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.

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.

Aromatic Foldamer Helices as α-Helix Extended Surface Mimetics.

Helically folded aromatic oligoamide foldamers have a size and geometrical parameters very distinct from those of α-helices and are not obvious candidates for α-helix mimicry. Nevertheless, they offer multiple sites for attaching side chains. It was found that some arrays of side chains at the surface of an aromatic helix make it possible to mimic extended α-helical surfaces. Synthetic methods were developed to produce quinoline monomers suitably functionalized for solid phase synthesis. A dodecamer was prepared. Its crystal structure validated the initial design and showed helix bundling involving the α-helix-like interface. These results open up new uses of aromatic helices to recognize protein surfaces and to program helix bundling in water.

Assessing Interactions between Helical Aromatic Oligoamide Foldamers and Protein Surfaces: A Tethering Approach.

Helically folded aromatic foldamers may constitute suitable candidates for the ab initio design of ligands for protein surfaces. As preliminary steps toward the exploration of this hypothesis, a tethering approach was developed to detect interactions between a protein and a foldamer by confining the former at the surface of the latter. Cysteine mutants of two therapeutically relevant enzymes, CypA and IL4, were produced. Two series of ten foldamers were synthesized bearing different proteinogenic side chains and either a long or a short linker functionalized with an activated disulfide. Disulfide exchange between the mutated cysteines and the activated disulfides yielded 20 foldamer-IL4 and 20 foldamer-CypA adducts. Effectiveness of the reaction was demonstrated by LC-MS, by MS analysis after proteolytic digestion, and by 2D NMR. Circular dichroism then revealed diastereoselective interactions between the proteins and the foldamers confined at their surface which resulted in a preferred handedness of the foldamer helix. Helix sense bias occurred sometimes with both the short and the long linkers and sometimes with only one of them. In a few cases, helix handedness preference is found to be close to quantitative. These cases constitute valid candidates for structural elucidation of the interactions involved.

Structure Elucidation of Helical Aromatic Foldamer-Protein Complexes with Large Contact Surface Areas.

The development of large synthetic ligands could be useful to target the sizeable surface areas involved in protein-protein interactions. Herein, we present long helical aromatic oligoamide foldamers bearing proteinogenic side chains that cover up to 450 Å2 of the human carbonic anhydrase II (HCA) surface. The foldamers are composed of aminoquinolinecarboxylic acids bearing proteinogenic side chains and of more flexible aminomethyl-pyridinecarboxylic acids that enhance helix handedness dynamics. Crystal structures of HCA-foldamer complexes were obtained with a 9- and a 14-mer both showing extensive protein-foldamer hydrophobic contacts. In addition, foldamer-foldamer interactions seem to be prevalent in the crystal packing, leading to the peculiar formation of an HCA superhelix wound around a rod of stacked foldamers. Solution studies confirm the positioning of the foldamer at the protein surface as well as a dimerization of the complexes.

Helical Oligourea Foldamers as Powerful Hydrogen Bonding Catalysts for Enantioselective C-C Bond-Forming Reactions.

Substantial progress has been made toward the development of metal-free catalysts of enantioselective transformations, yet the discovery of organic catalysts effective at low catalyst loadings remains a major challenge. Here we report a novel synergistic catalyst combination system consisting of a peptide-inspired chiral helical (thio)urea oligomer and a simple tertiary amine that is able to promote the Michael reaction between enolizable carbonyl compounds and nitroolefins with excellent enantioselectivities at exceptionally low (1/10 000) chiral catalyst/substrate molar ratios. In addition to high selectivity, which correlates strongly with helix folding, the system we report here is also highly amenable to optimization, as each of its components can be fine-tuned separately to increase reaction rates and/or selectivities. The predictability of the foldamer secondary structure coupled to the high level of control over the primary sequence results in a system with significant potential for future catalyst design.

Self-Assembled Protein-Aromatic Foldamer Complexes with 2:3 and 2:2:1 Stoichiometries.

