DNA mimic foldamers affect chromatin composition and disturb cell cycle progression.

The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Enhancing the Features of DNA Mimic Foldamers for Structural Investigations.

DNA mimic foldamers based on aromatic oligoamide helices bearing anionic phosphonate side chains have been shown to bind to DNA-binding proteins sometimes orders of magnitude better than DNA itself. Here, we introduce new features in the DNA mimic foldamers to facilitate structural investigations of their interactions with proteins. Thirteen new foldamer sequences have been synthesized and characterized using NMR, circular dichroism, molecular modeling, and X-ray crystallography. The results show that foldamer helix handedness can be quantitatively biased by means of a single stereogenic center, that the foldamer structure can be made C2-symmetrical as in palindromic B-DNA sequences, and that associations between foldamer helices can be promoted utilizing dedicated C-terminal residues that act as sticky ends in B-DNA structures.

Abiotic foldamer quaternary structures.

Abiotic aromatic foldamer sequences have been previously shown to fold in helix-turn-helix motifs in organic solvents. Using simple computational tools, a new helix-turn-helix motif was designed that bears additional hydrogen bond donor OH groups to promote its aggregation into a genuine, trimeric, abiotic quaternary structure. This sequence was synthesized and its self-assembly in solution was investigated by Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and Molecular Dynamics (MD) simulations. The existence of two stable discrete aggregates was evidenced, one assigned to the initially designed trimer, the other to a dimer including multiple water molecules. The two species may be quantitatively interconverted upon changing the water content of the solution or the temperature. These results represent important steps in the design of protein-like abiotic architectures.

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.

Cascading Macrocycle and Helix Motions in a Foldarotaxane Molecular Shuttle.

The design of a dynamically assembled foldarotaxane was envisioned with the aim of operating as a two cascading triggers-based molecular shuttle. In acidic conditions, both the macrocycle and helix were localized around their respective best molecular stations because they are far enough from each other not to alter the stability of complexes. The pH-dependent localization of the macrocycle along the encircled axle allowed us to modulate the association between the helical foldamer and its sites of interaction on the axle. Under kinetic control, at low concentration and room temperature, when the foldarotaxane supramolecular architecture is kinetically stable, the pH-responsive translation of the macrocycle along the thread triggered the gliding of the helix away from its initial best station. At higher concentration, when helix assembly/disassembly process is accelerated, the system reached the equilibrium state. A new foldarotaxane isomer then appeared through the change of the relative position of the helix and macrocycle along the thread. In this isomer, the helix segregated the macrocycle away from its best station. The fine control of the kinetic and thermodynamic processes, combined with the control of pH, allowed the reciprocal segregation of the helix or the ring away from their respective best sites of interaction.

(Re-)Directing Oligomerization of a Single Building Block into Two Specific Dynamic Covalent Foldamers through pH.

Dynamic foldamers are synthetic folded molecules which can change their conformation in response to an external stimulus and are currently at the forefront of foldamer chemistry. However, constitutionally dynamic foldamers, which can change not only their conformation but also their molecular constitution in response to their environment, are without precedent. We now report a size- and shape-switching small dynamic covalent foldamer network which responds to changes in pH. Specifically, acidic conditions direct the oligomerization of a dipeptide-based building block into a 16-subunit macrocycle with well-defined conformation and with high selectivity. At higher pH the same building block yields another cyclic foldamer with a smaller ring size (9mer). The two foldamers readily and repeatedly interconvert upon adjustment of the pH of the solution. We have previously shown that addition of a template can direct oligomerization of the same building block to yet other rings sizes (including a 12mer and a 13mer, accompanied by a minor amount of 14mer). This brings the total number of discrete foldamers that can be accessed from a single building block to five. For a single building block system to exhibit such highly diverse structure space is unique and sets this system of foldamers apart from proteins. Furthermore, the emergence of constitutional dynamicity opens up new avenues to foldamers with adaptive behavior.

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.

Foldaxanes: Rotaxane-like Architectures from Foldamers.

