Beyond de novo protein design — de novo design of non-natural folded oligomers

Beyond de novo protein design — de novo design of non-natural folded oligomers Richard P Cheng The mystery of how a protein sequence specifies a unique structure has intrigued chemists, leading to the design and study of foldamers, non-natural oligomeric molecules that adopt well-defined structures. Recently, the sequence specificity of the various regular repeating structures has been revealed for bioinspired foldamers and such foldamers have been created to adopt helical bundle tertiary structures. One major strategy for the generation of abiotic foldamers has involved molecular design of the monomer geometry. These advances in foldamer research may lead to future applications in biomedical and materials science. Addresses Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA e-mail: [email protected]

Current Opinion in Structural Biology 2004, 14:512–520 This review comes from a themed issue on Engineering and design Edited by Lars Baltzer and William F DeGrado

mers to form secondary structures such as helices, sheets or turns has become a very active area of research [2,3]. Recently, multiple secondary structure elements have been combined to form tertiary structures (also known as tyligomers) [2]. Foldamers can be divided into two categories: bioinspired and abiotic. Whereas most bioinspired foldamers chemically resemble proteins (Figure 1a), most abiotic foldamers include aromatic rings in the backbone (Figure 2). Recent developments concerning bioinspired foldamers include elucidating the sequence specificity of various secondary structures, stabilizing secondary structures in water and creating tertiary structures such as helical bundles. Efforts in abiotic foldamer research have mainly focused on monomer geometry design for modulating the structure of the homo-oligomers. Conformationally constrained monomers have also been used in bioinspired foldamers. This review intends to capture some of the recent highlights in foldamer research, focusing on cases with finite chain length (i.e. oligomers).

Available online 29th July 2004 0959-440X/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2004.07.001

Abbreviations ANS 1-analinonaphthalene-8-sulfonate CD circular dichroism Dan 1,5-dialkoxynaphthalene DMSO dimethylsulfoxide Ndi 1,4,5,8-naphthalene-tetracarboxylic diimide NOE nuclear Overhauser effect PE phenylene ethynylene

Introduction The engineering and design of protein structures are often coupled to advances in our understanding of protein structure. The de novo design (designing from scratch) of a given structure is the ultimate demonstration of complete understanding of the non-covalent forces that specify the structure. In proteins, the combination of non-covalent forces is encoded in the primary structure, the sequence of amino acids. Because non-covalent forces (such as hydrophobic, hydrogen-bonding, electrostatic and van der Waals interactions) are universal, certain non-natural polymers/oligomers should also be able to adopt compact, well-defined three-dimensional structures. Such molecules are termed foldamers [1–3]. The design of foldaCurrent Opinion in Structural Biology 2004, 14:512–520

Bioinspired foldamers Peptoids

Peptoids (Figure 1a), poly-N-substituted glycines, have been shown to adopt a polyproline type I helix in solution and solid state [4]. Analogous to polyproline, peptoids lack backbone hydrogen-bond donors. The helical ‘handedness’ of peptoids is dictated by the stereochemistry of the sidechains; a-chiral aromatic and aliphatic sidechains [4] have both been shown to support the polyproline type I helix. With the aim of creating peptoids with tertiary structure, Zuckermann and co-workers [5

] generated a 15-mer amphiphilic peptoid library using a ‘mix-and-split’ strategy. The sidechains of four hydrophobic positions and three hydrophilic positions were varied randomly using twelve hydrophobic and two hydrophilic sidechains, respectively. The library was screened for binding to 1-analinonaphthalene-8-sulfonate (ANS), because ANS would exhibit strong fluorescence upon binding the hydrophobic pocket of the presumed multimeric helical assembly. The sequences that induced ANS fluorescence were identified by tandem electrospray ionization mass spectrometry (ESI-MS). One such (Figure 1 Legend) Chemical representations of various bioinspired foldamers. (a) Monomeric units. (b) Secondary structures of b-peptides and the constituting monomers. (c) Possible conformations of alternating a/b-peptides. (d) Secondary structures of g-peptides and the constituting monomers. (e) Helix formed by oligoureas and the constituting monomer. www.sciencedirect.com

De novo design of non-natural folded oligomers Cheng 513

Figure 1

(a)

α-Peptide

β-Peptide

Peptoid

R

O

O

N 3 H β3-residue

2 N H R β2-residue

O N H

N R

O

N H

O

γ-Peptide

Oligourea

N H

N H

O

H N O

(b) O

14-helix O N H

O N H

γ

O

O N H

N H

O

N H β3hVal (γ-branched) promotes 14-helix

N H

Constrained β-residue favoring 14-helix

O

12-helix O

O O

O N H

O N H

H N

O

H2 N

H2N

N H

O

Constrained β-residue favoring 12-helix

N H

R

N N H H Pyrrolidine-containing β-residues

N H

R = OMe, OPh, CH2NH3+ Functionalized cyclopentanecontaining β-residues

12/10-helix O

O

O

O

O

O

O O

O N H

O N H

R N H

O

N H

O N H

Alternating

O

β3/β2

O

MeO

H N

N H O O R' pattern Alternating pattern studied by Kessler [28]

8-helix O

R O

O N H

O

N H

N H

N H

R' O

(c)

N H

N H

N H

O

H N O

R

O N H

N H

O

H N

H N O

O

R

N H

13-helix

O

H N

R

O N H

Alternating α/β-peptide

O

14/15-helix

O O

O Oxanorbornene-containing residue

R α,β-syn-dialkyl residue prefers extended conformation

11-helix

R

NH

OH

N H

2,2-disubstituted-pyrrolidine4-carboxylic acid residue

Alternating α/β-peptide N H

O

H N

O

H N

N H

O

COOMe

N H

O

COOMe

O

COOMe

14-helix

Strand structure

O

O

H N

N H

(e)

