Design, Synthesis, and Structural Analysis of Turn Modified - American

Article pubs.acs.org/crystal

Design, Synthesis, and Structural Analysis of Turn Modified cyclo(αβ3αβ2α)2 Peptide Derivatives toward Crystalline Hexagon-Shaped Cationic Nanochannel Assemblies José M. Otero,†,§ Matthijs van der Knaap,† Antonio L. Llamas-Saiz,‡ Mark J. van Raaij,δ Manuel Amorín,φ Juan R. Granja,φ Dmitri V. Filippov,† Gijsbert A. van der Marel,† Herman S. Overkleeft,† and Mark Overhand*,† †

Bioorganic Synthesis Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands Departamento de Bioquímica y Biología Molecular Facultad de Farmacia and Centro Singular de Investigación en Química Biológica y Materiales Moleculares, Universidad de Santiago de Compostela Campus Vida, E-15782 Santiago de Compostela, Spain ‡ Unidad de Rayos X RIAIDT, Edificio CACTUS, Universidad de Santiago de Compostela Campus Vida, E-15782 Santiago de Compostela, Spain δ Centro Nacional de Biotecnología (CNB-CSIC), c/Darwin 3, Campus Cantoblanco, E-28049 Madrid, Spain φ Departamento de Química Orgánica Centro Singular de Investigación en Química Biológica y Materiales Moleculares and Unidad Asociada al CSIC, Universidad de Santiago de Compostela Campus Vida, E-15782 Santiago de Compostela, Spain §

S Supporting Information *

ABSTRACT: Intrigued by the not fully extended hairpin monomeric structure of the previously reported cyclo(αβ3αβ2α)2 peptide 2, as well as its nanochannel crystallographic assembly, we here investigate the structural properties of a series of turn derivatives (3−17). Five crystallographic monomeric structures of the symmetric and asymmetric cyclo(αβ3αβ2α)2 peptides 3−17 are found; the novel saddlelike (4 and 16) and the twisted hairpin (6) conformers, as well as the not fully extended hairpins 7 and 14. The pentafluorophenyl/ 1-naphthyl and pentafluorophenyl/9-phenanthryl derivatives 7 and 14, respectively, adopt the anticipated hexagon-shaped crystalline nanotube assemblies, resembling the crystal packing of the parent peptide 2. The structural analysis of the compounds as described here can serve as a basis for biological applications, such as the design of β-sheet mimics or for the development of functional nanomaterials.

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residues and six α-amino acid residues, four of which have the (R)-configuration. Compound 2 provided single crystals, and X-ray crystallography showed a cyclic7a,b hairpinlike structure with a predetermined alternative intramolecular hydrogenbonding pattern7c−k of the strand regions (compare Figure 1, panels b and d). Apart from the four planned intramolecular hydrogen bonds per monomer, we did not anticipate that each βαβ-strand in the crystal packing of molecule 2 interacts with lateral neighboring molecules to form a hydrogen-bonded network of infinite rows of aligned strands (Figure 1e). These rows interact with each other via aromatic π−π stacking interactions (Figure 1f) of the turn residues at an angle of 60°, resulting in a cationic nanotube with a hexagonal cross section. The exterior of the nanotube is hydrophobic and each of its six

INTRODUCTION Hydrogen-bonding interactions play an important role in the stability of engineered peptide structures. A remarkable example of peptide engineering is the design of short cyclic peptides composed of alternating (S)- and (R)-α-amino acids (corresponding to L- and D-amino acids in the Fisher notation)1 with intriguing material and biological properties.2 These designed peptide macrocycles stack on top of each other via an intended intermolecular hydrogen-bonding network, thereby creating nanotubes. Related nanotube structures can be obtained, having an alternative intermolecular hydrogenbonding network between the cyclic peptide sequences by using β-amino acids3 or combining α- with γ-amino acid residues.4 We recently reported a series of macrocycles that have both β-amino acid and (R)-α-amino acid residues at strategic positions in the sequence.5 The most prominent member out of this series, a derivative of the antibiotic gramicidin S (1, Figure 1a),6 is the cyclo-(αβ3αβ2α)2 peptide 2 (Figure 1c).5 Cyclic decapeptide 2 contains four β-amino acid © 2013 American Chemical Society

