On Crystal versus Fiber Formation in Dipeptide Hydrogelator Systems

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On Crystal versus Fiber Formation in Dipeptide Hydrogelator Systems Kelly A. Houton,† Kyle L. Morris,‡ Lin Chen,† Marc Schmidtmann,† James T. A. Jones,† Louise C. Serpell,‡ Gareth O. Lloyd,*,§ and Dave J. Adams*,† †

Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, U.K. Chichester 2 Building, School of Life Sciences, University of Sussex, Falmer BN19QG, U.K. § Department of Chemistry, University of Cambridge. Lensfield Road, Cambridge CB2 1EW, U.K. ‡

S Supporting Information *

ABSTRACT: Naphthalene dipeptides have been shown to be useful low-molecular-weight gelators. Here we have used a library to explore the relationship between the dipeptide sequence and the hydrogelation efficiency. A number of the naphthalene dipeptides are crystallizable from water, enabling us to investigate the comparison between the gel/fiber phase and the crystal phase. We succeeded in crystallizing one example directly from the gel phase. Using X-ray crystallography, molecular modeling, and X-ray fiber diffraction, we show that the molecular packing of this crystal structure differs from the structure of the gel/fiber phase. Although the crystal structures may provide important insights into stabilizing interactions, our analysis indicates a rearrangement of structural packing within the fibers. These observations are consistent with the fibrillar interactions and interatomic separations promoting 1D assembly whereas in the crystals the peptides are aligned along multiple axes, allowing 3D growth. This observation has an impact on the use of crystal structures to determine supramolecular synthons for gelators.

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INTRODUCTION Amino acid- and dipeptide-based low-molecular-weight gelators (LMWGs) are currently receiving significant attention.1−5 The simplicity and relatively low cost of the molecules are undoubtedly two reasons. In addition, these gelators have been shown to have utility in a range of applications from cell culturing4,6−9 to drug delivery,10−12 energy transfer,13 and conductivity.14 Gels are formed as the LMWGs self-assemble into fibrous structures that entangle and form the matrix.1,15−20 As for other classes of LMWGs, a key issue is how to control the competition between gelation and crystallization; it is currently poorly understood how to predict which molecules will form fibers and thus gels and which will crystallize. Even within this specific subclass of LMWGs, a large number of structures have been described, but no link between the molecular structure and the gelation ability has yet been shown. A small number of studies are beginning to show differences between small families of structurally similar molecules.21−26 However, extrapolating to allow prediction is not yet possible. The packing of the molecules in the fibers forming the matrix is also currently not clearly understood, although recently a small number of reports have attempted to link the packing in the fibers to that in crystals formed from the gel.27−29 Hence, it is clear that there is a need for further insight into the design of hydrogelators. Generally, two approaches are used to overcome this.30 First, libraries are generated from modifications of previously described LMWGs. Second, a crystal engineering approach has been taken, where crystal structures of LMWGs © 2012 American Chemical Society

are examined, with the aim of identifying intermolecular interactions and hence supramolecular synthons that result in a successful gelator.30,31 With regard to this, Mura-Small et al. suggested that the strength of the intermolecular interactions is more important than their directionality on the basis of the observed trend between the dissolution parameters and the gelation ability.32 For dipeptides functionalized at the N terminus with large aromatic groups such as naphthalene21,25,33 or fluorenylmethoxycarbonyl (Fmoc),8,34−39 gels can be formed in a number of ways. A trigger is required; this can be the solvent quality,8,35 temperature,40 addition of salts,41,42 enzymatic cleavage,43 or pH.33,37,39,44 We and others have shown for both Fmocdipeptides and naphthalene dipeptides that the apparent pKa of the C terminus of the dipeptide is much higher than might be expected.21,22,36,45 This apparent pKa correlates with the hydrophobicity of the molecule (the higher the hydrophobicity, the higher the pKa), and the pH at which gels form is just below the pKa of the molecule.21 Although this does not allow a prediction of the ability of a molecule to form a gel, the pH at which gels will form can be predicted. Because many of the applications for which these gels may be useful are pH-limited, this is a valuable tool. Received: April 3, 2012 Revised: May 23, 2012 Published: May 31, 2012 9797

