Molecular Recognition within the Cavity of a Foldamer Helix Bundle

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Molecular Recognition within the Cavity of a Foldamer Helix Bundle: Encapsulation of Primary Alcohols in Aqueous Conditions Gavin W. Collie,¶,† Remy Bailly,‡ Karolina Pulka-Ziach,∥,† Caterina M. Lombardo,† Laura Mauran,†,§ Nada Taib-Maamar,‡ Jean Dessolin,‡ Cameron D. Mackereth,# and Gilles Guichard*,† †

Univ. Bordeaux, CNRS, CBMN, UMR 5248, Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac, France Univ. Bordeaux, CNRS, CBMN, UMR 5248, All. Geoffroy Saint-Hilaire, 33600 Pessac, France § UREkA, Sarl, 2 rue Robert Escarpit, 33607 Pessac, France # Univ. Bordeaux, Inserm, CNRS, ARNA Laboratory, U1212, UMR 5320, Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, 33076 Pessac, France ‡

S Supporting Information *

ABSTRACT: Artificial synthetic molecules able to adopt welldefined stable secondary structures comparable to those found in nature (“foldamers”) have considerable potential for use in a range of applications such as biomaterials, biorecognition, nanomachines and as therapeutic agents. The development of foldamers with the ability to bind and encapsulate “guest” molecules is of particular interest; as such an ability is a key step toward the development of artificial sensors, receptors and drug-delivery vectors. Although significant progress has been reported within this context, foldamer capsules reported thus far are largely restricted to organic solvent systems, and it is likely that the move to aqueous conditions will prove challenging. Toward this end, we report here structural studies into the ability of a recently reported water-soluble self-assembled foldamer helix bundle to encapsulate simple guest molecules within an internal cavity. Seven high-resolution aqueous crystal structures are reported, accompanied by molecular dynamics and high-field NMR solution data, showing for the first time that encapsulation of guests by a complex self-assembled foldamer in aqueous conditions is possible. The findings also provide ample insight for the future functional development of this system.

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INTRODUCTION

Until recently, examples of atomic-level structural elucidation of water-soluble foldamer quaternary assemblies were largely limited to β-amino acid containing backbones.8−13 However, we recently reported the strong propensity of short aliphatic oligourea foldamers to self-assemble in aqueous conditions into unique, precise quaternary structures, encompassing discrete helix bundles and extended tubular structures with water-filled pores.14−16 Of particular note was the surprise discovery of an isolated hydrophobic cavity with a volume of around 500 Å3 within the helix bundle arrangements reported (Figure 1). Although this volume is much smaller than that of nanocages built from the self-assembly of de novo designed proteins,17−20 it compares favorably with cavities engineered into peptide coiled-coils for small guest recognition.21−24 Cavities formed from water-soluble constructs represent a particularly enticing structural motif, as one could envisage the use of such a feature for, eventually, (drug) delivery, biosensing or catalysis. Over the past decade, supramolecular chemists have made remarkable progress toward the preparation of sophisticated organic and metal−organic containers, including covalent and self-

Foldamers are artificial folded oligomers designed to mimic specific structural aspects of natural biomolecules (such as proteins and nucleic acids).1−3 A key principle of foldamer research is not to exactly copy natural biomolecules but rather to use biomolecules as inspiration for the design and development of molecules with functions and capabilities beyond those found in nature, such as catalysts or artificial bioreceptors with tailored ligand specificity. As function is intimately linked with structure, the creation of new and unique foldamer architectures is a necessary step toward the goal of developing foldamers with tailored/preternatural functions. However, the construction of novel foldamer structures can be challenging, particularly the creation of multicomponent architectures, which require controlled, precise self-assembly. A seemingly even greater challenge than this is presented by the development of multimeric foldamer systems with the ability to self-assemble in aqueous conditions into precise, well-defined arrangements. Indeed, there are few reports of such findings, yet the development of aqueous self-assembling foldamer systems is an important step toward the creation of foldamers with true biofunctions, such as biocatalysis.4−7 © 2017 American Chemical Society

Received: January 6, 2017 Published: February 24, 2017 6128

DOI: 10.1021/jacs.7b00181 J. Am. Chem. Soc. 2017, 139, 6128−6137

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Journal of the American Chemical Society

Figure 1. (a,b) Details of oligourea H1. (c−e) Details of the isolated cavity present in the crystal structure of the hexameric helix bundle formed from the self-assembly of oligourea H1 (H1-bundle). Black arrows in panel e indicate the carbonyl groups of Leuu6 residues which are available for hydrogen bonding within the otherwise hydrophobic cavity. (f) 1.18 Å crystal structure of H1 with three isopropyl alcohol guest molecules bound within the hydrophobic cavity of the helix bundle (structure 1). Each isopropyl alcohol molecule is hydrogen bonded to a carbonyl group of a Leuu6 residue. All hydrogen bonds shown here are 2.9 Å.

generally provide high-resolution structural insight into the entrapment of guest molecules in aqueous conditions by an artificial cavity formed by a self-assembled foldamer.