Received: May 7, 2013 Revised: July 16, 2013 Published: September 23, 2013 4355

dx.doi.org/10.1021/cg4007108 | Cryst. Growth Des. 2013, 13, 4355−4367

Crystal Growth & Design

Article

Figure 1. (a) Gramicidin S (1) structure. (b) Crystallographic structure of 1 (top view); side chains are omitted for clarity (red dotted lines represent intramolecular hydrogen bonds).6a (c) Structure of the cyclo-(αβ3αβ2α)2 peptide 2.5 (d) Crystallographic structure of 2 (top view), side chains are omitted for clarity (red dotted lines represent intramolecular hydrogen bonds). (e) Inter- and intramolecular hydrogen bonds (blue and red dotted lines, respectively) and aromatic π−π stacking (black dotted lines) interactions in the crystallographic hexagon-shaped channel selfassembly of 2. (f) Details of the aromatic π−π stacking interactions, side-view (above), and top-view (below) of the three proximate aromatic rings. (g) Hexagon-shaped nanochannel assemblies forming a honeycomb-type structure, longitudinal view along the channel direction with detail of molecular surface in one of the channels.

crystallographic assembly, we wanted to investigate whether derivatives can be prepared with similar structural properties. Previously, we reported the synthesis of amphiphilic positional isomers of 2,5,8 in which the β3- and β2-amino acid residues

sides provides a honeycomb-type molecular assembly via hydrophobic interactions (Figure 1g). Intrigued by the monomeric structure of the previously reported cyclo-(αβ3αβ2α)2 peptide 2, as well as its nanochannel 4356



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Figure 2. Structures of the projected cyclo-(αβ3αβ2α)2 peptide derivatives 3−17.

Figure 3. Views of crystallographic monomeric structures of compounds 4 (top), 16 (middle), and 188 (bottom). Primary structures have been depicted for clarity (left). Top views (b, e, and h) are shown, indicating the four intramolecular hydrogen-bonding interactions (red dotted lines). Most side-chains are omitted for clarity. Side views (c, f, and i) are also shown, depicting the side-chain positioning.

were swapped in the sequence to yield a series of cyclo(αβαβα)2 derivatives. One of the thus obtained strand-modified

derivatives of 2 was shown to exert modest antibacterial activity8 but no further structural insight was obtained from 4357



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conditions using a homemade set of crystallization buffers and solvents. Compounds 4, 6, 7, 14, 16, and 18 provided single crystals suitable for X-ray diffraction analysis and their high-resolution structures were solved (for details see the Supporting Information).

these compounds. From these previous studies, we conclude that modifications of the strand regions have a large effect on both structural and biological properties of cyclo-(αβαβα)2 peptides, and we therefore decided to focus on turn-modified derivatives of peptide 2. We here report on the design, synthesis, and structural (X-ray, NMR, CD) evaluation of a series of turn-modified derivatives of 2, the symmetric and asymmetric cyclo-(αβ3αβ2α)2 derivatives 3−17 (Figure 2). We show that out of this series, two previously unprecedented monomeric cyclo-(αβαβα)2 conformers were identified and moreover that the asymmetric derivative 7 and 14 featuring both a pentafluorophenyl in combination with a 1-naphthyl group and a 9-phenanthryl group, respectively, adopt hexagonshaped crystalline nanotube assemblies that resemble the crystal packing of the lead structure 2.

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RESULTS AND DISCUSSION Crystallographic Analysis. Compound 4, containing the 1-naphthyl group, adopts a saddlelike structure (Figure 3, panels a−c) having the predetermined four intramolecular hydrogen-bonding pattern as observed for peptide 2 (Figure 1d).5 By comparing the θ angle values12 (Table 1) of the Table 1. Hairpin Twist Angles13 and Dihedral Angles of the β-Amino Acid Residues θ12 in the Peptides 2,5 4, 6, 7, 14, and 16 (Based on the Crystallographic Data)