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g·cm−3, μ = 0.100 mm−1, F(000) = 728, crystal size = 0.09 × 0.06 × 0.01 mm3, T = 100 K. 16 543 reflections (2.19 < Θ < 26.36°) measured, 3440 unique (Rint = 0.0676), 2675 observed (I > 2σ(I)), R1 = 0.0436 for the observed and R1 = 0.0676 for all reflections, max/min residual electron densities 0.454 and −0.263 e·Å−3, respectively, data/ restraints/parameters = 3440/0/240, GOF = 1.018. Crystal Data for Dipeptide 3. C20H24N2O5·1.5H2O, formula C20H27N2O6.50, M = 399.44 g·mol−1, monoclinic space group C2, a = 25.003(2), b = 6.8390(6), c = 12.0710(12) Å, β = 94.207(3)°, V = 2058.5(3) Å3, Z = 4, ρ = 1.289 g·cm−3, μ = 0.097 mm−1, F(000) = 852, crystal size = 0.09 × 0.06 × 0.02 mm3, T = 100 K. 12 150 reflections (2.26 < Θ < 26.37°) measured, 3711 unique (Rint = 0.0477), 2905 observed (I > 2σ(I)), R1 = 0.0421 for the observed and R1 = 0.0620 for all reflections, max/min residual electron densities 0.161 and −0.197 e·Å−3, data/restraints/parameters = 3711/1/288, GOF = 1.016. Crystal Data for Dipeptide 10. C17H18N2O5·H2O, formula C17H20N2O6, M = 348.35 g·mol−1, orthorhombic space group P212121, a = 4.7658(3), b = 5.8982(3), c = 61.176(3) Å, V = 1719.65(17) Å3, Z = 4, ρ = 1.346 g·cm−3, μ = 0.103 mm−1, F(000) = 736, crystal size = 0.12 × 0.11 × 0.005 mm3, T = 100 K. 8903 reflections (2.00 < Θ < 22.72°) measured, 2251 unique (Rint = 0.0436), 1997 observed (I > 2σ(I)), R1 = 0.0653 for the observed and R1 = 0.0732 for all reflections, max/min residual electron densities 0.324 and −0.297 e·Å−3, data/restraints/parameters = 2251/0/234, GOF = 1.120. All H atoms (with the exception of those associated with water molecule O6) were fixed to geometric positions using the riding model. Crystal Data for Dipeptide 11. C18H20N2O5·H2O, formula C18H22N2O6, M = 362.38 g·mol−1, orthorhombic space group P212121, a = 5.7838(7), b = 8.6133(11), c = 36.312(5) Å, V = 1809.0(4) Å3, Z = 4, ρ = 1.331 g·cm−3, μ = 0.101 mm−1, F(000) = 768, crystal size = 0.18 × 0.15 × 0.020 mm3, T = 100 K. 33 375 reflections (2.24 < Θ < 30.50°) measured, 5504 unique (Rint = 0.0365), 5261 observed (I > 2σ(I)), R1 = 0.0290 for the observed and R1 = 0.0309 for all reflections, max/min residual electron densities 0.304 and −0.188 e·Å−3, data/restraints/parameters = 5504/0/257, GOF = 1.075. Crystal Data for Dipeptide 12. C20H24N2O5·1.5H2O, formula C20H27N2O6.50, M = 399.44 g·mol−1, monoclinic space group C2, a = 24.656(6), b = 7.0007(16), c = 12.561(3) Å, β = 101.288(6)°, V = 2126.1(9) Å3, Z = 4, ρ = 1.248 g·cm−3, μ = 0.094 mm−1, F(000) = 852, crystal size = 0.10 × 0.05 × 0.01 mm3, T = 100 K. 12 729 reflections (2.12 < Θ < 21.96°) measured, 2564 unique (Rint = 0.0697), 1757 observed (I > 2σ(I)), R1 = 0.0563 for the observed and R1 = 0.0927 for all reflections, max/min residual electron densities 0.220 and −0.179 e·Å−3, data/restraints/parameters = 2564/1/268, GOF = 1.059. The valine group is disordered over two sites. Because of the low data/ parameter ratio, the C atoms of the disordered sites have been refined isotropically, and all H atoms (with the exception of those associated with water molecule O6) were fixed to geometric positions using the riding model. Powder X-ray Diffraction. Powder X-ray diffraction data were collected on a PANalytical X’pert pro multipurpose diffractometer (MPD) in transmission Debye−Scherrer geometry operating with a Cu anode at 40 kV and 40 mA. For the dried gels, samples were ground and mounted as a loose powder onto a transparent film and spun at 2 s/rotation. For the gel phase, the gel was sealed between two transparent films in an airtight manner. PXRD patterns were collected in 4 × 1 h scans with a step size of 0.013° 2θ and a scan time of 115 s/ step over 5−50° 2θ. The incident X-ray beam was conditioned with 0.04 rad Soller slits and an antiscattering slit of 0.5°. The diffracted beam passed through 0.04 rad Soller slits before being processed by the PIXcel2 detector operating in scanning mode. Pawley fitting was carried out using the TOPAS-Academic software.49 X-ray Fiber Diffraction. Fiber diffraction samples were prepared in situ from dipeptide solutions after the addition of GdL (8.9 mg mL−1). By placing a 10 μL droplet between two wax-filled capillary tubes and allowing the solution to gel and air dry, a partially aligned fiber sample was formed (as previously described33). The fiber sample was mounted on a goniometer head, and X-ray diffraction data were collected at room temperature at the I24 MX microfocus beamline at the Diamond synchrotron radiation facility (Oxfordshire, U.K.) at a

Naphthalene dipeptides are interesting examples of this class of LMWGs.21,25,33 The range of commercially available or easily synthesized naphthols means that libraries of related molecules can easily be generated. Here, we describe an extended library of naphthalene dipeptides and their efficiency as LMWGs. A small library of these have been crystallized from water. This has allowed us to use both approaches described above in an attempt to understand the link between gelator ability and molecular structure or supramolecular synthon.

