Conformational Effects through Hydrogen Bonding in a Constrained Î³

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Conformational effects through hydrogen bonding in a constrained #-peptide template: from intra-residue 7-membered rings to a gel-forming sheet structure Hawraa Awada, Claire M. Grison, Florence Charnay-Pouget, Jean-Pierre Baltaze, François Brisset, Régis Guillot, Sylvie Robin, Ali Hachem, Nada Jaber, Daoud Naoufal, Ogaritte Yazbeck, and David J. Aitken J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b00494 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 13, 2017

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The Journal of Organic Chemistry

Conformational effects through hydrogen bonding in a constrained γ-peptide template: from intra-residue 7-membered rings to a gel-forming sheet structure Hawraà Awada,§,†,+ Claire M. Grison,§,+ Florence Charnay-Pouget,§ Jean-Pierre Baltaze,§ François Brisset,§ Régis Guillot,§ Sylvie Robin,§,‡ Ali Hachem,† Nada Jaber,† Daoud Naoufal,† Ogaritte Yazbeck,† and David J. Aitken§,*

§

CP3A Organic Synthesis Group and Services Communs, ICMMO, UMR 8182, CNRS,

Université Paris Sud, Université Paris Saclay, Bât. 420, 15 rue Georges Clemenceau, 91405 Orsay cedex, France †

Inorganic and Organometallic Coordination Chemistry Laboratory and Laboratory for

Medicinal Chemistry and Natural Products, Faculty of Sciences (I) & PRASE-EDST, Lebanese University, Hadath, Lebanon ‡

UFR Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 avenue de

l’Observatoire, 75270 Paris cedex 06, France +

These two authors contributed equally to this work and should be considered as having joint

first-author status

[email protected]

ACS Paragon Plus Environment


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Table of Contents

Abstract A series of three short oligomers (di- tri- and tetramers) of cis-2-(aminomethyl)cyclobutane carboxylic acid, a γ-amino acid featuring a cyclobutane ring constraint, were prepared and their conformational behavior was examined spectroscopically and by molecular modeling. In dilute solution, these peptides showed a number of low-energy conformers including ribbon-like structures pleated around a rarely-observed series of intramolecular 7-membered hydrogen bonds. In more concentrated solution, these interactions give deference to an organized supramolecular assembly, leading to thermoreversible organogel formation notably for the tripeptide, which produced fibrillar xerogels. In the solid state, the dipeptide adopted a fully-extended conformation featuring a 1D network of intermolecularly H-bonded molecules stacked in an antiparallel sheet alignment. This work provides a unique insight into the interplay between interand intramolecular H-bonded conformer topologies for the same peptide template.


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Introduction In Nature, peptides and proteins rely on secondary, tertiary and quaternary structural features to provide an exquisite array of ordered biomaterials constructed from simple building blocks.1 Privileged conformations are often stabilized by short- or long-range non-covalent interactions, leading to well-defined molecular architectures. This behavior is not only primordial for biological functions, but it is a rewarding source of inspiration for numerous design facets of nano-scale materials with functional and responsive features: organized self-assembly of synthetic peptides can provide higher-ordered architectures such as tubes, sheets, fibers, tapes, wires, and gels.2 The preparation of regularly-shaped synthetic peptidomimetic manifolds has been at the forefront of developments in foldamer science.3 Aromatic amino acids or homologated β-amino acids have been the most studied non-canonical building blocks, but there is a growing interest in the use of γ-amino acids.4 Through the judicious choice of building blocks, stable secondary structures built upon networks of C=O···H−N hydrogen bonds (Hbonds) can be induced. The most common regularly-folded architectures are helical: they include the 14-helix5 and the 9-helix6 for γ-peptides, the 13-helix7 and the 11/13-helix8 for β,γ-peptides, the 12-helix9 and the 12/10-helix8,10 for α,γ-peptides. These secondary structures along with other helical manifolds have been anticipated by theoretical calculations.11 Helical structures are also observed for γ-peptide/urea hybrids,12 while replacement of short α-peptide segments with β/γpeptide fragments in α-helical peptides has been carried out successfully.13 Short distance H-bonding may give rise to regular non-helical structures, although these are significantly rarer. Two examples of a 9-ribbon have been observed for γ-peptides,14 and a unique 9/8-ribbon was described recently for particular β,γ-peptides incorporating GABA as the γcomponent.15 Intra-residue H-bonding of a γ-amino acid in a peptide constitutes a C7 conformer



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feature (or C7 turn), which is quite rare in γ-peptides;16,17 only one example of successive C7 rings, leading to a short 7-ribbon topology, has been characterized.18 γ-Peptides having structural constraints which privilege extended conformations are sparse. In early work, it was shown that vinylogous dipeptides constructed from α,β-unsaturated γ-amino acids can adopt parallel or anti-parallel sheet structure in the solid state.19 Parallel sheet secondary structure has been observed between adjacent strands in hairpin architectures constructed from cyclically-constrained γ-amino acid residues.20,21 A parallel sheet in a novel γtripeptide was stabilized by both C=O···H−N and C=O···H−C interactions,22 while short γpeptides build from 4,4-dimethyl-GABA adopted parallel or anti-parallel sheets in the crystal state.23 Supramolecular self-assembly giving nano-tubes has been observed for rigid macrocyclic systems based on γ-peptides,24 α,γ-peptides,25 or other γ-peptide hybrids.26 Cyclic backbone constraints have been a prominent feature of the γ-amino acids employed in the organized structures described above (Figure 1). The cyclohexane-constrained motif A (or its 1R,2R,3R enantiomer) favors the formation of helices containing C=O(i)···H−N(i+3) H-bonds, enabling 14-, 13- and 12-helices in γ-, β/γ- and α/γ-peptides, respectively.7b,27,28 Motifs B, C, D and E have been shown experimentally to induce 12/10-helix conformers in α/γ-peptides,29,30,31,32 although contributions from 12-helix conformers are also noted for B.29 Theoretical studies suggest that γ-peptide homo-oligomers of C might adopt both 12- and 14-helices,33 while homooligomers of F and of G should adopt a 14-helix structure.34 The rigid trans-(1R,2R)cyclopropane motif H precludes formation of secondary structure and oligomers of this residue self-assemble to form sheets;22 the factors which stabilize the parallel sheets were examined in a recent theoretical study.35 The only cyclobutane γ-residue examined to date is the highly rigid 3aminocyclobutane carboxylate I (or its 1R,3S enantiomer). Oligomers of this structure appear to adopt extended conformations bereft of intramolecular H-bonds,36 while hybrid γ-peptides of I


