INTRODUCTION:

Life relies on polymers that are able to form a well-defined hierarchy of self-assembling structural levels. An essential attribute of biological macromolecules such as proteins and nucleic acids is their masterful control over the non-covalent forces that govern folding and self-assembly processes. For a long time only biopolymers were known to have such properties, whereas non-natural folded polymers (foldamers) have the potential for being similarly or even more versatile¹. A foldamer-a discrete chain molecule or oligomer-folds into a conformationally ordered state in solution, and its structure is stabilized by a collection of noncovalent interactions between adjacent monomer units. It is easy to admit that foldamer research has been motivated by the highly-ordered structures of the well-known biopolymers and the fact that the folding into a specific regular structure is the key to their functions such as molecular recognition, catalysis, and information storage². Therefore it is obvious that foldamers ability in mimicing the attributes of proteins, nucleic acids, and polysacharides can be interpreted with their folding into well-defined conformations, such as helices, sheets, turns, as seen in biological macromolecules.

Figure-1

Foldamers can be roughly classified into three categories with regard to their monomer types: peptidomimetic foldamers, nucleotidomimetic foldamers, and abiotic foldamers. The former two are inspired by the structures of proteins and nucleic acids, and are mainly based on the modification of the chemical structure of the monomer (amino acids and nucleotides), while the latter one utilizes aromatic interactions, charge-transfer interactions, and others, that are not general in the nature. Among them, inspired by sophisticated structures and functions of proteins, the peptidomimetic foldamers have been most actively investigated so far³. The major advantage in peptidomimetic foldamers is that the amide groups, that combine monomers into the chain, also act as cross-linking points via hydrogen bonding between the amide proton and the carbonyl oxygen to fold the chain into a regular structure. Therefore these structures are able to easily form secondary structures (various helix-types, strand-like conformations, and turns) and are capable of forming higher-order self-assemblies too. That’s why they belong among the most intriguing models of unnatural polymers. The importance of foldamers in chemistry and biochemistry can be verified by a number of interesting supramolecular properties including molecular self-assembly, molecular recognition, and host-guest chemistry. They have been studied as models of biological molecules and have been shown to display antimicrobial activity. They also have great potential application to the development of new functional materials⁴.

Chemistry:

A foldamer or tyligomer as Moore proposed (deriving from Greek tyligos, meaning “to fold”), is a discrete chain molecule or oligomer that adopts a secondary structure stabilized by non-covalent interactions⁵. They are artificial molecules that mimic the ability of proteins, nucleic acids, and polysaccharides to fold into well-defined conformations, such as helices and β-sheets. Foldamers have been demonstrated to display a number of interesting supramolecular properties including molecular self-assembly, molecular recognition, and host-guest chemistry. They are studied as models of biological molecules and have been shown to display antimicrobial activity. They also have great potential application to the development of new functional materials⁶.

Examples:

· m-Phenylene ethynylene oligomers are driven to fold into a helical conformation by solvophobic forces and aromatic stacking interactions.

· β-peptides are composed of amino acids containing an additional methylene unit between the amine and carboxylic acid. They are more stable to enzymatic degradation and have been demonstrated to have antimicrobial activity.

· Peptoids are N-substituted polyglycines that utilize steric interactions to fold into polyproline type-I-like helical structures.

· Aedamers that fold in aqueous solutions driven by hydrophobic and aromatic stacking interactions.

The main objective of the action is to develop peptidomimetic foldamers into a technology platform in drug discovery and biomedical applications. The goal is to relay the ideas, pharmacophore models and requirements among the potential biomedical applications (e.g., inhibition of protein-protein interactions, self-assembling nanostructured drug delivery systems, functional biomimetic materials, etc.), to the laboratories involved in foldamer design and synthesis, and the researchers who are continuously extending the pool of homologated amino acidss. This parallel top-down and bottom-up information handling is expected to boost the application oriented foldamer research in Europe⁷. The functions carried out by proteins and nucleic acids provide the foundation for life, and chemists have begun to ask whether it is possible to design synthetic oligomers that approach the structural and functional complexity of these biopolymers. The study of foldamers, non-natural oligomers with discrete folding propensities, has demonstrated that a variety of synthetic backbones can show biopolymer-like conformational behavior. Early work in this area focused on oligomers comprised of a single type of monomer subunit, but recent efforts have highlighted the potential of mixed or “heterogeneous” backbones to expand the structural and functional repertoire of foldamers. In this Account, we illustrate the promise of heterogeneous backbone foldamers by focusing on examples containing both α- and β-amino acid residues. The use of heterogeneous backbone foldamers offers advantages over homogeneous backbone counterparts, including access to many new molecular shapes, based on variations in the stoichiometries and patterns of subunit combination, and improved prospects for side chain diversification. Recent efforts to develop α/β-peptide foldamers can be divided into two conceptually distinct classes.

