Lucile Fischer

The Canonical Helix of Urea Oligomers at Atomic Resolution: Insights Into Folding‐Induced Axial Organization

Foldamers are discrete artificial oligomers with defined and predictable folding patterns akin to naturally occurring helices, turns, and linear strands. Because of their diversity in size, shape, and side chain appendages, and also their resistance to enzymatic degradation, peptidomimetic helical foldamers are unique scaffolds for use in a range of biological and biomedical applications. Characterizing such helical folds at atomic resolution is of prime importance if molecules are to be designed that can target biological surfaces and for reliable structure–function analysis. To date, extensive crystallographic data sets have been gathered on aliphatic (band a/b-peptides) and aromatic oligoamides, thus providing a detailed picture of the structural diversity within these foldamer families. Few other helical peptidomimetic backbones have been characterized by crystallographic analysis. Crystal structures are also central to gain precise insight into axial and lateral 13] self-assembling properties of helical foldamers, en route to new tertiary and quaternary structural motifs and more sophisticated self-assembled nanostructures. Notable achievements include the atomic structure determination of large (> 8 kDa) aromatic oligoamide foldamers and helix-bundle quaternary structures formed by designed band a/b-peptides. 13] Oligomers consisting of N,N’-linked urea bridging units are receiving increasing attention as folding backbones. Peptidomimetic oligoureas belonging to the g-peptide lineage, (-NH-CH(R)-CH2N’H-CO)n-, have a remarkable propensity to fold into helical secondary structures in solution and show promise for interaction with biologi-

Condensation Approach to Aliphatic Oligourea Foldamers: Helices with N‐(Pyrrolidin‐2‐ylmethyl)ureido Junctions

The design and synthesis of large and complex folded structures resembling those of natural biopolymers is one of the current challenges in the field of foldamers. Despite the difficulty, significant progress in this direction has been made over the last few years. Remarkably long helical segments, tertiary-type structures, and quaternary arrangements (helix bundles) constructed from aliphatic b peptides, a/b-peptide hybrids, or aromatic oligoamides, have been characterized at atomic resolution. Long helical foldamers also show promise as a-helical mimics to inhibit protein–protein interactions. For example, 33-residue-long helical a/b peptides designed to mimic the heptad repeat 2 domain of the HIV protein gp41 are potent inhibitors of virus fusion and display significant improvement in proteolytic stability over corresponding a peptides. Also promising is the use of proteins in which elements of the secondary structure have been replaced by synthetic foldamers to address the role of individual folded segments and to replicate or modulate protein topology and function. Nevertheless, a prerequisite to accessing highmolecular-weight foldamers is the development of a robust synthetic methodology. In the case of aliphatic and aromatic oligoamides, optimized procedures involving convergent condensation of activated segments, 7, 8a] and stepwise solidphase synthesis (eventually assisted by microwave irradiation), have proven particularly useful. However, such methods have hardly been applied to the construction of long non-oligoamide segments. Aliphatic oligoureas of the general formula [NHCH(R)CH2NHCO]n represent an interesting class of peptidomimetic foldamers with potential for interacting with bio-macromolecules. High resolution structural studies in solution and in the crystal state have shown that these aza analogues of g peptides form well-defined 2.5-helical structures stabilized by three-centered hydrogen bonds. Although aliphatic oligoureas can be prepared by solidphase techniques, the need for long coupling times and the limitations imposed by the choice of the N-protecting group have so far limited the synthesis of oligourea helices to short segments of about 10 units long. 12] To decrease the number of synthetic steps and thus evolve more rapidly towards longer oligomers, we now introduce an iterative segment condensation approach to oligourea foldamers. Our initial plan was to activate short oligoureas bearing an amino terminus with succinimidyl carbonate to yield the corresponding activated segment A. However, the competitive formation of cyclic biuret (B) resulting from the attack of the activated succinimidyl carbamate by the nearest urea NH was found to significantly reduce the yield of A (Scheme 1a).

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.

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.

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.

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.

Experimental and Theoretical Study of the Vibrational Spectra of Oligoureas: Helical versus β-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.

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.

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.

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.