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EXPERIMENTAL PROCEDURES Design and Synthesis. Upon close inspection of the phenyl side-chain interactions of the turn regions in the crystal structural assembly of compound 2 (Figure 1f), we envisioned that the presence of additional aromatic rings could lead to improved aromatic π−π stacking interactions. As we do not know whether single crystals can be obtained let alone the formation of a similar hexagon-shaped crystal structure assembly can be achieved, we decided to prepare a series of turn modified derivatives having di- and tricyclic aromatic moieties. For this purpose, derivatives 4−6 and 11−13 were prepared (Figure 2). In perfluorinated aromatic rings, the electron π polarity is inverted, which leads to stronger aromatic π−π stacking interactions with nonfunctionalized aromatic rings.9 We decided to make the asymmetric derivatives 3, 7 and 8, and 14 and 15. The derivatives 9 and 10, and 16 and 17, containing the 9H-fluorenyl and the octanyl moieties, respectively, were chosen as control substances. The linear precursors of the cyclo-(αβ3αβ2α)2 peptides 3−17 were synthesized using suitably protected α- and β-amino acid building blocks, following a solid-phase Fmoc peptide chemistry strategy. The required Fmoc-α-amino acids of which the (R)-ornithine residue has its side chain protected with a Boc-group, (S)-Fmoc-β3-homoleucine, (R)-Fmoc-(1naphthyl)alanine, (R)-Fmoc-(2-naphthyl)alanine, (R)-Fmoc(pentafluorophenyl)alanine, and (R)-Fmoc-2-aminodecanoic acid are commercially available. (R)-Fmoc-β2-homovaline, (R)-Fmoc-(9-fluorenyl)alanine, (R)-Fmoc-(9-anthracyl)alanine, and (R)-Fmoc-(9-phenanthryl)alanine were synthesized following adaptations of literature procedures.10 The hyper-acid labile HMPB-MBHA resin11 was functionalized with (R)-Fmoc-phenylalanine or the required (R)-Fmoc-(aryl)alanine in the presence of the coupling reagent DIC and catalytic DMAP and elongated using standard repetitive piperidine deprotection/HCTU coupling conditions. The assembled partially protected decameric peptides were cleaved from the resin by repetitive mild acid treatment and cyclization of the C- → N-terminus, realized in DMF under high dilution (0.01 M) in the presence of PyBOP/HOBt/DiPEA. Removal of reagents was affected by LH-20 gel filtration, the Boc-groups of the ornithine residues of the cyclic peptides were removed using 50% TFA in DCM and the fully deprotected peptides 3− 17 were purified using preparative RP-HPLC (15−63% overall yield). Crystallography. The cyclopeptides 3−17 reported here (Figure 2) and the previously described8 control compound 18 (Figure 3g) were subjected to controlled evaporation

peptide

hairpin twist angle (deg)

residue

θ (deg)

2

2

4

37

6

55

7

5

14

1

16

1

(S)-β3hLeu(2) (R)-β2hVal(4) (S)-β3hLeu(7) (R)-β2hVal(9) (S)-β3hLeu(2) (R)-β2hVal(4) (S)-β3hLeu(7) (R)-β2hVal(9) (S)-β3hLeu(2) (R)-β2hVal(4) (S)-β3hLeu(7) (R)-β2hVal(9) (S)-β3hLeu(2) (R)-β2hVal(4) (S)-β3hLeu(7) (R)-β2hVal(9) (S)-β3hLeu(2) (R)-β2hVal(4) (S)-β3hLeu(7) (R)-β2hVal(9) (S)-β3hLeu(2) (R)-β2hVal(4) (S)-β3hLeu(7) (R)-β2hVal(9)

160 177 156 175 58 83 72 124 175 78 170 80 169 170 171 164 165 178 163 172 75 76 87 90

residues of 2 and 4, a large difference in backbone structure is apparent: θ angle values for the not completely extended βαβsheet structure in the parent peptide 2 are 156/160° and 175/ 177° for the two (S)-β3-homoleucine and the two (R)-β2homovaline amino acids, respectively.12 In peptide 4 the two (S)-β3-homoleucine and two (R)-β2-homovaline θ-angles are 58/72° and 83/124°, respectively. Compound 16 also crystallized in this novel saddlelike structure (Figure 3, panels d−f). The β-hairpin twist angle13 in this case is 1° (37° in compound 4), and the θ angles in the β-amino acids pairs are 75/87° and 76/90°, respectively (Table 1). Several other macrocyclic molecules have been reported to form (horse) saddle motifs. Examples include, patellamide D, metalcomplexed forms of antamanide and patellamide-A derivatives, and the depsipeptide surfactin.14 The crystallographic structures of peptides 4 and 16 resemble the rigid saddle structure of the previously synthesized8 gramicidin S derivative 18, containing ten α-amino acid residues, four of which have the (R)-configuration (Figure 3, panels g−i). Besides the alternative hydrogen-bonding pattern in peptides 4 and 16, other 4358