Figure-2

The first includes entities prepared by a “block” strategy, in which α-peptide segments and β-peptide segments are combined to form a hybrid oligomer⁸. The second class encompasses designs in which α- and β-amino acid monomers are interspersed in a regular pattern throughout an oligomer sequence. A variety of secondary structures has been generated from α/β-peptides via these approaches. Helical secondary structures available to α/β-peptides have recently been parlayed into higher order structure, specifically, helix bundle quaternary structure. Desirable biological functions have been elicited from α/β-peptide foldamers. Efforts to mimic naturally occurring host-defense α-peptides have yielded new antimicrobial agents and led to a re-examination of the long-held views regarding structure-activity relationships among α-peptides and other amphiphilic oligomers. Foldamers offer new platforms for mimicry of molecular surfaces involved in specific protein-protein recognition events; recent achievements with α/β-peptide inhibitors of protein-protein interactions involved in apoptotic signaling have revealed benefits of heterogeneous backbones relative to homogeneous backbones for foldamer-based designs. These initial successes in the development of α/β-peptides with specific biological activities highlight the potential of heterogeneous backbone foldamers for biomedical applications and provide guidelines for the future work on new target functions⁹.

Foldamers are unnatural oligomers that display conformational propensities akin to those of proteins and nucleic acids, the oligomers that play starring roles in living systems¹⁰. The relationship between folding and function among proteins has long been a source of fascination to the molecularly inclined scientist. The interplay between α-amino acid residue sequence and the three-dimensional arrangement of these subunits that results from adoption of a specific conformation enables proteins to manifest an extraordinary range of functions. Chemists have been drawn to ask whether other backbones, containing subunits that were not selected by biological evolution, might be competent to support recognition, catalysis or assembly activities comparable to those displayed by folding biopolymers. Since many of these activities appear to require precise spatial positioning of key functional groups (“side chains”), foldamer studies often begin with an effort to determine whether a particular family of oligomers has any tendency to adopt specific shapes.

Many oligomeric backbones have been evaluated as potential foldamers since the mid-1990s. A majority of the foldamers studied to date are analogous to their biopolymer progenitors in that all subunits fall within a single class. Thus, for example, β-peptide foldamers contain exclusively β-amino acid residues, and m-phenyleneethynylene foldamers contain exclusively meta-linked phenylacetylene subunits. We refer to these systems as having “homogeneous” backbones. Recently, a number of research groups have turned their attention to foldamers with “heterogeneous” backbones, i.e., foldamers that contain more than one type of subunit. This Account is intended to highlight the promise of heterogeneous backbones for foldamer design and application. We will focus on examples that feature combinations of α- and β-amino acid residues (Figure-3), collectively referred to as “α/β-peptides,” because these are presently the best understood foldamers with heterogeneous backbones.

Figure-3-Structures of an α-residue along with various β-residues

Nature occasionally slips a β-amino acid residue in among α-residues, as in the cyclic depsipeptide Dolastatin¹¹ (Figure-4), which contains a β-residue (and a γ-residue) along with the α-residues11. Analogously, several research groups have designed peptides in which one or two β-residues are placed among α-residues. Karle et al. reported one of the earliest examples, a cyclo-tetrapeptide with an α-β-α-β backbone pattern¹².

