Comparison of the Self-Assembly Behavior of Fmoc-Phenylalanine

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Comparison of the Self-Assembly Behavior of FmocPhenylalanine and Corresponding Peptoid Derivatives Annada Rajbhandary, William W. Brennessel, and Bradley L. Nilsson Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00709 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Crystal Growth & Design

Comparison of the Self-Assembly Behavior of Fmoc-Phenylalanine and Corresponding Peptoid Derivatives

Annada Rajbhandary, William W. Brennessel, 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

1 ACS Paragon Plus Environment

Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Fluorenylmethoxycarbonyl-protected phenylalanine (Fmoc-Phe) derivatives are a privileged class of low molecular weight amino acid hydrogelator that undergo spontaneous self-assembly in water to form one-dimensional (1D) fibril networks. Structural studies indicate that these fibrils feature unidirectional hydrogen bonding and parallel π–π interactions (Fmoc–Fmoc and side chain benzyl–benzyl), which stabilize the 1D fibrils. However, the relative contribution of hydrogen bonding vs π–π interactions in these assemblies is not understood. Herein, we compare self-assembly of Fmoc-Phe amino acids with corresponding Fmoc-protected peptoid derivatives. The N-benzyl glycine-derived peptoid analogs exhibit altered hydrogen bonding ability and benzyl side chain presentation geometry relative to the parent Fmoc-Phe molecules. We found that Fmoc-peptoid analogs preferentially assemble into two-dimensional (2D) nano- and microsheets that ultimately adopt crystalline states, whereas the Fmoc-Phe amino acids assemble into 1D nanofibrils. Crystal diffraction analysis suggests that hydrogen bonding of the carbamate group within Fmoc-Phe assemblies may be crucial for directing unilateral 1D growth of fibrils, while alteration of these interactions in the peptoid analogs removes the possibility for hydrogen bonds involving the carbamate moiety and limits intermolecular interactions to π–π bonding, which favors assembly into 2D architectures.


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Introduction Supramolecular self-assembly of peptides and functionalized amino acids into onedimensional fibril structures has garnered significant research interest due to the relevance of these processes to amyloid disorders and based on the recognition that the resulting selfassembled biomaterials can be exploited for diverse applications.1-6 Hydrogels formed from entangled networks of supramolecular nanofibrils are an important class of material that have been designed for use as wound healing, tissue engineering, and drug delivery agents.7-13 Low molecular weight (LMW) amino acid and dipeptide hydrogelators have advantages over longer peptide-based systems in terms of cost of production, but most LMW hydrogel systems lack elements of the ideal array of properties displayed by peptide assemblies that are required for advanced biological applications.14 These properties include optical transparency, shear responsiveness, appropriate viscoelasticity, and compatibility with cells.4 Most supramolecular hydrogels are discovered through serendipity or empirical approaches. A lack of understanding regarding the mechanisms of self-assembly and the relationship between self-assembly and emergent hydrogelation has complicated efforts to rationally design ideal LMW supramolecular hydrogels. Fluorenylmethoxycarbonyl-protected phenylalanine (Fmoc-Phe) (1a, Figure 1) and its derivatives represent a privileged class of low molecular weight amino acid hydrogelator that undergo spontaneous self-assembly in water to form one-dimensional (1D) fibril networks.15-17 Research in the development of Fmoc-Phe based hydrogels has focused on empirical engineering of these networks to exhibit properties that make them useful functional biomaterials.18-20 Additional studies have provided phenomenological insight into how the structure of the Fmoc-



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Phe gelator influences self-assembly and the properties of the resultant gels.21-23 Most of these studies lack information regarding the packing structure of the fibril network, which has complicated detailed understanding of these materials towards facilitating practical rational design. Recently, we reported the serendipitous discovery of Fmoc-Phe derivatives that rapidly form one-dimensional fibril networks in which these fibrils, over time, evolve into high aspect ratio crystals.17, 24 These crystals have facilitated structural analyses that have given insight into the probable packing architecture within fibrils.17,

24

In one case, this structural insight was

sufficient to enable a design strategy to stabilize the hydrogel fibril state by prevention of a specific set of hydrogen-bond interactions that were inferred to be unique to the crystal state relative to the fibril state.17 Based on these findings, it is evident that the self-assembly of Fmoc-Phe derivatives into hydrogel networks relies on noncovalent interactions that bias assembly into 1D fibrils as opposed to two- or three-dimensional sheet or crystal states.17,

24

The primary intermolecular

interactions in Fmoc-Phe derived fibrils are π–π interactions and putative hydrogen bonds.25 The previously reported crystallographic studies give detailed insight into the role that π–π interactions between Fmoc and benzyl aromatic groups have on enforcing 1D structures.17, 24 It seems apparent, however, that hydrogen bond interactions may also play a critical role. In order to probe the role of both hydrogen bonds and π–π interactions in facilitating 1D assembly of Fmoc-Phe derivatives, we compare herein the self-assembly of Fmoc-Phe amino acids that have been previously demonstrated to self-assemble into hydrogel networks15-17 (1a-4a, Figure 1) with corresponding Fmoc-Nphe peptoid constitutional isomers (1b-4b, Figure 1). Peptoids are achiral peptidomimetics in which the side chain functionality is shifted from the α-carbon to the nitrogen, thus altering both the hydrogen bond capacity at the amide nitrogen and the geometry 4 ACS Paragon Plus Environment

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of presentation of the side chain functionality. Herein, we report that this subtle structural change significantly alters the observed assemblies. While the Fmoc-Phe derivatives undergo selfassembly into 1D fibrils as expected, the Fmoc-Nphe derivatives instead assemble into two- and three-dimensional structures, favoring crystallization over fibril formation. The resulting crystals have facilitated structural comparisons between Fmoc-Phe 1D and Fmoc-Nphe 2D assemblies that give insight into the potential importance of hydrogen bonding in directing the formation of 1D assemblies. This insight offers design cues for next-generation Fmoc-Phe hydrogels by suggesting strategies to avoid crystal states by enforcing assembly into 1D fibril networks.



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Figure 1. Chemical structures of Fmoc-Phe derivatives (1a-4a) and the corresponding Fmoc-Nphe peptoid analogs (1b-4b). 1a Fmoc-Phe, 2a Fmoc-3F-Phe, 3a Fmoc-4-NO2-Phe, 4a Fmoc-F5-Phe, 1b Fmoc-Nphe, 2b Fmoc-3F-Nphe, 3b Fmoc-4-NO2-Nphe, 4b Fmoc-F5-Nphe.