assembled capsules, with a wide range of physicochemical properties (such as water solubility) and functions (such as dynamic systems able to adapt to the nature of the guest).25−29 Systems exploiting secondary structural folding have also been developed as capsules, and in particular, helical foldamers able to form internal cavities upon folding have emerged as a new and promising class of synthetic receptor.30−40 Such capsule systems permit the optimization of guest recognition to be achieved through rational sequence variation, allowing the volume and chemical nature of the cavity to be modified by the incorporation of appropriate residual building blocks. A recent example of such a fine-tuning process describes the iterative design of helical foldamers with high selectivity for specific monosaccharides.41 The vast majority of existing foldamer capsules, however, are restricted to monomeric helical foldamers with internal cavities, with few reports of foldamer capsules formed by the self-assembly of multimolecular components.30 Furthermore, foldamer capsule systems have not generally been employed and studied in aqueous environments.36 Thus, there is a need for further development and diversification of foldameric capsule systems. Toward the goal of investigating the principles of guestencapsulation in aqueous conditions by a self-assembled foldameric capsule, we report here structural studies into a series of host−guest complexes involving a self-assembled oligourea helix bundle bound by an array of primary alcoholbased guests within its internal cavity. We describe seven highresolution aqueous crystal structures of oligourea helix bundles bound by different guest molecules, which are supported by indepth molecular dynamics simulations and complementary solution data (including high-field NMR studies). These findings provide the basis for the future functional development of this recently reported foldamer helix bundle, and more

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RESULTS AND DISCUSSION Investigating a Self-Assembled Foldamer Helix Bundle as a Tool for Guest Encapsulation. H1 is an aliphatic oligourea foldamer composed of 11 residues, all bearing proteinogenic side-chains. The folding of this molecule into a canonical 2.5-helix in aqueous conditions distributes the charged and uncharged side-chains such that the resulting helix has a strong global amphiphilic character, with distinct hydrophilic (charged) and hydrophobic regions (Figure 1). The amphiphilic nature of H1 imbues this foldamer with a strong tendency to self-assemble. Indeed, it was recently shown that H1 readily self-assembles in aqueous conditions into a discrete, hexameric helix bundle, with a leucine-rich core and a hydrated charged exterior (referred to from hereon as “H1bundle”) (Figure 1).16 An X-ray crystal structure revealed the presence of an isolated internal cavity within the hydrophobic core of this foldamer helix bundle, with a volume of approximately 500 Å3. The hexameric helix stoichiometry and innate symmetry of the bundle results in the cavity possessing a unique 3-fold symmetry, composed of a roughly spherical central chamber from which three “tunnels” project at 120° relative to one another (Figure 1c−e). As the cavity is located within the core of the helix bundle, it is predominantly hydrophobic in nature. However, there exists two hydrogen bond accepting groups (specifically, the urea carbonyl groups of the Leuu6 residues [superscript “u” denotes urea-based residue]) at the extremity (relative to the central chamber) of each “tunnel”. In an attempt to explore the potential of the H16129


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Journal of the American Chemical Society bundle cavity to bind guest molecules, we sought to obtain structural data as a starting point for further studies. As the previously reported crystal structure of H1 was determined from crystals grown in the presence of isopropyl alcohol, this small secondary alcohol seemed a logical starting point for investigations into the capability of the H1-bundle cavity to encapsulate guests. Aqueous cocrystallization trials of H1 in the presence of varying concentrations of isopropyl alcohol and at different equilibration rates eventually led to the determination of a high-resolution crystal structure revealing the presence of well-resolved isopropyl alcohol molecules localized to the internal cavity of the H1-bundle (Figure 1f). Three isopropyl alcohol molecules are bound within a single internal cavity, with each molecule localized to a separate tunnel, hydrogen bonded to the urea carbonyl groups of Leuu6 residues situated at the extremity of each tunnel. The isopropyl carbons are thus orientated toward the hydrophobic central chamber. However, the majority of the cavity volume is predominantly unoccupied (cavity analysis using SURFNET42 indicates the cavity to be 47.7% occupied). Because of their small size, the isopropyl alcohol guests are unconstrained and consequently are somewhat mobile in their binding sites. Thus, from this crystal structure it was evident that (1) aliphatic alcohol groups are suitable moieties for binding to the hydrogen bonding groups present within the cavity, and (2) there was considerable scope for optimization of the alkyl chain to satisfy the unoccupied volume of the cavity. Consequently, we used circular dichroism methods to screen a range of molecules as potential guest for binding to the H1-bundle in aqueous conditions. Screening of Alcohol-Based Molecules for Binding to a Foldamer Helix Bundle Cavity by Circular Dichroism. A range of aliphatic compounds differing in chain length and chemical composition yet sharing the common feature of possessing at least one primary alcohol group were chosen as potential guests for binding to the H1-bundle cavity (listed in Table 1).