Advantages of heterogeneous backbones:

Foldamer design based on heterogeneous backbones offers at least two benefits relative to an exclusive reliance on homogeneous backbones. First, for a given set of monomer classes, the number of candidate foldamer backbones is vastly larger if we include heterogeneous backbones than if we are limited to homogeneous backbones. If, for example, we consider only α- and β-amino acids as building blocks, then the homogeneous approach limits us to α-peptides or β-peptides. The heterogeneous approach, in contrast, allows many different combinations (e.g., α-β-α-β-α-β, α-α-β-α-α-β, α-β-β-α-β-β, α-α-β-β, to name just a few). Each of these heterogeneous backbones offers a potentially distinctive way to project sets of side chains in space. By analogy to proteins, interesting and valuable foldamer activities are likely to depend upon achieving a specific three-dimensional arrangement of functional groups; therefore, the more distinct shapes we can generate with foldamers, the better our prospects for realizing any particular activity.

Figure-4 The natural product Dolastatin 11; α- and β-residues are highlighted yellow and blue, respectively.

The availability of multiple, complementary oligomeric skeletons may be necessary for generating a broad array of foldamer functions, but scaffold variability is not sufficient for this goal; one must also be able to decorate the scaffolds with diverse side chains. The heterogeneous backbone approach can greatly facilitate the generation of foldamer sets with broad side chain diversity. This advantage is particularly evident when α-amino acids constitute one of the building block types in a foldamer backbone, because a wide variety of protected α-amino acids is commercially available. Having to synthesize every new building block, especially if the building blocks must be enantiomerically pure, represents a substantial practical barrier to function-oriented development of new foldamers. Combining α-amino acids with other building blocks offers the prospect of purchasing some or all of the side chain diversity in a ready-to-use form. One can envision using a few types of rigidified unnatural building blocks to promote a specific folded conformation while relying on α-residues to provide many different side chain functional groups¹³.

New secondary structures from systematic combination of α- and β-residues:

Several research groups have undertaken conformational analysis and function-based design with α/β-peptides in the past few years. Early on we recognized two distinct approaches to heterogeneous backbone foldamers¹⁴. One approach involves oligomers comprised of multiple “blocks,” each containing different subunits, and each fulfilling different structural and/or functional roles. The other approach is to examine backbones with a regular pattern of subunit alternation. As discussed below, these two approaches can be combined in various ways.

The “block” approach to combining α and β subunits arose from our efforts to study sheet secondary structure in pure β-peptide foldamers. α-Peptide research had shown that the minimum increment of sheet secondary structure is a hairpin, in which strand-forming segments are connected via a reverse turn.

Figure-5 (A), (B) Chemical structures of two α/β-peptide hairpins. (C) The crystal structure of the hairpin depicted in (A); some atoms are omitted for clarity.

The European team used 2D NMR analysis to document helix formation by α/β-peptides such as 1, containing L-Ala residues alternating with 3-substituted cis-2-aminocyclopropanecarboxylic acid residues. Heptamer 1 in methanol was shown to adopt a helix defined by i→i-2 C=O···H–N H-bonds (Figure-6A). This structural work was inspired by earlier pharmacological studies, which showed that incorporation of a single cis-2- aminocyclopropanecarboxylic acid residue into a peptide corresponding to the C-terminus of neuropeptide Y gave rise to substantial affinity and unique selectivity toward the natural receptor proteins¹⁵. Interestingly, this biological activity required the β-residue absolute configuration that did not give rise to a discrete folding propensity when paired with L-α-residues in the subsequent structural studies.

Figure-6 H-bonding patterns observed in different helices formed by α/β-peptides with 1:1 backbone alternation: the (A) 13-helix, (B) 11-helix, (C) 14/15-helix, and (D) 9/11-helix. α- and β-residues are highlighted yellow and blue

Initial studies of alternating α/β-peptides arose from a desire to expand the range of foldamer scaffolds beyond those previously shown for homogeneous β-peptide backbones. Our β-peptide work had demonstrated the unique ability of conformationally preorganized residues (Figure-3) to promote stable secondary structures in short oligomers, with distinct β-peptide helices generated by sequences containing either trans-2-aminocyclohexanecarboxylic acid (ACHC) or trans-2-aminocyclopentanecarboxylic acid (ACPC). We wondered whether combining these preorganized β-residues with α-residues would lead to new foldamers. This question was addressed by preparing hexamers and octamers containing (S,S)-ACPC or (S,S)-ACHC residues alternating with either L- or D-α-amino acid residues. Evaluation of these oligomers by 2D NMR in methanol solution revealed that only the combination of (S,S)-ACPC and L-α-amino acid residues, as in 2, gave rise to NOEs between backbone protons from residues that are not adjacent in sequence¹⁶. Such non-sequential NOEs provide the clearest evidence of folding in solution.