Results and Discussion It is well known that select fluorenylmethoxycarbonyl-protected phenylalanine (FmocPhe) derivatives undergo spontaneous self-assembly into 1D fibrils that form emergent hydrogels.15, 22, 26 In the studies reported herein, we compare the self-assembly of several of these previously reported derivatives (1a-4a, Figure 1)15-17 with their peptoid counterparts (1b-4b, Figure 1) in order to gain insight into the role of hydrogen bonding and π-π interactions in


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facilitating fibrillization. Specifically, Phe-derived Nphe peptoids have the benzyl side chain shifted from the α-carbon to the amino acid nitrogen. This altered side chain orientation changes the hydrogen-bonding capacity of the carbamate nitrogen and also subtly modifies the geometry of side chain benzyl presentation relative to the N-terminal Fmoc group. Comparing the selfassembly of Fmoc-Phe-derived amino acids with peptoid analogs facilitates an assessment of how hydrogen bonding and π–π interactions enforce the 1D assembly that is required for fibril hydrogel network formation in this subclass of LMW gelator. Synthesis of Fmoc-Nphe peptoid analogs. While the required Fmoc-Phe derivatives 1a4a are commercially available, the corresponding Fmoc-Nphe peptoid derivatives (1b-4b) are not commercially available. Accordingly, we synthesized derivative compounds 1b-4b by adapting a previously reported protocol.27-28 Detailed procedures and characterization data for all novel compounds can be found in the Supporting Information (Schemes S1–S3, Figures S1– S11). Self-assembly studies. We initiated our comparative self-assembly analysis of Fmoc-Phe (1a-4a) and Fmoc-Nphe (1b-4b) derivatives under conditions in which the Fmoc-Phe derivatives have been shown to undergo self-assembly and hydrogelation.15-17 Specifically, each derivative was dissolved as a concentrated stock solution in DMSO (247 mM) which was then diluted into water to a concentration of 4.9 mM in a final solution of 2% DMSO/H2O (v/v). As previously reported, each Fmoc-Phe derivative (1a-4a) formed an opaque suspension immediately upon dilution into water; these opaque suspensions became optically transparent, self-supporting hydrogels within 10 minutes.15-17 Compound 1a was an exception: under these conditions, FmocPhe 1a does not form an optically transparent hydrogel, but forms an amorphous precipitate.16 TEM images of the self-supported hydrogels indicate that the hydrogel networks were composed



of 1D nanofibrils that are identical to those previously described for these compounds (Figure S12, Supporting Information). TEM images taken of the precipitate formed by compound 1a show mostly amorphous aggregate, but a few fibril assemblies are observed within the amorphous aggregate (Figure S12A). In contrast, Fmoc-Nphe derivatives 1b-4b did not form self-supporting hydrogels under these conditions. At concentrations of 4.9 mM in 2% DMSO/H2O (v/v), compounds 1b-4b are notably less soluble than their 1a-4a counterparts. Upon dilution from a 247 mM DMSO stock solution into 2% DMSO/H2O, compounds 1b-4b do not form self-supporting hydrogel networks, but instead precipitate from solution as solids (Figure S13, Supporting Information). TEM images were obtained of these precipitates after 3 hours and after 24 hours (Figure S14, Supporting Information). After 3 hours, TEM images revealed micelle structures that were 0.6– 2.5 µm in diameter (Figure S14A, D, G, and J). After 24 hours, these microspheres coalesced into distinct two-dimensional sheet structures that ranged from nanometer (Figure S14B, E, H, K) to micrometer (Figure S14C, F, I, and L) dimensions. The observed micelles are similar to those observed by us at early time points in the assembly of Fmoc-Phe-derivative fibrils22 and in the assembly of peptoid polymers as reported by Zuckermann and coworkers.29 In the case of Fmoc-Phe derivatives, these micelles were observed to be nucleation products that eventually matured into 1D fibrils. In the case of peptoid polymers, Zuckermann reported that the micelles observed from these materials often aggregated further into undefined aggregates in competition with sheet formation. In the case of peptoid analogs 1b-4b, these early micelles are nucleation products that evolve from micelle to sheet aggregates. The solubility of the sheet aggregates of compounds 1b-4b are significantly lower than the fibrils formed by compounds 1a-4a as indicated by their precipitation from solution under these conditions.


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Based on these findings, we explored the assembly properties of Fmoc-Phe and FmocNphe derivatives at lower concentrations where precipitation of the compounds and/or their aggregates would be avoided. No precipitation of either Fmoc-Phe (1a-4a) or Fmoc-Nphe (1b4b) analogs was observed at 200 µM concentrations (0.17% DMSO/H2O, v/v) (see Figure S14, Supporting Information for images of Fmoc-Nphe solutions under these conditions). Under these conditions, the Fmoc-Phe derivatives 1a-4a self-assembled into one-dimensional fibrils ~10 nm in diameter, similar to those observed at 4.9 mM (Figure 2). However, at 200 µM concentrations, these fibrils were not sufficiently dense to form a three-dimensional hydrogel network. TEM images of Fmoc-Nphe peptoids 1b-4b reveal that microsheet structures 0.1–1.5 µm wide are also formed at these lower concentrations (Figure 2).



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Figure 2. TEM image of fibrils formed by compounds 1a-4a and microsheets formed by peptoids 1b4b at 200 µM monomer in 0.17% DMSO/H2O.

These studies show that the subtle change in structure between the Fmoc-Phe and FmocNphe constitutional isomers fundamentally alters the self-assembly pathways accessed by these compounds. While Fmoc-Phe derivatives uniformly assembly into 1D fibrils, the corresponding Fmoc-Nphe analogs preferentially assemble into 2D sheets. Zuckermann and coworkers have previously shown that peptoid polymers form 2D nanosheets for sequences in which the corresponding polypeptides form 1D fibrils. 29-31 Zuckermann et al. explained that formation of 2D nanosheets by peptoid polymers occurs due to the lack of interstrand hydrogen bonds that constrain self-assembly of the corresponding peptides to one-dimensional assembly. While the fundamental properties of peptoid polymers and the Fmoc-Nphe derivatives considered herein differ to a significant extent, intermolecular hydrogen bonding interactions involving the


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carbamate N–H of Fmoc-Phe derivatives also appears to play a critical role in 1D fibrillization of these functionalized amino acids.17,

25

While altered hydrogen bonding in the Fmoc-Nphe

derivatives is likely to play a fundamental role in microsheet formation by these compounds, the subtle change in Fmoc and benzyl group geometry may also have an effect. The altered presentation of side chain benzyl groups relative to the N-terminal Fmoc functionality may also alter the geometry of intermolecular interactions in self-assembly pathways as may the achiral nature of these peptoid derivatives compared to the Fmoc-Phe amino acids.29-30,