Mechanically interlocked molecules such as rotaxanes and catenanes contain free-moving components that cannot dissociate and have enabled the investigation and control of various translational and rotational molecular motions. The architecture of pseudo-rotaxanes and of some kinetically labile rotaxanes is comparable to that of rotaxanes but their components are reversibly associated and not irreversibly interlocked. In other words, pseudo-rotaxanes may fall apart. This Account focuses on a peculiar family of rotaxane-like architectures termed foldaxanes.Foldaxanes consist of a helically folded oligomer wound around a rod-like dumbbell-shaped guest. Winding of the helix around the rod thus entails an unwinding-rewinding process that creates a kinetic barrier. It follows that foldaxanes, albeit reversibly assembled, have significant lifetimes and may not fall apart while defined molecular motions are triggered. Foldaxanes based on helically folded aromatic oligoamide hosts and oligo(alkyl carbamate) guests can be designed rationally through the inclusion of complementary binding motifs on the rod and at the inner rim of the helix so that helix length and rod length match. Single helical foldaxanes (bimolecular species) and double helical foldaxanes (trimolecular species) have thus been produced as well as poly[n]foldaxanes, in which several helices bind to long rods with multiple binding stations. When the binding stations differ and are organized in a certain sequence, a complementary sequence of different stacked helices, each matching with their binding station, can be assembled, thus reproducing in an artificial system a sort of translation process.Foldaxane helix handedness may be controlled by stereogenic centers on the rod-like guest. Handedness can also be transmitted from helix to helix in polyfoldaxanes. Foldaxane formation has drastic consequences for the rod properties, including its stiffening and the restriction of the mobility of a macrocycle already interlocked on the rod. Fast translation (without dissociation) of helices along rod-like guests has been demonstrated. Because of the helical nature of the hosts, translation may be accompanied by rotation in various sorts of screw-like motions. The possibility, on longer time scales, for the helix to dissociate from and reassociate to the rod has allowed for the design of complex, kinetically controlled supramolecular pathways of a helix on a rod. Furthermore, the design of helices with a directionality, that is, with two distinct termini, that bind to nonsymmetrical rod-like guests in a defined orientation makes it possible to also control the orientation of molecular motion. Altogether, foldaxanes constitute a distinct and full-of-potential family of rotaxane-like architectures that possess designer structures and allow orchestration of the time scales of various supramolecular events.

Optimization and Automation of Helical Aromatic Oligoamide Foldamer Solid-Phase Synthesis.

Helically folded oligoamides of 8-amino-2-quinolinecarboxylic acid composed of up to 41 units were prepared using optimized manual solid-phase synthesis (SPS). The high yield and purity of the final products places these SPS protocols among the most efficient known to date. Furthermore, analytical methods allowing for the clear identification and purity assessment of the products were validated, including 1 H NMR, a seldom used method for such large molecules. Adaption of the SPS protocols, in particular using in situ acid chloride activation under Appel's conditions, made it possible to efficiently implement SPS on a commercial peptide synthesizer, leading to a dramatic reduction of the laboratory work required to produce long sequences. Automation constitutes a breakthrough for the development of helical aromatic oligoamide foldamers.

Combining local conformational preferences and solvophobic effects in helical aromatic oligoamide foldamers.

Aromatic oligoamide foldamers were designed using a newly-developed monomer so that helical folding was promoted by both local conformation preferences and solvophobic effects. Solid phase synthesis provided quick access to the desired sequences. Sharp solvent-driven conformational transitions that depended on sequence length were evidenced by both NMR and UV absorption spectroscopies.

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.

High-Affinity Hybridization of Complementary Aromatic Oligoamide Strands in Water.

We prepared a series of water-soluble aromatic oligoamide sequences all composed of a segment prone to form a single helix and a segment prone to dimerize into a double helix. These sequences exclusively assemble as antiparallel duplexes. The modification of the duplex inner rim by varying the nature of the substituents borne by the aromatic monomers allowed us to identify sequences that can hybridize by combining two chemically different strands, with high affinity and complete selectivity in water. X-ray crystallography confirmed the expected antiparallel configuration of the duplexes whereas NMR spectroscopy and mass spectrometry allowed us to assess precisely the extent of the hybridization. The hybridization kinetics of the aromatic strands was shown to depend on both the nature of the substituents responsible for strand complementarity and the length of the aromatic strand. These results highlight the great potential of aromatic hetero-duplex as a tool to construct non-symmetrical dynamic supramolecular assemblies.