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and cis-4-(Boc-amino)-L-proline present more compact conformations combining intra- and inter-residue H-bonding in which the Boc function (non-backbone) competes with the cyclobutane carbonyl (backbone) as an H-bond acceptor.37

O

O

O R S

N H

S S

N H

S

R

R S

A

C O

R R

N H

R B

O

R

N H

S

O

R R

S

N H

N H

S S

F

E

D

O

O O N H

R R

N H

S S

S

G

N H

H O

R

I

J

S R

N H

studied in this work

Figure 1. Alicyclic γ-amino acid residues studied for their ability to induce conformational preferences in peptides.

The study of rationally designed building blocks remains a central requirement in order to expand the array of organized secondary and/or supramolecular γ-peptide architectonics. To this end, we identified the (1R,2S)-2-(aminomethyl)cyclobutane carboxylate motif J as an appealing candidate for study. As a homologue of 2-aminocyclobutane carboxylate, a building block which plays a significant role in determining conformational preferences in β-peptides,38 it has more flexibility



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and pursues the trend of vicinal backbone restriction prevalent in motifs A-H. The cis cyclobutane geometry of J neither precludes nor enforces any specific intra-molecular H-bonding mode, yet the four-membered ring restriction can be expected to have some effect on conformational preferences. Herein we describe the detailed study of the behavior of short oligomers of this γ-amino acid.

Results and discussion Synthesis The N-protected (1R,2S)-γ-amino acid building block 1a was obtained in single enantiomer form using literature procedures39 and was converted smoothly into its benzyl ester 1b (Scheme 1). Standard solution-state peptide coupling procedures using HATU were then used to prepare the short series of N- and C-capped oligomers 2, 3 and 4. The solvent system employed was a CH2Cl2/DMF mixture, which ensured that all peptide material remained soluble during the synthesis. Indeed, solubility became a significant issue with increasing peptide length. In the course of the synthetic and chromatographic purification procedures, it became evident that gels were being formed; once the three peptides were in hand, we examined this phenomenon in more detail.


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Scheme 1. Synthesis of the γ-peptides studied in this work.

Gelation and xerogel morphology studies Gels formed by low molecular weight molecules are of interest due to their physicochemical properties and potential applications.40,41 Small peptides can be propitious gelling agents, principally via formation of intermolecular H-bond networks, although other non-covalent interactions such as π-stacking, van der Waals or electrostatic forces may also play a role.42 Only recently, however, was a γ-peptide gelator described.23 The gel-forming ability of the peptides 24 was therefore investigated on a large panel of common solvents, ranging from hydrocarbons to water; four industrial/domestic liquids were also examined: PEG-400, red wine, olive oil, and maple syrup (Table 1).



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Table 1. Gelation properties of peptides 2-4.[a] Solvent

2

3

4

Cyclohexane Toluene THF Chloroform 1,2-Dichloroethane Dichloromethane Methanol Isopropanol Ethanol Ethyl acetate Acetonitrile DMF Acetone Diethyl ether Water PEG-400 Red Wine Olive Oil Maple syrup

I* S S S S S S S S S I S S PG I* I* I* I* I*

I G (7.6) G (62.5) I PG S S G (62.5) PG G (9.8) G (30.0) S PG I* I* I* I* I* I*

I* PG I* PG I* I* I I* I* I* I* I* I* I* I* I* I* I* I*

[a] Results are given for samples with c = 50 mg/mL, except for 3 in THF and isopropanol for which c = 75 mg/mL. S = soluble, I = insoluble, I* = insoluble at c = 10 mg/mL, PG = partial gel, G = gel. For gels, the mgc value (in mg/mL) is given in parentheses.

Dipeptide 2 was soluble in most solvents, at least to a concentration of 50 mg/mL, with exceptions being noted at the extreme ends of the polarity scale. Lower solubility in acetonitrile was observed but gelation did not occur with more dilute solutions. A sample at c = 50 mg/mL in diethyl ether gave a partial gel but samples at c = 75 mg/mL simply precipitated. Conversely, tetrapeptide 4 was insoluble in most solvents, even at near-reflux temperatures, for 50 mg/mL samples. Toluene and chloroform did however provide partial gelation at this concentration. Tripeptide 3 proved to be an excellent gelator, giving well defined, transparent gels in toluene, THF, isopropanol, ethyl acetate and acetonitrile. The gels were stable at room temperature for days and their formation was thermoreversible. Three other solvents – 1,2-dichloroethane,


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ethanol and acetone – gave partial gelation. This solvent panel encompasses a considerable range of solvent dielectric constants and polarities, although peptide 3 was insoluble in water and in the highly polar domestic liquids. The minimum gel concentration (mgc) for 3 was determined in each of the five first-noted solvents. With THF and isopropanol mgc values just over 60 mg/mL were observed, while for toluene the value was half of that. Lower values were observed for toluene (7.6 mg/mL) and ethyl acetate (9.8 mg/mL), equating to 14 mM and 18 mM respectively making tripeptide 3 comparable with other small peptide systems which show good gelation properties.43 The gel melting temperature was determined for the gels formed by 3 in toluene and ethyl acetate. In both cases, the gel melting temperature increased significantly as the concentration was increased from the mgc, reaching a plateau at a concentration which was only 2- to 3-fold the mgc value (Figure 2). These temperatures were 70 °C for toluene and 67 °C for ethyl acetate; the latter is only 10 °C below the boiling point of the solvent, attesting to the remarkable thermal stability of the gel. It is interesting to recall here that many proteins are denatured thermally in the range 60-70 °C, through rupture of their H-bonding networks.

