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Substituent Effects on the Self-Assembly/Coassembly and Hydrogelation of Phenylalanine Derivatives Wathsala Liyanage, and Bradley L. Nilsson Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03227 • Publication Date (Web): 30 Dec 2015 Downloaded from http://pubs.acs.org on January 5, 2016

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Substituent Effects on the Self-Assembly/Coassembly and Hydrogelation of Phenylalanine Derivatives Wathsala Liyanage and Bradley L. Nilsson* Department of Chemistry, University of Rochester, Rochester, NY, 14627-0216, USA.

E-mail: [email protected] Fax: +1 585 276-0205; Tel. +1 585 276-3053

ACS Paragon Plus Environment

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2 TOC graphic


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3 Abstract Supramolecular hydrogels derived from the self-assembly of organic molecules have been exploited for applications ranging from drug delivery to tissue engineering. The relationship between the structure of the assembly motif and the emergent properties of the resulting materials are often poorly understood, impeding rational approaches for the creation of nextgeneration materials. Aromatic π−π interactions play a significant role in the self-assembly of many supramolecular hydrogelators, but the exact nature of these interactions lack definition. Conventional models that describe π−π interactions rely on quadrupolar electrostatic interactions between neighboring aryl groups in the π-system. However, recent experimental and computational studies reveal the potential importance of local dipolar interactions between elements of neighboring aromatic rings in stabilizing π−π interactions. Herein we examine the nature of π−π interactions in the self- and coassembly of Fmoc-Phe-derived hydrogelators by systematically varying the electron-donating or electron-withdrawing nature of the side chain benzyl substituents and correlating these effects to the emergent assembly and gelation properties of the systems. These studies indicate a significant role for stabilizing dipolar interactions between neighboring benzyl groups in the assembled materials. Additional evidence for specific dipolar interactions is provided by high-resolution crystal structures obtained from dynamic transition of gel fibrils to crystals for several of the self-assembled/coassembled Fmoc-Phe derivatives. In addition to electronic effects, steric properties also have a significant effect on the interaction between neighboring benzyl groups in these assembled systems. These findings provide significant insight into the structure-function relationship for Fmoc-Phe derived hydrogelators and give cues for the design of next-generation materials with desired emergent properties.


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Introduction The self-assembly of functionalized amino acids into functional hydrogels has attracted great

attention

due

to

their

promise

for

application

in

biomedical

research.1-5

Fluorenylmethoxycarbonyl (Fmoc) protected amino acids are a privileged motif that has been shown to form hydrogel networks through spontaneous self-assembly.6-13 However, the development of Fmoc-amino acid hydrogelators relies primarily on empirical approaches or serendipity due to the lack of understanding regarding the mechanisms of self-assembly and how the structure of the assembly motif relates to the emergent properties of the resulting hydrogel network. Insight into the nature of the molecular interactions that drive supramolecular assembly and hydrogelation will facilitate rational approaches to the development of more sophisticated hydrogel biomaterials. Coassembly of two or more distinct monomeric units through complementary intermolecular interactions is of great interest due to the potential to form materials with more sophisticated emergent properties.11-19 However in a multicomponent system, the molecular assembly process is complex and unpredictable since molecules compete between coassembly and potentially orthogonal self-sorting processes.19-21 Despite the challenge associated with coassembly techniques, careful tuning of complementary noncovalent interactions can induce the assembly of two or more different structural motifs into a single fibril architecture. For example, complementary charge-charge interactions have been exploited as an effective driving force to promote coassembly into amyloid-like fibrils.22,23 In addition, complementary π−π interactions have also been used to promote coassembly and hydrogelation of binary supramolecular


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5 systems.11,13,16,24-26 The structural and mechanistic basis for how π−π interactions elicit coassembly is, as yet, poorly understood. Herein we examine the mechanistic and structural basis for assembly of Fmoc-Phe derivatives into fibrillar hydrogel networks. Specifically, the self-assembly/coassembly and hydrogelation properties of side chain-substituted Fmoc-Phe derivatives was studied in order to understand the contribution of aromatic π−π interactions on assembly and functional hydrogel formation of these derivatives. Fmoc-Phe and related molecules have been widely exploited as self-assembling hydrogelators.8,10,16 It has been found that the assembly properties of these molecules can be profoundly enhanced by the incorporation of various substituents, including halogens, on the benzyl side chain.8,9,11 Halogenated Fmoc-Phe derivatives have a much higher propensity for spontaneous self-assembly into hydrogel fibril networks compared to the parent Fmoc-Phe.8,9,11 However, Fmoc-Phe is more efficiently incorporated into hydrogel fibrils when coassembled with halogenated Fmoc-Phe analogs.11 The mechanistic basis for efficient coassembly of Fmoc-Phe with halogenated Fmoc-Phe derivatives is not understood, but it has been hypothesized that complementary aromatic interactions between the benzyl side chain functional groups mediate coassembly. The specific nature of these aromatic interactions has been unclear due to a lack of high-resolution structural information regarding both the self- and coassembled materials. Recently, we discovered that Fmoc-para-nitrophenylalanine (Fmoc-4-NO2-Phe) selfassembles into a fibillar hydrogel network that then spontaneously rearranges to form crystalline microtubes.27 This discovery has facilitated high-resolution structural interrogation of these materials (Figure 1) that provide insight into the nature of π−π interactions in the assembled state. Fmoc groups stack in an offset edge-to-face mode while the benzyl side chain groups


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6 interact via complementary dipolar interactions between the aromatic groups. This implies that in coassembled Fmoc-Phe-derived materials, interactions between the aromatic benzyl side chain groups are not based on bulk quadrupolar effects, but are more consistent with complementary dipolar interactions between specific atoms in the neighboring aromatic groups (Figure 1B).

Figure 1. Molecular packing of Fmoc-4-NO2-Phe in assembled fibrils and crystals.27 A. Packing architecture in the basic fibril unit that feature Fmoc-Fmoc and benzyl-benzyl aromatic interactions. B. Local dipole interaction between neighboring benzene rings in the assembled state. C. Chemical structure of Fmoc-Phe derivatives studied herein. Based on these findings, we initiated studies to understand the generality of dipolar π−π effects between benzyl aromatic groups in the self-assembly and coassembly of various side chain-substituted Fmoc-Phe derivatives. Specifically, Fmoc-Phe analogs with either electron withdrawing or electron donating groups in the para position of the benzyl aromatic ring were


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7 studied (Figure 1B). Each derivative was assessed for the effects of side chain substituents on either the self-assembly of the derivative or the coassembly of the derivative in combination with other analogs. It was found that both self-assembly and coassembly occurred most efficiently when Fmoc-Phe derivatives possessed complementary dipolar charges consistent with the packing mode shown in Figure 1B. High-resolution structures were also obtained when self-assembled fibril hydrogels of Fmoc-para-methylphenylalanine (Fmoc-4-CH3-Phe) and coassembled fibrils of Fmoc-4-NO2Phe and Fmoc-para-cyanophenylalanine (Fmoc-4-CN-Phe) underwent gel fibril to crystal transitions as has previously been reported for Fmoc-4-NO2-Phe.27 The resulting structural data is consistent with packing of the side chain benzyl groups stabilized by π−π interactions that feature similar complementary dipolar interactions to those found in Fmoc-4-NO2-Phe materials. Collectively, these findings clarify the π−π effects that promote self-assembly of Fmoc-Phe derivatives and provide cues for the design of next-generation materials from these types of molecules.28 The influence of π−π interactions on self-assembly processes. Rational design of selfassembling hydrogelators remains a major challenge due to a lack of understanding regarding the precise relationship between the structure of the assembly motif and the noncovalent forces that elicit self-assembly.2,29 Among these noncovalent forces, the specific contribution of aromatic π−π interactions to functional hydrogel formation is poorly understood. Recent studies on the coassembly of quadrupole phenylalanine and pentafluorophenylalanine (F5-Phe), which have complementary benzyl side chain quadrupoles, indicates that complementary π−π interactions can be exploited to promote assembly of distinct monomeric units into multicomponent fibrils.3032