R'

N

O

(d)

O

R

Pyrrolidine-3-carboxylic acid residue

O

Strand structure

Non-hydrogen-bonded structure

N

N H

Alternating chirality studied by Sharma et al. [29]

R = CH3, (CH3)2CH, (CH3)2CHCH2 (2R,3S)-3-amino-2-hydroxy acid residues

α-Aminoxy acid residue

1-(aminomethyl)cyclopropanecarboxylic acid residue

OMe O

O

N H

O

N H

O

R N H

O

R

N H

N H

O γ4-residue

R''

R' O γ2,3,4-residue

N H

O

14-helix O N H

H N

H N O

O N H

R N H

N H

H N O Current Opinion in Structural Biology

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Current Opinion in Structural Biology 2004, 14:512–520

514 Engineering and design

peptoid was studied by sedimentation equilibrium, showing a monomer-dimer-tetramer equilibrium. However, the authors noted that ‘‘there was little evidence of cooperativity’’ in folding, suggesting the presence of many isoenergetic folded states. Nonetheless, the study demonstrated that foldamers such as helical peptoids can associate to form well-defined higher order structures [5

]. b-Peptides

b-Peptides (Figure 1a,b), oligo(b-amino acids), can adopt a wide variety of secondary structures, including helices, sheets and turns [1,6–8]. The sidechain substitution pattern of the constituting b-amino acids can bias the local backbone conformational preference and thus the overall secondary structure [1,6–8]. Although some design guidelines (sequence specificity) for secondary structures have been revealed [8], the construction of b-peptides with tertiary structure has been limited due to the difficulty in synthesizing sequences longer than eight residues using solid-phase peptide chemistry. Seebach and co-workers [9] showed that coupling is particularly difficult for the fifth to eighth residues from the solid support. Coupling and deprotection procedures for these residues were avoided by convergent synthesis involving protected tripeptide fragments and dipeptide fragments for b3-peptides and b2/b3-mixed peptides, respectively [9]. Also, two b-peptides were linked by native ligation [10], enabling the generation of long b-peptides. Among the b-peptide helical conformations, the 14-helix is the best studied [1,6,7]. The 14-helix has 14 atoms in the hydrogen-bonded ring N–H(i)


O=C(i+2) (Figure 1b) and an approximately three-residue repeat. The 14helix macrodipole places partial negative charge on the N terminus and partial positive charge on the C terminus. In aqueous solution, the 14-helix is stabilized by electrostatic interactions between oppositely charged residues one turn apart (positions i and i+3) [11–13]. The 14-helix is further stabilized by placing complementary charged residues to interact favorably with the helix macrodipole [14
]. Unprotected termini also stabilize the 14-helix conformation by interacting with the helix macrodipole [14
]. Gellman [15], Schepartz [14
] and colleagues showed independently that g-branched b3-amino acids (such as b3hVal and b3hIle) can be used to promote the formation of the 14-helix. Gellman and co-workers [15] also showed that a single conformationally constrained cyclic residue in the center of the sequence is sufficient to bias a 10-mer to adopt a 14-helix. Furthermore, a 12-mer with four cyclic residues evenly distributed in the sequence was sufficient to stabilize the 14-helix between pH 2 and 12, even though the remainder of the b-peptide comprised charged acylic residues to potentially stabilize the 14-helix through electrostatic interactions [16]. A monomeric 14-helical b-peptide in methanol was Current Opinion in Structural Biology 2004, 14:512–520

unfolded by adding water and the process was monitored by NMR [17]. The non-abrupt, gradual unfolding [17,18

] suggests that the 14-helix resembles the natural a-helix in having multiple partially folded intermediates during the folding/unfolding transition. In order to create b-peptides with tertiary structure, Gellman [19

], DeGrado [18

], Diederichsen [20

] and co-workers have generated helical bundles based on the 14-helix using different design strategies. Gellman and colleagues [19

] designed an amphiphilic 14-helix using cyclic residues with six-membered rings and b3hLys; the cyclic residues stabilize the 14-helix in water and the amphiphilic nature of the helix promotes association. Indeed, the b-peptides exhibited tetrameric and hexameric helical bundles in TRIS and acetate buffers, respectively. Cheng and DeGrado [18

] designed a disulfide-linked parallel two-helix bundle using shape complementarity at the helix–helix interface based on a 3.33 residue per turn helix geometry. The resulting motif exhibited cooperative thermal unfolding, analogous to proteins. Gellman, Diederichsen and co-workers [20

] designed 14-helical b-peptides containing nucleobase sidechains to mediate antiparallel dimerization by Watson–Crick base pairing. Thermal denaturation experiments revealed less entropy loss for dimerization compared to DNA strand pairing, suggesting that preorganization of the 14-helix facilitated association. Recently, Kimmerlin and Seebach [21] designed an amphiphilic 14-helix with 20 different residues and an N-terminal b3hCys to potentially form a disulfide-linked two-helix bundle. The b-peptide 12-helix had previously been formed with only constrained cyclic residues containing fivemembered rings [1,6,7]. Various pyrrolidine-containing [22,23] and functionalized cyclopentane-containing [24] cyclic b-amino acids were incorporated into 12-helical b-peptides to improve solubility in aqueous solution and to provide sequence diversity. Furthermore, heptamers with up to two b2- or b3-residues remained significantly 12-helical [25,26], providing even more sequence diversity. b-Peptides with alternating b2- and b3-monosubstituted amino acids can adopt the 10/12-helix conformation [2,7,27

]. The 10/12-helix consists of an intertwined network of 10- and 12-membered hydrogen-bonded rings. Seebach and co-workers [27

