Domain Swapping in Abiotic Foldamers.

Foldamer sequences that adopt tertiary helix-turn-helix folds mediated by helix-helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain-swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

Molecular Sensing and Manipulation of Protein Oligomerization in Membrane Nanotubes with Bolaamphiphilic Foldamers.

Adaptive and reversible self-assembly of supramolecular protein structures is a fundamental characteristic of dynamic living matter. However, the quantitative detection and assessment of the emergence of mesoscale protein complexes from small and dynamic oligomeric precursors remains highly challenging. Here, we present a novel approach utilizing a short membrane nanotube (sNT) pulled from a planar membrane reservoir as nanotemplates for molecular reconstruction, manipulation, and sensing of protein oligomerization and self-assembly at the mesoscale. The sNT reports changes in membrane shape and rigidity caused by membrane-bound proteins as variations of the ionic conductivity of the sNT lumen. To confine oligomerization to the sNT, we have designed and synthesized rigid oligoamide foldamer tapes (ROFTs). Charged ROFTs incorporate into the planar and sNT membranes, mediate protein binding to the membranes, and, driven by the luminal electric field, shuttle the bound proteins between the sNT and planar membranes. Using Annexin-V (AnV) as a prototype, we show that the sNT detects AnV oligomers shuttled into the nanotube by ROFTs. Accumulation of AnV on the sNT induces its self-assembly into a curved lattice, restricting the sNT geometry and inhibiting the material uptake from the reservoir during the sNT extension, leading to the sNT fission. By comparing the spontaneous and ROFT-mediated entry of AnV into the sNT, we reveal how intricate membrane curvature sensing by small AnV oligomers controls the lattice self-assembly. These results establish sNT-ROFT as a powerful tool for molecular reconstruction and functional analyses of protein oligomerization and self-assembly, with broad application to various membrane processes.

Molecular torsion springs: alteration of helix curvature in frustrated tertiary folds.

The first abiotic foldamer tertiary structures have been recently reported in the form of aromatic helix-turn-helix motifs based on oligo-quinolinecarboxamides held together by intramolecular hydrogen bonds. Tertiary folds were predicted by computational modelling of the hydrogen-bonding interfaces between helices and later verified by X-ray crystallography. However, the prognosis of how the conformational preference inherent to each helix influences the tertiary structure warranted further investigation. Several new helix-turn-helix sequences were synthesised in which some hydrogen bonds have been removed. Contrary to expectations, this change did not strongly destabilise the tertiary folds. On closer inspection, a new crystal structure revealed that helices adopt their natural curvature when some hydrogen bonds are missing and undergo some spring torsion upon forming the said hydrogen bonds, thus potentially giving rise to a conformational frustration. This phenomenon sheds light on the aggregation behaviour of the helices when they are not linked by a turn unit.

Homochiral versus Heterochiral Dimeric Helical Foldamer Bundles: Chlorinated-Solvent-Dependent Self-Sorting.

Aromatic oligoamide sequences programmed to fold into stable helical conformations were designed to display a linear array of hydrogen-bond donors and acceptors at their surface. Sequences were prepared by solid-phase synthesis. Solution 1 H NMR spectroscopic studies and solid-state crystallographic structures demonstrated the formation of stable hydrogen-bond-mediated dimeric helix bundles that could be either heterochiral (with a P and an M helix) or homochiral (with two P or two M helices). Formation of the hetero- or homochiral dimers could be driven quantitatively using different chlorinated solvents-exemplifying a remarkable case of either social or narcissistic chiral self-sorting or upon imposing absolute handedness to the helices to forbid PM species.

Selective and Cooperative Photocycloadditions within Multistranded Aromatic Sheets.