32

Additional

structural analyses discussed in the following sections provide further insight into these effects. In an effort to gain additional insight into the dissimilar self-assembly properties of Fmoc-Phe and Fmoc-Nphe analogs, we explored conditions at which self-assembly would occur at higher concentrations without immediate precipitation of the Fmoc-Nphe analogs. It was found that increasing the DMSO content of DMSO/H2O solutions maintained initial solubility of Fmoc-Nphe derivatives upon dilution from DMSO stock solutions into the two-solvent mixture. Specifically, self-assembly of compounds 1a-4a and compounds 1b-4b was compared at 4.9 mM concentrations in 1:1 DMSO/H2O (v/v). Amino acids 1a and 2a precipitated in less than 3 minutes of dissolution from DMSO into 1:1 DMSO/H2O. TEM images of the precipitates showed self-assembled fibrils that were 55-75 nm in diameter (Figure 3, 1a and 2a). Compound 3a formed worm-like micelles (Figure 3, 3a) and thin needle-like crystals within 1 minute of dissolution into 1:1 DMSO/H2O. Compound 4a formed an opaque hydrogel within 1 minute of dissolving in 1:1 DMSO/H2O; the hydrogel network was comprised of fibrils 40 nm in diameter (Figure 3 (4a) and Figure S15, Supporting Information). Rheological analysis of the hydrogel formed by 4a in 50% DMSO/H2O indicates a gel with high rigidity, having a storage modulus (G') of 10923 ± 1212 Pa and a loss modulus (G") of (1386 ± 236 Pa) (See Figure S16,



Supporting Information for all rheological data). It should be noted that changes from 20–80% DMSO in DMSO/H2O ratios resulted in similar outcomes, but that increasing DMSO ratios to above 80% DMSO (v/v) resulted in impeded assembly due to enhanced solubility. While compound 3a formed thin needle-like crystals within minutes of dissolution in DMSO/H2O, compounds 1a and 2a, which initially assembled into wide fibrils, eventually formed thin, needle-like crystals after 1 month (Figure S15, 1a–3a). Of the crystals formed by compounds 1a-3a under these conditions, only those formed by compound 1a were of sufficient quality for high resolution X-ray diffraction analysis. The crystals of compounds 2a and 3a were extremely thin, and efforts to isolate them for powder diffraction analysis resulted in rapid transformation of the unstable crystals into oils. These oils proved unsuitable for X-ray analysis using either high-resolution or powder diffraction methods. However, the crystals of Fmoc-Phe 1a provided an important assessment of the packing architecture in these materials that is discussed in the following section. In comparison, peptoid analogs 1b-3b formed crystalline structures after 24 hours and compound 4b formed crystals after 7 days in 1:1 DMSO/H2O (4.9 mM). Optical microscopy of these crystals revealed that these crystals had plate-like appearances (Figure S15, Supporting Information). SEM images indicate that these plates are composed of laminated sheets (Figure 3, 1b-3b). For peptoid 4b, the crystals adopted both block-like and needle-like morphologies (Figure 3, 4b), indicating that this derivative has several possible assembly modes. These crystals facilitated high-resolution structural analysis of the packing architecture of Fmoc-Nphe assemblies, which is discussed in the following section.


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Figure 3. TEM and SEM images of fibrils and crystals formed by compounds 1a-4a and peptoid

analogs 1b-4b at 4.94 mM in 50% DMSO/H2O (v/v). This initial comparison of Fmoc-Phe (1a-4a) and Fmoc-Nphe (1b-4b) indicates that movement of the benzyl side chain from the α-carbon of the Fmoc-Phe parent compounds to the nitrogen alters the favored assembly pathways between Fmoc-Phe and Fmoc-Nphe derivatives. Table 1 summarizes the observed morphologies of self-assembled structures for each of these compounds under the solvent conditions utilized. It is striking that the Fmoc-Phe amino acids form high-aspect ratio, one-dimensional fibrils and crystals whereas the Fmoc-Nphe peptoids form 2D sheets that, at higher concentrations, laminate into three-dimensional crystals. The packing architecture of monomers within these crystals provides insight into the structural basis for the differences in self-assembly between these molecules.



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Table 1. Observed morphology for self-assembled structures of compounds 1a-4a and 1b-4b at various concentrations of monomer and ratios of DMSO/H2O in the solvent. 200 µM (0.17%

4.9 mM (2%

4.9 mM (50%

DMSO/H2O, v/v)

DMSO/H2O, v/v)

DMSO/H2O, v/v)

Compound

Observed structures

Observed structures

Observed structures

1a

Fibrils

Hydrogel fibrils

Crystals (needle)

2a

Fibrils

Hydrogel fibrils

Crystals (needle)

3a

Fibrils

Hydrogel fibrils

Crystals (needle)

4a

Fibrils

Hydrogel fibrils

Hydrogel fibrils

1b

Microsheets

Microsheets

Crystals (sheet)

2b

Microsheets

Microsheets

Crystals (sheet)

3b

Microsheets

Microsheets

Crystals (sheet)

4b

Microsheets

Microsheets

Crystals (needle and block)

X-ray diffraction analysis of crystals of Fmoc-Phe (1a) and Fmoc-Nphe analogs (1b4b). X-ray diffraction studies facilitated structural insight into the effects that drive assembly of Fmoc-Phe derivatives into high-aspect ratio materials and Fmoc-Nphe derivatives into twodimensional sheet structures. Fmoc peptoid analogs 1b-3b rapidly (within 1 day) crystallized in


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1:1 DMSO/H2O while 4b crystallized in 7 days at 4.9 mM. In contrast, crystallization of the Fmoc-Phe derivatives occurred slowly under these conditions, and only crystals of 1a were suitable for high-resolution diffraction studies. The needlelike crystals of compound 1a that were sufficiently large to enable X-ray diffraction analysis required nearly one month of incubation. The diffraction intensity of the data obtained was very weak for 1a, but the structure obtained is unambiguous. While compounds 2a and 3a did form crystals after extended incubation, the crystals were not of sufficient quality to perform X-ray diffraction studies; attempts to isolate these extremely thin crystals resulted in the formation of oils, perhaps due to the incorporation of DMSO within the crystals. The preference of Fmoc-Phe analogs to rapidly assemble into 1D fibrils undoubtedly contributes to the inefficient crystallization of these compounds. This is consistent with previous analyses of these molecules, for which examples of crystal formation under conditions that are similar to fibril formation and gelation are uncommon.17, 24-25, 33 The packing architecture for Fmoc-Phe 1a is unique compared to the Fmoc-Nphe derivatives. The packing architecture for 1a does, however bear striking similarity to previously reported structures of Fmoc-Phe derivatives Fmoc-4-NO2-Phe (3a), Fmoc-4-CH3-Phe, and coassembled Fmoc-4-NO2-Phe and Fmoc-4-CN-Phe (Figure 4).17,