] showed that the alternating b2/b3-sequence can adopt a 10/12-helix and the alternating b3/b2-sequence can adopt a 12/10-helix. Solution structures of such mixed b2/b3-oligomers up to nine residues in length have been studied in detail to devise a convenient schematic representation for future design efforts [27

]. Kessler and co-workers [28] designed a 10/12-helix in acetonitrile using a different alternating residue pattern: bhGly with no sidechain and www.sciencedirect.com

De novo design of non-natural folded oligomers Cheng 515

Figure 2

Pyridine oligoamide (Lehn [44] and Huc [45]) O N H

N

Hydrazone-linked pyrimidines (Lehn [47])

O

N H

N

N

N

OiBu

N N

N

N

N

N N

R OO N

H

H

O

N

Aryl oligoamide (Gong [48])

R N

O O

R O

O R=

H N

O

H N

O

O O

H N R O

N

R N

H N

O

H

N

O O

O

H

N H

O R O

O

O O

N

N

H N

O R O R

O R

R

H

O

N O

N H

O R

H O R

O

N

N N H O

oPE (Tew [63])

R

N H

H O R

O

O

O

R

O

O

R

N

O

O

O

R = H,CH3

3

O

O

R

O

H N

R

O

3

O

R

pPE (Moore [53])

3

O

R = CH3, C8H17, (CH2CH2O)3CH3 RR OO

N

O

O

O

RR OO

N

O H N

N

H N

O

H N

O

H

N

Oligo-ureidophthalimide (Meijer [52])

R

O

H N

N N

N

O

R O

N

N

R O H N

N

N

N

N O

O

N

N N

N H

N H

N

N

N

H

R N

O

N N

N N

Quinoline oligoamide (Huc [50])

N H

R

O

H

O

N R

Pyr/pym (Lehn [65])

Heteroduplex (Iverson [68])

SnPr

O O

O

N

N

O N

RO

R = H, Ph

R=

N

N O

O

O

R

O

H N

Oligo-Ndi

RO

O R

N

N

N N

N

O O

R

O

Oligo-Dan

N

O

H N

O

N N

N H

N H

OR N

RO

N R

Monomers for duplex design (Gong [70]) R O

R O

H N

H N O

O

H N

H N

O O

O

O N H O

O R

O

R O

N H

N H

R = C8H17

O R

Triazine-based duplex (Krische [72]) R R

H N

N

O

N

H N

O

N H

H

N

N

H NH O

R R

O N N HNH

O

HNH

N

N N

H

N

N HNH N

NH O

N N

R R

NH O R R

R = 4-decyloxybenzyl

Current Opinion in Structural Biology

Chemical representations of the repeating units of various abiotic foldamers. A representation of one helical turn or the duplex is also depicted. www.sciencedirect.com

Current Opinion in Structural Biology 2004, 14:512–520

516 Engineering and design

a constrained cyclic b-amino acid with a carbohydratederived five-membered ring. It appears that the constrained cyclic residue and bhGly play the roles of the b3- and b2-residues, respectively. Sharma et al. [29
] showed that b3-peptides with alternating chirality can also adopt the 10/12-helix in chloroform and DMSO, as predicted theoretically by Wu and Wang [30]. The b3peptides studied by Sharma et al. [29
] contained C-linked carbohydrate sidechains, extending the variety of sidechain functionality. Previous studies on oligomers of 1-(aminomethyl)cyclopropanecarboxylic acid and a-aminoxy acids have suggested the formation of the b-peptide 8-helix [7]. Recent efforts involving (2R,3S)-3-amino-2-hydroxy acids [31
] and cyclic oxanorbornene b-amino acid [32
] have provided more sequence diversity for the 8-helix. The oligo(3-amino-2-hydroxy acid) stabilized the 8-helix in methanol through hydrogen bonding between the sidechain hydroxyl and backbone carbonyl [31
]. According to NMR studies and theoretical calculations, the oxanorbornene-containing residues favored the 8-helix conformation in chloroform due to constraints imposed by the oxanorbornene ring [32
]. Homo-oligomers of pyrrolidine-3-carboxylic acid exhibited multiple amide rotamers. To enable the design of a b-peptide structure without backbone hydrogen bonds, Carlson, Gellman and co-workers [33] studied a 2,2-disubstituted-pyrrolidine-4-carboxylic acid, which adopts a single rotamer in chloroform, by NMR. The corresponding dimer exhibited a Z conformation of the linking amide bond, analogous to the polyproline type I helix. Although the corresponding oligomers appeared to form a regular structure in methanol based on circular dichroism (CD) data, multiple conformations were revealed by NMR and molecular modeling studies [33]. In addition to the known b-peptide antiparallel hairpins, Gellman and co-workers [34] studied a hairpin structure with parallel sheets composed of b-amino acids. Whereas the a,b-syn-dialkyl configuration of the b-amino acid was critical for parallel hairpin formation, the stereochemistry of the reverse turn was less important than for a-peptide hairpin formation [34]. Alternating a/b-peptides

The emergence of b-peptides has raised curiosity about mixed a/b-peptides (Figure 1c) [3]. Thomasson, Gellman and co-workers [35] studied the conformational preferences of alternating a/b-peptides in which the b-residues contained conformationally constraining five-membered rings; Figure 1c). Based on NMR studies in CD3OH, peptides with L-a-residues exhibited long-range NOEs (nuclear Overhauser effects), but peptides with D-aresidues showed no such NOEs, suggesting the importance of stereochemistry for forming well-defined strucCurrent Opinion in Structural Biology 2004, 14:512–520

tures. As both the 14/15-helix and the 11-helix were consistent with the NMR data, more studies are necessary to specify one predominant conformation. Additionally, Reiser and co-workers [36] studied an alternating a/bpeptide in which the b-residues contained conformationally constrained three-membered rings. NMR studies showed that a 13-helix is formed in CD3OH, but not in water. Balaram and co-workers [37] also introduced b-amino acids into an a-peptide hairpin, occupying positions in the turn, hydrogen-bonded pairs and non-hydrogen-bonded pairs. NMR and crystallography studies confirm the formation of hairpin structures, demonstrating the compatibility of b-amino acids with a-peptide-based hairpins. g-Peptides