A series of aromatic helix-sheet-helix oligoamide foldamers composed of several different photosensitive diazaanthracene units have been designed and synthesized. Molecular objects up to 7 kDa were straightforwardly produced on a 100 mg scale. Nuclear magnetic resonance and crystallographic investigations revealed that helix-sheet-helix architectures can adopt one or two distinct conformations. Sequences composed of an even number of turn units were found to fold in a canonical symmetrical conformation with two helices of identical handedness stacked above and below the sheet segment. Sequences composed of an odd number of turns revealed a coexistence between a canonical fold with helices of opposite handedness and an alternate fold with a twist within the sheet and two helices of identical handedness. The proportions between these species could be manipulated, in some cases quantitatively, being dependent on solvent, temperature, and absolute control of helix handedness. Diazaanthracene units were shown to display distinct reactivity toward [4 + 4] photocycloadditions according to the substituent in position 9. Their organization within the sequences was programmed to allow photoreactions to take place in a specific order. Reaction pathways and kinetics were deciphered and product characterized, demonstrating the possibility to orchestrate successive photoreactions so as to avoid orphan units or to deliberately produce orphan units at precise locations. Strong cooperative effects were observed in which the photoreaction rate was influenced by the presence (or absence) of photoadducts in the structure. Multiple photoreactions within the aromatic sheet eventually lead to structure lengthening and stiffening, locking conformational equilibria. Photoproducts could be thermally reverted.

Directing the Self-Assembly of Aromatic Foldamer Helices using Acridine Appendages and Metal Coordination.

Folded molecules provide complex interaction interfaces amenable to sophisticated self-assembly motifs. Because of their high conformational stability, aromatic foldamers constitute suitable candidates for the rational elaboration of self-assembled architectures. Several multiturn helical aromatic oligoamides have been synthesized that possess arrays of acridine appendages pointing in one or two directions. The acridine units were shown to direct self-assembly in the solid state via aromatic stacking leading to recurrent helix-helix association patterns under the form of discrete dimers or extended arrays. In the presence of Pd(II), metal coordination of the acridine units overwhelms other forces and generates new metal-mediated multihelical self-assemblies, including macrocycles. These observations demonstrate simple access to different types of foldamer-containing architectures, ranging from discrete objects to 1D and, by extension, 2D and 3D arrays.

Oligo-Quinolylene-Vinylene Foldamers.

Quinoline based aromatic amide foldamers are known to adopt stable folded conformations. We have developed a synthetic approach to produce similar oligomers where all amide bonds, or part of them, have been replaced by an isosteric vinylene group. The results of solution and solid state structural studies show that oligomers exclusively containing vinylene linkages are not well folded, and adopt predominantly flat conformations. In contrast, a vinylene segment flanked by helical oligoamides also folds in a helix, albeit with a slightly lower curvature. The presence of vinylene functions also result in an extension of π-conjugation across the oligomer that may change charge transport properties. Altogether, these results pave the way to foldamers in which both structural control and specific electronic properties may be engineered.

Large-Amplitude Conformational Changes in Self-Assembled Multi-Stranded Aromatic Sheets.

The orchestration of ever larger conformational changes is made possible by the development of increasingly complex foldamers. Aromatic sheets, a rare motif in synthetic foldamer structures, have been designed so as to form discrete stacks of intercalated aromatic strands through the self-assembly of two identical subunits. Ion-mobility ESI-MS confirms the formation of compact dimers. X-ray crystallography reveals the existence of two distinct conformational dimeric states that require large changes to interconvert. Molecular dynamics simulation validates the stability of the two conformations and the possibility of their interconversion.

Loading Linear Arrays of CuII Inside Aromatic Amide Helices.

The very stable helices of 8-amino-2-quinolinecarboxylic acid oligoamides are shown to uptake CuII ions in their cavity through deprotonation of their amide functions with minimal alteration of their shape, unlike most metallo-organic structures which generally differ from their organic precursors. The outcome is the formation of intramolecular linear arrays of a defined number of CuII centers (up to sixteen in this study) at a 3 Šdistance, forming a molecular mimic of a metal wire completely surrounded by an organic sheath. The helices pack in the solid state so that the arrays of CuII extend intermolecularly. Conductive-AFM and cyclic voltammetry suggest that electrons are transported throughout the metal-loaded helices in contrast with hole transport observed for analogous foldamers devoid of metal ions.