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Figure 4. Crystallographic structure of compound 6. (a) Primary structure. (b) Top view showing the four intramolecular hydrogen-bonding interactions (red dotted lines), side-chains are omitted for clarity. (c) Side-view. (d) Longitudinal view along the channel direction of the crystallographic packing with detail of molecular surface in one of the channels. (e) Intramolecular hydrogen bonding (red dotted lines) and Tshaped edge-to-face aromatic interactions (black dotted lines) in the channel assembly. The β-sheets are oriented almost longitudinally to the sixthorder crystallographic axis. Side chains and coordinated solvent molecules are omitted for clarity.

core and 14 vs 27 Å for the hydrophilic channel), although in both structures, six peptide molecules are related by a 6n screw crystallographic axis (n = 2 in derivative 2 and n = 1 in derivative 6). This means that nanotubes obtained with derivative 6 have a hexagon-shaped disposition of the monomers, although the nanotube shape is pseudocylindrical. The main intermolecular interaction between the peptide subunits observed in these nanotubes is a weak T-shaped edgeto-face aromatic interaction between two 2-naphthalenyl groups from neighboring molecules. No intermolecular hydrogen-bonding interactions are observed in the crystallographic assembly of 6. The crystallographic structure of the asymmetric compound 7, containing a 1-naphthyl and a pentafluorophenyl moiety, showed a not fully extended hairpin structure similar to the parent derivative 2. The hairpin twist of this compound is 5°, and the θ angles of the β-amino acids pairs are 169/171° and 170/164°, respectively (Table 1). Compound 7 crystallized forming similar hexagon-shaped cationic nanochannels as 2 (Figure 5). The ornithine side chains are located in the interior

important structural differences are their concave positioning of the (R)-ornithine side chains, as compared to the convex positioning of these side chains in peptide 18 (see Figure 3, panels c, f, and i, respectively). The crystallographic packings of peptides 4, 16, and 18 are depicted in Figure 10. The crystallographic structure of compound 6 (Figure 4), containing the 2-naphthyl group in each turn region, showed a higher hairpin twist angle as compared with the parent structure of compound 2,5 that is 55° versus 2° (Table 1). This means that the four intramolecular hydrogen-bonding interactions of 6 in its crystallographic structure are less ideal than those in 2 (Table 2). The new structure of 6 resembles twisted cyclic hairpin structures of gramicidin S derivatives previously reported by us.15 Such twisted cyclic hairpin structures are also named “twisted figure of eight”.14a The θ angles in the β-amino acid pairs of 6 are 175/170° and 78/80°, respectively (Table 1). Peptide 6 forms amphiphilic channels with a cylindrical shape in its crystallographic packing (Figure 4d). These nanotubes are thinner than those observed with the parent derivative 2 (approximately 30 vs 50 Å for the external 4359



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Table 2. Intra- and Inter-Molecular Interactions Involved in the Crystallographic Assembly of Compounds 2,5 4, 6, 7, 14 and 16 (Based on the Crystallographic Data) peptide

interaction

donor

acceptor

d(D···A) (Å)

d(H···A) (Å)

D···OC (deg)

2

intramolecular H bond intramolecular H bond intermolecular H bond intramolecular H bond intramolecular H bond intermolecular H bond parallel displaced π-stacking intramolecular H bond intramolecular H bond intramolecular H bond intramolecular H-bond intramolecular H-bond intramolecular H bond intramolecular H bond intramolecular H bond T-shaped edge-to-face π-stacking intramolecular H bond intramolecular H bond intermolecular H bond intermolecular H bond intramolecular H bond intramolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond parallel displaced π-stacking intramolecular H bond intramolecular H bond intermolecular H bond intermolecular H bond intramolecular H bond intramolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond intermolecular H bond parallel displaced π-stacking intramolecular H bond intramolecular H bond intramolecular H bond intramolecular H bond

N(2) N(3) N(4) N(7) N(8) N(9) Ph(5) N(2) N(3) N(7) N(8) N(2) N(3) N(7) N(8) H(5) N(2) N(3) N(4) N(5) N(7) N(8) N(9) N(10) O(W106) O(W102) O(W102) O(W104) O(W101) O(W104) F5−Phe(5) N(2) N(3) N(4) N(5) N(7) N(8) N(9) N(10) O(W101) O(W103) O(W103) O(W102) O(W104) O(W104) F5−Phe(5) N(2) N(3) N(7) N(8)