We have also recently demonstrated that Fmoc-Phe and Fmoc-F5-Phe effectively


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8 coassemble.11 It may be inferred that this coassembly effect is promoted by the complementary quadrupoles of the Phe and F5-Phe benzyl side chains. However, the observation that monofluorinated Fmoc-Phe derivatives also coassemble with Fmoc-Phe casts this inference into doubt, since these more moderately substituted monofluororinated Phe side chains have quadrupole moments that are more similar to the parent Phe.11 Thus, it is also possible that the influence of direct substituent-π or dispersion interactions (direct dipole-interaction model) between substituted and unsubstituted aryl rings stabilize coassembly rather than more dramatic quadrupole involvement.30 It is appreciated that the electronic nature of substituents appended to aromatic rings can be used to tune the strength and geometry of intermolecular aromatic interactions.33-36 Among the major energetic contributions of aromatic-aromatic interactions (which include hydrophobic, van der Waals, and electrostatic effects),37 the electrostatic component arises, in part, due to the quadrupole moment of the aromatic ring, which largely determines the geometric preference for π−π interactions.38-40 The quadrupole moment of the benzene ring can be explained by its inherent polarity due to the positively charged σ−framework between two regions of π-electron density on the face of the ring.41-43 The favored geometries of intermolecular π−π interactions (edge-to-face or offset-stacked) between benzene rings with similar quadrupole moments and the favorable face-to-face interactions between benzene and reversely polarized hexafluorobenzene are clearly quadrupole effects.44-46 Aromatic substituent effects also influence π−π interactions by the polarization of the πsystem and the resulting perturbation in quadrupole moment. According to the Cozzi and Siegel polar/π model47-49 and the Hunter and Sanders model,50-53 electron-withdrawing substituents enhance intermolecular π−π interactions by lowering electrostatic repulsion between electron


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9 clouds, whereas electron-donating substituents hinder aromatic interactions by increasing these repulsive effects. According to Hunter and Sanders, binary aromatic systems with an electron withdrawing component on one ring and an electron donating component on the other ring, simultaneously adjust the effect of the neighboring ring to form face centered pairings.54,55 This favorable “π orbital mixing” creates an alternative stacking of electron-rich and electron-poor aromatic rings to form a wider variety of molecular architectures.56 However, Cozzi and Siegel argue that the Coulombic repulsion between electron-poor rings should be lower compared to interactions between electron-rich rings; electron-rich and electron-poor pairs should be energetically intermediate.45,46 Wheeler and Houk challenged the π−polarization model57-59 with theoretical studies that suggest that substituent-induced changes in π−π interactions is due to the direct interaction between local dipoles induced by the substituents and complementary dipoles in neighboring aromatic rings (and not more global quadrupole effects), regardless of the electronic-donating or electron-withdrawing nature of substituents. The specific dipolar interactions are of primary importance, and thus each novel system needs to be considered on this basis.29,60,61 Further, Wheeler and Houk argued that in a binary aromatic system containing electron withdrawing or electron donating substituents, stabilizing interactions between aromatic groups depend on the relative position of substituents on opposing rings and that the total contribution from substituents to π−π interactions will be additive and transferable. These theoretical predictions have been supported experimentally.62-64 These competing explanations regarding the fundamental nature of aromatic π−π interactions complicate understanding of the specific role of aromatic interactions in promoting self-assembly processes. Recent structural data from Fmoc-4-NO2-Phe crystalline nanotubes


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10 derived from hydrogel fibrils suggests that π−π interactions that are both quadrupolar and dipolar in nature may play a role in stabilizing these materials.27 Fmoc-Fmoc interactions adopt an edgeto-face orientation that is consistent with favorable quadrupole overlap of these molecules (Figure 1A). However, the side chain benzyl groups interact in a manner that is consistent with favorable dipolar overlap between atoms in the σ-system of neighboring rings (Figure 1B). In an effort to understand the role of aromatic π−π interactions involving the benzyl side chain groups of Fmoc-Phe derivatives on molecular assembly processes, we report herein an assessment of the effect of side chain substitution on the self- and coassembly of various FmocPhe analogs. Specifically, Fmoc-Phe derivatives containing electron withdrawing groups (NO2, CN, F) and electron donating groups (NH2, OH, CH3) at the para position of the benzyl aromatic ring were studied (Figure 2). These substituents exert variable effects on the aromatic ring via inductive (through-σ-bond), π-resonance, and field (through-space) effects. Each derivative was assessed for the effects of side chain substituents on either the self-assembly of the derivative or the coassembly of the derivative in combination with other analogs. It was found that both selfassembly and coassembly occurred most efficiently when Fmoc-Phe derivatives possessed complementary dipolar charges consistent with the packing mode shown in Figure 1B. High-resolution structures were also obtained when self-assembled fibril hydrogels of Fmoc-para-methylphenylalanine (Fmoc-4-CH3-Phe) and coassembled fibrils of Fmoc-4-NO2Phe and Fmoc-para-cyanophenylalanine (Fmoc-4-CN-Phe) underwent gel fibril to crystal transitions as has previously been reported for Fmoc-4-NO2-Phe.27 The resulting structural data is consistent with packing of the side chain benzyl groups stabilized by π−π interactions that feature similar complementary dipolar interactions to those found in Fmoc-4-NO2-Phe materials. Collectively, these findings clarify the π−π effects that promote self-assembly of Fmoc-Phe


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11 derivatives and provide cues for the design of next-generation materials from these types of molecules.28

Results and Discussion Electrostatic potentials (ESPs) of substituted toluenes (para X-toluene) that are analogous to the benzyl side chain of Fmoc-para-X-Phe analogs were generated to illustrate the electronic perturbation effects of these substitutions on the aromatic ring (Figure 2). Molecular volumes were also calculated in order to provide insight into the potential effects that the sterics of the various substituents may have on intermolecular interactions. The ESPs indicate that substitution with electron withdrawing groups (NO2, CN, F) perturbs the quadrupole electronics via πresonance and local dipole electronics inductively through the σ-bond framework and via through-space field effects. The primary effect of the electron withdrawing substituents on the quadrupole is to reduce the overall negative charge of the π-system. Conversely, substitution with electron donating groups (NH2, OH, CH3) enhances the negative quadrupole of the aromatic ring in addition to exerting local dipole effects.


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Figure 2.

Computed electrostatic potentials and molecular volumes of para-X-toluene

derivatives (Hartree-Fock, 6-31G* basis set with a scale of -100 to +100 kJ mol-1 using Spartan software). Red indicates areas of greater electron density and blue indicates areas of lower electron density.

Wheeler et al. have suggested that the substituent-induced changes in ESP do not necessarily reflect the local changes in the π-electron distribution.65 Instead, ESPs are most useful in understanding the through-space effects of the substituents. Similarly, Politzer et al. have shown that in nitrobenzene the π-resonance component of the ESP is negligible compared to inductive/field effects.66 This is consistent with strong inductive electron withdrawing ability of NO2 substituent appended to aromatic ring. Thus, it is also useful to consider the magnitude of the effects of C-X bond polarization on the atoms in the aromatic system in order to gain insight


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13 into possible dipolar effects of these substituents on intermolecular π-π interactions. C-X bond polarization effects were quantitatively analyzed using the Hartree-Fock method (6-31G* basis set) (Table 1). The calculations indicated that the magnitude of δ- for the X substituent and δ+ for the attached C (ipso) is greatest for the C-NO2 bond relative to other electron withdrawing substituents and for the C-NH2 bond relative to the other electron donating substituents. The effects of these substituents on the local charges of the atoms in the σ-framework of the aromatic rings may have significant impacts on local dipole effects in π-π interactions between neighboring rings, as suggested by the orientation of benzyl groups in the Fmoc-4-NO2-Phe structure (Figure 1). The trend observed for C-X bond polarization is roughly proportional to calculated molecular volumes for substituted toluene. According our previous studies on Fmoc4-X-Phe variants, the significant changes in molecular volumes exert effects in both rate of molecular self-assembly and fibril morphology.48 The data from these calculations will be correlated with self- and coassembly properties of the Fmoc-Phe analogs studied herein to gain further insight into the nature of π-π interactions involving the benzyl side chain.

Table 1. Calculated C-X bond polarization in 4-X-toluene derivatives (Hartree-Fock, 6-31G* basis set).