Simplification in the Acquisition and Analysis of Fluorescence Decays Acquired with Polarized Emission for Time-Resolved Fluorescence Anisotropy Measurements.

This study introduces a global fluorescence decay analysis that substantially simplifies the acquisition and analysis of time-resolved fluorescence decays acquired with a vertically polarized excitation and vertically (IVV(t)) and horizontally (IVH(t)) polarized emission for time-resolved fluorescence anisotropy (TRFA) measurements. TRFA measurements were conducted whereby the IVV(t) and IVH(t) fluorescence decays of a series of oligoquinolines labeled at one end with an oligo(phenylenevinylene) dye (OPV-Qn with n = 4, 7, 17, 24, 33) were acquired according to the standard protocol that is currently accepted in the scientific literature which involves toggling the emission polarizer before fitting linear combinations of the IVV(t) and IVH(t) decays or acquiring the IVV(t) and IVH(t) decays with static polarizers before fitting them globally. The rotational time (ϕ) and initial anisotropy (r0) retrieved from these analyses were identical within experimental error regardless of whether the decays were acquired with toggling or static polarizers and fitted according to the standard protocol or globally. These experimental results were further supported by retrieving the parameters used to generate mono-, bi-, and tri-exponential TRFAs from the global analysis of simulated IVV(t) and IVH(t) fluorescence decays which were found to match perfectly the values that were inputted. Together, these experiments and simulations demonstrated that the parameters describing any type of TRFA can be extracted directly from the analysis of the IVV(t) and IVH(t) fluorescence decays acquired with a standard time-resolved fluorometer, a substantial simplification compared to the protocols currently in place to determine the TRFA.

Parallel Homochiral and Anti-Parallel Heterochiral Hydrogen-Bonding Interfaces in Multi-Helical Abiotic Foldamers.

A hydrogen-bonding interface between helical aromatic oligoamide foldamers has been designed to promote the folding of a helix-turn-helix motif with a head-to-tail arrangement of two helices of opposite handedness. This design complements an earlier helix-turn-helix motif with a head-to-head arrangement of two helices of identical handedness interface. The two motifs were shown to have comparable stability and were combined in a unimolecular tetra-helix fold constituting the largest abiotic tertiary structure to date.

Light-Controlled Conformational Switch of an Aromatic Oligoamide Foldamer.

An aromatic oligoamide sequence composed of a light-responsive diazaanthracene-based aromatic ß-sheet flanked by two variable diameter helical segments was prepared. Structural investigations revealed that such oligomers adopt two distinct conformations: a canonical symmetrical conformation with the two helices stacked above and below the sheet, and an unanticipated unsymmetrical conformation in which one helix has flipped to directly stack with the first helix. Photoirradiation of the foldamer led to the quantitative, and thermally reversible, formation of a single photoproduct resulting from the [4+4] cycloaddition of two diazaanthracenes within the aromatic ß-sheet. NMR and crystallographic studies revealed a parallel arrangement of the diazaanthracene photoproduct and a complete conversion into a symmetrical conformation requiring a rearrangement of all unsymmetrical conformers. These results highlight the potential of foldamers, with structures more complex than isolated helices, for the design of photoswitches showing nontrivial nanometer scale shape changes.

Controlling Dipole Orientation through Curvature: Aromatic Foldamer Bent ß-Sheets and Helix-Sheet-Helix Architectures.