The medium-range NOEs observed for α/β-peptide 2 in methanol could be explained by two distinct hypotheses. (1) The α/β-peptides adopt a single helical conformation containing unusual bifurcated H-bonds, with each backbone C=O group simultaneously interacting with two backbone N–H groups and vice versa (simultaneous i→i+3 and i→i+4 C=O···H–N H-bonding). (2) The α/β-peptides interconvert rapidly on the NMR time scale between two different helical conformations, one involving i→i+3 and the other i→i+4 C=O···H–N H-bonds; these conformations are designated the 11-helix and the 14/15-helix, respectively, based on H-bonded ring size (Figure-6B, C). We favored hypothesis (2) in light of the folding behavior of α-peptides and proteins. The i→i+3 and i→i+4 C=O···H–N H-bonding patterns are commonly observed for the α-peptide backbone, corresponding to 310- and α-helical secondary structure, respectively¹⁷. There is evidence that these two H-bonding patterns can rapidly interconvert in helix-forming α-peptides, but bifurcated H-bonds are rare in α-peptide helices.

Crystallographic analysis of many α/β-peptides, including 3–5, has provided strong support for the hypothesis of interconverting helical conformations (Figure-7)¹⁸. Octamer 3 displays purely 11-helical secondary structure in the solid state, while nonamer 5 displays purely 14/15-helical secondary structure. Octamer 4, which differs subtly from 3, is mostly 11-helical in the crystalline form, but the N-terminal Boc carbonyl engages in a 14-membered ring H-bond. Thus, although 4 seems to be torn between the 11- and 14/15-helical conformations in the solid state, the observed helix does not contain any bifurcated H-bonds. Two-dimensional NMR data acquired for 15-mer α/β-peptides indicate that at this length the 14/15-helix is favored over the 11-helix¹⁹. Thus, these 1:1 α/β-peptides behave comparably to α-peptides, in which both 310- and α-helical conformations populated among shorter oligomers, but the α-helix is favored when the backbone is lengthened²⁰.

Figure-7 (A) Chemical structures and (B) crystal structures of 3–5. Note the change in the hydrogen bonding behavior of the Boc group (grey carbons) from an i→i+3 H-bond in 3 to an i→i+4 H-bond in 4. Some atoms are omitted for clarity.

The variety of helical secondary structures available to α/β-peptides with 1:1 α:β alternation has been expanded by Sharma, Kunwar et al., who provided evidence for a helix with a “mixed” H-bonding pattern in oligomers such as 6 in chloroform solution^22,23. α/β-Peptide 6 is heterochiral because it contains D-α-amino acid residues and β3-residues derived from L-α-residues; by analogy, α/β-peptide 2 is homochiral. The 9/11-helix identified by this group contains two distinct types of backbone C=O···H–N H-bonds that have opposite orientations relative to the backbone direction (Figure 6D). Based on 2D NMR analysis of α/β-peptides containing β-residues and α-aminoisobutyric acid in methanol, Seebach et al. concluded that this type of α/β-peptide can adopt a 14/15-helix-like conformation that, curiously, lacks intramolecular H-bonds²⁴. Foldameric behavior is not limited to α/β-peptides with a 1:1 α:β residue alternation. We recently found that both 2:1 and 1:2 α:β backbone patterns (such as 8 and 9, respectively) support helix formation in short oligomers²⁵. Medium-range NOEs observed for these α/β-peptides in methanol are consistent with population of two helical conformations in each case, one helix containing i→i+3 and the other i→i+4 C=O···H–N H-bonds. Crystallographic analysis of short 1:2 and 2:1 α/β-peptides has provided high-resolution data for the i→i+3 C=O···H–N H-bonded helices (Figure 8), but not yet for the i→i+4 H-bonded counterparts.

Figure-8 Crystal structures of short α/β-peptides with (A) an ααβ repeat and (B) an αββ repeat.