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EXPERIMENTAL SECTION

Materials. All chemicals and solvents were purchased from SigmaAldrich and used as received. Millipore water was used throughout. The functionalized peptides were synthesized as described previously.21 Full synthesis and characterization details for all intermediates and final dipeptides are given in the Supporting Information. NMR. 1H NMR spectra were recorded at 400.13 MHz using a Bruker Avance 400 NMR spectrometer. 13C NMR spectra were recorded at 100.6 MHz. Determination of the Apparent pKa. An FC200 pH probe (Hanna Instruments) with a 6 mm × 10 mm conical tip was employed for all pH measurements. The stated accuracy of the pH measurements is ±0.1. The pKa values of the dipeptide solutions were determined by titration via the addition of aliquots of a 0.1 M HCl solution. pH values were recorded until a stable value was reached after each addition in the titration process. To prevent gel formation during the titration, the solutions were thoroughly stirred. Gelation Studies. The dipeptide (25.0 mg) was suspended in deionized water (5.0 mL). An equimolar amount of NaOH (0.1 M, aq) was added, and the solution was gently stirred until a clear solution was formed. The pH of the solution was checked. To form gels, aliquots of the solution were added to weighed amounts of glucono-δlactone (GdL) and the samples were gently swirled to dissolve the GdL before being left to stand for 24 h without stirring. After this time, the samples were examined by the vial inversion test to indicate gelation. Rheology. Dynamic rheological experiments were performed using an Anton Paar Physica MCR101 rheometer. For the oscillatory shear measurements, a sandblasted top plate with a 50 mm diameter and a 1.0 mm gap distance was used. Gels for rheological experiments were prepared on the bottom plate of the rheometer by loading a 2.0 mL aliquot of a solution (prepared as described above) immediately after GdL addition. At this point, the sample was a free-flowing liquid, and hence the sample uniformity and reproducibility are high. Evaporation of water from the sample was minimized by covering the sides of the plate with a low-viscosity mineral oil. The measurements of the shear moduli (storage modulus G′ and loss modulus G″) were carried out using a cup and vane system. All gels were formed directly in 7 mL Sterilin cups and left overnight (at least 20 h) at room temperature to gel before the measurements. Frequency scans were performed from 1 to 100 rad/s under a strain of 0.5%. The shear moduli (G′ and G″) were measured at a frequency of 10 rad/s. The strain amplitude measurements were also performed within the linear viscoelastic region, where G′ and G″ are independent of the strain amplitude. Single-Crystal Structure Determination. Single-crystal X-ray data were measured on a Rigaku MicroMax-007 HF rotating anode diffractometer (Mo Kα radiation, λ = 0.71073 Å, Kappa 4 circle goniometer, Rigaku Saturn724+ detector). Empirical absorption corrections using equivalent reflections were performed with the SADABS program;46 the structures were solved with the SHELXD program47 and refined with SHELXL47 using OLEX2 GUI.48 Unless specified otherwise, all non-H atoms were refined anisotropically, H atoms bonded to C were fixed to geometric positions using the riding model, and H atoms involved in hydrogen bonding (amide, carboxylic acid, and water H atoms) were refined freely. Crystal Data for Dipeptide 2. Formula C18H20N2O5, M = 344.36 g·mol−1, orthorhombic space group P212121, a = 4.9680(6), b = 9.6339(14), c = 35.173(5) Å, V = 1683.4(4) Å3, Z = 4, ρ = 1.359 9798