Figure 2. Circular dichroism (CD) studies into the effect of a series of alcohol-based guests on the helicity of H1. Alcohols studied by CD include 1-butanol, 1-pentanol, 1-hexanol (a), and 2-ethoxyethanol, 2propoxyethanol, 1,4-butanediol and isopropyl alcohol (b). Full spectrum and thermal melting data were recorded for H1 at 200 μM in pure water plus 1% guest. Full spectra show that the helicity of H1 is not significantly destabilized by any of the guests studied here. Melting data indicate that 1-pentanol and 1-hexanol elicit a noticeable stabilizing effect on the helicity of H1 in these conditions. Melting profiles were fit to a simple 2-state unfolding model, the results of which are shown in Table 1. [θ]: molar elipticity, with units of deg· cm2·dmol−1. [θ]202: molar elipticity at 202 nm. [θ] and [θ]202 values have been divided by 10 000 for the purposes of visual clarity.

negatively affected by the presence of the alcohols, implying that the quaternary structure of H1 is unperturbed by these potential guests (Figure 2). Significantly, the melting profiles of H1 in the presence of 1pentanol and 1-hexanol showed a clear increase in thermal stability, with pronounced melting transitions strongly indicative of quaternary structure unfolding. Thermal melting values determined from these melting profiles demonstrate that 1-pentanol and 1-hexanol (at a concentration of 1% v/v) increase the Tm of H1 by 13.4 and 11.5 °C, respectively. It should of course be noted that due the relatively high guest concentrations used in these studies (1% v/v), the presence of nonspecific host−guest interactions cannot be ruled out. It should also be noted that the somewhat moderate melting transition exhibited by many of the H1−alcohol mixtures necessitates that Tm values derived from such data should be viewed as semiquantitative (or semiqualitative) at best. Nevertheless, the clear right-shifting of the alcohol-free H1 melting profile in response to the addition of 1-pentanol and 1hexanol can be interpreted as a strong indication of potentially interesting host−guest interactions. To investigate further these preliminary findings, we turned to X-ray crystallographic studies as a means to provide atomic scale details of the potential host−guest assemblies. X-ray Crystallographic Studies of Aqueous H1-Bundle Host−Guest Complexes. Although it was apparent from the CD data that 1-pentanol and 1-hexanol elicited the greatest stabilizing effect on the helicity of H1 in aqueous solution, we

Table 1. Circular Dichroism (CD) Studies into the Effect of Various Primary and Secondary Alcohols (at a concentration of 1% v/v) on the Helicity of H1 (at a concentration of 200 μM in pure water)

a

guesta

MW

[θ]202

Tm (°C)

adj. R2

ΔTm (°C)b

no guest isopropyl alcohol 1-butanol 1-pentanol 1-hexanol 2-ethoxyethanol 2-propoxyethanol 1,4-butanediol

60.10 74.12 88.15 102.17 90.12 104.15 90.12

575000 592000 595000 681000 660000 579000 603000 551000

41.5 46.4 50.8 54.9 53.0 44.5 47.1 43.6

0.99875 0.99840 0.99847 0.99893 0.99918 0.99877 0.99877 0.99881

+4.9 +9.3 +13.4 +11.5 +3.0 +5.6 +2.1

Used at a concentration of 1% (v/v). b“No guest” used as reference.

The effect of these compounds on the helicity of H1 in aqueous conditions was investigated by circular dichroism (CD). H1 at a concentration of 200 μM in pure water was supplemented with each guest listed in Table 1 at a concentration of 1% (v/v). Spectra recorded over a wavelength range of 185−250 nm indicated that the helicity of H1 was not perturbed by the addition of any of the guests (Figure 2). CDmonitored thermal melting data confirmed this observation, and showed that the melting profile of the guest-free H1 is not 6130


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Journal of the American Chemical Society Table 2. Details and Analysis of Seven Crystal Structures of H1-Bundle−Alcohol Host−Guest Complexes cavity volumec (Å3)

B-factor (Å2) structure structure structure structure structure structure structure

1 2 3 4 5 6 7

a

host

guest

res. (Å)

guest:bundle

guest

overall

ratio

H1 H1 H1 H1 H1 H1 H1

isopropyl alcohol 1-butanol 1-pentanol 1-hexanol 2-ethoxyethanol 2-propoxyethanol 1,4-butanediol

1.18 1.35 1.52 1.15 1.45 1.62 1.40

3:1 3:1 3:1 3:1 3:1 3:1 3:1

24.24 27.27 21.97 24.60 32.35 26.43 43.29

14.50 26.05 24.70 19.27 18.46 25.22 29.39

1.67 1.05 0.89 1.28 1.75 1.05 1.47

RSCC

b

0.853 0.960 0.953 0.895 0.797 0.949 0.741

w/o guest

w/guest

% occupied

RMSDd (Å)

513.0 517.4 526.4 543.0 502.9 484.8 500.5

268.5 139.8 48.3 62.5 37.1 34.6 24.1

47.7 73.0 90.8 88.5 92.6 92.9 95.2

0.352 0.535 0.449 0.884 0.864 0.538

Ratio = guest alcohol B-factor/overall B-factor. bRSCC: real-space correlation coefficient (correlation between 2mFobs−DFcalc map and Fcalc map59). Cavity volumes calculated using SURFNET using a 1.4 Å probe radius.42 Please note: “% occupied” refers to the percentage of cavity volume occupied by guest molecules, and does not refer to the crystallographic atomic occupancy. dRMSD: root-mean square deviation. Structure 1 used as reference. Alignments performed in PyMOL, with 732 to 732 atoms aligned in all cases. Structural alignment of structures 1−7 is depicted visually in Figure S1. a c

Figure 3. Crystal structures of H1-bundle−alcohol host−guest complexes showing guest alcohols bound to cavities. (a−f) 1-Butanol (structure 2), 1pentanol (structure 3), 1-hexanol (structure 4), 2-ethoxyethanol (structure 5), 2-propoxyethanol (structure 6) and 1,4-butanediol (structure 7). Hydrogen bonds to the carbonyl groups of Leuu6 residues are shown as black dashes. Additional interguest hydrogen bonds are present in panel f.