There have been several studies on g-peptides, revealing helix and turn conformations [2]. Whereas g4-peptides form a 14-helix in solution (Figure 1d), g2,4-disubstituted residues with the appropriate stereochemistry form a reverse turn. Seebach et al. [38] performed NMR and crystallography studies on oligomers of g2,3,4-trisubstituted residues, revealing a 14-helix in methanol. Using NMR and crystallography, Smith, Gellman and colleagues [39] showed that g-residues with conformationally constrained five-membered rings form strands in a hairpin. Guichard and co-workers [40] studied oligoureas that also form the 14-helix (Figure 1e), similar to g-peptides. NMR and crystallography studies revealed a more sophisticated pattern of hydrogen-bonded rings involving 12 and 14 atoms due to the urea functionality.

Abiotic non-natural foldamers Abiotic foldamers typically contain aromatic rings in the backbone; this is distinctly different from bioinspired foldamer backbones, which resemble the protein backbone. The major strategy for designing abiotic foldamers has mostly emphasized monomer design. Abiotic foldamers with helical conformations have been designed using oligoarylamides and ureas, oligoaromatics and phenylene ethynylene oligomers (Figure 2). The helix radius and repeat are determined by the collective curvature of the constituting monomers and the linking functionality (amide or urea). The curvature/bend of the monomers can be modulated by several factors [41,42

,43]: substitution patterns on the aromatic ring (ortho, meta, para); placement of hydrogen-bond donor/ acceptor for backbone rigidification; and identity of the aromatic ring, such as benzene, pyridine, pyrimidine, naphthalene and so on. Independently, Lehn [44], Huc [45] and co-workers performed NMR and CD studies on different protonation states of oligoamides containing 2,6-disubstituted pyridines. Diamino and dicarbonyl substitution of alternating pyridine rings resulted in varying pKa values for the www.sciencedirect.com

De novo design of non-natural folded oligomers Cheng 517

pyridine nitrogens. This caused conformational change in a two-stage manner upon protonation. Whereas the nonprotonated form and the fully protonated form exhibited helical conformations in solution, the partially protonated form was proposed to adopt an extended conformation [45]. Huc et al. [46] also appended benzyloxy, hydroxy and hydroxylate functionalities onto the pyridine-containing oligomer; the hydroxylate derivative was fully soluble in water. NMR and crystallography studies confirmed the helical conformation. Lehn and co-workers [47] also devised helical oligomers composed of hydrazone-linked pyrimidines. NMR and crystallography studies revealed that aromatic sidechains promoted more regular helices with better pyrimidine ring stacking, most probably due to sidechain aromatic stacking. Gong and co-workers [48] have studied oligoamides composed of 1,3-disubstituted benzene rings. A threecenter hydrogen bond rigidifies the local conformation to generate a bend in the backbone. Nonamers exhibited one helical turn, as characterized by crystallography and NMR. Incorporation of 1,4-disubstituted benzene rings served to decrease the backbone curvature and increase the radius of the helical conformation [49

]. Huc and coworkers [50] have studied a helical oligomer of quinolinederived amino acids by NMR and crystallography. Two quinoline nitrogens hydrogen bond with one backbone amide hydrogen to increase the curvature of the monomer, resulting in a helical oligomer with approximately 2.5 residues per turn. Chiral induction was achieved by incorporating a chiral R-phenethylamino group at the C terminus [51]. NMR studies in chloroform, DMSO and toluene revealed a 10:1 ratio for the two helical handedness [51]. Meijer and co-workers [52] designed an aromatic helix using para-substituted aromatic units in an oligo ureidophthalimide. The phthalimide functionality hydrogen bonds to the backbone urea and rigidifies the backbone conformation. Also, the incorporation of chiral amines into the phthalimide induced helical handedness. Molecular modeling and CD studies showed that there are 6–8 repeating units in each helical turn. Interestingly, the helical conformation is observed in THF, but not in chloroform. Further studies are necessary to understand this solvent-dependent behavior. m-Phenylene ethynylene (mPE) oligomers exhibit helical conformations in a solvent-dependent manner [53

]. In polar solvents such as acetonitrile, each helical turn consists of six repeating units, as shown by electron spin resonance (ESR) studies of double spin labeled oligomers [54]. Although the helix is mainly stabilized by p-stacking and solvophobic effects, Moore [55], Gong [56] and coworkers independently showed that hydrogen-bonding interactions can be used to restrict bond rotation of the ethynylene unit and favor the helical conformation by 1.2 kcal
mol1. Moore and co-workers [57,58] also showed that the imine functionality, analogous to the www.sciencedirect.com

ethynylene moiety, is compatible with the helical conformation. Furthermore, a reversible acid-catalyzed imine metathesis reaction was carried out in a helix-promoting solvent to bias the assembly of oligomers longer than eight repeating units through heterodimerization [57,58]. As eight repeating units is the minimal length for helix formation, the synthesis and folding of the mPE oligomer are linked. High molecular weight poly(m-phenylene ethynylene imine)s were also synthesized by performing the reversible imine metathesis reaction on bisfunctionalized oligomers [59]. The product distribution was dependent on solvent, temperature and starting oligomer (length and sequence) [60]. In the helical conformation, mPE oligomers can bind non-polar ligands within the tubular hydrophobic cavity. In the presence of a dumbbell-shaped non-polar ligand, the reversible acidcatalyzed imine metathesis reaction was used to preferentially generate mPE oligomers of appropriate length to bind the ligand [61]. Water-soluble mPE oligomers were obtained by substituting the triethylene glycol sidechains with hexaethylene glycol sidechains [62]. Tew and co-workers [63] synthesized alkoxy-substituted o-phenylene ethynylene oligomers (oPE), which may form helices with three units per turn [64]. The alkoxy substituents make the oligomers soluble in common organic solvents such as chloroform, enabling future solution studies of oPE. Lehn and co-workers [65,66