The helix, turn, and ß-strand motifs of biopolymer folded structures have been found to prevail also in non-natural backbones. In contrast, foldamers with aryl rings in their main chains possess distinct conformations that may give access to folded objects beyond the reach of peptidic and nucleotidic backbones. In search of such original architectures, we have explored the effect of bending aromatic amide ß-sheets using building blocks that impart curvature. Cyclic and multiturn noncyclic sequences were synthesized, and their structures were characterized in solution and in the solid state. Stable bent-sheet conformations were shown to prevail in chlorinated solvents. In these structures, folding overcomes intramolecular electrostatic repulsions and forces local dipoles in each layer of the stacked strands to align in a parallel fashion. Sequences having helical segments flanking a central bent aromatic ß-sheet were then synthesized and shown to form well-defined helix-turn-helix architectures in which helical and sheet subcomponents conserve their respective integrity. These objects have a unique basket shape; they possess a cavity the depth and width of which reflects the curvature of the ß-sheet segment. They can be compared to previously described helical closed-shell receptors in which a window has been open, thus providing a means to control guest binding and release pathways and kinetics. As a proof of concept, guest binding to one of the helix-sheet-helix structures is indeed found to be fast on the NMR time scale while it is generally slow in the case of helical capsules.

Photoinduced Electron Transfer and Hole Migration in Nanosized Helical Aromatic Oligoamide Foldamers.

A series of photoactive triads have been synthesized and investigated in order to elucidate photoinduced electron transfer and hole migration mechanism across nanosized, rigid helical foldamers. The triads are comprised of a central helical oligoamide foldamer bridge with 9, 14, 18, 19, or 34 8-amino-2-quinolinecarboxylic acid repeat units, and of two chromophores, an N-terminal oligo(para-phenylenevinylene) electron donor and a C-terminal perylene bis-imide electron acceptor. Time-resolved fluorescence and transient absorption spectroscopic studies showed that, following photoexcitation of the electron acceptor, fast electron transfer occurs initially from the oligoquinoline bridge to the acceptor chromophore on the picosecond time scale. The oligo(para-phenylenevinylene) electron donor is oxidized after a time delay during which the hole migrates across the foldamer from the acceptor to the donor. The charge separated state that is finally generated was found to be remarkably long-lived (>80 µs). While the initial charge injection rate is largely invariant for all foldamer lengths (ca. 60 ps), the subsequent hole transfer to the donor varies from 1 × 109 s-1 for the longest sequence to 17 × 109 s-1 for the shortest. In all cases, charge transfer is very fast considering the foldamer length. Detailed analysis of the process in different media and at varying temperatures is consistent with a hopping mechanism of hole transport through the foldamer helix, with individual hops occurring on the subpicosecond time scale (kET = 2.5 × 1012 s-1 in CH2Cl2). This work demonstrates the possibility of fast long-range hole transfer over 300 Å (through bonds) across a synthetic modular bridge, an achievement that had been previously observed principally with DNA structures.

Selective Dynamic Assembly of Disulfide Macrocyclic Helical Foldamers with Remote Communication of Handedness.

Disulfide bridge formation was investigated in helical aromatic oligoamide foldamers. Depending on the position of thiol-bearing side chains, exclusive intramolecular or intermolecular disulfide bridging may occur. The two processes are capable of self-sorting, presumably by dynamic exchange. Quantitative assessment of helix handedness inversion rates showed that bridging stabilizes the folded structures. Intermolecular disulfide bridging serendipitously yielded a well-defined, C2 -symmetrical, two-helix bundle-like macrocyclic structure in which complete control over relative handedness, that is, helix-helix handedness communication, is mediated remotely by the disulfide bridged side chains in the absence of contacts between helices. MM calculations suggest that this phenomenon is specific to a given side chain length and requires disulfide functions.

Aromatic oligoamide ß-sheet foldamers.

A rational approach for the construction of multi-stranded artificial ß-sheets based not on hydrogen bonding, but rather on π-π aromatic stacking, is presented. Using 4,6-dinitro-1,3-phenylenediamine units, rigid turns were designed that allow face-to-face π-π interactions between appended linear aromatic segments to be strong enough for folding in an organic solvent, but weak enough to prevent aggregation and precipitation. Solution and solid-state studies on a series of turn units showed that the desired degree of rigidity, resulting from hindered bond rotation, could be fine-tuned by the inclusion of additional methyl substituents on the aromatic rings. The high degree of preorganization afforded by these qualities further allowed the facile preparation of macrocyclic sheet structures from their noncyclic precursors. These macrocycles were shown to have slow internal dynamics and defined conformational preferences. Using this background, three- and five-stranded artificial ß-sheets were synthesized and their folded conformations extensively characterized in solution by NMR. The solid-state structures of the three- and five-stranded sheets were also elucidated in the solid state by X-ray crystallography and confirmed intramolecular π-π aromatic stacking.