24, 34

It should be noted

examples of crystallization of Fmoc-Phe derivatives that form hydrogel fibril networks is exceptionally rare. Our previously reported examples of Fmoc-Phe derivative crystallization occurred serendipitously; in all cases, fibril formation preceded crystal formation.17,

24, 34

Specifically, solutions of Fmoc-4-NO2-Phe (3a),17 Fmoc-4-CH3-Phe,24 and Fmoc-4-NO2Phe/Fmoc-4-CN-Phe24 were diluted from DMSO into water (2% DSMO/H2O, 4.9 mM) whereupon they rapidly self-assembled into one-dimensional fibrils that entangled for form selfsupporting hydrogels. Over time (hours to weeks, depending on the derivative), crystals formed



spontaneously from these fibrils by alignment and fusion of fibrils under gelation conditions (2% DSMO/H2O, 4.9 mM). This process occurred identically to crystal formation observed with our previously Fmoc-4-NO2-Phe and related systems.17,24 The crystals that formed from these various derivatives also showed nearly identical packing architectures in which Fmoc-Fmoc and benzyl-benzyl aromatic stacking interactions are the dominant intermolecular forces. It is expected that the packing architecture observed in these crystals is similar to that observed in the precursor fibrils. Since the crystals were observed to arise directly from the initially formed fibrils it is reasonable to assume that the major elements of the packing architecture between fibril and crystal are similar. This assumption is also supported by the structural similarity of Fmoc-Phe 1a to Fmoc-Phe derivatives that we have previously shown to undergo fibril to crystal transitions. Detailed discussion of this reasoning behind these inferences can be found in the previously published work.17,

24, 34

While the precedent for similarity between the fundament

packing architecture observed in gel and crystal states is strong, there is a possibility that differences do exist. These differences will require additional high-resolution structural analyses using emerging techniques that include solid state NMR. This will be the topic of future reports for these materials.


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Figure 4. Chemical structures and packing architectures of previously reported Fmoc-Phe derivatives that undergo spontaneous transition from hydrogel fibrils to crystalline microtubes.17, 24, 34

A) Fmoc-4-NO2-Phe (3a),17 B) Fmoc-4-CH3-Phe,24 C) Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe.24 The crystals of Fmoc-Phe 1a reported herein share a common packing architecture with

those of the previously reported structures described above. The Fmoc-Phe (1a) crystals reported herein crystallized in a triclinic space group P1 (Figure S17 and Appendix 1, Supporting Information). The asymmetric unit contains two Fmoc-Phe molecules and two cocrystallized dimethylsulfoxide (DMSO) solvent molecules, all in general positions, and the carboxylate group of each Fmoc-Phe molecule is hydrogen-bonded (O–H…O) to one DMSO molecule



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(Figure 5A) instead of bonding to an adjacent Fmoc-Phe molecule as had previously been reported for 3a.25 The packing for 1a also shows intermolecular hydrogen bonds (2.9 Å) between neighboring carbamate groups (N–H…O) linking the Fmoc-Phe molecules along the [100] direction (Figure 5B). Previously reported structures for 3a (Figure 4A) and Fmoc-4-CH3-Phe (Figure 4B) show this carbamate group to be twisted such that this hydrogen bond does not exist, but we have hypothesized that it might exist in the pre-crystal fibril state for these molecules as an anchor interaction that confines the assemblies to primarily one dimension.17,

24

Adams’

structure of Fmoc-Phe formed using a “pH switch” method in a low amount of glucono-δ-lactone (GdL), shows a similar hydrogen bond to that displayed in the Fmoc-Phe crystals herein.25 Offset face-to-face π-π interactions along the a-axis between the neighboring Fmoc groups (FmocFmoc) and benzyl side chain groups (phenyl-phenyl) also stabilize the packing architecture (Figure 4B) as was previously observed in crystals of 3a, Fmoc-4-CH3-Phe, and Fmoc-4-NO2Phe/Fmoc-4-CN-Phe.17, 24-25


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Figure 5. Packing of Fmoc-Phe 1a from X-ray diffraction analysis. A) Packing arrangement including DMSO, B) View of packing structure highlighting offset face-to-face aromatic interactions between neighboring side chain phenyl groups and N-terminal Fmoc groups as well as hydrogen bonds (N–H…O) between neighboring carbamate groups.

X-ray diffraction data were also obtained for the Fmoc-Nphe analogs 1b-4b. Peptoid 1b, crystallizes in monoclinic space group P21/c with two independent molecules in the asymmetric unit (Figure 6A (extended packing structure) and Figure S18, Supporting Information (unit cell)) (see also Supporting Information, Appendix 2 for complete details and coordinates). The packing shows moderately strong classical hydrogen bonding between the hydroxyls of the carboxylic acids and the carbonyls of the carbamate groups of adjacent symmetry equivalent molecules with 19 ACS Paragon Plus Environment


donor-acceptor distances of 2.664(3) and 2.686(3) Å (Figure 7A). These collective hydrogen bonds zigzag along the c-axis direction. Numerous weak non-classical (C–H…O) intermolecular hydrogen bonds with C...O distances ranging from 3.261(3) to 3.393(4) Å also link the molecules along the c-axis direction, and additionally along the direction of the a-axis, thus promoting the assembly of 2D sheets. Also, notable intermolecular π–π interactions between the neighboring benzyl side chain phenyl groups in a displaced face-to-face arrangement along the a-axis direction at distances of 4.8 Å are observed (Figure 7B). An apparent intramolecular edgeto-face (T-shaped) π–π interaction between the Fmoc group and the phenyl group of the benzyl side chain at a distance of about 5.0 Å appears to confine the shape of each molecule (Figure 7C). This intramolecular Fmoc-benzyl interaction is not observed in the Fmoc-Phe analog 1a, probably due to less favorable geometry for this type of an interaction with the benzyl group oriented at the α-carbon and also due to the formation of the directing hydrogen bond in the Fmoc-Phe 1a assembly. Thus, this intramolecular π–π effect may play a critical role in diverting assembly of Fmoc-Nphe from 1D fibril/high-aspect ratio crystals (as seen in Fmoc-Phe) into sheet structures. This intramolecular interaction is likely favored since no possibility for intermolecular hydrogen bonding occurs, facilitating this molecular rearrangement in FmocNphe compared to Fmoc-Phe. Finally, an intermolecular edge-to-face T-shaped interaction was also observed between the Fmoc group of one molecule with the phenyl ring of the benzyl side chain of the neighboring molecule at a distance of 5.5 Å along the c-axis (Figure 7D).