] have studied helical oligoheterocyclic strands, composed of alternating pyridine and pyrimidine rings, by molecular modeling, NMR and crystallography. Upon binding Pb2+ in acetonitrile or chloroform, the original helical oligomer adopted an extended conformation [65]. The conformational change was readily reversed by introducing crytand [2,2,2], which sequestered the Pb2+ from the extended oligomer. Upon the addition of Ag+, the monomeric helical oligomers dimerized to give a double helix. The Ag+ bound the terminal bipyridyl moieties to give an Ag2(oligomer)2 complex [66

]. The double helix was stabilized by favorable face-to-face stacking of the aromatic rings involving dipole–dipole or dipole–complexation-induced dipole interactions. Dimerization was also reversed by subjecting the double-helical complex to cryptand [2,2,2], which sequestered the Ag+ ion from the double helix. Aedamers are molecules that form a pleated secondary structure based on the stacking of alternating electronrich (1,5-dialkoxynaphthalene, Dan) and electrondeficient (1,4,5,8-naphthalene-tetracarboxylic diimide, Ndi) aromatic units. Folding is driven by desolvation/ hydrophobics coupled with electrostatic complementarity for face-centered stacking. Based on chemical shift data, the linkers determined the aromatic stacking geometry (center versus off-center) [67], indicating conformational modularity for aedamers. In order to develop higher order structures, a heteroduplex composed of oligo-Ndi and Current Opinion in Structural Biology 2004, 14:512–520

518 Engineering and design

oligo-Dan was designed and studied [68]. Interestingly, the heat capacity (DCp) for unfolding was found to be temperature dependent (50 to 94 cal
mol1
K1 over 288–318 K). Gong and co-workers [69,70
] have devised heterodimerizing and homodimerizing oligoamides using 1,3-disubstituted benzene rings linked by glycine. Alkoxy groups were introduced to form intramolecular hydrogen bonds that rigidify the conformation and mask certain hydrogenbond donors. NMR experiments indicated that sequencespecific dimerization occurred in chloroform between oligoamides with complementary hydrogen-bond donating and accepting patterns. The oligoamides adopted different topologies to facilitate dimerization [69,70
]. Analogous oligoamides were used to template the formation of an antiparallel b-sheet with three amino acid strands [71]. Archer and Krische [72

] designed homodimerizing oligomers by a covalent casting strategy. The strategy involved introducing optimal covalent linkers to connect hydrogen-bonded aminotriazines. An amino alcohol linker was found to be optimal for supporting the aminotriazine hydrogen-bonding array and for forming highaffinity duplexes in chloroform.

Conclusions There have been major advances in foldamer research in recent years. In the early stages of investigating b-peptides and other bioinspired foldamers, it was quite surprising that backbones with more conformational flexibility (than the protein backbone) could exhibit welldefined secondary structures. Furthermore, the extra backbone conformational flexibility has provided the malleability for bioinspired foldamers to adopt a wider variety of secondary structures compared to proteins. The sequence specificity of various secondary structures has been elucidated and should be expanded further to other secondary structures. Although several novel secondary structures have been revealed, more should be discovered in the future. Efforts to stabilize the secondary structures in water have led to the development of bioactive foldamers [73]. Although there has been some success in creating foldamer helical bundles, designing tertiary structure remains extremely challenging. For abiotic foldamers, one main strategy involves designing monomers with discrete geometries, which affect the topology of the homo-oligomers; however, sequence specificity and water compatibility remain to be fully explored. Abiotic foldamers have also been shown to be diverse in secondary structure, but well-defined tertiary structures are yet to be demonstrated. Nonetheless, applications of abiotic foldamers in materials science have been actively pursued. Both bioinspired and abiotic foldamers have been shown to form secondary structures in the absence of long-range interactions, suggesting that much smaller ‘tyligomers’ Current Opinion in Structural Biology 2004, 14:512–520

(foldamers with tertiary structure) can be formed compared to proteins.

Acknowledgements The author gratefully thanks support from the New York State Office of Technology and Academic Research for the James D Watson New Investigator Award, University at Buffalo for start-up funds and John Kapoor for endowment funds. The author would like to thank Raheel Ahmad, Marc Koyack, Prashant Girinath and Chia-Wen Kuo for assistance in preparing the manuscript. The author would also like to thank Bing Gong, Janet Morrow and John Richard for constructive comments on the manuscript.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
of special interest

of outstanding interest 1.

Gellman SH: Foldamers: a manifesto. Acc Chem Res 1998, 31:173-180.

2.

Hill DJ, Mio MJ, Prince RB, Hughes TS, Moore JS: A field guide to foldamers. Chem Rev 2001, 101:3893-4012.

3.

Cubberley MS, Iverson BL: Models of higher-order structure: foldamers and beyond. Curr Opin Chem Biol 2001, 5:650-653.

4.

Wu CW, Kirshenbaum K, Sanborn TJ, Patch JA, Huang K, Dill KA, Zuckermann RN, Barron AE: Structural and spectroscopic studies of peptoid oligomers with a-chiral aliphatic side chains. J Am Chem Soc 2003, 125:13525-13530.