Folding of a linear array of α-amino acids within a helical aromatic oligoamide frame.

Control of the spatial organization of proteinogenic side chains is critical for the development of protein mimics with selective recognition properties toward target protein surfaces. We present a novel methodology for producing a linear array of proteinogenic residues based on the incorporation of α-amino acids into sequences of rigid, helically folded oligoamides of 8-amino-2-quinolinecarboxylic acid (Q). When L-leucine (L) was alternated with dimer Q2, the resulting sequence adopted a right-handed helical conformation, as deduced in solution from the CD spectra of L-(LQ2)n (n = 2, 4) and in the solid state from X-ray crystallographic analysis of (±)-(LQ2)4. Each LQ2 segment spanned just one helix turn (pitch of 3.5 Å), and consequently, the four leucine side chains of (LQ2)4 formed a linear array. In solution, NMR analysis showed that both L-(LQ2)2 and L-(LQ2)4 exist as a mixture of two slowly equilibrating folded conformers, the proportion of which strongly varies with the solvent.

An abiotic, tetrameric, eight-helix bundle.

Four helically folded aromatic oligoamide sequences containing either a chiral monomer based on 2-(2-aminophenoxy)-propionic acid, an N-terminal (1H)-camphanyl group, or both, were synthesized. Spectroscopic solution investigations using 1H NMR and circular dichroism (CD) demonstrated that the 2-(2-aminophenoxy)-propionic acid unit biases helix handedness quantitatively in chloroform and dichloromethane. It even quantitatively overcomes an opposing effect of the camphanyl group and thus ensures reliable helix handedness control. A series of nine sequences composed of two helically folded aromatic oligoamide segments separated by a flexible linker based on a di-, tri- or tetraethylene glycol unit were then synthesized. In these sequences, helix handedness was controlled by means of an N-terminal (1H)-camphanyl group or a 2-(2-aminophenoxy)-propionic acid units in either both helical segments, or only in the N-terminal segment, or in none of the segments. The helical segments all displayed hydroxy and carbonyl groups at their surfaces as hydrogen bond donors and acceptors so as to promote helix-to-helix hydrogen bonding. NMR and CD spectroscopic studies showed that, in some cases, well-defined, stable, discrete abiotic helix-turn-helix tertiary folds form in organic solvents. Molecular modelling suggests that these correspond to structures in which the two helix axes are at an angle. In one case, the absence of handedness control resulted in a complex and large aggregate. A solid state structure obtained by single crystal X-ray diffraction analysis revealed a tetrameric assembly composed of eight helices with both right and left handedness arranged in three subdomains consisting of two hydrogen-bonded three-helix bundles and one two-helix-bundle. Several helix-to-helix hydrogen bonds were mediated by bridging water molecules. This structure constitutes an important milestone in the construction of abiotic protein-like architectures.

Coaxial assembly of helical aromatic foldamers by metal coordination.

Deprotonation of acid-terminated helical aromatic foldamers with a mineral base in chlorinated solvents led to their dimerization through the coordination of a metal cation (Li+, Na+, K+, Ag+, or Hg2+) with the terminal carboxylate functions. This new ligation method was applied to oligomerize diacid-functionalized foldamers.

Structural adaptations of electrosprayed aromatic oligoamide foldamers on Ag(111).

Aromatic foldamers are promising for applications such as molecular recognition and molecular machinery. For many of these, defect free, 2D-crystaline monolayers are needed. To this end, submonolayers were prepared in ultra-high vacuum (UHV) on Ag(111) via electrospray controlled ion beam deposition (ES-CIBD). On the surface, the unfolded state is unambiguously identified by real-space single-molecule imaging using scanning tunnelling microscopy (STM) and it is found to assemble in regular structures.