Compound 4NO2-toluene 4CN-toluene 4F-toluene 4NH2-toluene

C-X (Y) bond polarization δ+/- of C δ+/- of X and (Y) -0.119 0.055 0.423 0.504

0.888 (-0.492) 0.274 (-0.429) -0.243 -0.927 (0.371)

C-H bond polarization δ+/- of Co and Cm δ+/- of Ho and Hm -0.073 -0.133 -0.321 -0.361

-0.345 -0.345 -0.241 -0.257

0.168 0.175 0.198 0.189


0.187 0.188 0.179 0.177

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14 4OH-toluene 0.498 -0.660 (0.450) -0.271 -0.358 4CH3-toluene 0.381 -0.729 (0.192) -0.293 -0.293 4H-toluene -0.197 0.141 -0.077 -0.344 Co ortho carbon, Ho ortho hydrogen, Cm meta carbon, Hm meta hydrogen

0.197 0.177 0.131

0.205 0.177 0.174

Self-assembly/coassembly conditions. The self-assembly properties of each Fmoc-Phe derivative and the coassembly properties of various mixtures of these derivatives were assessed in order to correlate changes in the aromatic benzyl group to assembly propensity. Coassembly was assessed for equimolar mixtures of electron withdrawing-electron withdrawing, electron donating-electron donating, and electron donating-electron withdrawing pairing patterns. Also, the coassembly of the parent Fmoc-Phe with each Fmoc-Phe derivative was also assessed. As a control study, self-assembly of both EWG and EDG substituted Fmoc-Phe into supramolecular fibril architectures was studied. Self-assembly was initiated by dilution of a concentrated DMSO solution of the Fmoc-Phe derivative (247 mM) into unbuffered water (final concentration was 4.9 mM Fmoc-Phe derivative in 2% DMSO/H2O, v/v). Upon dilution, formation of a colloidal suspension is observed; for Fmoc-Phe derivatives that assemble, this suspension transforms into an optically transparent hydrogel within minutes. For Fmoc-Phe derivatives that do not effectively assemble, formation of an amorphous precipitate is observed over time. Effective coassembly was characterized by formation of transparent hydrogels as described for selfassembly. Self-assembly of Fmoc-4-X-Phe derivatives. Self-assembly of the Fmoc-4-X-Phe derivatives shown in Figure 1C was assessed in order to gain insight into benzyl π-π effects on assembly propensity. We found that each of these derivatives effectively self-assembled into dense networks of amyloid-like fibrils at 4.9 mM. With the exception of Fmoc-Phe, each of the derivatives formed optically transparent hydrogels. The time required for transition from a


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15 colloidal suspension to a transparent gel ranged from ~30 sec (Fmoc-4-NO2-Phe and Fmoc-4-FPhe) to ~24 h (Fmoc-4-NH2-Phe). Fmoc-Phe formed opaque (as opposed to optically transparent) gels after ~30 min that were significantly weaker than those formed by the other Fmoc-Phe derivatives studies herein. Table 2. Clarification times, fibril dimensions, and rheological viscoelasticity for selfassembly/hydrogelation of Fmoc-4-X-Phe derivatives. Compound

Clarification time (min)a

appearance b

Fibril diameter (nm)

G′ (Pa)

G″ (Pa)

Fmoc-4-NO2-Phe Fmoc-4-CN-Phe Fmoc-4-F-Phe Fmoc-4-NH2-Phe Fmoc-Tyr (4-OH) Fmoc-4-CH3-Phe Fmoc-Phe

~0.5 5 ~0.5 > 24 h 15 10 25

TG TG TG TG TG TG OG

12 ± 2 25 ± 4 26 ± 3 11 ± 2 13 ± 2 21 ± 2 295 ± 84

410 ± 18 140 ± 21 102 ± 7 527 ± 47 506 ± 55 280 ± 26 39 ± 3

66 ± 4 17 ± 6 9±3 61 ± 5 59 ±13 53 ±11 5±1

a

Time required for optical transition from a opaque suspension to a transparent or opaque

hydrogel. bTG, transparent gel (stable to vial inversion); OG, opaque gel (stable to vial inversion). TEM images reveal the morphology of the resulting self-assembled fibrils (Figure 3). The average dimensions of the smallest observable fibrils for each self-assembly mixture are summarized in Table 2. The fibrils formed ranged in size from ~10–300 nm in diameter. It was found that prolonged incubation led to bundling of the fibrils into structures with wider diameters. Interestingly, the relatively monodisperse Fmoc-4-NO2-Phe has previously been shown to undergo a spontaneous transition from hydrogel fibrils to a crystalline state.27 Fmoc-4CH3-Phe fibrils also undergo a similar fibril lamination process over hours to days following hydrogelation that results in a transition from gel fibrils to crystalline structures (Figure S1). TEM images obtained over time show a gradual transition from the initially formed narrow hydrogel fibrils to larger aggregates by alignment and lamination of the fibrils, culminating in


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16 the formation of crystalline microtubes, exactly as previously observed for Fmoc-4-NO2-Phe.27 High-resolution X-ray diffraction (XRD) analysis of these crystals gives significant insight into the nature of benzyl π-π interactions and will be discussed in detail in a later section.

Figure 3. A. Chemical structure of Fmoc-4-X-Phe derivatives. B–H. TEM images of fibrils formed by the self-assembly of each Fmoc-4-X-Phe derivative. B. Fmoc-4-NO2-Phe; C. Fmoc-4CN-Phe; D. Fmoc-4-F-Phe; E. Fmoc-Phe; F. Fmoc-4-NH2-Phe; G. Fmoc-Tyr; H. Fmoc-4-CH3Phe. The unsubstituted parent Fmoc-Phe exhibited unique assembly characteristics among the analogs studied herein. In contrast to the other derivatives, which formed optically transparent hydrogels upon dilution into water, Fmoc-Phe instead formed weak opaque hydrogels in which the network of assembled material failed to become fully solvated. These assembled materials were notably larger than the fibrils formed from the other derivatives, with average diameters of nearly 300 nm (Figure 3E). These large structures are inflexible in appearance, and the weakness


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17 of the resulting gel network can most likely be attributed to the inability of these structures to entangle into a network to efficiently entrap solvent. Fmoc-4-F-Phe fibrils also underwent distinct morphological changes over time. After short incubation times (1-2 h) Fmoc-4-F-Phe self-assembles into both helical fibrils with a mean diameter of 62 ± 6 nm and non-helical fibrils with diameters of 150 ± 16 nm. The helical fibrils display left handed periodicity with a pitch of 251 ± 27 nm. In addition, these fibrils were also found to undergo extensive bundling over time, but the resulting structures failed to transition to crystalline states, as was observed for Fmoc-4-NO2-Phe and Fmoc-4-CH3-Phe. The degree of fibril bundling and the transition to crystalline forms did not correlate with the electron donating/withdrawing nature of the substituent. Thus, it is expected that the varying higher order aggregation behavior of the initially formed fibrils may be due to steric effects exerted by the para substituents. We have previously reported that the position of halogen substitution on the benzyl group of Fmoc-Phe derivatives correlates with the rate of assembly and hydrogelation for these derivatives.67 Halogen substitution at the para position resulted in more rapid rates of assembly compared to ortho or meta substitution. Here we find that the rates of assembly (as approximated by the times required for transition from a colloidal suspension to an assembled hydrogel, Table 2) for the Fmoc-Phe derivatives assessed correlates generally with the electron withdrawing or donating nature of the substituent. Derivatives substituted with electron withdrawing groups (NO2, CN, F) were found to form hydrogels more rapidly (0.5–5 min) than Fmoc-Phe (25 min) and the electron rich derivatives (NH2, OH, CH3) (10 min–24 h). The extremely rapid selfassembly and hydrogelation of the electron deficient Fmoc-Phe analogs may be due to the


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18 lowering of electrostatic repulsion between aromatic π-systems in neighboring benzyl groups within assembled fibrils, consistent with the Hunter/Sanders electrostatic model. The trends for the rates of hydrogelation of these derivatives may also be explained by local dipole effects due to polarization of the σ-framework by the C-X(Y) substituent (Table 1 provides calculated charges for the atoms in the benzyl ring). Our recent study detailing the dynamic transition of Fmoc-4-NO2-Phe hydrogel fibrils into crystalline microtubes provided structural data indicating that a 4-NO2 O (δ−) on the benzyl side chain specifically interacts with the electropositive ortho H (Ho) (local C···H dipole) on the nearby vertex of the opposing ring ((δ-)O···(δ+)H-C) (Figure 1B).27 In addition to substituent-associated local dipole interactions, the ortho C-H dipole (Ho) interacts with the meta C-H dipole (Cm) on the opposing ring. These interactions stabilize a parallel but offset arrangement between the benzyl side chain groups that is propagated along the fibrillar axis in the context of the assembled material. These interactions indicated by the Fmoc-4-NO2-Phe crystal structure27 are consistent with specific dipolar effects as predicted by the Wheeler and Houk local interaction model. If this packing architecture is common to each of the Fmoc-Phe derivatives studied herein, local dipole effects may more accurately explain the relative rates of hydrogelation. The relatively slow hydrogelation of Fmoc-4-NH2-Phe may be due to unfavorable interactions between the local dipole of the N-H (H is +0.371) and the meta C-H (Hm is +0.177) of opposing benzyl rings in the assembled state. This δ+/δ+ interaction is common for each of the electronrich Fmoc-Phe derivatives. In contrast, stabilizing interactions between the benzyl substituent (δ) and Hm (δ+) (see Table 1 for calculated charges) may accelerate the more rapid gelation of the electron deficient derivatives.