5.



Burkoth TS, Beausoleil E, Kaur S, Tang DZ, Cohen FE, Zuckermann RN: Toward the synthesis of artificial proteins: the discovery of an amphiphilic helical peptoid assembly. Chem Biol 2002, 9:647-654. This paper presents a combinatorial approach to obtaining a stable peptoid helical bundle by screening for binding to ANS. Optimal combinations of the sidechains for four hydrophobic and three hydrophilic positions were obtained. Although the resulting motif did not exhibit cooperative folding or a single association state, this work demonstrated that foldamers could associate and form higher order structures. 6.

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Arvidsson PI, Frackenpohl J, Seebach D: Syntheses and CD-spectroscopic investigations of longer-chain b-peptides: preparation by solid-phase couplings of single amino acids, dipeptides, and tripeptides. Helv Chim Acta 2003, 86:1522-1553.

10. Kimmerlin T, Seebach D, Hilvert D: Synthesis of b3-peptides and mixed a/b3-peptides by thioligation. Helv Chim Acta 2002, 85:1812-1826. 11. Arvidsson PI, Rueping M, Seebach D: Design, machine synthesis, and NMR-solution structure of a b-heptapeptide forming a salt-bridge stabilized 314-helix in methanol and in water. Chem Commun 2001, 7:649-650. 12. Cheng RP, DeGrado WF: De novo design of a monomeric helical b-peptide stabilized by electrostatic interactions. J Am Chem Soc 2001, 123:5162-5163. 13. Rueping M, Mahajan YR, Jaun B, Seebach D: Design, synthesis and structural investigations of a b-peptide forming a 314-helix stabilized by electrostatic interactions. Chemistry 2004, 10:1607-1615. 14. Hart SA, Bahadoor ABF, Matthews EE, Qiu XJ, Schepartz A: Helix
macrodipole control of b3-peptide 14-helix stability in water. J Am Chem Soc 2003, 125:4022-4023. A brief communication on the stabilization of the b-peptide 14-helix in aqueous solution. The effects of sidechain interaction and terminal www.sciencedirect.com

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15. Raguse TL, Lai JR, Gellman SH: Evidence that the b-peptide 14-helix is stabilized by b3-residues with side-chain branching adjacent to the b-carbon atom. Helv Chim Acta 2002, 85:4154-4164.

This paper describes NMR and CD studies on mixed b3/b2- and b2/b3peptides in methanol. Whereas b3/b2-peptides exhibited 12/10-helices, b2/b3-peptides showed 10/12-helices in solution. Such mixed b-peptides up to nine residues long still adopt the corresponding helices. The authors provide a convenient schematic representation for designing the 12/10helix. Arvidsson et al. [74] used the schematic representation to design an antibiotic 12/10-helix.

16. Raguse TL, Lai JR, Gellman SH: Environment-independent 14-helix formation in short b-peptides: striking a balance between shape control and functional diversity. J Am Chem Soc 2003, 125:5592-5593.

28. Gruner SAW, Truffault V, Voll G, Locardi E, Sto¨ ckle M, Kessler H: Design, synthesis, and NMR structure of linear and cyclic oligomers containing novel furanoid sugar amino acids. Chemistry 2002, 8:4365-4376.

17. Etezady-Esfarjani T, Hilty C, Wu¨ thrich K, Rueping M, Schreiber J, Seebach D: NMR-structural investigations of a b3dodecapeptide with proteinogenic side chains in methanol and in aqueous solutions. Helv Chim Acta 2002, 85:1197-1209.

29. Sharma GVM, Reddy KR, Krishna PR, Sankar AR, Narsimulu K,
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charge interaction with the helix dipole are studied by CD. Also, the authors discovered that g-branched b3-residues stabilize the 14-helix.

18. Cheng RP, DeGrado WF: Long-range interactions stabilize the

fold of a non-natural oligomer. J Am Chem Soc 2002, 124:11564-11565. This paper describes the design of a disulfide-linked b-peptide two-helix bundle. The design is based on a 3.33 residue per turn repeat for the 14helix. The design strategy for the helix–helix interface involved size complementarity. Based on CD data, the two-helix bundle showed cooperative thermal denaturation and the individual helix exhibited gradual structure loss. 19. Raguse TL, Lai JR, LePlae PR, Gellman SH: Toward b-peptide

tertiary structure: self-association of an amphiphilic 14-helix in aqueous solution. Org Lett 2001, 3:3963-3966. This paper describes the design and characterization of associating bpeptide 14-helices. The design of the amphiphilic 14-helix used cyclic residues to ensure the helical structure, and b3hLys for amphiphilicity and aqueous solubility. Sedimentation equilibrium data showed that the designed b-peptide associated into tetramers and hexamers, whereas an analogous non-amphiphilic control peptide did not form higher order aggregates. 20. Bru¨ ckner AM, Chakraborty P, Gellman SH, Diederichsen U:

Molecular architecture with functionalized b-peptide helices. Angew Chem Int Ed Engl 2003, 42:4395-4399. This paper describes the design of antiparallel heterodimeric two-helix bundles based on the b-peptide 14-helix. The incorporation of nucleobase-containing sidechains allows Watson–Crick pairing between two helices and specifies the antiparallel topology. UV absorption spectroscopy was used to monitor the thermal denaturation, which revealed less entropy loss per base pair compared to DNA. This suggested that the 14helix scaffold is preorganized optimally for the sidechain base-pairing interactions. 21. Kimmerlin T, Seebach D: Solid-phase synthesis of a b3icosapeptide containing the homologs of the twenty common proteinaceous amino acids. Helv Chim Acta 2003, 86:2098-2103. 22. Lee H-S, Syud FA, Wang X, Gellman SH: Diversity in short b-peptide 12-helices: high-resolution structural analysis in aqueous solution of a hexamer containing sulfonylated pyrrolidine residues. J Am Chem Soc 2001, 123:7721-7722. 23. Porter EA, Wang X, Schmitt MA, Gellman SH: Synthesis and 12helical secondary structure of b-peptides containing (2R,3R)aminoproline. Org Lett 2002, 4:3317-3319. 24. Woll MG, Fisk JD, LePlae PR, Gellman SH: Stereoselective synthesis of 3-substituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into short 12-helical bpeptides that fold in water. J Am Chem Soc 2002, 124:12447-12452.

