Periodic Reporting for period 1 - FOLDASYNBIO (Bioinspired Nanostructures by Self-assembly of Amphiphilic Non-peptide Helical Foldamers in Aqueous Environment)
Berichtszeitraum: 2015-09-01 bis 2017-08-31
Many of the promising applications of (bio)nanotechnology rely on extending synthetic organic chemistry at the nanometer length-scale to deliver materials with distinctive properties and function. Sequence-specific molecules, exhibiting a water soluble potential (e.g. biopolymers) have advantages as design elements for construction of these types of nanoscale materials in that correlations can be drawn between primary structure (i.e. the sequence), secondary structure (folded elements) and further assembly into higher order (tertiary and quaternary) structure, potentially affording ordered architectures with emergent functions tailored to various biotechnology and medical applications (e.g. encapsulation and release, sensing, storage, catalysis). Non-biological synthetic folded oligomers – i.e. foldamers – can be used to create both complex and atomically precise nanostructures, and allow the exploitation of a wide range of building units, enabling the creation of self-assembled supramolecular architectures that would differ fundamentally from nature in terms of topologies and emerging functions.
The general aim of this project was thus to create such protein-like quaternary structures by using (i) non-natural sequence-specific urea-based folded oligomers, and (ii) a fabrication process in aqueous conditions mimicking those at work in proteins (i.e. hierarchical structure formation). These oligourea foldamers possess specific advantages such as synthetic accessibility, high folding fidelity and stability to enzymatic proteolysis. In line with this general objective, the project was seeking a) to delineate the design principles leading to the formation of homogeneous assemblies of oligourea helices, b) to structurally elucidate the resulting nanostructures at the atomic level and c) to explore molecular recognition processes within such foldamer quaternary structures. The project capitalized on the discovery (published in Nature Chemistry in 2015) by the host group that properly designed water-soluble oligourea sequences were able to form well-defined compact or extended self-assembled nanostructures such as the six-helix bundle which was studied in more details in this project.
The general aim of this project was thus to create such protein-like quaternary structures by using (i) non-natural sequence-specific urea-based folded oligomers, and (ii) a fabrication process in aqueous conditions mimicking those at work in proteins (i.e. hierarchical structure formation). These oligourea foldamers possess specific advantages such as synthetic accessibility, high folding fidelity and stability to enzymatic proteolysis. In line with this general objective, the project was seeking a) to delineate the design principles leading to the formation of homogeneous assemblies of oligourea helices, b) to structurally elucidate the resulting nanostructures at the atomic level and c) to explore molecular recognition processes within such foldamer quaternary structures. The project capitalized on the discovery (published in Nature Chemistry in 2015) by the host group that properly designed water-soluble oligourea sequences were able to form well-defined compact or extended self-assembled nanostructures such as the six-helix bundle which was studied in more details in this project.
The workplan of the FOLDASYNBIO project was divided in three work packages (WPs).
- The first work package (WP1) was dedicated to organic synthesis including building block synthesis and oligomer synthesis and was centred on primary sequence variations and manipulations to delineate the requirements for the six-helix bundle formation by the reference sequene (H1). During the duration of the project, a total of eight building blocks have been prepared in multi-gram scale with the necessary side chains (hydrophobic, negatively and positively charged) to create water soluble amphiphilic oligourea sequences of interest. With these building blocks in hand, H1 and a series of new oligourea sequences (11-residue long) derived from the parent sequence H1 and containing single point mutations or simple permutations were then prepared and these molecules obtained in good purities and quantities ranging from 10-50 mg. Most of sequences were designed to modulate the polar face of the helix in terms of charged distribution to better understand the requirements for the assembly of H1 and eventually create new assembly modes. One additional sequence was obtained by replacing one hydrophobic side chain by a cysteine type residue in view of creating a covalent dimer that could then further assemble into more stable or new nanostructures.