endeavored to cocrystallize H1 in the presence of all guests listed in Table 1, as, importantly, the CD data also indicated that none of the guests studied actively destabilized H1. Crystallization experiments were performed in standard aqueous hanging drops containing H1 plus 1.5−8% (v/v) of the guests listed in Table 1. Crystals were obtained and structures successfully determined for all H1−guest complexes (structures 2−7, Tables 2 and S1), with resolutions in the range of 1.15−1.62 Å. All six H1−guest complexes are isomorphous, belonging to the same space group as the unbound and isopropyl alcohol-bound crystal structures (P63). Consequently, these crystal structures all reveal highly similar discrete hexameric helix bundles, with structural alignments (using structure 1, corresponding to the H1−isopropyl alcohol

complex, as a reference) generating RMSD values of 20%. Importantly, for all structures, alcohol-based guest molecules are well-resolved in electron density, with all guests localizing with full crystallographic occupancy to the three equivalent “tunnel” binding sites of the internal helix bundle cavity (Figures 3 and 4). Thus, all six H1−guest complexes possess a 3:1 guest:bundle ratio (equivalent to a 1:2 6131


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Figure 4. Electron density surrounding 1-butanol (a), 1-pentanol (b) and 1-hexanol (c) guests within H1-bundle cavities. 2Fo−Fc maps are shown at σ levels of 1.2, 1.5 and 1.0 for panels a−c, respectively. All three hydrogen bond distances (black dashes) are 2.8 Å.

Figure 5. Axial (top row) and side views (bottom row) of the H1-bundle cavity bound by increasingly larger alcohol guests ((a−e) unoccupied cavity, isopropyl alcohol, 1-butanol, 1-pentanol and 1-hexanol). Blue surface corresponds to unoccupied cavity volume. Cavity analysis performed using SURFNET42 using a 1.4 Å probe radius. Occupation of >80% of the cavity volume as in the case of panels d and e results in noticeable stabilization of the H1-bundle as measured by CD (Figure 2a).

Based on comparison of B-factor ratios (Table 2), from a purely structural perspective, 1-pentanol could arguably be considered as the optimal candidate of those studied here for binding to the H1-bundle cavity. Surprisingly, this finding correlates well with solution CD data, which indicate 1-pentanol to elicit the greatest positive effect on the thermal stability of H1 (Figure 2a). Increasing the alkyl chain length further, in the case of the H1−1-hexanol cocrystal structure (structure 4), results in an almost paradoxical reduction in the volume of cavity occupied by guest molecules: from 90.8% for the H1−1-pentanol complex to 88.5% for the H1−1-hexanol complex. Analysis of the total cavity volumes provides the answer: the cavity of the H1−1-hexanol complex is appreciably larger than that of the H1−1-pentanol complex. Indeed, cavity volumes appear to increase in size in direct correlation to increasing guest size. This finding has two implications: (1) the self-assembled H1bundle is not a rigid entity, but evidently has a significant degree of structural flexibility and, consequently, (2) the selfassembled cavity can, to some degree, adapt to accommodate the binding of suboptimal guests (such as 1-hexanol). Why could 1-hexanol be described as a suboptimal guest? With a higher guest:overall B-factor ratio (compared to the 1-pentanol complex), crystallographically, 1-hexanol is a poorer binder than 1-pentanol. This is supported by solution CD data, which indicate that, although 1-hexanol does indeed exert a stabilizing influence on the helicity of H1 in solution, it does so to a slightly less degree than 1-pentanol.

guest:helix ratio), with the guests adopting an isomorphous mode of binding to the cavity, involving hydrogen bonding to the carbonyl groups of Leuu6 residues, with the alkyl (or equivalent) tail projecting into the central chamber. Guest binding, however, is not entirely uniform, with differences in cavity volume occupation, guest orientation and hydrogen bonding correlating surprisingly well with solution CD data, and providing useful clues for the future development of the H1-bundle as a functional capsule/vector. Crystal Structures of H1-Bundle Host−Guest Complexes with Primary Alcohols. Structures 2−4. As for all seven host−guest complex cocrystal structures determined in this work, the H1−1-butanol host−guest complex (structure 2) reveals three molecules of 1-butanol bound to equivalent binding sites within the internal cavity of the helix bundle, with the primary alcohol groups hydrogen bonding to the available urea carbonyl groups of the Leuu6 residues located at the extremity of each “tunnel” (Figure 3a). The alkyl chains project into the central cavity, and although the volume of the cavity occupied by the guest molecules is increased significantly compared to the isopropyl alcohol-bound complex (from 47.7% to 73.0%), there remains considerable unoccupied cavity volume (Figure 5). As the alkyl chain length is increased to five carbons, in the case of the H1−1-pentanol host−guest complex (structure 3), the unoccupied space of the cavity is comfortably satisfied, with an increase in cavity volume occupation to 90.8% (Figure 5d). 6132