30. Wu Y-D, Wang D-P: Theoretical study on side-chain control of the 14-helix and the 10/12-helix of b-peptides. J Am Chem Soc 1999, 121:9352-9362. 31. Gademann K, Ha¨ ne A, Rueping M, Jaun B, Seebach D: The fourth
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conformationally-constrained b-peptides characterized by 1 H NMR chemical shifts. Chem Commun 2003: 2534-2535. This paper describes the formation of the b-peptide 8-helix using conformationally constrained residues containing oxanorbornene moieties. 33. Huck BR, Fisk JD, Guzei IA, Carlson HA, Gellman SH: Secondary structural preferences of 2,2-disubstituted pyrrolidine-4carboxylic acid oligomers: b-peptide foldamers that cannot form internal hydrogen bonds. J Am Chem Soc 2003, 125:9035-9037. 34. Langenhan JM, Guzei IA, Gellman SH: Parallel sheet secondary structure in b-peptides. Angew Chem Int Ed Engl 2003, 42:2402-2405. 35. Hayen A, Schmitt MA, Ngassa FN, Thomasson KA, Gellman SH: Two helical conformations from a single foldamer backbone: ‘‘split personality’’ in short a/b-peptides. Angew Chem Int Ed Engl 2004, 43:505-510. 36. De Pol S, Zorn C, Klein CD, Zerbe O, Reiser O: Surprisingly stable helical conformations in a/b-peptides by incorporation of cisb-aminocyclopropane carboxylic acids. Angew Chem Int Ed Engl 2004, 43:511-514. 37. Gopi HN, Roy RS, Raghothama SR, Karle IL, Balaram P: bHairpins generated from hybrid peptide sequences containing both a- and b-amino acids. Helv Chim Acta 2002, 85:3313-3330. 38. Seebach D, Brenner M, Rueping M, Jaun B: g2-, g3-, and g2,3,4Amino acids, coupling to g-hexapeptides: CD spectra, NMR solution and X-ray crystal structures of g-peptides. Chemistry 2002, 8:573-584. 39. Woll MG, Lai JR, Guzei IA, Taylor SJC, Smith MEB, Gellman SH: Parallel sheet secondary structure in g-peptides. J Am Chem Soc 2001, 123:11077-11078.

25. LePlae PR, Fisk JD, Porter EA, Weisblum B, Gellman SH: Tolerance of acyclic residues in the b-peptide 12-helix: access to diverse side-chain arrays for biological applications. J Am Chem Soc 2002, 124:6820-6821.

40. Semetey V, Rognan D, Hemmerlin C, Graff R, Briand J-P, Marraud M, Guichard G: Stable helical secondary structure in shortchain N,N0 -linked oligoureas bearing proteinogenic side chains. Angew Chem Int Ed Engl 2002, 41:1893-1895.

26. Park J-S, Lee H-S, Lai JR, Kim BM, Gellman SH: Accommodation of a-substituted residues in the b-peptide 12-helix: expanding the range of substitution patterns available to a foldamer scaffold. J Am Chem Soc 2003, 125:8539-8545.

41. Odriozola L, Kyritsakas N, Lehn J-M: Structural codons: linearity/helicity interconversion by pyridine/pyrimidine exchange in molecular strands. Chem Commun 2004: 62-63.

27. Rueping M, Schreiber JV, Lelais G, Jaun B, Seebach D: Mixed

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42. Huc I: Aromatic oligoamide foldamers. Eur J Org Chem

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43. Sanford AR, Gong B: Evolution of helical foldamers. Curr Org Chem 2003, 7:1649-1659. 44. Kolomiets E, Berl V, Odriozola I, Stadler A-M, Kyritsakas N, Lehn J-M: Contraction/extension molecular motion by protonation/deprotonation induced structural switching of pyridine derived oligoamides. Chem Commun 2003: 2868-2869. 45. Dolain C, Maurizot V, Huc I: Protonation-induced transition between two distinct helical conformations of a synthetic oligomer via linear intermediate. Angew Chem Int Ed Engl 2003, 42:2738-2740. 46. Huc I, Maurizot V, Gornitzka H, Le´ ger J-M: Hydroxy-substituted oligopyridine dicarboxamide helical foldamers. Chem Commun 2002: 578-579. 47. Schmitt J-L, Stadler A-M, Kyritsakas N, Lehn JM: Helicityencoded molecular strands: efficient access by the hydrazone route and structural features. Helv Chim Acta 2003, 86:1598-1624. 48. Gong B: Crescent oligoamides: from acyclic ‘‘macrocycles’’ to folding nanotubes. Chemistry 2001, 7:4336-4342. 49. Gong B, Zeng H, Zhu J, Yuan L, Han Y, Cheng S, Furukawa M,

Parra RD, Kovalevsky AY, Mills JL et al.: Creating nanocavities of tunable sizes: hollow helices. Proc Natl Acad Sci USA 2002, 99:11583-11588. This paper describes the design of oligoamides with varying helical diameters. This was achieved by the judicious use of meta- and parasubstituted aromatic moieties. The work is one of the few examples of sequence specificity and diversity. 50. Jiang H, Le´ ger J-M, Huc I: Aromatic d-peptides. J Am Chem Soc 2003, 125:3448-3449. 51. Jiang H, Dolain C, Le´ ger J-M, Gornitzka H, Huc I: Switching of chiral induction in helical aromatic oligoamides using solid state-solution state equilibrium. J Am Chem Soc 2004, 126:1034-1035.