Figure 2. Gel melting temperature curves for tripeptide 3 in EtOAc and in toluene. The concentration range is from mgc to 3× this value in each case.



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Scanning electron microscopy (SEM) analyses were carried out to investigate the morphology of xerogel samples of peptides 3 and 4 obtained from different solvents. Selected images are shown in Figure 3. Tripeptide 3, for the most part, furnished fibrillar material. Entangled networks of slim nanofibers resembling puff candy were obtained from toluene and acetonitrile, whereas more distinct interlaced fiber bundles, with a bundle width in the range 50–200 nm, were obtained from 1,2-dichloroethane. In contrast, from the polar protic solvent isopropanol, a more porous sponge-like xerogel was obtained with irregular-sized voids in evidence. This phenomenon may be the result of a morphology transition during the evaporation process, as has been observed in previous examples of ethanolic gels for a dipeptide.44 The morphology of the two xerogels from tetrapeptide 4 was also fibrillar. From toluene, small agglomerates of fibers of fairly uniform dimensions (50 nm wide, 600 nm long) were obtained, in aligned yet off-set arrays. From chloroform, elegant, dense, straw-like assemblies of long thin fibers (50 nm wide, several µm long) were observed.


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(a)

(b)

(c)

(d)

(e)

(f)

Figure 3. SEM images of xerogels obtained from tripeptide 3 in (a) toluene, (b) acetonitrile, (c) 1,2-dichloroethane and (d) isopropanol, and of xerogels obtained from tetrapeptide 4 in (e) toluene and (f) chloroform.

X-Ray crystal structures In the search for clues to the molecular organization in the above described gels, we considered single crystal x-ray diffraction. This technique has often been used to examine the intramolecular H-bonding patterns which define helical folded topology of γ-peptides.4 Single crystals of dipeptide 2 were obtained by slow evaporation of a chloroform solution; the X-ray structure is shown in Figure 4. The molecules adopt fully-extended conformations presenting two



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intermolecular H-bond donors and two H-bond acceptors, and are stacked with an anti-parallel alignment. Inspection of the backbone conformation around the N-terminal residue showed that while the ζ (Cγ–Cβ–Cα–C’) torsional angle (27.2°) is imposed by the cyclobutane ring, the θ (N– Cγ–Cβ–Cα) torsion angle (163.3°) turns the backbone away from the C-terminal and enables near-alignment of the i–1 and i C=O groups and, in an opposite orientation, the i and i+1 N–H groups. This facilitates the formation of an infinite intermolecular 1D H-bond network resembling a polar sheet. The C-terminal ester C=O is not implicated in the H-bond network and the anti-parallel alignment obviates aromatic π-stacking. In the lattice, the adjacent H-bond network has the opposite orientation and is packed with van der Waals distances between the hydrophobic termini, but with no apparent π-stacking or other non-covalent interaction. Although we were unable to obtain suitable crystals from 3 or 4, the monomeric precursor 1b crystallized upon slow evaporation of a chloroform solution. From the X-ray diffraction study, it emerged that each molecule adopted a tweezer-like shape, with near parallel alignment of the two fragments borne by the cyclobutane ring (Figure 5). Despite their proximity, the ester carbonyl and the urethane H atom do not interact; indeed, they are oriented in opposite directions from each other, allowing instead the formation of an infinite intermolecular H-bonding network along the a-axis implicating only the urethane functions.


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Figure 4. Single crystal x-ray structure of peptide 2, showing the anti-parallel arrangement and the infinite 1D H-bonding network from the front (left) and the side (right), and two adjacent Hbonding networks (lower) highlighting the opposite network orientation. Hydrogen atoms have been removed from the images for clarity.



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Figure 5. Single crystal x-ray structure of 1b, showing the tweezer-shaped molecule with no intramolecular bonding (left) and the intermolecular H-bonding network in the lattice (right).

NMR and FTIR experiments We moved our attention to the solution-state behavior of peptides 2-4. 1H and 13C NMR spectra were recorded in CDCl3 (10 mM). Standard 1D and 2D NMR sequences were used to attribute all resonances, although the 1H spectra showed considerable signal superposition and sharp signals were lacking (particularly for the amide NH protons), a first indication that no single, welldefined conformer was present. The 1H spectra showed no change upon ten-fold dilution. NOESY experiments were conducted on each peptide, but meaningful interpretation of the correlation plots was difficult. Considerable ambiguity arose from signal superposition, and the only correlations which could be made with confidence implicated short-range interactions. There was no conclusive evidence for long-range interactions nor of a repeating correlation portfolio between successive residues, as would have been expected for regular helically-folded conformers.5-10 More detailed analysis was conducted on the NH protons in peptides 2-4. The temperature coefficients for the 1H NMR NH signals of peptides 2-4 were determined (Table 2). In all cases,


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the values were no greater than –5 ppb/K, which suggests that the NH protons are involved to a significant extent in hydrogen bonding.45 Titration of CDCl3 solutions of peptides 2-4 was carried out with DMSO-d6 over the range 0→50% solvent composition; the ∆δ values for 10% added DMSO-d6 were determined (Table 2) to allow comparison with relevant literature.13c,46 For each peptide, the urethane NH signals had low titration coefficients (in the range 0.13-0.19 ppm), supporting the contention of their involvement in hydrogen-bonding. The internal amide signals – NH(2) for peptide 3, NH(2) and NH(3) for peptides 4 – had even lower coefficients (in the range 0.01-0.01 ppm), likewise indicating a strong implication in hydrogen bonding. In contrast, the Cterminal amide NH signals showed significantly higher titration coefficients (in the range 0.390.43 ppm). A 10% DMSO-d6 titration generally gives ∆δ values in the range 0.5-1.0 ppm for solvent exposed (non H-bonded) NH signals,13c,46 so our data suggest that, despite the results of the variable temperature studies described above, the C-terminal residues of peptides 2-4 are at least partially solvent exposed. These interpretations are corroborated by the chemical shift values of the amide signals in CDCl3 solution: NH(3) for peptide 3 and NH(4) in peptide 4 resonate at around 6.1 ppm, while the other H-bonded amide signals appear further downfield, in the range 7.0-7.2 ppm. A further observation was that although chemical shift values in the 1H NMR spectra shifted concomitantly with progressive addition of DMSO-d6 aliquots (up to 50% solvent composition), no abrupt change in the general features was noted, neither was there any improvement in signal definition or separation. Whatever the conformational landscape might be in chloroform, it was not simplified nor significantly modified when a more polar solvent medium was employed. Overall, the NMR data provided some clues but was less helpful than we had hoped, or at least it appeared that no clear-cut conformational preferences for peptides 2-4 could be deduced. The



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lack of well-resolved proton NMR signals, a general propensity for hydrogen bonding which does not evolve significantly with dilution or variation of temperature or solvent polarity, yet without a clear-cut conformational preference, led us to hypothesize that peptides 2-4 may adopt (and interconvert regularly between) several intra-molecularly hydrogen-bonded conformers whose precise structures are not identified at this stage.