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Figure 6. Titration of H1 hexamer bundles with isopropyl alcohol, 1-butanol, 1-pentanol or 1-hexanol. The methyl region of 1D 1H spectra are shown to specifically include methyl groups located within the hexamer cavity such as Leuu6. Full spectra are shown in Figure S2. The top 1D 1H NMR spectrum in each series corresponds to 33.3 μM H1-bundle (200 μM monomer H1 oligourea) in D2O. The next six spectra have added 33.3, 66.7, 100, 133.3, 166.7 and 200 μM alcohol guest, equivalent to guest:host stoichiometric ratios of 1:1, 2:1, 3:1, 4:1, 5:1 and 6:1, respectively. The final spectrum in each series is the isolated alcohol in D2O at the same concentration as the end titration point (200 μM).

the primary alcohol series (structures 2−4), indicating further that the H1-bundle cavity has a noticeable degree of structural flexibility. Curiously, the cavity volumes of the H1-bundles for the three primary alcohol guests with polar chains are all smaller than that of the H1-bundles bound by the pure n-alkyl primary alcohols. The precise cause of this effect is not known, although it likely to be linked to the increased hydrophilicity of the diol and alkyl glycol guests. Regardless of the cause of this cavity-shrinking effect, 1,4-butanediol and the alkyl glycol guests occupy surprisingly large percentages of the H1-bundle cavity volumes−over 95% in the case of 1,4-butanediol, indicating clearly that, in certain conditions, the H1-bundle cavity is able to bind guests containing multiple hydrogen bonding groups and, furthermore, that hydrogen bonding groups are tolerated within the highly hydrophobic central chamber of the cavity. In addition, the H1−1,4-butanediol host−guest complex (structure 7) reveals interguest hydrogen bonding between all three guest molecules within the heart of the cavity, providing a positive indication for the future encapsulation of guests with a higher degree of complexity. Observation of Host−Guest Interaction by NMR Spectroscopy. The series of crystal structures described above present a clear stoichiometry of 3:1 guest molecules per H1-bundle host. Solution studies with CD spectroscopy had already demonstrated a stabilizing consequence of the bound alcohols, and we next wanted to see if aspects of stoichiometry seen in the crystal structures could also be observed in solution. Using NMR spectroscopy, we therefore followed the addition of isopropyl alcohol, 1-butanol, 1-pentanol or 1-hexanol to H1bundles in D2O (Figure 6; Figure S2). To perform the analyses, the alcohol guest molecules were added in increments from 1 to 6 equivalents of alcohol to one H1-bundle. The effect of alcohol binding was monitored by changes in the chemical shift values. From the crystal structures, it was observed that the alcohols bind proximal to Leuu side-

Structural alignment of the 1-butanol, 1-pentanol and 1hexanol cocrystal structures (using the isopropyl alcohol structure as a reference) reveals remarkably little difference in the oligourea foldamer component of these complexes, despite the substantial changes in cavity volumes across this series. This implies that the changes in cavity volume in response to differing guest size is a consequence of local rearrangements of the side-chains directly involved in forming the cavity environment, rather than gross alterations in interhelix packing. Close analysis of structural alignments supports this conclusion, as moderate variations in orientations of the side-chains of key leucine-type urea residues (Leuu4, Leuu6, Leuu9) are indeed evident (Figure S1). Crystal Structures of H1-Bundle Host−Guest Complexes with Chemically Diverse Primary Alcohols. Structures 5−7. Because of the predominantly hydrophobic nature of the H1-bundle cavity, hydrophobic forces are likely to play a key role in host−guest interactions, forces which the primary alcohol series described above show can be effectively exploited for guest binding. However, to increase guest diversity, we sought to investigate whether guests bearing polar groups in addition to the alcohol moiety could be encapsulated by the H1-bundle cavity. CD data indicated that potential guest molecules containing ether groups (2ethoxyethanol and 2-propoxyethanol) or multiple primary alcohol groups (1,4-butanediol) had little effect on the helicity or thermal stability of H1 in solution (Figure 2b). Highresolution crystal structures determined of H1-bundle−guest complexes involving each of these three molecules (structures 5−7, Table 2) all reveal an isomorphous mode of host−guest binding as observed for the primary alcohol structures described above (structures 2−4), with the alcohol groups hydrogen bonded to Leuu6 carbonyls, and the guest “tails” projecting into the central cavity chamber (Figure 3d−f). All three structures exhibit variations in cavity volumes, as seen for 6133


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Figure 7. RMSD profiles of alcohol-based guest molecules located (initially) within the H1-helix bundle cavity over the course of 100 ns molecular dynamics simulations. RMSD values above 0.2 nm indicate ejection of a guest molecule from the cavity of the H1-bundle.

disorder and a change in chemical environment following alcohol encapsulation. The fact that the NMR spectra change gradually upon addition of the alcohol also indicates that the host−guest interaction occurs with a fast exchange regime. As a consequence, it is probable that the interaction of alcohol molecules with the assembled H1-bundle is of a dynamic nature. Exploring Guest Encapsulation through Molecular Dynamics Simulations. To provide further insight into dynamic aspects of guest encapsulation by the H1-bundle, a series of X-ray guided dynamics simulations were performed on H1 in the presence of either isopropyl alcohol, 1-butanol, 1pentanol or 1-hexanol. For these simulations, the H1-bundle was placed in an 8 nm3 box with explicit water molecules, with three copies of each guest alcohol placed in the central cavity of the H1-bundle, with their hydroxyl groups orientated toward the carbonyl groups of the Leuu6 residues. Following an equilibration phase, 100 ns molecular dynamic simulations were then performed for each of the four systems. Analysis of the alcohol RMSD values over the simulations provides a clear indication of guest retention within the H1-bundle cavity, with RMSD values above 0.2 nm indicating the ejection of a single guest molecule from the cavity (Figure 7). In the case of the H1-bundle−isopropyl alcohol simulation, all three isopropyl alcohol molecules are rapidly ejected from the H1-bundle cavity (all three guests are ejected within 20 ns), resulting in significant compression of the helix bundle and loss of the internal cavity. Similarly, all three 1-butanol molecules are also ejected from the H1-bundle over the course of the 100 ns simulation, although the overall retention time of the 1butanol molecules is significantly extended compared to that of isopropyl alcohol. Guest retention is extended further in the