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2 position of the naphthalene rings. As described previously,21 apparent pKa values were determined by titration, minimizing gel formation (where this occurred) by vigorous stirring. Previously unreported dipeptides 1−9 and 13−15 showed apparent pKa values in line with expected values from our previous work (Table S1, Supporting Information). As in our previous reports, the apparent pKa values were higher than predicted for the C terminus of a dipeptide, all being in the range of 4.0 to 6.7. Figure 2 shows these apparent pKa values plotted against clogP for dipeptides 1−18. The pKa can shift dramatically in hydrophobic environments for proteins and peptides.53 Hydrogen bonding between molecules has also been shown to lead to an increase in the apparent pKa.54 High apparent pKa values for a number of fatty acid soaps with high alkyl chain lengths have been reported;55,56 the pKa was found to increase with the chain length. This was explained by the fact that aggregation leads to the formation of strong ion−dipole interactions between RCOO− and RCOOH, stabilizing the charge and disfavoring the deprotonation of more carboxyl groups. Urry et al. have also argued that the magnitude of the pKa shifts in ionizable groups in polypeptides arises from the competition for fully hydrated shells between polar groups and hydrophobic groups in close proximity.53 We have overlaid in Figure 2 our previously reported data21 for a number of related dipeptides. The correlation between clogP and the apparent pKa clearly still holds despite the greater diversity in structure and hence presumably the packing of the molecules. Although the correlation is not perfect, this again demonstrates that at least some properties of these molecules can be predicted to some degree from the molecular structure. We reported previously that the pH at which gelation occurs (if it does, see below) is related to the apparent pKa of the C terminus. This correlation therefore allows the choice of an appropriate gelator if gelation is required at a predefined pH. However, there is no clear link between the molecular structure and the ability of dipeptides 1−18 to form hydrogels. To probe the gel formation, the pH decrease was affected using glucono-δ-lactone (GdL) as described previously from an initial pH of 10.5.33,44 In water, GdL hydrolyzes to gluconic acid, allowing a slow and uniform pH change throughout the system. We examined a dipeptide concentration of 0.5 wt % (Table 1). For the dipeptides substituted at the 1 position of the naphthalene ring, only 4 and 6−9 formed gels. Some of these were optically transparent, and others were turbid (examples shown in Figure 3a). Scanning electron microscopy of those systems producing hydrogels showed the presence of well-defined fibers with uniform diameters as expected (example data shown in Figure 3b). All other 1-position dipeptides formed crystalline precipitates. For those substituted at the 2 position, 11, 13, 16, 17, and 18 formed gels, with all others again resulting in crystalline precipitates. We note that the observation regarding gelation also holds for gels where a mineral acid (HCl) was used to lower the pH. As noted previously, however,44 for the more hydrophobic molecules, this method results in lesshomogeneous gels than when GdL is used. The rheological properties of the gels were measured and are summarized in Table 1 (see also Figure S1, Supporting Information). Linking molecular structure to the ability of a molecule to form a gel is difficult, and it is often noted that LMWGs are discovered serendipitously.32 Closely related molecules can show very different abilities to form gels; indeed, a simple variation in the length of alkyl chains can cause drastic changes

wavelength of 0.9778 Å. Diffraction data was recorded using a MarCCD detector at a specimen-to-detector distances of 312.2 mm. Fiber samples were rotated about their long axes to ensure that cylindrical averaging was present. The diffraction patterns were converted using MOSFLM50 and examined, processed, and analyzed using CLEARER.51 Signal positions and intensities were automatically measured by defining the equator and sampling signal intensities within an angular search width of 60° as a function of the distance from the center of the pattern in pixels. Simulated fiber diffraction patterns from crystal structures were calculated using CLEARER.51 Unit cell dimensions, space groups, and atomic coordinates were used to generate a “fiber” texture (cylindrically averaged around the fiber axis). The atomic coordinates were used to calculate the intensity and position of each reflection in reciprocal space for a true crystal and of those that are intercepted by the Ewald sphere. The fiber axis direction, crystalline size, rotation, angular fiber disorder, and diffraction geometry were then accounted for to trace diffraction intensities from each reciprocal lattice point to the plane of the detector. The diffraction settings were equivalent to the fiber collection parameters (i.e., specimen-to-detector distance, wavelength, and detector parameters). The structural lattice settings were consistent with the unit cell of the crystal structure of 11, the fiber disorder was σθ 0.2 and σϕ ∞ radians, and the crystallite size was set to 400 Å3 with a pattern-sampling interval of 1 pixel whereas all other simulation parameters used were default values.

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RESULTS AND DISCUSSION The functionalized dipeptides were prepared in a stepwise fashion from 1-naphthol in an analogous fashion to our previous work21 based on 2-naphthol (full details found in the ESI). Additionally, we increased the library size of the 2naphthol family to allow a more thorough comparison of the effect of substitution on assembly. The functionalized dipeptides examined here are shown in Figure 1, with the numbering shown in Table 1.

Figure 1. Generic structures of the functionalized dipeptides (top, 1− 9; bottom, 10−18). For R1 and R2, see Table 1.

Tang et al. first described that the apparent pKa of Fmocdiphenylalanine was significantly higher than might be expected for the C terminus of a peptide.45 We showed that the apparent pKa's of the C terminus of a range of naphthalene dipeptides and Fmoc-dipeptides could be correlated with the hydrophobicity of the molecules.21,36 To estimate the hydrophobicity, we used an online predictive tool52 and showed that the hydrophobicity (as estimated by clogP, the calculated partition coefficient) of the entire molecule correlates with the measured apparent pKa. This correlation held whether or not the dipeptide was an effective hydrogelator; however, where gelation occurred, this began at a pH slightly lower than the apparent pKa. Recently, Tang et al. validated these observations for specific Fmoc-dipeptides.22 clogP was calculated for dipeptides 1−18 (Table S1, Supporting Information). The clogP values are slightly different depending on whether the dipeptides are positioned at the 1 or 9799