60. Zhao D, Moore JS: Folding-driven reversible polymerization of oligo(m-phenylene ethynylene) imines: solvent and starter sequence studies. Macromolecules 2003, 36:2712-2720. 61. Nishinaga T, Tanatani A, Oh K, Moore JS: The size-selective synthesis of folded oligomers by dynamic templation. J Am Chem Soc 2002, 124:5934-5935. 62. Stone MT, Moore JS: A water-soluble m-phenylene ethynylene foldamer. Org Lett 2004, 6:469-472. 63. Jones TV, Blatchly RA, Tew GN: Synthesis of alkoxy-substituted ortho-phenylene ethynylene oligomers. Org Lett 2003, 5:3297-3299. 64. Blatchly RA, Tew GN: Theoretical study of helix formation in substituted phenylene ethynylene oligomers. J Org Chem 2003, 68:8780-8785. 65. Barboiu M, Lehn J-M: Dynamic chemical devices: modulation of contraction/extension molecular motion by coupled-ion binding/pH change-induced structural switching. Proc Natl Acad Sci USA 2002, 99:5201-5206. 66. Barboiu M, Vaughan G, Kyritsakas N, Lehn J-M: Dynamic

chemical devices: Generation of reversible extension/ contraction molecular motion by ion-triggered single/double helix interconversion. Chemistry 2003, 9:763-769. This paper describes the use of metal ions to alter the conformation and association state of a foldamer. NMR and crystallographic studies revealed that Ag+ induced the dimerization of a foldamer with alternating pyridine/pyrimidine units. The foldamer transformed from a monomeric helix to a double helix. Such a system represents a dynamic device modulated by ion concentration. 67. Zych AJ, Iverson BL: Conformational modularity of an abiotic secondary-structure motif in aqueous solution. Helv Chim Acta 2002, 85:3294-3300. 68. Gabriel GJ, Iverson BL: Aromatic oligomers that form hetero duplexes in aqueous solution. J Am Chem Soc 2002, 124:15174-15175.

52. van Gorp JJ, Vekemans JAJM, Meijer EW: Facile synthesis of a chiral polymeric helix; folding by intramolecular hydrogen bonding. Chem Commun 2004: 60-61.

69. Zeng H, Yang X, Brown AL, Martinovic S, Smith RD, Gong B: An extremely stable, self-complementary hydrogen-bonded duplex. Chem Commun 2003: 1556-1557.

53. Hill DJ, Moore JS: Helicogenicity of solvents in the

conformational equilibrium of oligo(m-phenylene ethynylene)s: Implications for foldamer research. Proc Natl Acad Sci USA 2002, 99:5053-5057. This paper describes the study of the folding of mPE in various solvents. UV and CD studies showed that the helical conformation is more stabilized in polar solvents compared to non-polar solvents. The solvophobic effect appears to be critical for mPE helix formation, analogous to the hydrophobic effect for protein folding.

70. Yang X, Martinovic S, Smith RD, Gong B: Duplex foldamers from
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54. Matsuda K, Stone MT, Moore JS: Helical pitch of m-phenylene ethynylene foldamers by double spin labeling. J Am Chem Soc 2002, 124:11836-11837. 55. Cary JM, Moore JS: Hydrogen bond-stabilized helix formation of a m-phenylene ethynylene oligomer. Org Lett 2002, 4:4663-4666. 56. Yang X, Brown AL, Furukawa M, Li S, Gardinier WE, Bukowski EJ, Bright FV, Zheng C, Zeng XC, Gong B: A new strategy for folding oligo(m-phenylene ethynylenes). Chem Commun 2003: 56-57. 57. Oh K, Jeong K-S, Moore JS: Folding-driven synthesis of oligomers. Nature 2001, 414:889-893. 58. Oh K, Jeong K-S, Moore JS: m-Phenylene ethynylene sequences joined by imine linkages: dynamic covalent oligomers. J Org Chem 2003, 68:8397-8403. 59. Zhao D, Moore JS: Reversible polymerization driven by folding. J Am Chem Soc 2002, 124:9996-9997.

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71. Zeng H, Yang X, Flowers RA, Gong B: A noncovalent approach to antiparallel b-sheet formation. J Am Chem Soc 2002, 124:2903-2910. 72. Archer EA, Krische MJ: Duplex oligomers defined via covalent

casting of a one-dimensional hydrogen-bonding motif. J Am Chem Soc 2002, 124:5074-5083. This paper describes the utilization of covalent casting to design duplexforming foldamers. Linkers were designed based on the crystal structure of the triazine unit. A hydrogen-bonded amino alcohol linker was found to be most optimal for stabilizing a duplex-forming foldamer in chloroform. Isothermal titration calorimetry studies were performed on duplexes of varying lengths, revealing that three repeating units are most ideal for duplex formation because longer lengths may lead to strong intramolecular interactions. 73. Patch JA, Barron AE: Mimicry of bioactive peptides via nonnatural, sequence-specific peptidomimetic oligomers. Curr Opin Chem Biol 2002, 6:872-877. 74. Arvidsson PI, Ryder NS, Weiss HM, Gross G, Kretz O, Woessner R, Seebach D: Antibiotic and hemolytic activity of a b2/b3 peptide capable of folding into a 12/10-helical secondary structure. ChemBioChem 2003, 4:1345-1347.

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