chains within the cavity of the H1 hexamer bundle, and we therefore focused on the 1H spectral region corresponding to side-chain methyl groups (Figure 6). In all four cases, the final spectrum (shaded black) differs from the original spectrum of H1 (shaded dark gray), thus supporting an interaction between the alcohol and oligourea at high micromolar concentrations. In particular, the Leuu side-chain methyl group 1H resonances between 0.8 and 0.9 ppm experience a subtle change in chemical shift and a noticeable dispersion of resonances indicative of an altered chemical environment of the interior cavity. Although the spectral changes are gradual, the pattern at a 3:1 ratio of guest:host (shaded medium gray in Figure 6) closely resembles that of the final titration point (in black). Comparison of the spectra at 3:1 and 6:1 guest:host ratios therefore supports a saturation of the H1-bundles by three alcohol molecules. This similarity is most evident for titration by 1-pentanol and 1-hexanol, likely reflecting an increased affinity for the H1-bundle as compared to isopropyl alcohol and 1-butanol. A general retention of the 1H peak pattern argues for a conservation of overall structure upon interaction with the alcohols. For the initial H1-bundles, the interior Leuu side chains are likely flexible around the unfilled interior cavity, with dynamic Leuu side-chain interactions that average to broadened methyl peaks with few distinct chemical shift values. The interaction with internal bound alcohols should result in chemical shift perturbation. However, most contacts involve aliphatic parts of the alcohols that resemble the previously neighboring Leuu side-chains. Despite this lack of a dramatic chemical shift change, the increased dispersion of Leuu methyl chemical shifts is consistent with a reduction in side chain 6134


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guest complexes has two important consequences: (1) three copies of the largest guest studied here equates to 312.45 Da, which theoretically permits the encapsulation of a guest large enough to be functionally interesting (e.g., a pharmaceutically active compound or a biosubstrate), and (2) multiple guestbinding results in a surprisingly high percentages of cavity occupation. These occupancy levels are far higher than those described for optimal encapsulation of guests by molecular containers in organic solvent conditions,43 and thus implies that the rules governing the encapsulation of relatively large, monomolecular guests in nonaqueous solvents may not all apply to the encapsulation of multiple small guests in aqueous self-assembled systems. Although the development of the foldamer helix bundle described here as a potential capsule for drug/cargo delivery is indeed a key future goal, the engineering of alternative capabilities into the H1-bundle, such as catalytic properties, is of equal interest. A cavity provides an ideal isolated compartment for (catalytic) reactions to take place, and, although the current chemical nature of the H1-bundle cavity needs to be altered significantly in order to resemble a catalytic site, the H1−1,4-butanediol crystal structure reported here indicates that the introduction of multiple polar groups within the helix bundle core is at least feasible. Along similar lines, the use of the H1-bundle hydrophobic cavity as an enclosed environment for “confined reactions”44,45 may be a more attainable goal, as this would permit the direct application of H1 without further modification.

case of both 1-pentanol and 1-hexanol, with the H1-bundle retaining one 1-pentanol molecule and two 1-hexanol molecules over the course of the 100 ns simulation. In all cases, guest ejection from the H1-bundle cavity involved the H1-bundle rearranging significantly to expose the central cavity (and therefore the entrapped guests) to the bulk solvent, followed by, surprisingly, refolding and reformation of the H1bundle, albeit into a new structural conformation (Figure S3). Although these results appear to suggest that the H1-bundle cavity does not tolerate the presence of these potential guests, it should be noted that (1) retention of a guest molecule on the multi-nanosecond time scale is significant (as in the case 1pentanol and 1-hexanol), and (2) as the concentration of alcohol in the bulk solvent surrounding the H1-bundle is zero (a chosen initial simulation condition), re-entry of these guests into the cavity is unlikely. Although caution must be taken when interpreting computational results such as these, two conclusions at least can be drawn with some confidence: (1) encapsulation of alcoholbased guests by the H1-bundle is an energetically feasible yet highly dynamic process, and (2) 1-pentanol and 1-hexanol bind more favorably to the H1-bunde cavity than isopropyl alcohol and 1-butanol, a tentative conclusion that appears to correlate well with CD-monitored thermal melting data (Figure 2 and Table 1). Further details and analyses of these molecular dynamics simulations can be found in the Supporting Information (Figures S4 and S5).