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Table 1. Substituents and Summary of Rheological Properties of Hydrogels Formed Where Appropriate at a Concentration of 0.5 wt %a

a

dipeptide

R1

R2

structure formed

1 2 3 4 5 6 7 8 9 10b 11b 12b 13 14 15 16b 17b 18b

CH3 CH3 CH3 CH(CH3)2 CH(CH3)2 CH(CH3)2 CH2Ph CH2Ph CH2Ph CH3 CH3 CH3 CH(CH3)2 CH(CH3)2 CH(CH3)2 CH2Ph CH2Ph CH2Ph

H CH3 CH(CH3)2 H CH3 CH(CH3)2 H CH(CH3)2 CH2Ph H CH3 CH(CH3)2 H CH3 CH(CH3)2 H CH(CH3)2 CH2Ph

crystal crystal crystal G crystal TG TG TG G crystal G crystal G crystal crystal G TG G

G′/Pa

G″/Pa

2.5 × 104 4.8 1.2 4.2 1.8

× × × ×

103 103 103 104

3.2 × 103 3.9 1.0 3.7 2.5

× × × ×

102 102 102 103

8.3 × 104

9.3 × 102

3.6 × 104

8.1 × 103

2.8 × 104 2.0 × 106 7.2 × 104

2.1 × 103 1.1 × 104 1.2 × 103

G, clear gel; TG, turbid gel. bData previously reported by Chen et al.21

substitution position of the dipeptide sequence on the naphthalene ring unsurprisingly did have an effect on the ability of the molecules to gel, presumably because of differences in the packing ability and awkwardness. All of the dipeptides had phenylalanine at the N-terminus gel, whether substitution is at the 1 or 2 position on the naphthalene ring. For other examples, only the ValGly sequence forms gels for both substitution positions. Changing the substitution pattern from 2 (11) to 1 (2) results in the sequence AlaAla becoming a nongelator. It has recently been reported that dissolution enthalpies can be used to indicate whether a molecule will be a gelator or nongelator.32 This method was used for a number of functionalized dipeptides, with gelating dipeptides having higher dissolution enthalpies (ΔHdiss) and entropies (ΔSdiss) than nongelators. Using this methodology for dipeptides 1, 2, 3, 10, 11, and 12 (where only 11 forms gels), we found for our data that the values of ΔHdiss and entropies ΔSdiss were similar to the values reported elsewhere. However, 11 did not have the highest values of ΔHdiss and ΔSdiss (Table S2, Supporting Information). We note, however, that gelation was induced by Muro-Small et al. using a thermal trigger that differs from the pH trigger used here.32 We also note that Muro-Small observed that dipeptides that were reported previously as nongelators

Figure 2. Plot of clogP against apparent pKa for dipeptides 1−9 (red), 13−15 (blue), and previously reported data for a range of naphthalene dipeptides21 (black). The line represents a linear regression to all data.

in the gelation ability. This is highlighted again by the results presented here. For example, the small chemical functionality differences in 4−6 induce different gelation abilities. 4 formed transparent gels. Increasing the steric bulk at the C-terminal amino acid to a methyl group (5) resulted in a crystalline solid being formed, but increasing the bulk further to an isopropyl group (6) again resulted in a gel, albeit a turbid gel. The

Figure 3. (a) SEM of a transparent gel formed from 4. (b) SEM of a turbid gel formed from 8. (b) The scale bars represent 200 nm. (Insets) Photographs of the gels formed. 9800

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Figure 4. (a) One-dimensional stacking interactions for 1. Broken red lines represent hydrogen bonding. Broken yellow lines represent C−H···π interactions. Broken green lines represent face-to-face π−π interactions. (b) One-dimensional stacking interactions for 10. (c) Extended packing in 1. (d) Extended packing in 10 through water hydrogen bonding in hydrophilic regions and π−π interactions in hydrophobic regions.

Figure 5. (a) 2 1D stacking interactions. Broken red lines represent hydrogen bonding. Broken yellow lines represent C−H···π interactions. Broken green lines represent face-to-face π−π interactions. (b) 11 1D stack formation through hydrogen bonding of amide to water to carboxylic acid groups. (c) 2 packing. (d) Interdigitation of stacks of 11 through π−π interactions. Red spheres represent oxygen atoms of the water molecules.

dipeptides, only 11 effectively forms a gel. Since our first report, we have since found that 12 can form a gel, but only under very specific conditions (between a pH of 3.9 and 4.2, with gel formation occurring after approximately 2 days; we currently have no explanation for this unusual behavior). For 11, crystallization from the gel phase occurred slowly for samples using increased quantities of GdL; when we used HCl to adjust the pH, crystals formed more quickly (over a day). By careful control over the amount of GdL used, crystals suitable for diffraction were grown directly from the gel phase. This is still an unusual case for LMWG.28,58−63 As noted above, for most examples where crystal structures are determined, the crystals are most often grown from a solvent different from that where gels are formed. Some examples do exist where good evidence

using a pH trigger25 could become gelators using a thermal approach. This further demonstrates the complexity of this area. For the examples discussed here, crystals suitable for singlecrystal diffraction were grown for 1−3 as well as the direct analogues substituted at the 2 position on the naphthalene ring (10−12). The structure for 1 has been previously reported.57 In all cases, crystals were grown in aqueous solutions by lowering the pH with GdL. Our previous work implies that the lactone or hydrolysis products are not incorporated in or associated with the dipeptide self-assembled structures.44 Indeed, the same structures could be grown using HCl to adjust the pH (Figure S2, Supporting Information), but the quality of the crystals was generally poorer. Out of these six 9801