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Figure 6. Extended packing arrangements from X-ray diffraction analysis for crystals of A) FmocNphe (1b), B) Fmoc-3F-Nphe (2b), C) Fmoc-4-NO2-Phe (3b).

Figure 7. Significant intermolecular and intramolecular interactions within crystals of FmocNphe (1b).

A face-indexing experiment done on the crystals indicated that the two long dimensions of the crystalline plate correspond to the shortest dimensions of the unit cell [100] and [001] with unit cell distances 9.33 and 12.07 Å, respectively, while the shortest dimension of the plate corresponding to the [010] direction is the longest dimension of the unit cell with a distance of



34.65 Å (Figure S18, Supporting Information). This data is consistent with the observed hydrogen bonding and π–π interactions that link molecules in the ac plane to form two dimensions in bilayer sheets. Presumably these interactions in the ac plane possibly cause faster crystal growth in the two dimensions relative to the third (Figure S18 and S19). The sheet bilayers as formed in aqueous solvents may be defined by confinement of the hydrophobic Fmoc and benzyl groups to a hydrophobic interior face while the hydrophilic COOH groups define the exterior face and mediate interactions with neighboring sheets via a hydrogen bond network to facilitate stacking of 2D bilayer sheets (Figure S19). Peptoid crystals of 2b and 3b (Figures S20, S21 and Appendices 2 and 3, Supporting Information) are isostructural with those of 1b as observed in the common packing arrangements (Figure 6) as well as in the cell axis lengths that are ~9, 12, and 35 Å, in some order, depending upon the specific structure. While crystals of 1b and 2b have both crystallized in the monoclinic space group type P21/c, the unit cell lengths a, b, c are 9.327(2), 34.646(9), 12.074(3) Å and 12.093(3), 9.210(2), 35.336(8) Å with β angles of 91.120(4) and 100.314(4) degrees, respectively. Structure 3b crystallized in the orthorhombic space group Pbca with unit cell lengths of 12.115(3), 9.187(2), 36.754(9) Å. Thus all views in Figure 6 are perpendicular to the shortest unit cell dimension; that is, the planes shown are of the two largest cell dimensions. In all cases (1b, 2b, 3b) there are eight molecules in the unit cell: for 1b and 2b, there are two independent molecules in the asymmetric unit; for 3b, there is one molecule in the asymmetric unit. Consistent with the sheet morphologies observed by microscopy, all three structures 1b, 2b, and 3b have molecules arranged in similar bilayer sheets with classical hydrogen bonding (O–H…O) between the carboxylates, non-classical (C–H…O) hydrogen bonding between 22 ACS Paragon Plus Environment

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adjacent molecules, intermolecular T-shaped π–π interactions between neighboring Fmoc–benzyl groups, displaced face-to-face π–π interactions between neighboring benzyl side chain groups, and intramolecular edge-to-face π–π interactions between Fmoc and side chain benzyl groups (all as shown for 1a in Figure 7). Structure 2b has one additional weak intermolecular interaction due to the presence of the of the benzyl fluorine atom, C—H…F, with a donor-acceptor distance of 3.53 Å. Interestingly, peptoid 4b formed crystals of two distinct morphologies (needles and blocks) as can be seen in SEM images (Figure 3, 4b). X-ray diffraction analyses performed separately for each crystal type showed unique packing arrangements giving rise to the respective crystal morphologies. Block crystals have an asymmetric unit with one molecule in a general position in space group P21/c (Figure S22 and Appendix 5, Supporting Information). Pairs of inversion-related molecules are linked via classical hydrogen bonding (O–H…O) between the carboxylates with donor-acceptor distances of 2.632(1) Å and weak C–H…F intermolecular interactions exist along the [010] direction (Figure 8A and C). A displaced faceto-face π–π interaction exists between Fmoc–pentafluorophenyl group along c-axis with a distance of 4.9 Å (Figure 8A and 8B). Displaced π-π interactions between adjacent Fmoc groups (4.4 Å) and pentafluorophenyl groups (5.5 Å) are observed along the a-axis (Figure 8A and 8D). Interestingly, the intramolecular arrangement

of the Fmoc group

relative to

the

pentafluorophenyl group is rotated nearly 45º relative to the edge to face interaction observed in crystals of 1b-3b (Figure 8A and 8E). This altered orientation is most likely due to intramolecular complementary quadrupole π–π interactions between the Fmoc group and the pentafluorophenyl side chain. This is consistent with data that indicates that pentafluorophenyl and phenyl groups in Fmoc-Phe derivatives can coassemble due to complementary quadrupole



effects.24,

35-37

This particular π–π interaction is unique to 4b since the pentafluorophenyl

quadrupole electronics are unique relative to those found in the benzyl side chains of 1b-3b. Therefore, it is likely that this unique quadrupole interaction is responsible for the unique parallel stacking molecular arrangement of molecules in crystals of 4b.


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Figure 8. A) Extended packing arrangements from X-ray diffraction analysis for crystals of Fmoc-F5Nphe (4b) with block morphology. B) Intermolecular interactions between Fmoc and pentafluorophenyl groups in neighboring molecules. C) Intermolecular hydrogen bonding between carboxyl groups in neighboring molecules. D) Intermolecular Fmoc-Fmoc and pentafluorophenyl-pentafluorophenyl groups in neighboring molecules. E) Intramolecular face-to-face Fmoc-pentafluorophenyl interactions.

The other morphology observed in crystals of 4b had a higher aspect ratio and was more



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needlelike in appearance. These needles crystallized in the monoclinic space group I2/a (Figure 9A and Figure S23, Supporting Information). It has an asymmetric unit with one molecule of interest and partial occupancy of a water molecule. The hydrogen atoms of the partial occupancy cocrystallized water molecule were placed to avoid close contacts but it is understood that these may not be the true positions. The molecules are connected pairwise via hydrogen bonds between the carboxylates with donor-acceptor distances of 2.608(5) Å (Figure 9B). Face-to-face displaced π–π interactions between neighboring Fmoc groups (5.4 Å) and pentafluorophenyl groups (4.8 Å) are also observed along the a-axis (Figure 9C). Similar to compound 2b, crystalline needles of 4b also feature weaker intermolecular interactions between a pentafluorophenyl fluorine and a C–H donor from the analogous methylene group (C–H…F) with a donor-acceptor distance of 3.382(7) Å along the [001] direction (Figure 9D).