The promotion of protein dimerization using the aggregation properties of a protein ligand was explored and shown to produce complexes with unusual stoichiometries. Helical foldamer 2 was synthesized and bound to human carbonic anhydrase (HCA) using a nanomolar active site ligand. Crystal structures show that the hydrophobicity of 2 and interactions of its side chains lead to the formation of an HCA2-23 complex in which three helices of 2 are stacked, two of them being linked to an HCA molecule. The middle foldamer in the stack can be replaced by alternate sequences 3 or 5. Solution studies by CD and NMR confirm left-handedness of the helical foldamers as well as HCA dimerization.

Solution Observation of Dimerization and Helix Handedness Induction in a Human Carbonic Anhydrase-Helical Aromatic Amide Foldamer Complex.

The design of synthetic foldamers to selectively bind proteins is currently hindered by the limited availability of molecular data to establish key features of recognition. Previous work has described dimerization of human carbonic anhydrase II (HCA) through self-association of a quinoline oligoamide helical foldamer attached to a tightly binding HCA ligand. A crystal structure of the complex provided atomic details to explain the observed induction of single foldamer helix handedness and revealed an unexpected foldamer-mediated dimerization. Here, we investigated the detailed behavior of the HCA-foldamer complex in solution by using NMR spectroscopy. We found that the ability to dimerize is buffer-dependent and uses partially distinct intermolecular contacts. The use of a foldamer variant incapable of self-association confirmed the ability to induce helix handedness separately from dimer formation and provided insight into the dynamics of enantiomeric selection.

Anion Recognition by Aliphatic Helical Oligoureas.

Anion binding properties of neutral helical foldamers consisting of urea type units in their backbone have been investigated. 1 H NMR titration studies in various organic solvents including DMSO suggest that the interaction between aliphatic oligoureas and anions (CH3 COO- , H2 PO4- , Cl- ) is site-specific, as it largely involves the urea NHs located at the terminal end of the helix (positive pole of the helix), which do not participate to the helical intramolecular hydrogen-bonding network. This mode of binding parallels that found in proteins in which anion-binding sites are frequently found at the N-terminus of an α-helix. 1 H NMR studies suggest that the helix of oligoureas remains largely folded upon anion binding, even in the presence of a large excess of the anion. This study points to potentially useful applications of oligourea helices for the selective recognition of small guest molecules.

Structure of a complex formed by a protein and a helical aromatic oligoamide foldamer at 2.1 Å resolution.

In the search of molecules that could recognize sizeable areas of protein surfaces, a series of ten helical aromatic oligoamide foldamers was synthesized on solid phase. The foldamers comprise three to five monomers carrying various proteinogenic side chains, and exist as racemic mixtures of interconverting right-handed and left-handed helices. Functionalization of the foldamers by a nanomolar ligand of human carbonic anhydrase II (HCA) ensured that they would be held in close proximity to the protein surface. Foldamer-protein interactions were screened by circular dichroism (CD). One foldamer displayed intense CD bands indicating that a preferred helix handedness is induced upon interacting with the protein surface. The crystal structure of the complex between this foldamer and HCA could be resolved at 2.1 Å resolution and revealed a number of unanticipated protein-foldamer, foldamer-foldamer, and protein-protein interactions.

Structural characterization of short hybrid urea/carbamate (U/C) foldamers: A case of partial helix unwinding.