Overall, the structural data we and others have obtained with short oligomers suggest that the propensity to form i→i+3 and/or i→i+4 C=O···H–N H-bonded helices may be a common feature of foldamers that contain homochiral α- and β-amino acid residues, for most α:β proportions and sequence patterns. As already noted, these two intramolecular H-bonding patterns are well-known among α-peptides and proteins, where they are seen in 310- and α-helices²⁶. In light of the considerable intrinsic flexibility of β3-residues, we wondered whether homochiral oligomers containing both α- and β -residues might be induced to adopt α-helix-like i→i+4 C=O···H–N H-bonded secondary structure based on information encoded in a specific side chain sequence. We have explored this possibility in the context of α/β-peptide self-assembly.

Helix bundle quaternary structure formation involving α/β β-peptides:

Most foldamer research to date has focused on secondary structure, but creating foldamers with discrete tertiary structure has long been recognized as a major aim²⁷. Conformational order at this level is important not only as a fundamental structural goal, but also as a prelude to developing foldamers with sophisticated functions, such as catalysis, which, among proteins, generally require discrete tertiary folding. Efforts to generate foldamer tertiary structure have built upon the hierarchical design strategy put forward by DeGrado et al. for the de novo development of α-peptides with helix bundle tertiary structure²⁸. Several groups have reported homogeneous β-peptides that accomplish the first step of the hierarchical approach, self-assembly to discrete helix bundles in aqueous solution. In the realm of heterogeneous backbone foldamers, we have demonstrated helix bundle quaternary structure formation in two distinct systems²⁹. α/β-Peptides 10 and 11 (Figure-9) were designed by extrapolation from a natural self-assembling sequence embedded in the yeast protein GCN4. GCN4-p1, a 33-residue α-peptide segment from the native protein, folds to form a coiled-coil dimer³⁰. α/β-Peptide 10 displays the side chain sequence of GCN4-p1 on a heterogeneous ααβαααβ backbone; each β-residue bears the side chain of the α-residue it replaces. The pattern of α→β replacement in 10 is attuned to the heptad repeat that is commonly observed among α-peptide sequences that form helix bundles³¹. Such sequences usually contain hydrophobic side chains at the first and fourth positions of each heptad, which are conventionally designated positions a and d of an abcdefg repeat. Side chains from a/d heptad positions, upon α-helical folding, form of a hydrophobic “stripe” on one side of the helix and pack against one another in the core of the helix bundle, providing the driving force for self-assembly. The β3-residues in 10 are placed at the b and f positions of each heptad. Thus, upon formation of an i→i+4 C=O···H–N H-bonded helix, 10 displays a stripe of hydrophobic side chains provided by natural α-residues and, on the opposite side of the helix, a stripe of β-residues. The crystal structure of 10 shows a three-helix bundle with the a/d side chains in the core and the b/f β-residues at the periphery, as expected. The helical conformation of 10 overlays very well on the α-helical conformation of the GCN-p1 α-peptide; however, the assembly behaviors of α- and α/β-peptides diverge. The stoichiometry of assembly differs (dimer vs. trimer) as does the stability of the helix bundles, with much weaker self-association of the α/β-peptide relative to the α-peptide.

Figure-9 (A) Primary sequence and (B) helical wheel diagrams of α/β-peptides 10 and 11; (C) Each blue circle in (A) and (B) indicates substitution of the α-amino acid with the corresponding β3-amino acid. (D), (E) Helix bundles from the crystal structures of (D) 10 (PDB: 2OXJ) and (E) 11 (PDB: 2OXK); α- and β-residues are colored yellow and blue, respectively.

α/β-Peptide 11 arises from b/f α→β modification of GCN4-pLI, a GCN4-p1 mutant that forms a very stable four-helix bundle³². The crystal structure of 11 reveals a four-helix bundle that is very similar to the quaternary structure formed by GCN4-pLI in the crystalline state. The helix bundle of 11 is highly resistant to thermal disruption; however, unlike GCN4-pLI, which forms a tetramer in solution, α/β-peptide 11 appears to form a trimer in solution. Thus, for both 10 and 11, systematic α→β modification at selected positions yields an α/β-peptide with self-assembly behavior that is reminiscent of but not identical to that of the starting α-peptide.

The crystallographic data for α/β-peptides 10 and 11, along with those for a purely β-peptide helix bundle, provide the first high-resolution insight on foldamer quaternary structure. The behavior of 10 and 11 raises many questions that provide a basis for future studies of α/β-peptides and perhaps other heterogeneous backbone foldamers that bear the side chain sequence of an α-peptide prototype. In particular, it will be interesting to see whether “sequence-based” oligomer designs provide a general source of foldamers with interesting functions.