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19 The electronic structures of each Fmoc-Phe hydrogel were characterized using CD spectroscopy (Figure S2, Supporting Information). Each hydrogel exhibited characteristic CD signals between 200–220 nm (n-π* transitions due to benzyl-benzyl interaction) and 270–310 nm (π−π∗ transitions between offset face-to-face stacked Fmoc groups). These spectra are consistent with previously reported spectra for assembled Fmoc-Phe-derived hydrogels9,10 and indicate a packing architecture that is common to all the derivatives studied herein, despite some minor spectroscopic differences between the various derivatives. The Fmoc-4-CN-Phe and Fmoc-4-FPhe spectra exhibit a moderate red shift compared to other variants and the Fmoc-4-CN-Phe hydrogel spectrum had only a weak absorbance in the Fmoc-Fmoc region from 270–310 nm. A blue-shifted peak at 264 nm in the Fmoc-4-NH2-Phe hydrogel may indicate subtle perturbation in the Fmoc-Fmoc stacking interactions for this derivative. As has been previously observed, the positive and negative orientation of the various signals shifts in some cases; this has been seen to occur even as a function of concentration for hydrogels of the same assembly motif.8 Thus, while moderate differences observed in these spectra may indicate subtle perturbation in monomer packing for the various hydrogels, the general similarity of the spectra suggests a common packing structure for each of the hydrogels. The viscoelasticity of each hydrogel was characterized using oscillatory rheology dynamic frequency sweep experiments. The storage (or elastic) modulus (G′), indicating the rigidity of the gel, and loss modulus (G″), representing the viscous component of the sample, were measured in the linear viscoelastic region by an oscillatory frequency sweep from 0-50 rad s-1 at 0.2% strain (Figure S2.D-F, Supporting Information). The linear viscoelastic region was determined by a prior strain sweep experiment for each sample. The measured G′ and G″ of these hydrogels were independent of the applied frequency, consistent with rigid gel formation.


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20 It is apparent that the gel rigidity, fibril diameter, and rate of hydrogelation are loosely correlated (Table 2). The rate of hydrogelation was directly correlated to gel rigidity (faster rates gave generally weaker gels), while fibril diameter was inversely proportional to the observed mechanical rigidity of hydrogel (narrow fibrils gave generally more rigid gels). For example, the Fmoc-4-NH2-Phe hydrogel has the highest G′ value (527 ± 47 Pa), the narrowest fibril diameter (11 ± 2 nm), and the slowest rate of gelation (> 24 h). It is possible that at faster rates of gelation, efficient fibril-fibril entanglement is impaired partly because these fibrils were observed to undergo fibril bundling to a larger extent, giving assembled materials with reduced flexibility. The storage modulus (G′) for the Fmoc-4-X-Phe hydrogels appears to also correlate with the electronic nature of the substituent. Hydrogels of derivatives with electron donating substituents generally have higher G′ values compared to derivatives with electron withdrawing substituents. Within each series, the gel rigidity decreases as a function of electron donating capabilities (NH2 > OH > CH3) and the strength of electron withdrawing abilities (NO2 > CN > F). However, the overall trend for the observed gel rigidity as indicated by G′ (X = NH2 > OH > NO2 > CH3 > CN > F > H) does not perfectly correlate to the electrostatics of the substituent or the rate of hydrogelation (NO2 > F > CN > CH3 > OH > NH2). This implies that both steric and electronic effects exert significant effects in the self-assembly process and reinforces the notion that the relationship between self-assembly/hydrogelation propensity and the emergent properties of the resulting hydrogels are as yet poorly understood. Coassembly of Fmoc-Phe/Fmoc-4-X-Phe. We have previously reported the coassembly of Fmoc-Phe with halogenated Fmoc-Phe derivatives to form hybrid hydrogels. Fmoc-Phe was found to efficiently coassemble with the highly fluorinated Fmoc-F5-Phe and other monohalogenated Fmoc-Phe derivatives.68 Herein, we also assess equimolar (4.9 mM total amino


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21 acid) coassembly of Fmoc-Phe with each Fmoc-4-X-Phe derivative under consideration in order to gain further insight into how the electron-donating/electron-withdrawing capability of the substituent influences coassembly and hydrogelation. These studies provide further insight into the nature of benzyl π-π interactions in promoting self-assembly and hydrogelation of these molecules. As with self-assembly, coassembly is characterized by a transition from a colloidal suspension of Fmoc-Phe derivatives (formed upon dilution of a DMSO solution of the coassembly mixture into water) into a transparent gel (Table 3). With the exception of the FmocTyr/Fmoc-Phe coassembly pair, each of the Fmoc-Phe/Fmoc-4-X-Phe mixtures efficiently forms coassembled hydrogels. The electron deficient derivatives (X = NO2, CN, F) form hydrogels with Fmoc-Phe at significantly faster rates (5–75 min after dilution into water) than the electron rich derivative (X = NH2, CH3)/Fmoc-Phe pairs (75 min–12 h). The Fmoc-Tyr/Fmoc-Phe pair fails to form a self-supporting hydrogel, and assembled fibrils are only observed after weeks of incubation. These observations mirror the faster hydrogelation observed by self-assembly of the electron deficient derivatives relative to the electron rich derivatives.

Table 3. Coassembly behavior, fibril dimensions, and viscoelastic rheological properties of Fmoc-Phe/Fmoc-4-X-Phe pairs (4.9 mM total amino acid, equimolar stoichiometry). Compound


Appearanceb

Fmoc-4-NO2-Phe/Fmoc-Phe Fmoc-4-CN-Phe/Fmoc-Phe Fmoc-4-F-Phe/Fmoc-Phe Fmoc-4-NH2-Phe/Fmoc-Phe Fmoc-Tyr/Fmoc-Phe Fmoc-4-CH3-Phe/Fmoc-Phe

~75 ~12 ~5 >12 h N/A ~75

TG TG TG TG OS TG

Fibril diameter (nm) 21 ± 3 23 ± 3 20 ± 3 8±1 NA 18 ± 2

a

G′ (Pa)

G″ (Pa)

12 ± 1 35 ± 2 587 ± 30 1048 ± 23 NA 321 ± 14

0.6 ± 0.1 4±1 92 ± 22 51 ± 7 NA 26 ± 1

Time required for optical transition from a opaque suspension to a transparent hydrogel. bTG, transparent gel (stable to vial inversion); OS, opaque suspension (material fails to clarify into a self-supporting hydrogel).


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22 The fibrils that constitute the Fmoc-Phe/Fmoc-4-X-Phe hybrid hydrogel networks were characterized using TEM images (Figure 4). The fibrils formed were of similar morphology, with average diameters of ~20 nm (Table 3). There were a few exceptions to this general trend. The slowly assembling Fmoc-Phe/Fmoc-4-NH2-Phe pair formed narrower fibrils that were 8 nm in diameter. In addition, the Fmoc-Phe/Fmoc-Tyr pair that failed to form a hydrogel did not exhibit fibrils, but instead formed spherical, micelle-like aggregates (Figure 4D). After extended incubation (weeks) these micelles matured into fibrils (Figure S3). The initial micelle-like aggregates have been previously observed in slowly assembling Fmoc-Phe derivatives69 and are most likely an early, intermolecular molten globule-like aggregate that formed by hydrophobic collapse of the Fmoc-Phe derivatives and precedes fibril formation. The more rapid hydrogelation of the electron-poor/Fmoc-Phe pairs is consistent with dipolar interactions between the ortho C-H dipole (Ho) and the meta C-H dipole (Cm) and between the X-related dipole and the ortho H (Ho) (Table 1), consistent with a packing structure as shown in Figure 1.