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CONCLUSIONS We have shown here, for the first time, the ability of a selfassembled foldamer helix bundle to encapsulate guests within an internal cavity in aqueous conditions. The seven highresolution crystal structures reported here, coupled to NMR solution data, provide valuable atomic-level insight into the mode and precise details of host−guest binding, and will enable the future rational development of this foldamer capsule. Although a single crystal structure essentially provides a threedimensional snapshot of a molecule or complex, a series of isomorphous structures gives some insight into dynamic aspects of a system. The seven high-resolution cocrystal structures reported here reveal an unexpected variation in cavity volumes, apparently in response to the size and chemical nature of the guests, conjuring an image of a “breathing” helix bundle, expanding or contracting in response to specific (guest) chemical stimuli. This subtle structural flexibility of the bundle suggests the cavity can adapt to some degree in order to accommodate suboptimal guests (as in the case of 1-hexanol). Further insight into dynamic aspects of alcohol-binding by the H1-bundle was provided through computational molecular dynamics methods, which revealed guest-binding within the H1 cavity to be an energetically feasible yet rather dynamic process, in which the helix bundle transiently unfolds, exposing the central cavity to the bulk solvent and resulting in guest ejection. Considering the relatively low Tm values of H1 both in the absence and presence of potential guests (as measured by CD), the results of these molecular dynamics simulations are to be expected. Yet structural flexibility is not necessarily a drawback for a guest encapsulation system, as certain applications of such a system may require high guest turnover (e.g., in the context of catalysis) or ready guest release (e.g., for the purposes of cargo delivery). Although the guest molecules studied here are relatively simple, the multiplicity in binding observed for all seven host−

METHODS

Chemistry. Oligourea H1 was synthesized on solid support using azide-protected building blocks following previously reported procedures.46 For details of the synthesis and purification, see Collie et al.16 X-ray Crystallography. For crystallization experiments, oligourea H1 was used at a concentration of 10 mg/mL in double-distilled water (ddH2O). Crystallization trials were performed at 20 °C in standard aqueous hanging drops composed of 0.6−0.7 μL of oligourea H1 plus an equal volume of crystallization reagent. The compositions of the crystallization reagents used to obtain crystals of structures 1−7 were as follows: 15% isopropyl alcohol (structure 1); 8% 1-butanol plus 15% 1,4-dioxane (structure 2); 2.5% 1-pentanol plus 10% 1,4-dioxane (structure 3); 1.5% 1-hexanol plus 25% 1,4-dioxane (structure 4); 5% 2-ethoxyethanol plus 20% 1,4-dioxane (structure 5); 5% 2-propoxyethanol plus 15% 1,4-dioxane (structure 6); 5% 1,4-butanediol plus 20% 1,4-dioxane (structure 7). In addition, all crystallization conditions contained 100 mM sodium acetate buffer (pH 4.6) and 200 mM calcium chloride. All crystals were cryo-protected in a solution composed of 67% of the concentration of crystallization reagent plus 25% glycerol before flash freezing in liquid nitrogen. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (beamlines ID23-2 and ID29) and SOLEIL synchrotron (beamline PROXIMA 1). Crystals diffracted in the range of 1.15 to 1.62 Å. Diffraction data were processed using XDS47 and Scala48 from the CCP4 suite.49 All diffraction data collected for H1 crystals grown in the presence of the above alcohols belong to space group P63 with almost identical unit cell dimensions (specific details of unit cell values are provided in Table S1). All structures were solved by molecular replacement using Phaser50 using the previously reported crystal structure of H116 as a search model (with side-chains, terminal residues and water molecules removed). Model building and restrained refinement were performed in Coot51 and REFMAC5,52 respectively, with geometric restraints generated using the PRODRG server.53 Following completion of oligourea and water model building, the relevant alcohol molecules were modeled into appropriate residual electron density (from Fo−Fc 6135