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Figure 6. (a) 3 and (b) 12 exhibit two hydrogen bonds for the amide N−H group. (c) 3 and (d) 12 form dimers through hydrogen bond dimer formation between amide and carboxylic acid groups. The isopropyl group is not shown in the dimer structure of 12 for clarity. Broken red lines represent hydrogen bonding.

3 and 12 have two hydrogen bonds for this amide N−H group and are both hydrates (Figure 6a,b). The bifurcated hydrogen bond results in an antiparallel set of amides, which cannot hydrogen bond together. This is probably due to the combination of hydrate formation and the bulky size of the isopropyl group of the Val residue, adding up to the possible prevention of the formation of amide hydrogen bond stacks and therefore the directional stacking required for fiber formation. A notable interaction is the dimer between carboxylic acid groups and amides closest to the carboxylic acid group (Figure 6c,d). A search of the Cambridge Structural Database revealed only 25 unique crystal structure examples with this interaction. There is continued interest in the determination of supramolecular synthons, not only in crystal engineering but across a diverse set of research fields such as biochemistry and soft materials.30,67,68 The dimer formation between two very common functionalities, carboxylic acids and amides, is therefore of interest, and its formation appears to be common enough to be described as a synthon but it does not occur as often as the amide tape motif or eight-atom hydrogen bond ring carboxylic acid dimer, amide···amide and amide···carboxylic acid synthons. Water also ties the carboxylic acid groups to the carbonyl group of the other amide, further binding the dimers together.69 3 and 12 are so similar that they can almost be described as isomorphous. For these two specific materials, it appears that the isopropyl group is too bulky to allow simple, quick assembly of fibers, and dimerization is preferred. However, it is interesting that when 12 is substituted with a bromine on the naphthalene ring the molecule becomes a very effective gelator.33 In the above discussion, we emphasize that some of the structures are hydrates and others are not. It is therefore very difficult to separate water-inclusion-induced structural characteristics from those of molecular structure factor causes. Nonetheless, in comparing all of the data above, 1, 2, and 10 all show well-defined 1D stacking. These stacks are hydrophobic on one end and hydrophilic on the other, which might promote interactions between fibers (hydrophobic collapse), resulting in fibril formation. Stable fibers seen in other structures require the hydrophobic areas to be either symmetrically outside the fiber to induce phase separation from solution (a possibility with 1) or buried inside the fiber as seen in amphiphilic structures (also a possibility with 1). The stacking of compounds 1, 2, and 10 could suggest a tendency for gel fiber formation. However, despite some assertions that

has been provided to show that the structures of the gel phase and the crystalline phase match. For example, Weiss et al. have shown that cholesteryl anthraquinone-2-carboxylate has polymorphic character and that some of the crystalline forms match the gel phases.64,65 However, in general it is not clear to what extent the crystal structures are related to the packing in the gel phase. With this caveat in mind, we closely examined the crystal structures of 1−3 and 10−12. For 1, as previously described, there is a clear stacking of these molecules on top of each other through standard amide-based hydrogen bonding between dipeptide chains, some C−H···π interactions, and face-to-face π−π interactions to give an asymmetric 1D stack (Figure 4a). These stacks interact through edge-to-face π−π stacking end to end to give a slightly larger strand with hydrophilic ends (Figure 4c). For 10, the same AlaGly dipeptide but now attached to the 2 position of the naphthalene ring, there is again also a clear stacking of these molecules on top of each other through standard amide-based hydrogen bonding between chains, some C−H···π interactions, and face-to-face π−π interactions to give an asymmetric 1D stack (Figure 4b). This structure forms as a hydrate and has clear-cut hydrophobic and hydrophilic sections (Figure 4d). In the crystal structure of 2, there is also a clear stacking of these molecules on top of each other through standard amidebased hydrogen bonding between chains, some C−H···π interactions, and face-to-face π−π interactions to give an asymmetric 1D stack (Figure 5a). For 11, the only example where crystals formed directly from a gel phase, interestingly, the “expected” amide interactions (which would give an easy explanation of fiber growth) are not present, mostly because of the formation of the hydrate. In other crystalline gelling materials, water can often work as a binder between molecules.59,66 This water binding is also present in 11. Figure 6b shows how the hydrogen bonding through the amide to water to carboxylic acid can give a symmetric 1D stack. (This particular stack of molecules runs along the crystallographic [010] direction.) The stacks, which are polar in nature, hydrogen bond together, the hydrogenbonded stacks being all of the same polarity. The polar stacks can interdigitate through edge-to-face π−π interactions with oppositely polarized stacks (Figure 5d). In all of the structures shown here, there is intramolecular hydrogen bonding of the nearest amide N−H group to the naphthyl moiety ether group. 9802