In a second approach to helix bundle self-assembly, we have pursued a “block” strategy in which the quaternary structure contains both α-peptide and α/β-peptide components. Previous work of Kim et al. showed that acidic α-peptide 12 co-assembles with a complementary basic α-peptide to form 2:2 tetramer³³. The α/β-peptide 14/15-helix features an approximate heptad repeat (i.e., seven residues comprise about two helical turns). We therefore designed basic α/β-peptide 13 to co-assemble with acidic 12 into a hetero-helix bundle quaternary structure (Figure-10). We included cyclically constrained residues in α/β-peptide 13 to encourage formation of the 14/15-helical conformation. CD and AU measurements indicate that mixing 12 and 13 in aqueous solution leads to a cooperatively folded 2:2 tetrameric assembly with high thermal stability and extensive helicity in the individual molecules³⁴.

Figure-10 (A) Primary sequence and (B) helical wheel diagrams of α-peptide 12 and α/β-peptide 13 that form a cooperatively folded hetero-tetrameric helix bundle assembly. (C) Structures of the β-amino acids abbreviated in (A) and (B).

α/β-Peptides with biological functions: Systematic study of α/β-peptides with regular backbone repeat patterns is a recent phenomenon, as indicated above, but already these heterogeneous backbones have been used to develop foldamers with interesting biological activities. Biomedical application of α-peptides can be limited by rapid degradation in vivo. Foldamers composed exclusively of unnatural subunits, such as β-peptides, are highly resistant to proteases, but what about α/β-peptides? It has long been known that placing a single β-residue within an α-residue context can substantially retard proteolytic cleavage of nearby peptide bonds³⁵. Based on this precedent, it is not surprising that oligomers featuring a 1:1 α/β backbone are highly resistant to proteolysis, although slow cleavage of amide bonds between α and β residues can be detected in some cases³⁶.

First effort at eliciting function from α/β-peptides involved evaluating the 11- and the 14/15-helices of 1:1 α/β-peptides for their ability to mimic α-helical host-defense peptides³⁷. Natural host defense peptides are produced by eukaryotes as part of the innate immune response to microbial infection³⁸. When they encounter bacterial membrane surfaces, host-defense peptides such as the magainins adopt an α-helical conformation. This folding leads to global segregation of lipophilic and hydrophilic side chains, which is thought to be responsible for disruption of the bacterial membrane. We previously found that β-peptides that form globally amphiphilic helices mimic the antibacterial activity of natural host-defense peptides³⁹. Surprisingly, however, our α/β-peptide studies revealed that formation of a globally amphiphilic helix is not required for host-defense peptide mimicry. This discovery has important implications for development of new antibacterial materials because an oligomer that achieves global segregation of lipophilic and hydrophilic side chains in a helical conformation must be synthesized in stepwise fashion, which is expensive. Our α/β-peptide findings led us to suggest that random copolymers of lipophilic and hydrophilic subunits might be able to mimic host-defense peptides, and we have recently provided experimental support for this hypothesis⁴⁰. A major function-oriented goal of foldamer research has been to develop inhibitors of biomedically important interactions between specific proteins. In many cases, this goal has been difficult to achieve with the small molecule-based approach that is the staple of traditional medicinal chemistry⁴¹. Foldamers offer the prospect of mimicking the surface features of one of the interacting partners by appropriate placement of side chains on an unnatural folded backbone while enhancing resistance to enzymatic degradation relative to a conventional α-peptide. Protein-protein interactions in which one partner contributes a single α-helix to the interface are attractive for foldamer-based inhibitor development, because a variety of foldamer helices can be predictably generated by proper choice of subunits. Interactions between pro- and anti-apoptotic members of the Bcl-2 protein family are intriguing in this regard⁴². This protein network controls cellular responses to various death stimuli, and over-expression of anti-apoptotic Bcl-2 family members is associated with cancer. The anti-apoptotic family members, including Bcl-2, Bcl-xL and Mcl-1, present a long cleft that can accommodate an α-helical BH3 domain from a pro-apoptotic family member, such as Bak or Bad. BH3 domain sequences feature the heptad repeat discussed above, with hydrophobic side chains occurring in i+3/i+4 patterns. There are four highly conserved hydrophobic positions shared among BH3 domain sequences, and the side chains of these residues bind into pockets along the BH3-recognition clefts of complementary anti-apoptotic Bcl-2 family members⁴³.