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23

Figure 4. TEM images of the fibrils formed by the coassembly of Fmoc-Phe and electrondeficient or electron-rich Fmoc-Phe derivatives. A. Fmoc-Phe and Fmoc-4-NO2-Phe; B. FmocPhe and Fmoc-4-CN-Phe; C. Fmoc-Phe and Fmoc-4-F-Phe; D. Fmoc-Phe and Fmoc-Tyr; E. Fmoc-Phe and Fmoc-4NH2-Phe; F. Fmoc-Phe and Fmoc-4-CH3-Phe. It was also observed that the hydrogel formed by coassembly of Fmoc-Phe with Fmoc-4CH3-Phe exhibited precipitation after 12–24 h of incubation. This is consistent with our previous observation of a spontaneous transition from hydrogel fibrils to crystalline microtubes from hydrogels of Fmoc-4-NO2-Phe. Examination of the precipitate formed from Fmoc-Phe/Fmoc-4CH3-Phe hydrogels showed that crystal formation from the initial hydrogel fibrils had also occurred in this mixture (Figure S4). In contrast to Fmoc-4-NO2-Phe, the crystals derived from Fmoc-Phe/Fmoc-4-CH3-Phe hydrogels were, unfortunately, not of adequate quality to facilitate high-resolution structural analysis.


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24 Electronic structures of coassembled Fmoc-Phe/Fmoc-4X-Phe hydrogels networks were characterized by CD spectroscopy (Figure S5, Supporting Information). It was observed that each of the coassembly mixtures that form hydrogels had characteristic CD signatures at 270– 310 nm and 200–210 nm. This indicates that the coassembled structures possess similar structural arrangements within the fibril as observed for the self-assembly of individual Fmoc-4X-Phe variants. The magnitude of the CD signal of Fmoc-4-NH2-Phe/Fmoc-Phe was significantly weaker, perhaps due to the slow assembly kinetics. The Fmoc-Tyr-Phe/Fmoc-Phe pair exhibited an exceptionally weak CD signal, consistent with the absence of assembled cofibrils. Generally, the Fmoc-4-X-Phe/Fmoc-Phe hybrid hydrogels were significantly weaker than the previously discussed self-assembled hydrogels (Figure S6, Table 3). The observed gel rigidity within the Fmoc-Phe/Fmoc-4-X-Phe coassembled series is X = NH2 > F > CH3 > CN ~ NO2 > OH; the most rigid gel (Fmoc-Phe/Fmoc-4-NH2-Phe) has a G′ value of 1048 Pa and the weakest gel (Fmoc-Phe/Fmoc-4-NO2-Phe) has a G′ value of only 12 Pa. The trend for observed viscoelasticity does not strongly correlate with the electronic nature of the substituent. For example, among the electron-deficient coassembled materials Fmoc-4-NO2-Phe/Fmoc-Phe (G′ = 21 ± 3 Pa) and Fmoc-4-CN-Phe/Fmoc-Phe (G′ = 23 ± 3 Pa) are weaker gels while Fmoc-4-F-Phe /Fmoc-Phe (G′ = 587 ± 30 Pa) is significantly more rigid. The same lack of correlation exists in the electron rich coassembled gels, with Fmoc-4-NH2-Phe/Fmoc-Phe (G′ = 1048 ± 23 Pa) and Fmoc-4-CH3-Phe (G′ = 321 ± 14 Pa) forming stronger gels while Fmoc-Tyr/Fmoc-Phe fails to form hydrogels at all. The rate of hydrogelation correlates more strongly (and directly) with the electronic nature of the X substituent than do the emergent properties of the resulting hydrogels,


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25 which are, generally, inversely correlated to the rate of hydrogelation. With some exceptions, the faster forming coassembled gels tend to be significantly weaker. Coassembly of electron-deficient and electron-rich Fmoc-Phe derivatives. The coassembly of equimolar mixtures of electron-deficient and electron-rich Fmoc-4-X-Phe derivatives was also assessed in order to determine if quadrupolar effects may play a stabilizing role in the coassembly of electronically matched benzyl groups. Equimolar mixtures (4.9 mM total amino acid) of Fmoc-4-X-Phe derivatives with electron donating substituents (X = NH2, OH, CH3) and electron withdrawing substituents (X = NO2, CN, F) were coassembled in the combinations shown in Table 4. Table 4. Coassembly behavior, fibril morphology, and comparative rheological viscoelasticity of electron-deficient/electron-rich Fmoc-4-X-Phe pairs. Compound


Appearanceb

Fmoc-4-NO2-Phe/Fmoc-4-NH2-Phe Fmoc-4-NO2-Phe/Fmoc-Tyr Fmoc-4-NO2-Phe/Fmoc-4-CH3-Phe

~8 ~11 ~6

TG TG TG

Fmoc-4-CN-Phe/Fmoc-4-NH2-Phe Fmoc-4-CN-Phe/Fmoc-Tyr Fmoc-4-CN-Phe/Fmoc-4-CH3-Phe Fmoc-4-F-Phe/Fmoc-4-NH2-Phe Fmoc-4-F-Phe/Fmoc-Tyr Fmoc-4-F-Phe/Fmoc-4-CH3-Phe

> 12 h ~ 45 ~7 ~4 ~3 ~1

TG TG TG TG TG TG

Fibril diameter (nm) 8±1 18 ± 3 25 ± 3, 96 ± 21 21 ± 4 11 ± 2 18 ± 4 10 ± 2 12 ± 3 22 ± 3, 60 ± 4

G′ (Pa)

G″ (Pa)

680 ± 39 832 ± 90 74 ± 11

37 ± 10 124 ± 36 13 ± 6

1585 ± 80 199 ± 35 111 ±16 642 ± 44 585 ± 48 84 ± 5

62 ± 12 42 ± 20 13 ± 5 168 ± 7 78 ± 14 14±3

a

Time required for optical transition from a opaque suspension to a transparent hydrogel. bTG, transparent gel (stable to vial inversion).

Each of the electron-rich/electron-deficient Fmoc-4-X-Phe pairs underwent effective coassembly and hydrogelation. The rates of hydrogelation were fast for the majority of these pairs, with hydrogelation times ranging from 1–11 min. Exceptions were the Fmoc-4-CNPhe/Fmoc-Tyr pair and the Fmoc-4-CN-Phe/Fmoc-4-NH2-Phe pair, which required ~45 min and >12 h respectively to clarify from colloidal suspensions to optically transparent gels. The


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26 relatively fast hydrogel formation for coassembled electron-deficient/electron-rich Fmoc-4-XPhe variants is consistent with the formation of stabilizing π-π interactions between complementary pairs. This may be explained by both quadrupolar and dipolar effects. TEM images of each equimolar mixture of the various electron-deficient/electron-rich pairs were acquired to characterize the morphology of the hybrid cofibrils (Table 4, Figure 5). The fibrils formed generally ranged from 8 nm to 18 nm in diameter. The Fmoc-4-NO2Phe/Fmoc-4-CH3-Phe (Figure 5G) and Fmoc-4-F-Phe/Fmoc-4-CH3-Phe (Figure 5I) pairs showed several cofibril morphologies, narrow (~20 nm in diameter) and wide (60–100 nm).


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27 Figure 5. TEM images of the fibrils formed by the coassembly of electron-deficient and electron-rich Fmoc-4-X-Phe pairs. A. Fmoc-4-NO2-Phe/Fmoc-4-NH2-Phe; B. Fmoc-4-CNPhe/Fmoc-4-NH2-Phe; C. Fmoc-4-F-Phe/Fmoc-4-NH2-Phe; D. Fmoc-4-NO2-Phe/Fmoc-Tyr; E. Fmoc-4-CN-Phe/Fmoc-Tyr; F. Fmoc-4-NO2-Phe/Fmoc-Tyr; G. Fmoc-4-NO2-Phe/Fmoc-4-CH3Phe; H. Fmoc-4-CN-Phe/Fmoc-4-CH3-Phe; I. Fmoc-4-F-Phe/Fmoc-4-CH3-Phe. The electronic structures of these hybrid materials as characterized by CD spectroscopy are consistent with a common packing architecture for each of the hydrogel pairs (Figure S7.AC, Supporting Information). As for the previously discussed materials, the CD spectra for the electron-rich/electron-deficient pairs displayed characteristic absorptions from 200–220 nm and 270–310 nm. Some subtle perturbation in the stacking interactions between opposing rings may account for minor differences observed in the spectra of the various hydrogels. These differences include subtle red/blue peak shifts and alteration of directionality of the signal. CD measurements are sensitive to both the chiral environment and to the electronic environment. Thus, these slight differences do not necessarily reflect differences in the chiral twist of the hybrid fibrils, but may also be due to changes in the electronic interactions between the functional groups in the materials. The CD spectra are most useful in the indication that each of the cofibril hydrogels are similar in structure. The rheological strength of each coassembled hydrogel was determined by dynamic frequency sweep experiments (Table 4, Figure S7.D-F). These experiments were conducted in the linear viscoelastic region as determined by prior strain sweep experiments. In general, the hydrogels formed from the electron-rich/electron-deficient Fmoc-4-X-Phe pairs (G′ values from ~75–1600 Pa at 4.9 mM total amino acid) were significantly more rigid than those found by coassembly with Fmoc-Phe. The Fmoc-4-CN-Phe/Fmoc-4-NH2-Phe hydrogel displays the