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Figure 7. Experimental fiber diffraction collected from (a) an in-situ-prepared alignment of fibers of 11, compared to (b) a quadrant of the simulated fiber diffraction pattern of the crystal structure of 11. (c) An inspection of the whole simulated pattern reveals the occurrence of some reflections that are comparable in their position but are aligned with different axes than in the experimental pattern. The meridian corresponding to the fiber axis is vertical, and measurements are made in angstroms.

compound that is able to gel. Powder X-ray diffraction (pXRD) methods have been used in some cases.73−76 However, this is often carried out after drying, so it is difficult to ensure that this has not led to a change. pXRD of gel fibers in the solvated state suffers from significant scattering contributions from the solvent and the less-crystalline nature of the fibers, meaning that this data is often inconclusive. Ostuni et al. have used pXRD to examine the packing of a cholesteryl-based gelator in the gel state, subtracting the solvent scattering to obtain that of the gelator fibers.64 Indeed, for dipeptide-based gelators, pXRD has been reported for dried samples of gels.22,38,45 Moreover, it has been reported for dipeptide-based systems that pXRD of the wet gel was unsuccessful in at least one case and also that pXRD in some cases depended on the method of drying.32 Here, for gels formed from 11, pXRD of the gel phase showed only diffuse scattering associated with the solvent. On drying, scattering was observed. This data agrees with that calculated from the crystal structure of 11 as described above (Figures S4 and S5, Supporting Information). In a number of cases, crystallization of a gelator has been achieved from a solvent different from that in which gelation occurs. Here, it is not clear that the dominant interactions are not affected by the nature of the solvent. A small number of studies have shown that the X-ray fiber diffraction of gels can be carried out.33,57 For example, we showed that reflections in the fiber diffraction of a bromo-naphthalene dipeptide arose from interatomic separations that were consistent with the formation of stacks of β strands spaced at 4.51 Å running parallel to the fiber long axis and complex lateral associations of these on length scales of up to 60 Å.33 We and others have previously attempted to use computational approaches to predict the crystal structures of gelators.57,77 This was successful for an example that directly crystallized from solution.57 However, for a dipeptide that crystallized from a gel phase, the predicted crystal structure did not agree with the data; similarly, both the predicted and experimentally derived crystal structures differed from the experimental fiber diffraction data for the gel phase. The fiber diffraction pattern from a gel of 11 is shown in Figure 7a and reveals the interatomic distances between ordered repetitive structural features in the fiber phase and which axis they align to relative to the fiber long axis. We noted earlier that drying may lead to artifactual structure formation

1D stacking within crystal structures correlates with the formation of morphologies of needles/fibers,70 the crystal morphology of these three compounds consists of plates, indicating that there is a strong tendency to interact in two directions as opposed to one direction, resulting in crystallization as opposed to fiber formation. The close packing through the π−π interaction of the naphthyl moieties prevents any interdigitation of these groups, as is possible with the gelforming compound 11. 1, 2, and 10 can form stacks but do not gel, whereas 11 gels but does not π stack, implying that in this case π stacking may not be an important prerequisite for gel formation (as is commonly suggested for related gelators38,71). The absence of such a motif is surprising but the formation of a hydrate-mediated stack indicates that maybe this assembly is preferred, and the use of water in this structure also indicates that maybe gel formation involves water as well. It is clear the Val-containing compounds 3 and 12 are simply too bulky to form clear hydrogen bonding stacks. 11 is the only one of these six molecules that robustly forms a gel. From crystal structure analysis, we hypothesize that this is due to the specific 1D stacking arrangement in 11 allowing the 1D stacks to form quickly. As implied above, it is not clear that the structure adopted in the crystalline state necessarily correlates with that formed in the gel state. In an attempt to shed light on this, we measured the diffraction from the gel state using X-ray fiber diffraction. There is significant discussion in the literature as to the similarity in packing between LMWGs in the gel state and in the crystalline state.30 Where there is clear evidence that an LMWG is forming an ordered fibrous matrix, it is likely that the LMWG must exhibit strong and directional intermolecular interactions that promote assembly into 1D fibers. These gelforming fibers must form in preference to crystallization products.58 The directional interactions can be hydrogen bonding, π stacking, and so forth. These interactions must be anisotropic to ensure growth in one direction; the relative absence of such interactions in the other two dimensions prevents lateral growth and thus crystallization. On this basis, there is a significant body of work that attempts to correlate supramolecular self-assembly patterns of a molecule obtained from its single-crystal data with its gelling behavior.30,31,72 This is significantly compromised by the difficulty in actually obtaining the crystal structure for a 9803