Figure-11 (A) Chemical structure of 14, a chimeric (α/β+α)-peptide with nM affinity for Bcl-xL. (B) Binding model for 14 bound to Bcl-xL based on docking calculations. (C) NMR structure of Bak α-peptide bound to Bcl-xL (PDB: 1BXL).

Initial efforts to develop foldamers that could mimic a natural BH3 domain focused on helical β-peptides and 1:1 α/β-peptides. Despite extensive effort, we were unable to identify any 12-helical or 14-helical β-peptides or 11-helical α/β-peptides that bound tightly to the BH3-recognition cleft of Bcl-xL. 14/15-Helical designs with modest affinity for Bcl-xL could be generated. Efforts to enhance binding by modifying the side chains on a purely 14/15-helical scaffold were not productive, so we turned to a “diblock” design strategy. We found that α/β-peptides containing both a 1:1 α:β block and a pure α block could be very effective ligands for the BH3-recognition cleft of Bcl-xL. These studies ultimately led us to oligomer 14, which contains a nine-residue α/β segment followed by a six-residue α segment (Figure-11A)⁴⁴. Oligomer 14 has an affinity for Bcl-xL (Ki ~ 1 nM) that rivals the tightest-binding BH3-derived α-peptides. Control experiments revealed that the bulk of the binding energy comes from the α/β segment rather than the α segment, and that binding to Bcl-xL requires a specific 3D complementarity (the enantiomer of 14 does not bind). Docking calculations coupled with extensive side chain modification studies enabled us to propose a binding mode for the association of 14 with Bcl-xL (Figure-11B). Studies of a slightly modified α/β-peptide show that this type of foldamer can block interactions between Bcl-xL and complementary pro-apoptotic proteins in cell lysates⁴⁵.

The search for effective inhibitors of BH3 domain binding by Bcl-xL suggests important lessons for future efforts to design foldamers that antagonize specific protein-protein interactions. First, these results show that it is important to have access to many different foldamer scaffolds. In this example, only one of several foldameric helices was successful at mimicking the α-helical prototype, and this success was limited to the N-terminal portion of the target α-helix. Second, for mimicry of extended protein surfaces, such as that displayed by an α-helix of more than four turns, it may be necessary to combine multiple foldamer scaffolds. We would like to replace the α-peptide segment of 14 with an unnatural backbone, to eliminate the proteolytic susceptibility in this region, but so far this effort has not succeeded⁴⁶. Presumably we need to use a new type of foldamer helix, perhaps one that has not yet been discovered. In support of this idea, we have recently applied the sequence-based design strategy, outlined above in the context of helix bundle quaternary structure, to create α/β-peptide mimics of BH3 domains with Ki values in the low nM range for Bcl-xL and enhanced proteolytic stability⁴⁷. Thus, the third lesson (actually a restatement of the first) is that we must continue to explore new oligomers, both homogeneous and heterogeneous, for foldameric behavior.

Future Prospects:

The study of α/β-peptide foldamers is relatively recent, but already a range of interesting structural behavior has been documented. Our developing knowledge of α/β-peptide conformational propensities has provided a basis for development of examples with specific biological activity. It is obvious that many new patterns of α- and β-residues remain to be explored in terms of both structure and function. More broadly, the rapid development of α/β-peptide chemistry illustrates the utility of the heterogeneous backbone concept in foldamer development.

Folding oligomers with other subunit mixtures have been described, and these examples provide further evidence that the heterogeneous backbone approach to foldamer design is valuable. Some of the reported systems interweave α- with γ- or even larger amino acids⁴⁸. These combinations have allowed researchers to take advantage of established conformation propensities of the α subunits, as in the early α/γ-peptide work of Clardy, Schreiber et al., or to employ useful functional groups that are readily available among α-amino acids, as in the α/cholate-peptide metal ion sensors of Zhao and Zhong⁴⁹. Other researchers have combined different types of unnatural residues, as illustrated by the pioneering aromatic amide oligomers of Hamilton et al., the helices with tunable internal diameters of Gong et al. and the novel structures reported by Huc et al. Collectively, these and related studies suggest that a large universe of new foldamers with distinctive structural and functional properties awaits discovery⁵⁰.