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28 highest gel rigidity (G′ = 1585 ± 80 Pa and G′/G″ ratio is ~ 20), while Fmoc-4-NO2-Phe/Fmoc-4CH3-Phe (G′ = 74 ± 11 Pa and G′/G″ ratio is ~ 7) shows the lowest. The G′ and G″ measurements are independent of the applied frequency consistent with the formation of a rigid hydrogel. The weakest gel in this series, formed by Fmoc-4-NO2-Phe/Fmoc-4-CH3-Phe, was observed to precipitate at oscillatory frequency above 50 rad s-1, indicating a relatively unstable network. It is interesting to note that the weakest gels in this series all included Fmoc-4-CH3-Phe in the coassembly mixture. This observation implies that steric effects (CH3 is non-planar) in addition to electronic properties of the aromatic substituents influence the emergent properties of the resulting hydrogels. Coassembly of electron-deficient/electron-deficient and electron-rich/electron-rich Fmoc-4-X-Phe derivatives. Finally, we examined each of the possible combinations for coassembly of electron-deficient/electron-deficient and electron-rich/electron-rich Fmoc-4-XPhe derivatives. If quadrupolar effects dominate the π−π interactions between benzyl side chains, the pairing of derivatives with electron-withdrawing substituents may enhance intermolecular π−π interactions by lowering electrostatic repulsion between electron clouds. Conversely, electron-donating substituents may hinder aromatic interactions by increasing these repulsive quadrupolar effects. If dipolar interactions between specific elements of the benzyl side chain are the primary stabilizing π−π force then these “like” pairings will depend on the dipole alignments. If these alignments are similar to those observed for the self-assembly of Fmoc-4-NO2-Phe, then the effects of these pairings on coassembly and hydrogelation will be more subtle. Based on the observations discussed in the previous sections, dipolar effects may primarily influence coassembly and hydrogelation rates as opposed to exerting dramatic effects on overall assembly propensity.


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29 Each of the mixtures tested formed coassembled hydrogels (total Fmoc-4-X-Phe concentrations of 4.9 mM) (Table 5). The electron-deficient/electron-deficient pairs (combinations of Fmoc-4-NO2-Phe, Fmoc-4-CN-Phe, and Fmoc-4-F-Phe) clarified from colloidal suspensions to transparent, self-supporting hydrogels at significantly faster rates (1–17 min) than the electron-rich/electron rich pairs (Fmoc-4-NH2-Phe, Fmoc-Tyr, and Fmoc-4-CH3Phe) (140 min–24 h). Such dramatic differences in the rates of gelation likely indicate that quadrupolar effects play some role in the benzyl π−π interactions in these coassembled materials since these theories predict that electron-deficient aromatic rings interact more readily due to decreased electron repulsion between the π-systems of proximate aromatic groups. However, significant dipolar effects cannot be ruled out and structural evidence discussed in the next section clearly indicates that the alignment of these benzyl groups is more consistent with stabilizing dipolar interactions between the rings.

Table 5. Coassembly behavior, fibril morphology, and rheological viscoelasticity of electrondeficient/electron-deficient and electron-rich/electron-rich Fmoc-4-X-Phe pairs. Compound


Appearanceb

Fmoc-4NO2-Phe /Fmoc-4CN-Phe Fmoc-4NO2-Phe/Fmoc-4F-Phe Fmoc-4CN-Phe/Fmoc-4F-Phe

~2 ~ 17 ~1

TG TG TG

Fmoc-4NH2-Phe/Fmoc-Tyr Fmoc-4NH2-Phe/Fmoc-4CH3-Phe Fmoc-Tyr/Fmoc-4CH3-Phe

>24 h ~140 >12 h

TG TG TS

Fibril diameter (nm) 18 ± 3 23 ± 2 19 ± 3, 59 ± 11 11 ± 3 9±2 8 ± 1, 17 ± 3

a

G′ (Pa)

G″ (Pa)

249 ± 26 225 ± 22 266 ± 12

32 ± 5 20 ± 6 26 ± 5

247 ± 20 1034 ± 49 NA

27 ± 2 89 ± 1 NA

Time required for optical transition from an opaque suspension to a transparent hydrogel. bTG, transparent gel (stable to vial inversion); TS, transparent solution.


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30 TEM images confirm that these mixtures form coassembled fibrils (Figure 6). For each combination, narrow fibrils ranging from 8 nm to 23 nm in diameter are observed. Several of the pairings form wider fibrils, either as the major morphology or in combination with narrow fibrils. The Fmoc-4-CN-Phe/Fmoc-4-F-Phe pair assembled into both narrow fibrils (19 ± 3 nm in diameter) and wider fibrils (59 ± 11 nm in diameter) (Figure 6C). The Fmoc-4-CH3-Phe/FmocTyr also pair provided fibrils of two distinct morphologies: narrow fibrils 8 ± 1 nm in diameter and wider fibrils 17 ± 3 nm in diameter (Figure 6F). Interestingly, the Fmoc-4-CN-Phe/Fmoc-4NO2-Phe mixture formed abundant coassembled fibrils (19 ± 3 nm) within 2 min; after extended incubation, these hydrogel fibrils undergo a spontaneous transition into crystalline microtubes (Figure 7F, Figures S8-S10). This crystallization process is similar to that observed for selfassembled Fmoc-4-NO2-Phe self-assembled hydrogels and the resulting crystalline microtubes are also similar in morphology. The structural analysis of these cocrystals and the insight they give into the nature of benzyl π-π interactions in the assembly of these materials will be discussed in the following section.


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31

Figure 6. TEM images of the fibrils formed by the coassembly of electron-deficient/ electrondeficient (A-C) and electron-rich/electron-rich (D-F) Fmoc-4-X-Phe derivatives. A. Fmoc-4NO2-Phe/Fmoc-4-CN-Phe; B. Fmoc-4-NO2-Phe/Fmoc-4-F-Phe; C. Fmoc-4-CN-Phe/Fmoc-4-FPhe; D. Fmoc-4-NH2-Phe/Fmoc-Tyr; E. Fmoc-4-NH2-Phe/Fmoc-4-CH3-Phe; F. FmocTyr/Fmoc-4-CH3-Phe. CD spectra obtained for the electron-deficient/electron-deficient and electronrich/electron-rich mixtures were consistent with a common packing architecture for each of the resulting hydrogels. The spectra each exhibited the characteristic absportion at 200-220 nm and at 270-310 nm (Figure S11.A-C and S12.B, Supporting Information). No significant deviation in CD signature was observed for any of the coassembled hydrogels. The viscoelestic properties of electron-deficient/electron-deficient and electronrich/electron-rich Fmoc-4-X-Phe coassembled mixtures were characterized by rheology dynamic


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32 frequency sweep experiments (Table 5, Figure S11.D-F, Figure S12.B, Supporting Information). Each of these hydrogels exhibited strikingly similar viscoelastic behavior, with G′ values of ~250 Pa and G′′ values of ~30 Pa. An exception was the Fmoc-Tyr/Fmoc-4-CH3-Phe mixture, which assembled into fibrils slowly (>12 h) but these fibrils failed to promote gelation in water. A second exception was the slowly forming (> 24 h) Fmoc-4-NH2-Phe/Fmoc-4-CH3-Phe hybrid hydrogel, which was found to be significantly more rigid (G′ ~1034 ± 49 Pa) than the other combinations of Fmoc-4-X-Phe. Interestingly, all Fmoc-4-NH2-Phe containing coassembly mixtures (regardless of the electronic nature of the partner derivative) display the highest gel rigidity; the Fmoc-4-NH2-Phe are typically found to be significantly more rigid that the others under consideration. The rate of assembly for these derivatives is also significantly slower. Perhaps the slower rates of assembly and hydrogelation provide a more integrated fibril network. It is also possible that the appended NH2 group can participate in stabilizing hydrogen bond interactions, both with solvent and between fibrils, which may account for the enhanced emergent hydrogel properties for these derivatives. A third possibility is that the slower rate of assembly for Fmoc-4-NH2-Phecontaining hydrogels subtly perturbs the packing orientation of the benzyl side chain, leading to imperfections or defects in the hydrogel network. These defects may provide nucleation sites for fibril entanglement and rigidification. Structural insight from high-resolution analysis of crystals derived from self- and coassembled fibrils. We have previously exploited high-resolution X-ray diffraction analysis of crystals obtained from the spontaneous transition from hydrogel fibrils to gain structural insight into the gel state. We have recently reported that rapidly formed Fmoc-4-NO2-Phe hydrogel fibrils fuse to form crystalline microtubes over time (Figure 7A-B).27 Herein, we have identified