Display Selection of a Hybrid Foldamer-Peptide Macrocycle.

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

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.

Isotope Ratio Encoding of Sequence-Defined Oligomers.

Information storage at the molecular level commonly entails encoding in the form of ordered sequences of different monomers and subsequent fragmentation and tandem mass spectrometry analysis to read this information. Recent approaches also include the use of mixtures of distinct molecules noncovalently bonded to one another. Here, we present an alternate isotope ratio encoding approach utilizing deuterium-labeled monomers to produce hundreds of oligomers endowed with unique isotope distribution patterns. Mass spectrometric recognition of these patterns then allowed us to directly readout encoded information with high fidelity. Specifically, we show that all 256 tetramers composed of four different monomers of identical constitution can be distinguished by their mass fingerprint using mono-, di-, tri-, and tetradeuterated building blocks. The method is robust to experimental errors and does not require the most sophisticated mass spectrometry instrumentation. Such isotope ratio-encoded oligomers may serve as tags that carry information, but the method mainly opens up the capability to write information, for example, about molecular identity, directly into a pure compound via its isotopologue distribution obviating the need for additional tagging and avoiding the use of mixtures of different molecules.

Generalizing the Aromatic δ-Amino Acid Foldamer Helix.

A series of aromatic oligoamide foldamer sequences containing different proportions of three δ-amino acids derived from quinoline, pyridine, and benzene and possessing varying flexibility, for example due to methylene bridges, were synthesized. Crystallographic structures of two key sequences and 1 H NMR data in water concur to show that a canonical aromatic helix fold prevails in almost all cases and that helix stability critically depends on the ratio between rigid and flexible units. Notwithstanding subtle variations of curvature, i. e. the numbers of units per turn, the aromatic δ-peptide helix is therefore shown to be general and tolerant of a great number of sp3 centers. We also demonstrate canonical helical folding upon alternating two monomers that do not promote folding when taken separately: folding occurs with two methylenes between every other unit, not with one methylene between every unit. These findings highlight that a fine-tuning of helix handedness inversion kinetics, curvature, and side chain positioning in aromatic δ-peptidic foldamers can be realized by systematically combining different yet compatible δ-amino acids.

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.

Differential Peptide Multi-Macrocyclizations at the Surface of a Helical Foldamer Template.

Hybrid sequences comprising a peptide with several Cys residues and an aromatic foldamer helix with several chloroacetamide functions at its surface were synthesized. Such products may in principle form numerous macromulticyclic thioether products by intramolecularly combining all Cys residues and all chloroacetamide functions. However, we show that the reactive sites on the structurally defined helix can be placed at such locations that the peptide selectively stitches itself to form a series of different macrocycles within mostly one preferred product. Reactions were monitored by HPLC and products with two, three or four macrocycles were identified using LC-MS and NMR. The series of selective macrocyclizations define a sort of reaction trail where reaction sites otherwise identical are involved successively because of their precise positioning in space. The trails can be predicted to a large extent based on structural considerations and the assumption that smaller macrocycles form faster.

Discrete Stacked Dimers of Aromatic Oligoamide Helices.

Tight binding was observed between the C-terminal cross section of aromatic oligoamide helices in aqueous solution, leading to the formation of discrete head-to-head dimers in slow exchange on the NMR timescale with the corresponding monomers. The nature and structure of the dimers was evidenced by 2D NOESY and DOSY spectroscopy, mass spectrometry and X-ray crystallography. The binding interface involves a large hydrophobic aromatic surface and hydrogen bonding. Dimerization requires that helices have the same handedness and the presence of a C-terminal carboxy function. The protonation state of the carboxy group plays a crucial role, resulting in pH dependence of the association. Dimerization is also influenced by neighboring side chains and can be programmed to selectively produce heteromeric aggregates.