An

additional set of C-H…F (methylene…pentafluorophenyl fluorine) interactions link pairs of molecules at a donor-acceptor distance of 3.228(6) Å. Interestingly, this needle-like crystal morphology is unique among the Fmoc-Nphe peptoid derivatives in that it does not exhibit intramolecular Fmoc-side chain benzyl interactions, giving it an extended packing structure that is somewhat similar to that observed for Fmoc-Phe 1a highaspect ratio crystals. The needle crystals of 4b exhibit Fmoc-Fmoc and side chain benzyl-benzyl interactions similar to those seen in the crystals of Fmoc-Phe 1a and other Fmoc-Phe derivatives for which crystals have been previously reported.17,

24, 25, 34

It is significant that even in the

absence of hydrogen bonding, a parallel stacking arrangement within these molecules to enforce a more 1D type arrangement to form needle-like morphology is possible. This demonstrates that change in side presentation geometry that occurs in Fmoc-Nphe derivatives by moving the side chain benzyl group to the amine group is indeed a subtle geometric change that doesn’t preclude


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a similar packing structure in Fmoc-Nphe analogs compared to Fmoc-Phe derivatives. This strengthens the conclusions that can be arrived at from these studies. Notably, these needlelike crystals of 4b are much less abundant than the block morphology crystals of compound 4b, indicating that the molecular configuration that enables the needle-like 1D packing is less energetically favorable under these conditions. An intermolecular carbamate hydrogen bond, as is observed in Fmoc-Phe fibrils, is thus a significant energetic contributor to 1D self-assembly modes.



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Figure 9. A) Extended packing arrangements from X-ray diffraction analysis for crystals of Fmoc-F5Nphe (4b) with needlelike morphology. B) Intermolecular hydrogen bonding between carboxyl groups in neighboring molecules. C) Intermolecular Fmoc-Fmoc and pentafluorophenyl-pentafluorophenyl groups in neighboring molecules. D) Intermolecular pentafluorophenyl C–F...H interactions.

Powder X-ray diffraction (PXRD) analysis of microsheets from Fmoc peptoid monomers. In order to assess the structural similarities between the microsheets formed by compounds 1b-4b at low concentrations and low DMSO content and crystals formed by the same compounds at higher concentration and solvent DMSO content, powder X-ray diffraction analysis was done on samples of crystals and microsheets (Figure S24, Supporting Information).


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Powder X-ray diffraction confirmed that microsheets and crystals of each Fmoc-Nphe analog share similar packing structure. All samples revealed intense peaks indicating highly ordered structures. Compounds 1b–3b, which have strikingly similar packing architecture, also have very similar power X-ray diffraction profiles. For these compounds, both crystal and microsheet samples show an intense peak at 17.38–18.55 Å. This distance corresponds to the distance between the each molecule and their symmetry equivalent molecule within the crystalline packing structure (Figure S19, Supporting Information). Scattering intensities at 8.70–9.26 Å correspond to the second order reflection, scattering intensities at 4.38–4.75 Å correspond to the fourth order reflection, and scattering intensities at 2.9 Å correspond to sixth order reflections. Other signals near 2.5 Å correspond to hydrogen bonding between the carboxylate groups and signals 4.5–5.5 Å correspond to the π-π interaction distances within the crystal structures. The microsheets have remarkably similar packing arrangements to the crystals, indicating that they share common packing arrangements. The crystals are likely to arise from initial microsheet structures. Powder X-ray diffraction for 4b reveals a more complex structure, reflecting the two crystal morphologies observed (Figure S24D, Supporting Information). Peaks between 4.0–6.6 Å corresponds to numerous π-π stacking interactions in the block and needle type crystals. Various peaks around 3.0 Å correspond to the distances for weak (C–H…O) and (C–H…F) interactions and peaks from 2.2–2.7 Å correspond to hydrogen bonding networks found within the crystals. Once again, the powder X-ray diffraction profiles of both the microsheet and crystalline samples of 4b are similar, indicating that similar packing architecture is found with each sample type.



Discussion. The structural data obtained by comparison of self-assembly of Fmoc-Phe and Fmoc-Nphe derivatives gives significant insight into the possible molecular interactions that give rise to 1D assembly in the former materials and 2D and 3D assembly in the latter. Specifically, a carbamate hydrogen bond observed in Fmoc-Phe 1a crystals may be responsible for confinement of assembly pathways to 1D fibrillization or needlelike, high-aspect ratio crystals. Conversely, the lack of the possibility for this particular hydrogen bond in Fmoc-Nphe derivatives 1b–4b removes this interaction as a possible constraining force in the assembly of these materials, resulting in 2D sheet structures. This data is in agreement with the reports by various groups that have described on the importance of unidirectional (1D) interactions in molecular aggregation for initiation of fibrillization.38-39 Fmoc-Nphe analogs also exhibit altered π–π bonding patterns relative to the Fmoc-Phe materials, which may also impact the 2D versus 1D assembly of Fmoc-Nphe peptoid derivatives. In Fmoc-Phe crystals, all observed π–π interactions are intermolecular (Fmoc-Fmoc and benzylbenzyl) interactions. In the design of the studies, we considered that the geometric differences apparent in presenting the side chain benzyl groups at the amine (Fmoc-Nphe) rather than at the α-carbon (Fmoc-Phe) may fundamentally alter the self-assembly arrangements that are possible for these molecules to adopt, independent of hydrogen bonding capacity. This appears to not be the case. In Fmoc-Nphe crystals, there is clearly a significant intramolecular π–π interaction between the Fmoc and benzyl groups that alters the molecular conformation and favors selfassembly in two-dimensions. However, the needlelike crystals of 4b that were unique among the crystals formed by the Fmoc-Nphe derivatives studied herein, were found to feature 4b packing modes that have no intramolecular π–π interactions. The needle crystals of 4b crystals have only intermolecular π–π bonding (Fmoc-Fmoc and benzyl-benzyl interactions) that are similar to


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those observed by the 1D assemblies of Fmoc-Phe and its derivatives. Thus, the altered side chain presentation of Fmoc-Nphe peptoids does not inherently prevent 1D assembly. Since 1D assembly is theoretically and practically possible for Fmoc-Nphe derivatives, the lack of a constraining carbamate hydrogen bond interaction in these analogs is potentially responsible for the fundamental change in the orientation of π–π interactions leading to sheet-like assemblies as opposed to fibrils in these materials. These results may be consistent with hydrogen bond interactions playing a critical role in the self-assembly of Fmoc-Phe derivatives into 1D fibril networks as opposed to assembly into multidimensional crystalline structures. These studies also illustrate the difficulty in attempting to explain the complexities of supramolecular self-assembly phenomena. It is tempting to draw strong conclusions regarding the importance of a putative carbamate hydrogen bond network in constraining the self-assembly of Fmoc-Phe derivatives into 1D fibrils. The lack of this specific type of hydrogen bond in the Fmoc-Nphe assemblies seems to be consistent with this conclusion. However, this may be a dangerous oversimplification of the observations, especially in light of the data that shows that Fmoc-Nphe 4b, which has a pentafluorophenyl side chain with altered quadrupolar electrons relative to the other derivatives, is capable of forming assemblies that are similar to those formed by the other Fmoc-Nphe derivatives and 1D assemblies similar to those formed by Fmoc-Phe derivatives. As such, the intermolecular interactions that divert assembly into the various pathways are most likely influenced by complex energetic forces that depend on not only the atomic structure of the assembly motif, but also on differing hydrophobic, aromatic, and environmental (solvent, temperature, etc.) effects. The studies herein illustrate these complexities and also raise questions for additional study.