Table 2. Variable temperature coefficients and DMSO-d6 titration data for NH signals in 1H NMR spectra of 2-4 (10 mM in CDCl3)[a] signal

NH(1)

NH(2)

2

–2.8

–1.5

3

–4.0

–5.0

–2.9

4

–4.0

–4.9

–5.0

∆δ (ppm) 2 for 0→10% DMSO in 3 solvent

0.19

0.43

0.13

0.10

0.39

4

0.13

0.01

0.08

∆δ/∆T (ppb/K)

NH(3)

NH(4)

–1.9

0.43

[a] NH signals are numbered according to the residue number going from N- to C-terminal.

In the search for more revelatory data we turned our attention to FTIR spectroscopy, which is a powerful technique for the analysis of peptide secondary structure;47 indeed, it proved to be instructive in the study of peptides 2-4 (Table 3). The solid state FTIR spectra for each peptide showed a broad N–H absorption band centered around 3360 cm–1 and a second band near 3300


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cm–1; the intensity of the latter increased with increasing peptide length. These observations indicate fully H-bonded amide and urethane N–H functions, respectively. In the carbonyl absorption region, the ester C=O absorption appeared in the range 1720-1730 cm–1 denotive of a non-bonded ester, whereas the urethane and amide C=O absorption bands were close to 1683 and 1640 cm–1 respectively, attesting to firmly H-bonded functions. Collectively, these data are entirely consistent with a sheet-like organization of extended conformers engaged in exclusive intermolecular H-bonding for all three peptides, as observed in the crystal structure of 2 above.

Table 3. Significant absorption bands in FTIR spectra of 24.[a] Sample

Urethane or Amide N–H

Ester C=O

Urethane C=O

Amide C=O

2 solid

3355 3321

1723

1683

1640

3 solid

3364w 3314

1722

1684

1639

4 solid

3361w 3317

1725

1685

1642 1627w

3 gel[b]

3361w 3322

1721

1684

1640

3 xerogel[b]

3363w 3322

1721

1684

1640

2 solution[c]

3441 (3370br)

1706

1659

3 solution[c]

3445 (3370br)

1706

1648

4 solution[c]

3443 3364br

1708

1648

[a] Data were recorded at 20 °C; values are given for νmax in wavenumbers (cm–1); w = weak, br = broad; parentheses indicate absorptions which appear as shoulders on nearby bands. [b] From toluene (40 mM). [c] In CHCl3 (2.5 mM).



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FTIR spectra recorded for peptides 2-4 in dilute solution (2.5 mM, CHCl3) were recorded to better understand the solution-state behavior. All three peptides showed a N–H absorption band near 3443 cm–1, indicative of a contribution from a free urethane or amide function. This band for dipeptide 2 was widened by a shoulder around 3370 cm–1 suggesting some H-bonded contribution. The shoulder was more prominent for tripeptide 3 and became a clearly defined broad absorption band centered at 3364 cm–1 in tetrapeptide 4. In the carbonyl absorption region, with respect to the solid state data, the ester C=O absorption was red-shifted while the urethane C=O absorption was blue-shifted so that the absorptions of these functions appeared together as a single band close to 1707 cm–1 for all three peptides. This indicated that, in marked contrast to the solid state behavior, the ester is clearly involved in H-bonded interactions in solution whereas the urethane is not. The amide C=O band appeared in the range 1648-1659 cm–1 for all three peptides, suggesting some implication in H-bonding but perhaps less so than in the solid state. The intramolecular nature of the H-bonding phenomena in solution was indicated by variableconcentration experiments: no evolution in the free/bonded N–H absorption band profile was observed following a series of dilutions of the sample solutions (see supporting information). Collectively, the solution-state FTIR data point to conformer sets which feature significant intramolecular H-bonding, implicating the ester but not the urethane carbonyl function, while some conformer contributions may implicate a free urethane or amide N–H. This corroborates the significant C-terminal DMSO-d6 titration coefficients observed in the 1H NMR experiments described above, which suggested that the C-terminal amide NH is solvent exposed in each peptide. Finally, the gel formed by peptide 3 in toluene (40 mM) and the corresponding xerogel were examined by FTIR. The data were remarkably similar to those for solid samples, strongly


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suggesting that the same intermolecular interactions and molecular organization are implicated in the gel and in the solid state.

Molecular modelling To gain further insight into the solution state behavior and rationalize the spectroscopic data, a hybrid Monte Carlo Molecular Mechanics (MCMM) conformation search was conducted on peptides 2-4 in a chloroform medium; the low-energy conformations were sorted into family sets and examined. Dipeptide 2 displayed four low-energy conformer families, illustrated in Figure 6. The predominant family (82% statistical abundance) had two successive intra-residue 7-membered (C7) H-bonded rings. Such an arrangement is facilitated by the cis geometry of the vicinal substituents on the puckered cyclobutane ring. This conformer family features a distinctive extended molecular architecture. The second conformer family of 2 showed a bifurcated Hbonding system involving both N–H functions and the ester C=O. With combined intramolecular 12- and 7-membered (C12/7) H-bonded rings, this leads to a more folded architecture, in which both amide and urethane C=O functions are free. The third family showed a single C12 interaction between the urethane N–H and the ester C=O, leaving the central amide function free. The fourth family resembled the first, with the important difference that the C7 feature of the second residue was no longer present. Instead, a tweezer-shaped alignment of the two substituents on the cyclobutane was in evidence, with both the N–H and ester C=O of this residue oriented in a very similar manner to that observed for the monomer 1b in the solid state. This partial fraying of the C-terminal, with respect to the first family, may result from the fact that Hbonding for esters is slightly weaker than for amides. These four low-energy families are likely to coexist and interconvert rapidly at ambient temperature, and collectively they explain the solution



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Page 20 of 37

state FTIR and NMR data entirely. Notably, the ester C=O is partly implicated in H-bonding in preference to the urethane C=O, while conformers with a free amide N–H give rise to the absorption at 3441 cm–1 alongside the bonded urethane and amide N–H absorption at 3370 cm–1.