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unequivocal link between the molecular structure and the ability of dipeptides to form hydrogels. For all of the dipeptides with phenylalanine in the N-terminus gel, whether substitution is at the 1 or 2 position on the naphthalene ring, and for the other peptides, it is not clear why minor adjustments in molecular structure lead to significant differences in gelation ability. We note that this is further complicated by the observation elsewhere of functionalized dipeptides in which the method of gel formation can convert a nongelator into a gelator.32 In an attempt to address this gap in understanding, we investigated the crystal structures of a subset of these molecules. Examining the crystal structures of a subset of the dipeptides revealed that the AlaVal dipeptides show an unusual supramolecular synthon, which is an interaction between the carboxylic acid groups and the amide closest to the carboxylic acid group. For those examples that only crystallize, the close packing through the π−π interaction of the naphthyl moieties prevents any interdigitation of these groups. For the single example where we managed to grow crystals from the gel phase, π stacking did not occur, implying that for these systems this may not be an important prerequisite for gel formation. The formation of a hydrate-mediated stack indicates that maybe this assembly is preferred and also that maybe gel formation involves water. From the comparison of the crystal structure of 11 to the structural information available for this dipeptide in the fiber phase, we conclude that this crystal structure is principally different from the fiber phase, although they are perhaps subtly related. The lattice packing is different in the fiber phase and is less condensed than the crystal. The internal structural spacings and thus stabilizing interactions of the crystal may be present in the fiber, but if so, they are reorganized to align. These observations are consistent with the differences in crystal versus fiber formation, whereby the interactions and interatomic separations within a fiber are known to be aligned, thus promoting 1D growth, whereas in the crystal structure they are aligned to multiple axes allowing 3D growth.

and showed that the pXRD of a dried gel of 11 showed that an identical structure to the crystals formed from the gel was adopted (Figures S4 and S5, Supporting Information). For the fiber diffraction data shown here, this was found not to be the case. Probing the structure in fully hydrated, semihydrated, and dried states over the time course of drying reveals that the principal meridional and equatorial reflections are resolved in the hydrated state (Figure S6, ESI). The principal meridional and equatorial reflections indicate a repetitive separation along the fiber axis of 4.67 Å and perpendicular to the fiber axis repetitive separations of 40.6, 14.37, and 5.79 Å (Table S3, ESI). The dimensions of the orthorhombic unit cell of the crystal structure of 11 are a = 5.79, b = 8.61, and c = 36.39 Å, and thus it is tempting to compare these dimensions to those of the fiber diffraction pattern. In this case, the a and c dimensions would represent the lateral packing of the fiber structure and the b dimension would represent the stacking along the fiber axis. To investigate the relationship between the fiber phase and the crystal structure fully, simulated fiber diffraction patterns were calculated from the crystal structure of 11. As previously described, it is possible that the b crystallographic axis represents the fiber axis; however, this is not certain, so simulations were performed such that each axis could represent the fiber axis (Figure S6, Supporting Information). A close inspection of the patterns reveals that the simulation with b as the fiber axis most closely represents the experimental fiber diffraction pattern; however, it was observed that overall the crystal lattice packing and structure contained within did not fully reproduce the diffraction signals along the experimental fiber diffraction pattern. This supports the view that the crystal structure does not represent the fiber structure. They may, however, be related. We note that the simulated pattern shows diffraction signals that match experimental signals, though the axes differ. This implies that some structural spacings present in the gel phase may also be present within the crystal structure but aligned to different crystallographic axes. The simulated fiber diffraction pattern from the crystal structure, assuming that b represents the fiber axis, is shown in Figure 7b,c. The reflections arising from long-range spacings (>10 Å) are so weak that they are not observed. Overall, the meridional and equatorial reflections are fundamentally different. Where comparison can be made, the simulated reflections occur off-axis, indicative of the complex alignment of these structural features within the unit cell. The 5.79 Å equatorial is reproduced, corresponding to the dipeptide and naphthyl packing distance. The principal meridional reflection occurs at a much shorter distance of 4.31 Å, suggesting that the crystal structure is more compact along the fiber axis than in the true fiber phase.

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ASSOCIATED CONTENT

S Supporting Information *

Full synthesis and characterization of the dipeptides. Further fiber diffraction data and crystallographic information files. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or [email protected].

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Notes

CONCLUSIONS Establishing all of the design rules for hydrogelation is an extremely challenging task. Clearly, π−π stacking, hydrophobicity, and hydrogen bonding are all crucial factors in the assembly of small-molecule hydrogelators. Here, we have shown that different regioisomers behave differently for a range of naphthol dipeptides. As in our previous reports, the apparent pKa values were higher than predicted for the C terminus of a dipeptide, and there is a correlation between clogP and the apparent pKa that holds despite the diversity in structure and hence presumably the packing of the molecules. However, for the dipeptides discussed here, there is no clear

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Engineering and Research Council (EPSRC) for financial support under grants EP/G012741/1 and EP/ H000925/1. G.O.L. thanks the Herchel Smith Fund (Cambridge). We thank Tom McDonald for collecting the SEM data.

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REFERENCES

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