Figure-12

Figure-13 Schematic representation of the preferential solvation of a folded Oligocholate

Application:

A hexameric cholate foldamer functionalized with a 4-dialkylaminopyridyl group displayed solvent-sensitive catalysis for the acetylation of alcohols. The catalytic oligocholate that has a hydrophilic nanocavity upon folding. The catalyst folded in carbon tetrachloride containing a low percentage (<4%) of DMSO and unfolded as more DMSO was added. By increasing the effective concentration of the substrate near the catalytic group, the folded catalyst was more active than the unfolded catalyst toward small, hydrophilic alcohols. The longer and hydrophobic n-octanol, however, was more reactive in the presence of the unfolded catalyst. The highest selectivity (21:1) was observed for methanol/n-octanol with the folded oligocholate catalyst⁵¹.

Peptidomimetic foldamers were synthesized by oligomerizing derivatives of the d-amino acid analogue, 2-(2-aminophenoxy) alkanoic acid. Single-crystal analysis of the tetramer reveals a 21-helical secondary structure stabilized by hydrogen bonding and the coiled stacking of aromatic rings. The M-helicity of 2-aminophenoxyacetic acid oligomers was induced by the incorporation of only a single chiral carbon of the N-terminal (R)-2-(2-nitrophenoxy) propionamide moiety. The solution state CD spectra demonstrated that the resulting helix induced a substantial Cotton effect. The secondary structure was further characterized by IR and NMR spectroscopy⁵².

Figure-14 Folding of compound 13 by two motifs of hydron bonding and Crystal structure of 13: distances between centroids (green balls) of benzene rings and distances between peptidemimetic a-carbons (orange).

Four porphyrin-bridge-C60 dyads have been synthesized by covalently linking the chromophores at the opposite ends of a hydrogen bonded arylamide-derived foldamer bridge. For comparison, four C60-free porphyrin derivatives of the same frameworks have also been prepared.

Figure-15

The fully hydrogen bonded bridges enable the appended porphyrin and C60 moieties to contact in a face-to-face manner. 1H NMR, UV–vis and fluorescent investigations in chloroform indicate that such a structural matching remarkably facilitates the intramolecular energy and electron transfer and charge separation between the two chromophores and also retards the recombination of the charge-separated state. Removing one hydrogen bond considerably reduces the energy and electron transfer⁵³. The β-peptide field is now poised to make significant contributions to chemical biology. Our strategy for the design of well-folded β-peptide 14-helices generates relatively small molecules possessing a broad binding surface and nearly unlimited chemical diversity—molecules with great potential as protein–protein interaction inhibitors. Using a combination of rational design and well-established high throughput combinatorial methods, or perhaps evolution, It may soon be possible to quickly generate small, folded β-peptide ligands for some fraction of the 75% of the human proteome currently considered undruggable. As this approach is applied to more targets and tested for in vivo efficacy, we will evaluate the potential of functionalized β-peptides as biological tools and therapeutics. For example, we are currently targeting proteins in the Bcl-2 family that help regulate apoptosis. β-peptides that bind to Bcl-2 family members would be extremely useful as tools to control programmed cell death and even as potential cancer drugs. We have also designed ligands that target the HIV membrane fusion protein gp41. β-Peptides that bind in gp41_s hydrophobic pocket could inhibit membrane fusion and represent a cost-effective alternative to the current a peptide based fusion inhibitor Fuseon⁵⁴.

Figure-16 Binding of the free bisADD-GNPs with Ca++

Bisacridinedione-functionalized gold nanoparticles (bisADD-GNPs) were prepared and characterized. Bis-ADD is a flexible acyclic moiety and a specific Ca++ sensor. Signaling of the binding events is achieved by the cation-induced folding of the bisADD-GNPs and the resultant fluorescence enhancement and visual color change are attributed to the suppression of photoinduced electron transfer (PET) through space and nano-Au aggregation. The selective binding of Ca++ is clear from steady state fluorescence and TEM techniques⁵⁵.

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Received on 19.08.2010 Modified on 26.08.2010

Asian J. Research Chem. 4(1): January 2011; Page 13-23