O(9) O(8) O(7)d O(4) O(3) O(2)d Ph(10)d O(9) O(8) O(4) O(3) O(9) O(8) O(4) O(3) Nal(10)d O(9) O(8) O(W104) O(W101) O(4) O(3) O(W102) O(W106) O(1) O(1) O(2) O(6) O(6) O(7) Nal(10)d O(9) O(8) O(W104) O(W102) O(4) O(3) O(W103) O(W101) O(1) O(1) O(2) O(6) O(6) O(7) Phen(10)d O(9) O(8) O(4) O(3)

3.0 3.0 3.3 3.1 2.9 3.3 4.6a 3.0 2.8 3.0 2.8 3.1 2.9 3.0 2.8 5.3a 2.9 2.7 2.6 2.7 3.1 2.6 2.6 2.9 3.4 3.0 2.2 2.7 3.4 2.3 3.4a 3.0 2.9 2.6 2.8 3.1 2.9 2.5 2.9 2.8 3.0 2.1 2.9 3.1 2.1 3.3a 2.8 2.8 3.0 2.9

2.2 2.1 2.4 2.2 2.1 2.4  2.2 1.9 2.1 2.0 2.2 2.1 2.2 1.9 3.5c 2.1 1.9 2.0 1.8 2.4 1.7 1.8 2.0        2.2 2.0 1.9 2.0 2.3 2.1 1.7 2.0        2.0 2.0 2.1 2.0

132 164 166 134 170 162 2b 138 161 155 142 140 143 148 149 44‡ 133 160   136 163   121 136 113 136 120 119 3b 133 165   132 163   126 134 122 120 140 122 4b 149 156 142 160

4

6

7

14

16

For parallel displaced and T-shaped π-stacking interactions, d(D···A) denotes the arenes centroid distance. bFor parallel displaced and T-shaped πstacking interactions, the D···OC angle denotes the aromatic plane angle. cFor a T-shaped π-stacking interaction, d(H···A) denotes hydrogen-toarene centroid distance. dIndicates a residue from an adjacent molecule. a

of the nanotubes; the exterior is hydrophobic and forms a honeycomb-type assembly. Some significant differences were found in the crystallographic assembly of derivative 7, as compared to 2. The intermolecular hydrogen-bonding network of the nanotubes of 7 does not contain direct hydrogen bonds

between the CO and N−H groups from neighboring molecules, but water molecules are acting as bridges in the middle of the N−H → OC interactions (Table 2). Because of this feature the longitudinal distance between adjacent molecules along the nanotubes obtained with compound 7 is 4360



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Figure 5. Crystallographic structure of compound 7. (a) Primary structure. (b) Top view of the monomer structure (red dotted lines represent intramolecular hydrogen bonds), side-chains are omitted for clarity. (c) Side view. (d) Longitudinal view along the channel direction of the crystallographic packing with the detail of the molecular surface in one of the channels. (e) Inter- and intramolecular hydrogen bonding (blue and red dotted lines, respectively) and aromatic π−π stacking (black dotted lines) interactions in the hexagon-shaped channel self-assembly (water molecules are indicated as red dots). (f) Details of the aromatic side chains of the turn regions of three interacting subunits of 7 (side view). (g) Details of the aromatic side chains of the turn regions of three interacting subunits of 7 (top view).

10.5 Å, slightly higher than the 9.9 Å observed in the crystallographic assembly of 2. The π−π interaction distance and angle of the pentafluorophenyl group with the 1-naphthyl group in the crystallographic packing of compound 7 are 3.4 Å and 3°, respectively, while those of the two phenyl groups of compound 2 are 4.6 Å and 2°, respectively. The closer proximity of the aromatic planes observed in the pentafluorophenyl/1-naphthyl pairs of 7 with respect to the phenyl/ phenyl pairs of 2 is consistent with our assumption that the presence of an electron π polarity inverted pentafluorophenyl ring induces stronger aromatic π−π stacking intermolecular interactions with a nonfunctionalized aromatic ring. The molecules of compound 7 in the nanotubes are related by a 6-fold axis, while the molecules of compound 2 in the nanotubes are related by a 62-fold crystallographic axis. The crystallographic structure of asymmetric cyclopeptide 14, containing a 9-phenanthryl and a pentafluorophenyl moiety, showed a not fully extended hairpin structure equivalent to those observed for compounds 2 and 7 (Figure 6, panels a−c). The hairpin twist angle is 1°, and the θ angles of the β-amino

