artificial cage complexes.
- The worked-out assembly strategies are exploited to implement multiple functionalities within a single assembly product, with a focus on donor-acceptor combinations to realize directed excitation or charge transfer (with relevance for molecular diagnostics, electronics and photovoltaics research), the pairing of catalytic sites with control elements (e.g. chiral groups) as well as the anisotropic outside decoration to realize higher-order structures with application in the synthesis of new nano-structured materials.

In conclusion, we not only enhance fundamental knowledge about adjusting enthalpic, entropic and geometric factors within non-covalently assembled nano-architectures, but furthermore pave the way to application of the worked-out principles. We envision several future areas of application of the project-derived findings, all of which share significance with respect to environmental aspects, sustainable process development and new smart molecular systems and materials, thus showing the potential to contribute technological advances with economic benefits on the long term.
Major assembly strategies compared
Collection of team pictures
Examples: amphiphilic and fullerene-binding cages
Lab scene
Logo
Concept: evolution of cage assembly strategies
3D printed cage models