Aliphatic oligoureas−(NH−CH(R)−CH2−NH−CO)n− and oligocarbamates −(NH−CH(R)−CH2−O−CO)n− are two classes of synthetic peptidomimetic oligomers whose backbone is isosteric to that of γ-peptides. We have shown recently that the constituent units of these backbones (i.e., amide (A), carbamate (C) and urea (U) units) can be combined in various ways to generate new heterogeneous oligomers with well-defined secondary structures. For example, oligomers consisting of urea (U) and carbamate (C) linkages arranged in a 1:1 pattern adopt a helical conformation akin to that of urea homoligomers and γ-peptide foldamers. In this case, helix formation is mainly driven by U units whose propensity for folding surpasses that of C units. Here, we have investigated further the influence of the U/C ratio on the folding preference of such heterogeneous oligomers. We report the synthesis and the structural analysis of a short oligomer with a 2:3 U/C ratio and two consecutive carbamate linkages. X-ray diffraction analysis reveals a helical structure that unwinds at one end. In contrast, a cognate oligomer prepared for comparison and containing four contiguous urea units adopts a fully helical conformation in the crystalline state. These results which are supported by data in solution indicate that the balance between U and C units should be carefully adjusted and that consecutive C linkages should be avoided for optimal helix formation.

Cooperative, heparan sulfate-dependent cellular uptake of dimeric guanidinoglycosides.

Oligoarginine and guanidinium-rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell-surface heparan sulfate proteoglycans as part of their cell-entry pathway. Unlike for other guanidinium-rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell-surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell-surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild-type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin-phycoerythrin-Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell-surface receptors. Competition studies with chemically desulfated or carboxy-reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell-surface heparan sulfate proteoglycans.

Folding and self-assembly of aromatic and aliphatic urea oligomers: towards connecting structure and function.

Folding and self-assembly of biomacromolecules has inspired the development of discrete, non-natural oligomers that fold and/or self-assemble in a controlled manner. Though aromatic and aliphatic oligoamides remain unmatched for structural diversity and synthetic versatility, oligomers based on amide bond surrogates, such as urea backbones, also demonstrated a propensity for folding and self-assembly. In this Perspective, we review the advances in the design of oligomeric aromatic and aliphatic urea sequences (essentially N,N'-linked) that fold and/or self-assemble. Whenever applicable, the relationship between structure and function will be highlighted.

Anion-macrodipole interactions: self-assembling oligourea/amide macrocycles as anion transporters that respond to membrane polarization.

Macrocyclic urea/amide hybrids are introduced as functional, anion-selective membrane transporters in lipid bilayer membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. "Antiparallel" macrocycles that self-assemble into "antiparallel" nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive, respectively (positive and negative AMFE). The activity of antiparallel nanotubes decreases rapidly with increasing membrane polarization, whereas parallel nanotubes are inactivated at high and activated by membrane potentials at low concentration. Hill coefficients of parallel nanotubes decrease significantly with membrane polarization, whereas those of antiparallel nanotubes increase slightly. The overall unusual characteristics of parallel nanotubes call for a new transport mechanism, where macrodipole-potential interactions account for voltage sensitivity and anion-macrodipole interactions account for anion selectivity.

Control of duplex formation and columnar self-assembly with heterogeneous amide/urea macrocycles.

The perfect blend: A new class of self-assembling cyclooligomers with mixed urea/amide backbone is described (see figure). A high level of hierarchical and directional control is achieved: depending on the level of backbone preorganization, columnar or tubular arrangements with either parallel or antiparallel growing modes can be selected.

Experimental and theoretical study of the vibrational spectra of oligoureas: helical versus ß-sheet-type secondary structures.

Ab initio calculations of two oligoureas stabilized in helix and sheet organization have been performed. The hydrogen bond distances were found to be almost the same for both structures. The vibrational assignment of the two oligourea structures and the direction of the transition moment of each vibration have been determined. From these results, and using the experimental isotropic optical index determined for one oligourea, we have established the anisotropic infrared optical files for the two structures. Interestingly, most urea absorptions vibrate in only one principal direction. Also, the shift of the carbonyl band is weaker and inverse to what was reported for corresponding protein secondary structures. Finally, simulations of the Polarization Modulation Infrared Reflection Absorption Spectroscopy (PMIRRAS) and Attenuated Reflection Spectroscopy (ATR) infrared spectra demonstrate the possibility to determine the orientation of the oligoureas in thin or ultrathin films, even if in some cases it may be difficult to unambiguously assign their secondary structure.