(a) (b)

(c)

(d)

Figure 6. Representative members of the four low-energy conformer families for dipeptide 2 obtained from the molecular modelling study. The key difference between the families is their Hbonding pattern. Several conformers may exist within any one family, only one representative example is illustrated. (a) C7,C7; (b) C12/7; (c) C12; (d) C7. Note the resemblance of the tweezer-shaped fourth conformer with the crystal structure of 1b (in Figure 5).

Tripeptide 3 showed four low-energy conformer families. The most prevalent family (68% statistical abundance) presented a succession of three successive C7 rings. These 7-ribbons presented a range of topologies with variable degrees of curvature, reminiscent of the 9/8-ribbon conformer family sets discovered for a β/γ-peptide recently;15 however they generally present extended conformers and one such structure is represented in Figure 7a. The second family (26% statistical abundance) had a bifurcated C12/7 H-bonding interaction followed by a C7 ring at the


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C-terminal residue, giving rise to more compact folded conformer shapes. The less abundant third and fourth families were similar to the first two, with the exception that the C-terminal C7 feature had once again given way to the pincer-type structure, freeing the N–H. The solution-state FTIR and NMR data for 3 are fully consistent with this collected set of conformer families. Following the trend, tetrapeptide 4 had two main conformer families which together were essentially consistent with the FTIR and NMR data. The first conformer had a series of four consecutive C7 interactions (Figure 7b), while the second had three C7 interactions and a pincershaped C-terminal residue with a free N–H (55% and 26% statistical abundances, respectively). Minor contributions were made by 8 other folded conformer families implicating longer-range intramolecular H-bonding interactions.

(a)

(b) Figure 7. Representative members of the most abundant conformer families for peptides 3 and 4 obtained from the molecular modelling study: (a) a C7,C7,C7 conformer of 3; (b) a C7,C7,C7,C7 conformer of 4.



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Two important observations emerge from these calculations. The first is that, as the solution state spectroscopic data suggested, single molecules (typically in dilute solution) are capable of adopting low energy conformations featuring a series of C7 H-bonded rings, a feature which has only been reported once before for γ-peptides.18 The second is that these structures are not the only low-energy options available on the conformational landscape. This corroborates the solid state studies (both x-ray diffraction and FTIR) which suggested that molecular assembly privileges the formation of intermolecular H-bonding interactions, and that this phenomenon is also prevalent in organogels (from FTIR). To complement our understanding of the peptide assemblies we performed a molecular dynamics (MD) simulation of a six-molecule ensemble of peptide 3 in an explicit toluene medium. In analogy with the crystal structure arrangement of dipeptide 2 above, an anti-parallel H-bonded conformation set was used as the starting point to represent a small organized molecular assembly as part of a gelling system. The MD simulation was conducted with all restraints removed and was run for 1.2 ns with configurations saved at each 1.2 ps interval. Full details and a video are presented in the Supporting Information files. The intermolecular H-bonding network of the system persisted sufficiently during the simulation to sustain the anti-parallel arrangement in isothermal conditions at 300 K. However, the simulation revealed an irregular dynamic pattern of only partially H-bonded successive peptide molecules with sizeable contributions implicating residues which showed no H-bonding for variable lengths of time. Despite this, no significant appearance of intramolecular C7 features was in evidence; even the terminal peptide of the six-peptide ensemble, which was unable to behave as an intermolecular H-bond donor, showed only intermittent short-lived C7 conformers.


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Evidently, the adoption of C7 rings in the simulated toluene medium is not compelling, leaving the molecules free to self-assemble in a fashion which is compatible with a gelation phenomenon.

Conclusion Previous studies had shown that homo or hybrid peptides incorporating γ-amino acids can form a number of helically-folded secondary structures, while regular intermolecular assembly to form sheets has been described on a number of occasions. In contrast, the adoption of ribbon-like conformer topologies or the formation of organogels by γ-peptides is a much rarer phenomenon. In this work we have made a detailed study of a γ-peptide template which fulfils both these latter properties. Built from a previously unstudied cyclobutane γ-amino acid, the short oligomers 2-4 have sufficient flexibility to sustain almost unprecedented C7 intramolecular H-bond networks in isolated molecules, but display facile supramolecular assembly leading to sheet-like arrays which enable gel formation in some solvents. This duplicitous and reversible behavior, vacillating between intra- and inter-molecular organization, is of importance in the developing areas of bioinspired responsive foldamer and smart materials design, and provides insight for novel features for inclusion into de novo molecular architectures.