acids pairs are 165/163° and 178/172°, respectively (Table 1). The crystallographic assembly of 14 forms hexagon-shaped nanotubes with the cyclopeptides disposed following a 61 screw axis with four water molecules acting as bridges in the middle of the N−H → OC intermolecular interactions (Figure 6, panels d−e). The distance between adjacent “β-sheets” is 10.3 Å, and the π−π interaction between the 9-phenanthryl and pentafluorophenyl moieties is optimal, as is reflected by the 3.3 Å distance between both aromatic planes (Table 2). The presence of an additional aromatic ring in the 9-phenanthryl moiety of 14 with respect to the 1-naphthyl moiety of 7 apparently allows for a tighter packing of the aromatic rings (Figure 6, panels f−g). NMR and CD Analysis. Recently, we studied the solution structure of peptide 2 using NMR techniques in great detail.5 Our conclusion was that the NMR data obtained for peptide 2 agrees well with its crystallographic structure shown in Figure 1. The C2-symmetric peptides 4, 6, and 16, and the asymmetric peptides 7 and 14 dissolve readily in polar solvents and by using a combination of COSY and TOCSY spectra, the NMR 4361



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Figure 6. Crystallographic structure of compound 14. (a) Primary structure. (b) Top view of the monomer structure (red dotted lines represent intramolecular hydrogen bonds); side-chains are omitted for clarity. (c) Side view. (d) Longitudinal view along the channel direction of the crystallographic packing with details of the molecular surface in one of the channels. (e) Inter- and intramolecular hydrogen bonding (blue and red dotted lines, respectively) and aromatic π−π stacking (black dotted lines) interactions in the hexagon-shaped channel self-assembly (water molecules are indicated as red dots). (f) Details of the aromatic side chains of the turn regions of three interacting subunits of 14 (side view). (g) Details of the aromatic side chains of the turn regions of three interacting subunits of 14 (top view).

amino acid residues of 4, 6, 7, 14, and 16 are small (3−4 Hz) or cannot be measured. The NH of the amides of the ornithine and β3-homoleucine residues give doublets with J = 7−9 Hz, the NH signals of the amides of the β2-homovaline residues are generally broad (or give a doublet of doublets with one large5 J value). This NMR data agrees with those of the corresponding residues of compound 2 and are a strong indication that the ornithine and the β-amino acid residues are part of a “strand”

signals could be fully assigned (see the Supporting Information). No multiple signals per nucleus in their 1H and 13 C spectra were found. The amide regions of the peptides 4, 6, 7, 14, and 16 and of the earlier reported peptide 2 are depicted in Figure 7. The signals of the amide regions of the peptides 4, 6, and 16 as well as those of 7 and 14, taken in account their asymmetry, are very similar as those of the parent peptide 2. That is, the NHCαH coupling constants of the aromatic α4362



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Figure 7. The amide regions of the 600 MHz 1D 1H NMR spectra in CD3OH of derivatives 2, 4, 6, 7, 14, and 16. Only amide and amine proton signals are quoted.

region5 and that the aromatic α-amino acids are part of a turn region. The NMR data of the compounds 3, 5, 8, 9−13, 15, and 17 are very similar and no multiple signals per nucleus in their 1H and 13C spectra are found (see the Supporting Information). Additionally, CD spectra were recorded in methanol with compounds 3−17 to assess the conformational behavior of the secondary structures of these cyclo-(αβ3αβ2α)2 peptides in solution (Figure 8). CD spectra of the parent

are thus likely the result of a preferred conformation in the packing in their respective crystals.