- The second work package (WP2) was related to host-guest interactions in helix bundles. The hexameric helix stoichiometry and innate symmetry of the bundle formed by H1 results in a hydrophobic cavity (500 Å3) possessing a unique three-fold symmetry. The work performed during the duration of the project was intended to exploit this cavity and a variety of potential guests with different sizes and shapes from small molecules (solvent molecules) to long hydrocarbons have been evaluated for their capacity to interact with the hydrophobic cavities of aliphatic oligoureas by using circular dichroism (CD). Temperature dependent experiments were used to get a hint about small molecules that bind in the cavity of the H1 helix bundle. The finding that long hydrocarbons interact with H1 as well as with sequence H3 is one of the most remarkable results obtained during the project.
-The third workpackage (WP3) essentially consisted in a systematic effort to characterize the structures formed by amphiphilic oligourea sequences prepared in WP1 and complexes formed in the context of WP2. The ability of the sequences to form discrete assemblies was first evaluated by using mass spectrometry under native conditions. Crystallization screens were performed by the researcher on all compounds synthesized in WP1 (with and without guest molecules) and five different sequences gave crystals (H1, H3, H4, H5 and H7). All of these crystals have been mounted on loops and sent to synchrotron for X-ray diffraction studies and structure determination and remarkably, four structures at atomic resolution have been solved out of which three are from sequences in complex with guest molecules. It is noteworthy that the resolution of three structures of helix bundles with guest molecules is well above the expected deliverables originally proposed in the project.
- The first work package (WP1) was dedicated to organic synthesis including building block synthesis and oligomer synthesis and was centred on primary sequence variations and manipulations to delineate the requirements for the six-helix bundle formation by the reference sequene (H1). During the duration of the project, a total of eight building blocks have been prepared in multi-gram scale with the necessary side chains (hydrophobic, negatively and positively charged) to create water soluble amphiphilic oligourea sequences of interest. With these building blocks in hand, H1 and a series of new oligourea sequences (11-residue long) derived from the parent sequence H1 and containing single point mutations or simple permutations were then prepared and these molecules obtained in good purities and quantities ranging from 10-50 mg. Most of sequences were designed to modulate the polar face of the helix in terms of charged distribution to better understand the requirements for the assembly of H1 and eventually create new assembly modes. One additional sequence was obtained by replacing one hydrophobic side chain by a cysteine type residue in view of creating a covalent dimer that could then further assemble into more stable or new nanostructures.
- The second work package (WP2) was related to host-guest interactions in helix bundles. The hexameric helix stoichiometry and innate symmetry of the bundle formed by H1 results in a hydrophobic cavity (500 Å3) possessing a unique three-fold symmetry. The work performed during the duration of the project was intended to exploit this cavity and a variety of potential guests with different sizes and shapes from small molecules (solvent molecules) to long hydrocarbons have been evaluated for their capacity to interact with the hydrophobic cavities of aliphatic oligoureas by using circular dichroism (CD). Temperature dependent experiments were used to get a hint about small molecules that bind in the cavity of the H1 helix bundle. The finding that long hydrocarbons interact with H1 as well as with sequence H3 is one of the most remarkable results obtained during the project.
-The third workpackage (WP3) essentially consisted in a systematic effort to characterize the structures formed by amphiphilic oligourea sequences prepared in WP1 and complexes formed in the context of WP2. The ability of the sequences to form discrete assemblies was first evaluated by using mass spectrometry under native conditions. Crystallization screens were performed by the researcher on all compounds synthesized in WP1 (with and without guest molecules) and five different sequences gave crystals (H1, H3, H4, H5 and H7). All of these crystals have been mounted on loops and sent to synchrotron for X-ray diffraction studies and structure determination and remarkably, four structures at atomic resolution have been solved out of which three are from sequences in complex with guest molecules. It is noteworthy that the resolution of three structures of helix bundles with guest molecules is well above the expected deliverables originally proposed in the project.
It is worth mentioning that the resolution of three structures of helix bundles with guest molecules achieved during the project in less than 12months is well above the expected deliverables originally proposed in the project. Because the preliminary results obtained during the course of the project as discussed above (characterization of new host-guest interactions inside the helix bundles) are extremely encouraging, efforts are currently engaged to pursue this overall research programme after termination of the action. In terms of dissemination, the writing of at least one paper that will incorporate some of the results obtained by the researcher in FOLDASYNBIO is undergoing.