Experimental section General remarks Compound 1a was prepared according to the literature.39 DMF was distilled from CaH2, dichloromethane was dried over alumina. All other reagents and solvents were of commercial grade and were used without further purification. Preparative chromatography was performed on columns of silica gel (15˗40 µm mesh) using an automated Combiﬂash system (Teledyne ISCO). Analytical thin-layer chromatography (TLC) was performed on 0.25 mm silica gel plates and



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were visualized by UV at 254 nm then stained using a basic aqueous KMnO4 solution or a ninhydrin solution. Retention factors (Rf) are given for such analyses. Analytical HPLC was carried out on a Agilent 1260 Infinity HPLC apparatus equipped with a Phenomenex® lux cellulose-2 column (250 mm × 4,6 mm; particle size 5 µm) using hexane/EtOH (60/40) as the mobile phase flowing at a rate of 1.0 mL/min. The column and the mobile phase were thermostated at 35 ± 0.8 °C; detection was performed by UV absorbance at 210 nm. Melting points were obtained with a Büchi B-545 apparatus in open capillary tubes and are uncorrected. Optical rotations were measured on a Jasco P-1010 polarimeter using a 10 cm quartz cell; [α]TD values were obtained for the D-line of sodium at the indicated temperature T, using solutions of concentration (c) in units of g·100 mL−1. Fourier-transform infrared (IR) spectral data were obtained on a Perkin-Elmer Spectrum One spectrophotometer for solutions in CHCl3 at the specified concentrations using a 1 mm path-length in a NaCl solution cell, or on a Perkin-Elmer Spectrum Two spectrophotometer in ATR mode for solid or gel samples; maximum absorbances (ν) are given for significant bands in cm−1. 1H and

13

C nuclear magnetic resonance (NMR)

spectra were recorded for solutions in CDCl3 (10 mM) on Bruker DPX250 (250 and 62.5 MHz, respectively), Bruker AV360 (360 and 90.6 MHz, respectively) or Bruker AV400 (400 and 100.6 MHz, respectively) spectrometers. Chemical shifts (δ) are reported in ppm from tetramethylsilane. For

13

C data collection, either broadband decoupled (1b and 3) or JMOD (2

and 4) pulse sequences were used. Splitting patterns for 1H NMR signals are designated as: s (singlet), d (doublet), t (triplet), bs (broad singlet) or m (multiplet); coupling constants (J) are reported in Hz. Attributions of 1H signals for peptides 2-4 were made with the help of standard 2D correlation sequences (COSY, HMBC, HSQC and TOCSY). NOESY experiments (400 MHz) were conducted at 300 K in TPPI acquisition mode with a mixing time of 600 ms. High-


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resolution mass spectrometry (HRMS) data were recorded using on a Bruker Daltonics micrOTOF-Q instrument equipped with a positive mode electrospray ionization source and a tandem Q-TOF analyzer; theoretical values for the exact ion masses were calculated automatically by the instrument software.

Benzyl (1R,2S)-2-({[(tert-butoxy)carbonyl]amino}methyl)cyclobutane-1-carboxylate (1b) To a solution of 1a (0.20 g, 0.87 mmol) in dry CH2Cl2 (4 mL) at 0 °C, were added DMAP (0.016 g, 0.09 mmol), DCC (0.22 g, 1.05 mmol) and benzyl alcohol (0.27 mL, 2.62 mmol). The mixture was stirred at 0 °C for 1 h then at rt for 18 h, then filtered. The filtrate was concentrated under reduced pressure then EtOAc (20 mL) was added and the resulting organic solution was washed successively with aqueous 1 M HCl, 5% NaHCO3, and brine. The organic solution was then dried over MgSO4, filtered and concentrated. The crude product was purified by flash chromatography using EtOAc/petroleum ether as eluent (gradient from 0/100 to 100/0) to afford the benzyl ester 1b (0.25 g, 98%) as a white solid. Mp 61-63 °C; Rf 0.69 (EtOAc/petroleum ether = 3/7); [] –7 (c 0.99, CHCl3); IR (solid) ν 1175, 1249, 1517, 1680, 1725, 2975, 3349, 3388; 1H NMR ( 360 MHz, CDCl3) δ 7.30-7.42 (m, 5H, HAr), 5.09-5.19 (m, 2H, H11), 4.72 (bs, 0.86H, H4), 4.50 (bs, 0.13H, H4), 3.20-3.34 (m, 2H, H6+H5), 3.05-3.17 (m, 1H, H5’), 2.74-2.90 (m, 1H, H9), 2.27-2.40 (m, 1H, H8), 1.99-2.15 (m, 2H, H7+H8’), 1.69-1.83 (m, 1H, H7’), 1.42 (s, 9H, H1); 13C NMR (90 MHz, CDCl3) δ 173.9 (C10), 155.8 (C3), 135.7 (C12), 128.6 (2C14), 128.4 (2C13), 128.4 (C15), 79.0 (C2), 66.4 (C11), 42.0 (C5), 40.0 (C6), 37.8 (C9), 28.3 (3C1), 22.5 (C8), 20.9 (C7); HRMS Calcd for C18H25NNaO4 [M+Na]+: 342.1676, found 342.1667.



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Page 26 of 37

Benzyl (1R,2S)-2-({[(1R,2S)-2-({[(tertbutoxy)carbonyl]amino}methyl)cyclobutyl]formamido}methyl)cyclobutane-1-carboxylate (2) TFA (3.0 mL, 40 mmol) was added dropwise to a solution of 1b (0.42 g, 1.32 mmol) in CH2Cl2 (22 mL) at 0 °C and the solution was stirred for 30 min, by which time the reaction was complete (TLC analysis). The solvent was evaporated under reduced pressure and the residual trifluoroacetate salt was dissolved in a CH2Cl2/DMF (10/1) mixture (35 mL) and neutralized with DIPEA (0.69 mL, 3.96 mmol), to provide Mixture A. Separately, HATU (0.65 g,1.70 mmol) and DIPEA (0.46 mL, 2.64 mmol) were added to a solution of 1a (0.30 g, 1.32 mmol) in a CH2Cl2/DMF (20/1) mixture (35 mL). The solution was stirred at rt for 10 min, to provide Mixture B. Mixture A was added to Mixture B and the combined mixture was stirred at rt for 72 h, then concentrated under reduced pressure. The residual solid was washed with water then dissolved in Et2O. This solution was dried over MgSO4, filtered and then concentrated under reduced pressure. The crude product was purified by flash chromatography using EtOAc/cyclohexane as eluent (gradient from 5/95 to 100/0) to afford 2 (0.44 g, 77%) as a white solid. Mp 118-120 °C; Rf 0.35 (EtOAc/petroleum ether = 1/1); HPLC retention time 4.7 min; [] –30 (c 1.00, CHCl3); IR (solid) ν 1366, 1456, 1528, 1640, 1683, 1723, 2928, 2965, 3321, 3355; 1H NMR (250 MHz, CDCl3) δ 7.12-7.10 (5H, m, HAr), 5.94 (1H, t, J = 5.4 Hz, H11), 5.32 (1H, t, J = 5.7 Hz, H4), 5.20 (1H, d, J = 12.3 Hz, AB system, H18/H18’), 5.10 (1H, d, J = 12.3 Hz, AB system, H18/H18’), 3.52 (1H, ddd, J = 14.2 Hz, J = 7.5 Hz, J = 5.4 Hz, H12), 3.34-3.28 (1H, m, H16), 3.24-3.14 (2H, m, H5+H5’), 3.12-3.03 (1H, m, H12’), 2.91-2.78 (2H, m, H9+H13), 2.71-2.63 (1H, m, H6), 2.39-2.23 (1H, m, H15), 2.19-2.03 (3H, m, H8+H14+H15’), 2.02-1.9 (2H, m, H7+H8’), 1.83-1.71 (1H, m, H14’), 1.67-1.59 (1H, m, H7’), 1.42 (9H, s, H1); 13C NMR (62.5 MHz, CDCl3) δ 174.1 (C17), 173.1