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33 several additional Fmoc-Phe-derived systems that undergo a similar spontaneous gel to crystal transition: Fmoc-4-CH3-Phe (Figure 7C-D) and Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe (Figure 7DF) hydrogels were both observed to transform into crystalline microtubes over time. The rate of the Fmoc-4-CH3-Phe gel to crystal transition is exceptionally slow, requiring more than 5 months. The hybrid, coassembled Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe fibrils undergo a gel to crystal transition within 5–7 days. Each of these systems crystallizes significantly more slowly than the previously described Fmoc-4-NO2-Phe hydrogel,27 which transforms from fibrils to crystals in hours. Extrapolating packing interactions in hydrogel fibrils from crystal states of the same molecules must be approached carefully. Often, packing architectures between crystalline and gel states differ. For the hydrogelators examined herein, several factors suggest that the packing modes are similar (even if they are not identical). First, time-dependent TEM images indicate that the crystals are formed spontaneously by alignment and fusion and pre-existing hydrogel fibrils, not via fibril disassembly and reassembly into crystals. Second, the crystals and hydrogel fibrils are both formed in water without the need for solvent changes/alterations. Thus, it is likely that the basic packing architecture is preserved between the two states and that the crystals merely contain additional contacts that expand the growth of the one-dimensional fibrils in three dimensions. Regardless, without independent structures of fibril packing in the hydrogel state, the conclusions drawn from crystal states must be treated with some degree of caution. The coassembled crystals each exhibit similar hollow microtube morphology. This is presumably a function of the mechanism of crystallization, which occurs via alignment and lamination of the existing hydrogel fibrils as was observed in our previous work with Fmoc-4NO2-Phe.27 Previous studies have described the difficulty of crystallizing molecules that have


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34 hydrogelation properties.70,71 Crystal formation from the Fmoc-Phe derivatives studied herein is difficult to predict and was discovered serendipitously. Hydrogels formed from Fmoc-Phe derivatives are all observed to undergo destabilization (as evidenced by precipitation of the gel network) over extended periods ranging from weeks to months.72 The precipitates that form do not always exhibit crystalline properties and in cases in which the precipitates are crystalline, the resulting crystals are often not of adequate quality for high-resolution structural analysis (see Fmoc-4-CH3-Phe/Fmoc-Phe cocrystals described previously – Figure S4). Fmoc-4-NO2-Phe is especially prone to hydrogel-crystal transformation since the NO2 substituent participates in stabilizing interactions in the crystal. Perhaps the CH3 group in Fmoc-4-CH3-Phe sterically (although not electronically) exerts a similar effect.

Figure 7. A. TEM image of Fmoc-4-NO2-Phe self-assembled hydrogel fibrils. B. SEM image of Fmoc-4-NO2-Phe crystalline microtubes formed spontaneously from fibrils shown in A. C. TEM


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35 image of Fmoc-4-CH3-Phe self-assembled hydrogel fibrils. D. SEM image of Fmoc-4-CH3-Phe crystalline microtubes. E. TEM image of Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe coassembled hydrogel fibrils. F. SEM image of Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe coassembled crystalline microtubes. The structural topology and packing arrangement in Fmoc-4-NO2-Phe, Fmoc-4-CH3-Phe, and Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe crystals are strikingly similar (Figure 1 and Figure 8). High resolution X-ray diffraction analysis revealed the packing arrangement of Fmoc-4-CH3-Phe in the crystalline state (Figure 8A-B). The asymmetric unit (Figure 8A) contains one molecule of Fmoc-4-CH3-Phe, with all atoms in the general positions shown. The packing is accentuated by variety of contacts (see Supporting Information, Appendix 1 for a detailed analysis), including moderately strong hydrogen bonds (OH···O) with a distance 1.84 Å between carboxyl groups in laminated fibril units, weak hydrogen bonds (NH···O) within the strand (intramolecular interaction) with a distance 2.5 Å, and numerous weak non-conventional hydrogen bonds (CH···O) with distances 2.4 Å (Figure S13) In the previously described structure of Fmoc-4NO2-Phe, there are electrostatic contributions of O2N [δ+]···[δ-] O=C with a N···O distance of 2.844 Å, which is 0.23 Å shorter than the sum of the van der Waals radii. In the Fmoc-4-CH3Phe structure in which a methyl group has replaced the nitro group, that specific stabilizing contribution is not present. The analogous C···O distance in the Fmoc-4-CH3-Phe structure is 3.192 Å, which is slightly longer and essentially the sum of the van der Waals radii (rc + ro = 3.22 Å). The interaction between the methyl C-H and ortho C-H on adjacent benzyl rings has a distance 2.7 Å (Figure S13D) and is consistent with the local dipole interactions found in the Fmoc-4-NO2-Phe variant as shown in Figure 1B. This indicates that these specific intermolecular benzyl π-π interactions common to both structures. The recurrence of this packing motif is


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36 consistent with a dominant role for dipolar effects in the π-π interactions between benzyl groups in the self-assembly of Fmoc-Phe derivatives.

Figure 8. X-ray crystallographic data A. Single crystal unit cell of Fmoc-4-CH3-Phe; B. Basic Fmoc-4-CH3-Phe fibril unit; C. Single crystal unit cell of Fmoc-4-CN-Phe/Fmoc-4-NO2-Phe; B. Basic Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe fibril unit.


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37 The unit cell of the Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe cocrystals depicted in Figure 8C (see Appendix 2 in Supporting Information for a detailed analysis). The structure is formed by transition from fibrils composed of equimolar ratios of Fmoc-4-NO2-Phe and Fmoc-4-CN-Phe. The ratio of Fmoc-4-NO2-Phe and Fmoc-4-CN-Phe in the crystalline material, however, is 7:3 (NO2:CN). Thus, the incorporation of the monomeric units is not driven by specific complementary π-π interactions between the derivatives in this case, but occurs via more random insertion. This observation is not surprising, since the electronic character of these benzyl derivatives are similarly electron-deficient and there is no expected complementarity between the respective aromatic groups. The structure is similar to that of Fmoc-4-NO2-Phe in all respects. A variety of noncovalent interactions are observed, including moderately strong hydrogen bonds (OH···O) with a distance 1.81 Å, weak non-conventional hydrogen bonds (CH···O), and moderately strong intermolecular electrostatic interactions (O2N [δ+]···[δ-] O=C). (Figure S14) The benzyl-benzyl interactions involve contact between the electropositive ortho H (Ho) with the nearby vertex of the opposing ring ((δ-)O···(δ+)H-C) (identical to the arrangement shown in Figure 1B). The ortho C-H dipole (Ho) also interacts with the meta C-H dipole (Cm) on the opposing ring in the cocrystal as well. Collectively, these data strongly support a common packing architecture for all Fmoc-Phe assemblies in which benzyl-benzyl π-π interactions are consistent with specific stabilizing dipolar interactions between neighboring aromatic rings. Discussion. The self-assembly/coassembly and hydrogelation behavior of the Fmoc-4-XPhe derivatives reported herein provides meaningful insight into the nature of aromatic π-π interactions in the assembly of these molecules. Specifically, we have gained functional and structural insight regarding the specific role of π-π interactions between benzyl side chain functionality in Fmoc-Phe self-assembly. We have considered these effects in light of both