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CONCLUSIONS Intrigued both by the monomeric- and assembled crystallographic structure of the previously reported cyclo-(αβ3αβ2α)2 peptide 2, we investigated the structural properties of a series of turn derivatives. Controlled evaporation conditions using the synthesized cyclo-(αβ3αβ2α)2 peptides 3−17 provided single crystals of the compounds 4, 6, 7, 14, and 16. Besides the not fully extended cyclic hairpinlike monomer structure of peptides 7 and 14 (very similar to the monomeric structure of 2), two novel crystallographic monomeric structures having four intramolecular hydrogen bonds were found; the saddlelike (4 and 16) and the twisted hairpin structure (of 6) (Figure 9). On the basis of the positioning of the iso-propyl, iso-butyl and the 3-aminopropyl side chains, the twisted hairpin structure may be viewed as an intermediate conformation in between the not fully extended cyclic hairpinlike conformation and the saddlelike conformation. As judged by NMR and CD, we conclude that in solution these cyclo-(αβ3αβ2α)2 peptides may coexist as a rapidly interchanging mixture of conformers. The results as presented here therefore give an interesting snapshot of at least three possible conformers of this type of cyclo-(αβ3αβ2α)2 peptides. It is well-established that 2,3-disubstituted β-amino acid residues adopt restricted preferred conformations.17 It is thus not unlikely that replacement of the partially flexible monosubstituted β-amino acid residues by syn disubstituted β2,2-amino acid residues17 toward a cyclo-(αβ3,2αβ3,2α)2 peptide, as shown in Figure 9 (top right), affords a conformationally rigid saddlelike structure. Similarly, the use of anti disubstituted β3,2-amino acids17 could provide a rigid extended cyclic hairpinlike structure (Figure 9, bottom right).

Figure 8. CD spectra of gramicidin S (1) and cyclo-(αβ3αβ2α)2 derivatives (2−17) recorded in 0.1 mM in methanol at 298 K.

derivative 2 and native cyclo-(ααααα)2 peptide GS (1) were also recorded in the same conditions, showing the typical negative ellipticities at 220 and 205 nm of their β-sheet/βhairpin conformations.16 All the cyclo-(αβ3αβ2α)2 derivatives (2−17) show considerable negative ellipticities around 202− 208 nm comparable to those observed for GS (1), confirming their β-sheet/β-hairpin conformation in methanol. From the NMR and CD results, we conclude that the novel horse saddlelike structures of peptides 4 and 16 and the twisted hairpin structure of peptide 6 as observed using X-ray analysis 4363



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Figure 9. The three crystallographic conformations as found for the peptide derivatives (a) 4 and 16, (b) 6, and (c) 7 and 14. The yellow line indicates hairpin twist angle.13 Projected structures having syn- and anti-disubstituted β-amino acid residues.

The crystallographic packing of molecules 2,5 4, 6, 7, 14, 16 and the previously synthesized8 18 are depicted in Figure 10. Compounds 4, 16, and 18 do not form nanotubes in their crystal packing (Figure 10, panels a, b, and c, respectively). The crystallographic packing of control compound 16 may indicate that aromatic substitutions in the turn regions of the cyclo(αβ3αβ2α)2 peptides are a minimal requirement to allow the formation of nanotubes. Molecules of derivative 6 form nanotubes that are related by a 61 screw crystallographic axis, adopting a helical stacking motif. In the assembly, there are no hydrogen-bonding interactions between the peptide subunits, and the external core has a pseudocylindrical shape (Figure 10d). The pentafluorophenyl/1-naphthyl derivative 7 and the pentafluorophenyl/9-phenanthryl derivative 14 form hexagonshaped nanotubes in their crystallographic assemblies resembling the crystal packing of the parent peptide 2. The π−π interaction distance between the pentafluorophenyl- and the aromatic moieties of molecules 7 and 14 in the crystal assemblies are significantly shorter than the π−π interaction distance between the phenyl groups of molecules 2, thus confirming that the use of an electron π polarity inverted aromatic ring can lead to stronger aromatic π−π stacking9 with

a nonfunctionalized aromatic ring. Other differences include the involvement of water molecules in the hydrogen-bonding network of the peptide subunits. In the reference derivative 2, the molecules in the nanotubes are related by a 62 screw crystallographic axis, as the nanotubes arise from an infinite helical intertwining of two rows of aligned cyclopeptides linked by the aromatic π−π interactions (Figure 10e). The molecules of compound 7 in the nanotubes are related by a 6-fold crystallographic axis, as the result of the linear stacking of hexagon-shaped discs formed by six molecules of 7 assembled in the same plane by means of the aromatic π−π interactions (Figure 10f). The molecules of compound 14 in the nanotubes are related by a 61 screw crystallographic axis, as the result of a helical motive stacking of an infinite row of cyclopeptides linked by the aromatic π−π interactions (Figure 10g). In summary, by preparing a series of cyclo-(αβ3αβ2α)2 peptide turn-derivatives, we found two novel monomeric conformers: a saddlelike and a twisted hairpin structure. These and related structures can potentially serve as a basis for the development of novel rigid molecular scaffolds in a template-assembled approach toward molecules with tailormade properties,18 or for biological applications such as the 4364