Page 27 of 37

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(C10), 156.1 (C3), 135.6 (C19), 128.7 (2C21), 128.5 (2C20+C22), 78.8 (C2), 66.5 (C18), 41.8 (C5), 41.6 (C9), 40.9 (C12), 40.3 (C16), 38.1 (C6), 37.7 (C13), 28.4 (3C1), 22.7 (C14), 22.0 (C7), 21.4 (C8), 21.1 (C15); HRMS Calcd for C24H34N2NaO5 [M+Na]+: 453.2360, found 453.2365.

Benzyl (1R,2S)-2-({[(1R,2S)-2-({[(1R,2S)-2-({[(tert-butoxy)carbonyl]amino}methyl) cyclobutyl]formamido}methyl)cyclobutyl]formamido}methyl)cyclobutane-1-carboxylate (3) TFA (0.54 mL, 7.0 mmol) was added dropwise to a solution of 2 (0.10 g, 0.23 mmol) in CH2Cl2 (10 mL) at 0 °C and the solution was stirred for 2 h, by which time the reaction was complete (TLC analysis). The solvent was evaporated under reduced pressure and the residual trifluoroacetate salt was dissolved in a CH2Cl2/DMF (20/1) mixture (4 mL) and neutralized with DIPEA (82 µL, 0.47 mmol), to provide Mixture A. Separately, HATU (0.11 g, 0.30 mmol) and DIPEA (82 µL, 0.47 mmol) were added to a solution of 1a (0.05 g, 0.23 mmol) in a CH2Cl2/DMF (20/1) mixture (5 mL). The solution was stirred at rt for 10 min, to provide Mixture B. Mixture A was added to Mixture B and the combined mixture was stirred at rt for 72 h, then concentrated under reduced pressure. The residual solid was washed with water, then Et2O, and was then purified by flash chromatography using EtOAc/cyclohexane as eluent (gradient from 5/95 to 100/0) to afford 3 (0.10 g, 82%) as a white solid. Mp 182-184 °C; Rf 0.64 (MeOH/CH2Cl2 = 1/9); HPLC retention time 5.9 min; [] –40 (c 0.98, CHCl3); IR (solid) ν 1367, 1453, 1525, 1639, 1684, 1722, 2854, 2925, 2960, 3314, 3364; 1H NMR (250 MHz, CDCl3) δ 7.39-7.36 (5H, m, HAr), 6.99 (1H, bs, H11), 6.14 (1H, bs, H18), 5.44 (1H, bs, H4), 5.21 (1H, d, J = 12.2 Hz, AB system, H25/H25’), 5.11 (1H, d, J = 12.3 Hz, AB system, H25/H25’), 3.60-3.47 (2H, m, H12+H19), 3.34-3.18 (4H, m, 2H5+H12’+H23), 3.15-3.07 (2H, m, H9+H19’), 2.97-2.92 (1H, m, H16), 2.87-2.79 (1H, m, H20), 2.77-2.67 (2H, m, H6+H13), 2.38-2.30 (1H, m, H22), 2.27-2.21 (1H, m, H8), 2.20-



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1.96 (6H, m, H7+H8’+H14+2H15+H22’), 1.82-1.58 (4H, m, H7+H14+2H21), 1.43 (9H, s, H1);

Page 28 of 37

13

C

NMR (62.5 MHz, CDCl3) δ 174.2 (C24), 173.7 (C17), 173.1 (C10), 156.2 (C3), 135.6 (C26), 128.7, 128.6, 128.5 (2C27+2C28+C29), 78.7 (C2), 66.6 (C25), 42.0 (C9), 41.9 (C5), 41.8 (C16), 40.9 (C19), 40.6 (C12), 40.3 (C23), 38.2 (C6), 37.9 (C13), 37.7 (C20), 28.44 (3C1), 22.7 (C21), 22.1 (C7), 22.0 (C15), 21.9 (C14), 21.5 (C8), 21.3 (C22); HRMS Calcd for C30H43N3NaO6 [M+Na]+: 564.3044, found 564.3033.

Benzyl (1R,2S)-2-({[(1R,2S)-2-({[(1R,2S)-2-({[(1R,2S)-2-({[(tert-butoxy)carbonyl] amino}methyl)cyclobutyl]formamido}methyl)cyclobutyl]formamido}methyl)cyclobutyl]for mamido}methyl)cyclobutane-1-carboxylate (4) TFA (0.54 mL, 7.0 mmol) was added dropwise to a solution of 2 (0.16 g, 0.37 mmol) in CH2Cl2 (13 mL) at 0 °C and the solution was stirred for 2 h, by which time the reaction was complete (TLC analysis). The solvent was evaporated under reduced pressure and the residual trifluoroacetate salt was dissolved in a CH2Cl2/DMF (20/1) mixture (4 mL) and neutralized with DIPEA (0.32 mL, 1.84 mmol) to provide Mixture A. Separately, 10% Pd-C (41 mg) was added to a solution of 2 (0.16 g, 0.37 mmol) in CH2Cl2 (18 mL). The mixture was stirred vigorously under an atmosphere of hydrogen for 4 h, then filtered through a plug of celite and the filtrate evaporated. The residue was dissolved in a CH2Cl2/DMF (20/1) mixture (5 mL) and HATU (0.17 g, 0.46 mmol) and DIPEA (0.16 mL, 0.70 mmol) were added. The solution was stirred at rt for 10 min, to provide Mixture B. Mixture A was added to Mixture B and the combined mixture was stirred at rt for 72 h, then concentrated under reduced pressure. The residue was washed with water (6 mL), Et2O (6 mL), then purified by flash chromatography using MeOH/EtOAc as eluent (2/98) to afford 4 (0.16 g, 68%) as a white solid. Mp 213-215 °C; Rf 0.60 (MeOH/CH2Cl2 = 1/9); HPLC retention time 8.5