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38 quadrupolar and dipolar models for π-π interactions. Self-assembly and hydrogelation rates appear to be most consistent with dipolar effects. As shown in Figure 1B, structural data from Fmoc-4-NO2-Phe assembled materials indicate that the side chain benzyl groups are oriented so that the electropositive ortho H (Ho) interacts with the with the nearby electronegative O atom of the NO2 substituent ((δ-)O···(δ+)H-C). In addition, the ortho C-H dipole (Ho) interacts with the meta carbon dipole (Cm, δ-). The empirical data reported herein are consistent with these interactions being generally present in the assembly of Fmoc-Phe derivatives. The fastest rates of hydrogelation are observed when these respective dipoles are complementary in charge between neighboring aromatic groups. The slowest rates of hydrogelation are observed in instances where like charges interact in the positions of these dipoles. Structural data from crystals formed spontaneously by alignment and fusion of hydrogel fibrils support a central role for specific local dipolar effects in stabilizing the assembled structures. Structures from crystals of Fmoc-4-CH3-Phe and Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe are practically identical in packing architecture to that observed from Fmoc-4-NO2-Phe alone. In particular, the benzyl side chain groups are packed in an orientation that is consistent with dipolar interactions playing a more central role in the arrangement of the side chain groups than more global quadrupole effects. Quadrupole effects also clearly play a role in the stabilization of the Fmoc-Fmoc interactions based on the crystal structure data. In addition, pairs of electrondeficient/electron-deficient Fmoc-4-X-Phe derivatives coassemble and form hydrogels significantly more rapidly than electron-rich/electron-rich pairs. Specific dipole interactions alone cannot account for this behavior. Electronic effects derived from the electron-donating/electron-withdrawing character of para substituents on the benzyl group most closely correlate with rates of hydrogelation for the


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39 combinations examined herein. The emergent viscoelastic properties of the hydrogels are much more variable. There is no clear trend between the rigidity of the hydrogels and the electronics of the benzyl ring. Fmoc-4-NH2-Phe-containing gels were often found to be among the most rigid. This may be due to the much slower rate of assembly, although other hydrogels that formed slowly did not always result in stronger viscoelasticity. Fmoc-4-NH2-Phe hydrogels may be strong due to the hydrogen-bonding ability of the NH2 group or to the presence of defects in packing due to perturbation of dipolar interactions when this group is included. Other properties, such as energy of dissolution may also play a role in the observed trends in hydrogel rigidity.69,73,74 Additional effort is required to clearly understand the relationship between the emergent properties of hydrogels and the molecular structure of the assembly motif.

Conclusion Elucidation of the fundamental physicochemical properties that promote self-assembly and hydrogelation of simple organic molecules will facilitate movement from empirical discovery to rational design in the development of self-assembled materials. Herein we have presented empirical and structural data that lends insight into the nature of the role π-π interactions involving the benzyl side chain play in the self-assembly of Fmoc-Phe derivatives. The data is consistent with specific dipolar effects providing the major stabilizing interactions between adjacent benzyl groups, with global quadrupolar effects playing a subtler role. Highresolution structural data confirms the specific orientation of these interactions and lends confidence in a common packing architecture for all Fmoc-Phe derived hydrogels. This information provides significant design cues for the optimization of self-assembly of this class of hydrogelator by specific modification of the benzyl side chains. Future efforts will involve


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40 studies to more closely correlate the molecular structure of the hydrogel assembly motif to the emergent viscoelasticity of the resulting materials.

Acknowledgments We thank William Brennessel (University of Rochester, Department of Chemistry) for highresolution analysis and structure determination from crystal isoforms. We gratefully acknowledge Karen Bentley (University of Rochester Electron Microscopy Core) for her assistance with TEM and SEM imaging and Dr. Scott Kennedy for assistance with CD experiments. This work was supported by the NSF (DMR-1148836).

Supporting Information Experimental details and additional characterization data for hydrogels and crystals, including CD, rheology, additional TEM and SEM images, and supplementary data for X-ray crystallographic data are provided in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org.


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of Structure and Solvent in Molecular Gelation. Langmuir 2011, 27, 13248-13253. (62)

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the Structural Characterisation of Fibrous Proteins & Peptides using Fibre Diffraction. Chem. Soc. Rev. 2010, 39, 3445-3453. (63)

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Figure 1. Molecular packing of Fmoc-4-NO2-Phe in assembled fibrils and crystals. A. Packing architecture in the basic fibril unit that feature Fmoc-Fmoc and benzyl-benzyl aromatic interactions. B. Local dipole interaction between neighboring benzene rings in the assembled state. C. Chemical structures of Fmoc-Phe derivatives studied herein. 106x88mm (300 x 300 DPI)


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Figure 2. Computed electrostatic potentials and molecular volumes of para-X-toluene derivatives (HartreeFock, 6-31G* basis set with a scale of -100 to +100 kJ mol-1 using Spartan software). Red indicates areas of greater electron density and blue indicates areas of lower electron density. 160x152mm (300 x 300 DPI)


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Figure 3. A. Chemical structure of Fmoc-4-X-Phe derivatives. B–H. TEM images of fibrils formed by the selfassembly of each Fmoc-4-X-Phe derivative. B. Fmoc-4-NO2-Phe; C. Fmoc-4-CN-Phe; D. Fmoc-4-F-Phe; E. Fmoc-Phe; F. Fmoc-4-NH2-Phe; G. Fmoc-Tyr; H. Fmoc-4-CH3-Phe. 174x86mm (300 x 300 DPI)


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Figure 4. TEM images of the fibrils formed by the coassembly of Fmoc-Phe and electron-deficient or electron-rich Fmoc-Phe derivatives. A. Fmoc-Phe and Fmoc-4-NO2-Phe; B. Fmoc-Phe and Fmoc-4-CN-Phe; C. Fmoc-Phe and Fmoc-4-F-Phe; D. Fmoc-Phe and Fmoc-Tyr; E. Fmoc-Phe and Fmoc-4NH2-Phe; F. FmocPhe and Fmoc-4-CH3-Phe. 128x85mm (300 x 300 DPI)


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Figure 5. TEM images of the fibrils formed by the coassembly of electron-deficient and electron-rich Fmoc-4X-Phe pairs. A. Fmoc-4-NO2-Phe/Fmoc-4-NH2-Phe; B. Fmoc-4-CN-Phe/Fmoc-4-NH2-Phe; C. Fmoc-4-FPhe/Fmoc-4-NH2-Phe; D. Fmoc-4-NO2-Phe/Fmoc-Tyr; E. Fmoc-4-CN-Phe/Fmoc-Tyr; F. Fmoc-4-NO2Phe/Fmoc-Tyr; G. Fmoc-4-NO2-Phe/Fmoc-4-CH3-Phe; H. Fmoc-4-CN-Phe/Fmoc-4-CH3-Phe; I. Fmoc-4-FPhe/Fmoc-4-CH3-Phe. 127x128mm (300 x 300 DPI)


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Figure 6. TEM images of the fibrils formed by the coassembly of electron-deficient/ electron-deficient (A-C) and electron-rich/electron-rich (D-F) Fmoc-4-X-Phe derivatives. A. Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe; B. Fmoc-4-NO2-Phe/Fmoc-4-F-Phe; C. Fmoc-4-CN-Phe/Fmoc-4-F-Phe; D. Fmoc-4-NH2-Phe/Fmoc-Tyr; E. Fmoc4-NH2-Phe/Fmoc-4-CH3-Phe; F. Fmoc-Tyr/Fmoc-4-CH3-Phe. 130x86mm (300 x 300 DPI)


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Figure 7. A. TEM image of Fmoc-4-NO2-Phe self-assembled hydrogel fibrils. B. SEM image of Fmoc-4-NO2Phe crystalline microtubes formed spontaneously from fibrils shown in A. C. TEM image of Fmoc-4-CH3-Phe self-assembled hydrogel fibrils. D. SEM image of Fmoc-4-CH3-Phe crystalline microtubes. E. TEM image of Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe coassembled hydrogel fibrils. F. SEM image of Fmoc-4-NO2-Phe/Fmoc-4CN-Phe coassembled crystalline microtubes. 172x217mm (300 x 300 DPI)


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Figure 8. X-ray crystallographic data A. Single crystal unit cell of Fmoc-4-CH3-Phe; B. Basic Fmoc-4-CH3-Phe fibril unit; C. Single crystal unit cell of Fmoc-4-CN-Phe/Fmoc-4-NO2-Phe; B. Basic Fmoc-4-NO2-Phe/Fmoc-4CN-Phe fibril unit. 208x198mm (300 x 300 DPI)


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Table of Contents/Abstract Figure 210x58mm (300 x 300 DPI)


Coassembly and

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