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Journal of the American Chemical Society and 2Fo−Fc maps) evident within the hydrophobobic cavities of all structures. The asymmetric units of all crystal structures reported here contain two oligourea H1 molecules and one guest molecule, resulting in a total of three crystallographically identical guest molecules bound per hexameric helix bundle. Final refinement details can be found in Table S1. Cavity analysis was performed using SURFNET42 (using a 1.4 Å probe radius) and PyMOL.54 Structure validation (including RSCC calculation) was performed in Phenix.55 Atomic coordinates and structure factors for structures 1−7 have been deposited in the Cambridge Crystallographic Data Centre with accession codes: 1057608−1057614. Circular Dichroism. Circular dichroism experiments were performed on a Jasco J-815 spectrometer. Oligourea H1 was analyzed at a concentration of 200 μM in ddH2O supplemented with 1% of one of the seven primary alcohols listed in Table 1. Single temperature spectra were recorded at 20 °C over a wavelength range of 185 to 235 nm. For CD-monitored thermal melting experiments, samples were heated from 5 to 90 °C using a gradient of 1 °C·min−1. The CD signal at 202 nm was monitored for these experiments. Thermal melting (Tm) values were estimated by fitting CD-monitored thermal melting data to a simple two-state Boltzmann unfolding model using Origin 8.6. Accuracy of the fits (reduced R2 values) are indicated in Table 1. NMR Spectroscopy. Spectra were measured at a temperature of 293 K on a Bruker Avance 700 MHz spectrometer equipped with a triple resonance gradient probe. Data were processed and analyzed with the software Topspin 3.5. To prepare the samples, lyophilized oligourea H1 was dissolved in 2H2O at a concentration of 200 μM (equivalent to an H1 hexamer concentration of 33.3 μM) as determined by absorbance at 280 nm and an extinction coefficient of 1490 M−1 cm−1. For each titration series, an initial 1H 1D spectrum was measured for a 170 μL sample of 200 μM H1 in 2H2O using 256 scans and sweepwidth of 14 000 Hz (20 ppm). Subsequent 1H 1D spectra were acquired following addition of isopropyl alcohol, 1butanol, 1-pentanol or 1-hexanol at concentrations of 33.3, 66.7, 100, 133.3, 166.7 and 200 μM. Alcohol stocks were prepared by a 1:4000 dilution into 2H2O. Reference spectra were also collected for 200 μM samples of isopropyl alcohol, 1-butanol, 1-pentanol and 1-hexanol in 2 H2O. Molecular Dynamics Simulations. All simulations were performed using the GROMACS simulation package version 4.6,56 using the gromos53a6 force field and the SPC (single point charge) water model.57 Simulations were performed using periodic boundary conditions with an integration time step of 0.002 ps. The temperature and pressure were kept constant at 300 K and 1 atm with Berendsen coupling baths in equilibration steps. During production, a velocity rescale temperature coupling and a Parrinello−Rahman pressure coupling were applied. Two separate groups were created to couple to each temperature bath: one for the oligourea helices with the three associated alcohol ligands and a second one for the solvent. The pressure coupling constant was 2.0 ps and the temperature coupling constant was 0.1 ps. Electrostatic interactions were treated with a fast smooth particle mesh Ewald and a cutoff distance of 0.9 nm. The van der Waals interactions were considered between 0.9 and 1.4 nm. The neighbor lists were updated every 10 steps. All bonds were constrained with the LINCS algorithm and a force constant of 1000 kJ mol−1 nm−1 was applied for all the distance restraints defined along the helix backbones. A first steepest descent minimization of 5000 steps was performed. Then an NVT (number of molecules, volume and temperature) equilibration was performed with position restraints of 1000 kJ mol−1 nm−1 on the helix backbones and all the ligand atoms. Finally, an NPT (number of molecules, pressure and temperature) equilibration was carried out without positional restraints on the ligands (while they were still applied to the helix backbones). All simulations were run for 100 ns. The starting H1-bundle model was built by providing a single helix from the previously reported H1 crystal structure16 to the ATB server (Automated Topology Builder).58 A series of quantum mechanics simulations were performed to determine atomic partial charges and bonded interaction parameters and generate new topology files compatible with the gromos53a6 force field. Helix backbones were

defined by analogy to peptidic structures, with amide bonds replaced by urea linkages. Crystallographic data were used to set up a distance restraints network based on the observed hydrogen bonds between urea carbonyls from residue i toward i+2/i+3 urea NHs. The network force constant was set to a value of 100 kJ mol−1 nm−1 to allow a moderate degree of flexibility within the system. The secondary structure of an individual helix was thus imposed, without a full parametrization of the urea chain. Indeed, side chains were identical to natural amino acids, the parametrization of which are well-established within the gromos53a6 force field. Topologies for the isopropyl alcohol, 1-butanol, 1-pentanol and 1-hexanol ligands were also generated using the ATB server. Four systems, one for each ligand, were simulated with identical key characteristics. In each case, the ligand was placed in the central cavity of the H1-bundle with the hydroxyl functional group directed toward the “exterior”, at 0.4 nm between the urea carbonyl groups of two Leuu6 residues. Each system was placed in a cubic box of 8 nm3 and solvated with around 17 000 water molecules. The initial velocities randomly generated during the NVT equilibration were reapplied for the subsequent simulations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b00181. Supplemental Figures S1−S5, Tables S1 (PDF) Crystallographic data for 1 (CIF) Crystallographic data for 2 (CIF) Crystallographic data for 3 (CIF) Crystallographic data for 4 (CIF) Crystallographic data for 5 (CIF) Crystallographic data for 6 (CIF) Crystallographic data for 7 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Gavin W. Collie: 0000-0002-0406-922X Cameron D. Mackereth: 0000-0002-0776-7947 Gilles Guichard: 0000-0002-2584-7502 Present Addresses ¶

Division of Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, London SM2 5NG, United Kingdom ∥ Faculty of Chemistry, University of Warsaw, Pasteura 1, 02093 Warsaw, Poland Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded in part by the CNRS and Conseil Regional d’Aquitaine (Project #20091102003). Marie Curie FP7-PEOPLE-2010-IEF-273224 and FP7-PEOPLE-2012-IEF330825 postdoctoral fellowships (to K.P.-Z. and C.M.L.) are gratefully acknowledged. J.D. and N.T.-M. are supported in part by a Ligue Nationale Contre le Cancer Grant and an Institut National du Cancer Grant. CIFRE support from UREkA and ANRT to L.M. is gratefully acknowledged. We thank SOLEIL synchrotron and the European Synchrotron Radiation Facility for providing access to data collection facilities (beamlines PROXIMA 1, ID23-2 and ID29), and thank Pierre Legrand for 6136


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assistance on PROXIMA 1. Credits for Cover Image: G.W. Collie and C.D. Mackereth.

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Molecular Recognition within the Cavity of a Foldamer Helix Bundle

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