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min; [] –42 (c 0.14, CHCl3); IR (solid) ν 1365, 1435, 1527, 1627, 1642, 1685, 1725, 2869, 2930, 3317, 3361; 1H NMR (400 MHz, CDCl3): δ 7.38 (5H, s, HAr), 7.24 (1H, bs, H11), 7.15 (1H bs, H18), 6.18 (1H, bs, H25), 5.50 (1H, bs H4), 5,11 (1H, d, J = 12.2 Hz, AB system, H32/32’), 5.19 (1H, d, J = 12.2 Hz, AB system H32/32’), 3.58-3.50 (3H, m, H12+H19+H26), 3.32-3.09 (8H, m, H5+H5’+H9+H12’+H19’+H23+H26’+H30), H6+H13+H20+H27),

2.37-2.19

3.00-2.94

(3H,

m,

(1H,

m,

H16),

H8+H22+H28),

2.86-2.66

2.17-1.98

(4H, (9H,

m, m,

H7+H8’+H14+H14’+H15+H21+H22’+H28’+H29), 1.77-1.58 (4H, m, H7’+H15’+H21+H29’), 1.42 (9H, s, H1); 13C NMR (100 MHz, CDCl3): δ 176.0, 173.8, 173.7, 170.7 (C10+C17+C24+C31), 156.3 (C3), 135.7 (C33), 128.7, 128.5, 128.4 (2C34+2C35+C36), 79.8 (C2), 66.6 (C32), 42.6 (C5), 42.3, 42.1, 42.0 (C9+C16+C23), 41.0, 40.9, 40.8 (C12+C19+C26), 40.4 (C30), 38.3 (C13), 38.1 (C20+C6), 37.8 (C27), 28.5 (3C1), 22,7, 22.2, 22.1, 22.0, 21.9 (C7+C14+C15+C21+C29), 21.5 (C8), 21.4 (C22+C28); HRMS Calcd for C36H52N4NaO7 [M+Na]+: 675.3728, found 675.3743.

Gel formation Reagent grade solvents and PEG-400 (MW range 380-420 g/mol) were obtained commercially. Red wine (Bordeaux), maple syrup (Canada) and olive oil (South Lebanon) were obtained in supermarkets. An accurately-weighed amount of the peptide (∼1 mg) and the specified solvent (100 µL) were placed in a glass vial (7 mm i.d.) which was sealed, sonicated (2 min) then heated near the boiling point in a water bath for 5 min (with further sonication if required). A clear solution was obtained, otherwise the peptide was deemed insoluble. The solution was allowed to cool to 20 °C and was inspected after 30 min and again after 4 h (gels formed more or less slowly). A gel was considered to have formed when a homogeneous sample was obtained that exhibited no gravitational flow upon inversion of the vial. Limited gravitational flow was



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Page 30 of 37

considered the result of partial gelation. The gel was considered stable if it had not evolved after 24 h standing at 20 °C. The thermoreversibility of gel formation was demonstrated by reheating the gel in its original sealed vial until a clear solution was obtained, then allowing the solution to cool to 20 °C as before, which resulting in gel formation once again. This process was repeated twice. Determination of mgc values was done by progressive dilution of the sample and repetition of the gel formation protocol, until such dilution was achieved where no gel formed from the solution at 20 °C.

Gel melting temperature ‘‘Dropping ball’’ experiments were performed.48 A gel of a specified concentration (volume 200 µL) was prepared as above and a stainless steel ball (2.62 mg; 2 mm ø) was placed on top of the gel, in the center, avoiding wall contact. The sealed vial was heated slowly (1 °C / min). The gel melting temperature was that at which the ball dropped to the bottom of the vial. Measurements were made at varied gel concentrations to provide the temperature–concentration curve.

Scanning electron microscopy A small sample of a gel was deposited on a conductive aluminium sample holder. The sample was slowly air-dried to produce the xerogel then the sample holder was introduced inside the microscope chamber which was then put under a normal secondary high vacuum (10−5/10−6 mbar range). The SEM instrument was equipped with a Field Emission Gun. The electron beam high voltage was set to 1 kV and the current to a few pA in order to observe the non-conductive products without any conductive deposit on top.

Associated content


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The Supporting Information is available free of charge on the ACS Publications website. Copies of 1H and 13C NMR spectra for compounds 1b-4; copies of FTIR spectra and hplc chromatograms for compounds 2-4; crystallographic data for compounds 1b and 2; details of molecular modelling protocols and images of gels (PDF) Crystallographic data for compound 1b (CCDC 1500039) (CIF) Crystallographic data for compound 2 (CCDC 1500040) (CIF) Molecular Dynamics video (AVI)

Author information Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest.

Acknowledgments Postgraduate funding was provided by the French MESR via a doctoral research scholarship (to C.M.G.) and by the Azm & Saade Association as well as by EGIDE via an Eiffel grant (to H. A.). We thank Ms Marjolaine Gras (Master student) for some preliminary studies.

References 1

(a) Branden, C.; Tooze, J. Introduction to Protein Structure; Garland: New York, 1991. (b)

Petsko, G. A.; Ringe, D. Protein Structure and Function; New Science Press: London, 2004.



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2

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(a) Rad-Malekshahi, M.; Lempsink, L.; Amidi, M.; Hennink, W. E.; Mastrobattista, E.

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