Conclusion These studies illustrate the significant intermolecular interactions that make Fmoc-Phe deriviatives privileged structures for self-assembly into fibril networks with emergent functional hydrogel properties. The central Fmoc carbamate group apparently provides a critical hydrogen bond that plays a role in constraining assembly of Fmoc-Phe derivatives primarily into 1D fibrils that have a lower propensity to evolve into crystalline states. This hydrogen bond may provide either a thermodynamic advantage to fibrils versus multidimensional assemblies or it provides a kinetic barrier to evolution of 2D or 3D structures. These unidirectional hydrogen bonding interactions in Fmoc-Phe derivative assemblies may also enforce a pattern of intermolecular π–π interactions between neighboring Fmoc and phenyl groups that reinforces 1D assembly. In contrast, the lack of this specific type of hydrogen bond interaction in the Fmoc-carbamate of Fmoc-Nphe derivatives may facilitate an intramolecular π–π interaction between the Fmoc and phenyl groups that diverts assembly pathways to favor 2D structures in which π–π bonding and hydrophobic effects primarily define the packing architectures. These results lend insight into the importance of intermolecular interactions that can be exploited in the design of Fmoc-Phe based low molecular weight hydrogelators. Hydrogelation is often found to occur in competition with crystallization in low molecular weight gelators, and these studies suggest that hydrogen-bonding may be a critical a constraining force that tips the balance in these competitive pathways (crystal versus fibril) towards 1D assemblies that favor fibril networks that lead to hydrogels. This insight into the self-assembly processes of Fmoc-Phe and Fmoc-Nphe derivatives thus provides critical design cues for crystal and hydrogel engineering for preparation of next-generation functional materials. Supporting Information


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Supporting information and related appendices include experimental details, synthetic protocols and characterization data for all compounds, additional TEM images, rheological data for hydrogels, unit cell and coordinate data for all crystal diffraction analyses. Acknowledgments This work was supported by NSF (DMR-1148836). We gratefully acknowledge Karen Bentley (URMC Electron Microscope Research Core) for her assistance in TEM and SEM imaging and Dr. Maura Weathers (Cornell Center for Materials Research) for assistance with PXRD experiments. References 1.

2.

3. 4. 5.

6. 7. 8. 9.

10.

Tao, K.; Levin, A.; Adler-Abramovich, L.; Gazit, E. Fmoc-modified amino acids and short peptides: simple bio-inspired building blocks for the fabrication of functional materials. Chem. Soc. Rev 2016, 45, 3935-3953. Habibi, N.; Kamaly, N.; Memic, A.; Shafiee, H. Self-assembled peptide-based nanostructures: Smart nanomaterials toward targeted drug delivery. Nano Today 2016, 11, 41-60. Hamley, I. W. The Amyloid Beta Peptide: A Chemist’s Perspective. Role in Alzheimer’s and Fibrillization. Chem. Rev. 2012, 112, 5147-5192. Ryan, D. M.; Nilsson, B. L. Self-assembled amino acids and dipeptides as noncovalent hydrogels for tissue engineering. Poly. Chem. 2012, 3, 18-33. Tatman, P. D.; Muhonen, E. G.; Wickers, S. T.; Gee, A. O.; Kim, E.-S.; Kim, D.-H. Selfassembling peptides for stem cell and tissue engineering. Biomater. Sci. 2016, 4, 543554. Fleming, S.; Ulijn, R. V. Design of nanostructures based on aromatic peptide amphiphiles. Chem. Soc. Rev. 2014, 43, 8150-8177. Yuan, X.; He, B.; Lv, Z.; Luo, S. Fabrication of self-assembling peptide nanofiber hydrogels for myocardial repair. RSC Adv. 2014, 4, 53801-53811. Seow, W. Y.; Hauser, C. A. E. Short to ultrashort peptide hydrogels for biomedical uses. Mater. Today 2014, 17, 381-388. Rajbhandary, A.; Nilsson, B. L., Self-Assembling Hydrogels. In Gels HandbooK: Fundamentals, Properties and Applications; Khademhosseini, A.; Demirci, U., Ed.; World Scientific: 2016; pp 219-250. Tian, R.; Chen, J.; Niu, R. The development of low-molecular weight hydrogels for applications in cancer therapy. Nanoscale 2014, 6, 3474-3482.



11.

12.

13.

14. 15.

16.

17.

18.

19. 20.

21.

22.

23.

24. 25.

26. 27.