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Figure 10. Crystallographic assembly of compounds 2, 4, 6, 7, 14, 16, and 18. (a, b, and c, respectively) Molecules 4, 16, and 18 crystallized, adopting tight packing. (e and d) A double helical motive stacking is observed with molecules 2 and 6 (respectively), (f) a linear stacking of hexagonal plain discs is observed with molecules 7, and (g) a simple helical motive stacking is observed with molecules 14.

design of subtilisin-resistant antibiotics,8 or as a β-sheet mimic. 19 The latter was realized by using the cyclo(αβ3αβ2α)2 peptides 3−17 as inhibitors of amyloid-β fibril formation in vitro.20 In addition, it was found that the pentafluorophenyl/1naphthyl derivative 7 and the pentafluorophenyl/9-phenanthryl derivative 14 form hexagon-shaped nanotubes in their crystallographic assemblies. Besides the anticipated improved π−π stacking interactions, other differences in the crystal packing of molecules 2, 7, and 14 demonstrate that crystal engineering of this type of molecules is a trial and error process. We will investigate whether the cyclo-(αβ3αβ2α)2 peptides 2, 7, and 14 can be used toward functional nanomaterials. Such an application may be feasible as we readily obtained long and narrow microcrystalline fibers of compound 2 with an average length of 200−300 μm and approximately 500−600 nm wide (Figure 11, for experimental details see the Supporting Information). These microcrystalline fibers could be obtained readily by the controlled addition of dimethylsulfoxide to a solution of 2 in chloroform. After allowing the mixture to stand for several hours, a large amount of microcrystals of 2 were obtained. Optic and scanning probe electron microscopy studies were performed with these thin needles to elucidate their morphology. Optical microscope images (Figure 11a) revealed the formation of very narrow microcrystals with an average length of 200−300 μm. Generally, these needles adopted a completely straight disposition, but also slightly curved and twisted needles are found, which is an indication that these needles are flexible and consequently do not form a proper crystal structure. Scanning electron microscope (SEM) images showed the approximate size and shape of these fibers. It could be estimated that they have a hexagonal prism shape approximately 500−600 nm wide. This feature could be related to the nanotube-assembling ability of compound 2 revealed by its crystallographic analysis, in which the hydrophobic exterior of the hexagon-shaped molecular assemblies interacts with its neighbors with each of its six molecular walls via hydrophobic interactions.5 It is therefore tentatively proposed that these

Figure 11. Optical and scanning probe electron microscopic images of the microcrystals of compound 2. (a) Optical microscope image reveals thin needles with average length of 200−300 μm. (b) Scanning probe electron microscope image of several fibers. (c) Detail of one fiber, a hexagonal-shaped morphology was observed, with 600 nm width approximately. d) Proposed peptide nanotubes honeycomb selfassembly in a 25 nm width hexagonal-shaped portion of the fiber. Hydrophilic pores inner diameter is approximately 3 nm. (e) Detail of two parallel stacked fibers retaining the proposed hexagonal-shaped morphology.

fibers are formed by a honeycomb-like packing (Figure 11d) of hexagonal-shaped amphiphilic nanotubes, containing thousands of longitudinally disposed cationic pores each with a 3 nm inner diameter. SEM pictures also showed that these fibers occasionally interact with themselves, forming groups of two or more parallel displaced fibers but retaining the hexagonal prism shape of each one (Figure 11e). 4365



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ASSOCIATED CONTENT

S Supporting Information *

Complete synthesis details and characterization of compounds 3−17, X-ray crystallographic data of compounds 4, 6, 7, 14, 16, and 18, and preparation and analysis details of microcrystals of compound 2 are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (+)31 527 4307. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by grants from the Dutch technical science foundation (STW), the Spanish Ministry of Economy and Competitivity (BFU2011-24843 and a Ramón y Cajal contract to M.A.), the Xunta de Galicia (Angeles Alvariño fellowship to J.M.O.), and the European Social Fund (fellowship to J.M.O.). We thank Kees Erkelens, Hans van den Elst, Nico Meeuwenoord, and Gustavo Rama for their technical assistance with the analysis and purification of the peptides, and the ESRF (Grenoble, France) and the ALBA synchrotron (Barcelona, Spain) for the provision of beam time on beamlines BM30-A and BL13-XALOC, respectively.

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