Fichman, G.; Gazit, E. Self-assembly of short peptides to form hydrogels: design of building blocks, physical properties and technological applications. Acta biomater. 2014, 10, 1671-82. Liyanage, W.; Vats, K.; Rajbhandary, A.; Benoit, D. S. W.; Nilsson, B. L. Multicomponent dipeptide hydrogels as extracellular matrix-mimetic scaffolds for cell culture applications. Chem. Commun. 2015, 51, 11260-11263. Wang, H.; Feng, Z.; Xu, B. D-amino acid-containing supramolecular nanofibers for potential cancer therapeutics. Adv. Drug Deliv. Rev. 2016, doi: 10.1016/j.addr.2016.04.008. van Esch, J. H. We Can Design Molecular Gelators, But Do We Understand Them? Langmuir 2009, 25, 8392-8394. Ryan, D. M.; Anderson, S. B.; Nilsson, B. L. The influence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives. Soft Matter 2010, 6, 3220-3231. Ryan, D. M.; Anderson, S. B.; Senguen, F. T.; Youngman, R. E.; Nilsson, B. L. Selfassembly and hydrogelation promoted by F5-phenylalanine. Soft Matter 2010, 6, 475479. Liyanage, W.; Brennessel, W. W.; Nilsson, B. L. Spontaneous Transition of Selfassembled Hydrogel Fibrils into Crystalline Microtubes Enables a Rational Strategy To Stabilize the Hydrogel State. Langmuir 2015, 31, 9933-9942. Pappas, C. G.; Abul-Haija, Y. M.; Flack, A.; Frederix, P. W. J. M.; Ulijn, R. V. Tuneable Fmoc-Phe-(4-X)-Phe-NH2 nanostructures by variable electronic substitution. Chem. Commun. 2014, 50, 10630-10633. Shi, J.; Gao, Y.; Yang, Z.; Xu, B. Exceptionally small supramolecular hydrogelators based on aromatic–aromatic interactions. Beilstein J. Org. Chem. 2011, 7, 167-172. Roy, S.; Banerjee, A. Amino acid based smart hydrogel: formation, characterization and fluorescence properties of silver nanoclusters within the hydrogel matrix. Soft Matter 2011, 7, 5300-5308. Frith, W. J.; Donald, A. M.; Adams, D. J.; Aufderhorst-Roberts, A. Gels formed from amino-acid derivatives, their novel rheology as probed by bulk and particle tracking rheological methods. J. Non-Newton. Fluid 2015, 222, 104-111. Ryan, D. M.; Doran, T. M.; Anderson, S. B.; Nilsson, B. L. Effect of C-Terminal Modification on the Self-Assembly and Hydrogelation of Fluorinated Fmoc-Phe Derivatives. Langmuir 2011, 27, 4029-4039. Singh, V.; Snigdha, K.; Singh, C.; Sinha, N.; Thakur, A. K. Understanding the selfassembly of Fmoc-phenylalanine to hydrogel formation. Soft Matter 2015, 11, 53535364. Liyanage, W.; Nilsson, B. L. Substituent Effects on the Self-Assembly/Coassembly and Hydrogelation of Phenylalanine Derivatives. Langmuir 2016, 32, 787-799. Draper, E. R.; Morris, K. L.; Little, M. A.; Raeburn, J.; Colquhoun, C.; Cross, E. R.; McDonald, T. O.; Serpell, L. C.; Adams, D. J. Hydrogels formed from Fmoc amino acids. CrystEngComm. 2015, 17, 8047-8057. Ryan, D. M.; Doran, T. M.; Nilsson, B. L. Complementary π–π Interactions Induce Multicomponent Coassembly into Functional Fibrils. Langmuir 2011, 27, 11145-11156. Kruijtzer, J. A. W.; Hofmeyer, L. J. F.; Heerma, W.; Versluis, C.; Liskamp, R. M. J. Solid-Phase Syntheses of Peptoids using Fmoc-Protected N-Substituted Glycines: The 34 ACS Paragon Plus Environment

Page 34 of 46

Page 35 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


28.

29.

30.

31.

32.

33.

34. 35. 36. 37.

38. 39.

Synthesis of (Retro)Peptoids of Leu-Enkephalin and Substance P. Chem. Eur. J. 1998, 4, 1570-1580. Barth, A.; Martin, S. R.; Corrie, J. E. T. Decarboxylation is a significant reaction pathway for photolabile calcium chelators and related compounds. Photochem. Photobiol. Sci. 2006, 5, 107-115. Nam, K. T.; Shelby, S. A.; Choi, P. H.; Marciel, A. B.; Chen, R.; Tan, L.; Chu, T. K.; Mesch, R. A.; Lee, B.-C.; Connolly, M. D.; Kisielowski, C.; Zuckermann, R. N. Freefloating ultrathin two-dimensional crystals from sequence-specific peptoid polymers. Nat. Mater. 2010, 9, 454-460. Kudirka, R.; Tran, H.; Sanii, B.; Nam, K. T.; Choi, P. H.; Venkateswaran, N.; Chen, R.; Whitelam, S.; Zuckermann, R. N. Folding of a single-chain, information-rich polypeptoid sequence into a highly ordered nanosheet. Biopolymers 2011, 96, 586-95. Sanii, B.; Kudirka, R.; Cho, A.; Venkateswaran, N.; Olivier, G. K.; Olson, A. M.; Tran, H.; Harada, R. M.; Tan, L.; Zuckermann, R. N. Shaken, Not Stirred: Collapsing a Peptoid Monolayer To Produce Free-Floating, Stable Nanosheets. J. Am. Chem. Soc. 2011, 133, 20808-20815. Sanii, B.; Haxton, T. K.; Olivier, G. K.; Cho, A.; Barton, B.; Proulx, C.; Whitelam, S.; Zuckermann, R. N. Structure-Determining Step in the Hierarchical Assembly of Peptoid Nanosheets. ACS Nano 2014, 8, 11674-11684. Houton, K. A.; Morris, K. L.; Chen, L.; Schmidtmann, M.; Jones, J. T. A.; Serpell, L. C.; Lloyd, G. O.; Adams, D. J. On Crystal versus Fiber Formation in Dipeptide Hydrogelator Systems. Langmuir 2012, 28, 9797-9806. Liyanage, W.; Cogan, N. M. B.; Nilsson, B. L. Amyloid-Inspired Optical Waveguides from Multicomponent Crystalline Microtubes. ChemNanoMat 2016, 2, 800-804. Zheng, H.; Comeforo, K.; Gao, J. Expanding the Fluorous Arsenal: Tetrafluorinated Phenylalanines for Protein Design. J. Am. Chem. Soc. 2009, 131, 18-19. Zheng, H.; Gao, J. Highly Specific Heterodimerization Mediated by Quadrupole Interactions. Angew. Chem. Int. Ed. 2010, 49, 8635-8639. Hwang, J.; Li, P.; Carroll, W. R.; Smith, M. D.; Pellechia, P. J.; Shimizu, K. D. Additivity of Substituent Effects in Aromatic Stacking Interactions. J. Am. Chem. Soc. 2014, 136, 14060-14067. Zurcher, D. M.; McNeil, A. J. Tools for Identifying Gelator Scaffolds and Solvents. The J. Org. Chem. 2015, 80, 2473-2478. Ramos Sasselli, I.; Halling, P. J.; Ulijn, R. V.; Tuttle, T. Supramolecular Fibers in Gels Can Be at Thermodynamic Equilibrium: A Simple Packing Model Reveals Preferential Fibril Formation versus Crystallization. ACS Nano 2016, 10, 2661-2668.

TOC Image/Abstract



Synopsis: We compare self-assembly behavior of Fmoc-Phe amino acids with their corresponding Fmoc-protected peptoid derivatives. The peptoid derivatives exhibit altered hydrogen bonding ability and spatial arrangement of the benzyl side chain relative to the parent Fmoc-Phe counterparts. While Fmoc-Phe amino acid derivatives preferentially assemble into one-dimensional fibrils, the Fmoc-peptoid analogs form two-dimensional microsheets that ultimately adopt three-dimensional crystalline states.


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Figure 1. Chemical structures of Fmoc-Phe derivatives (1a-4a) and the corresponding peptoid analogs (1b4b). 1a Fmoc-Phe, 2a Fmoc-3F-Phe, 3a Fmoc-4-NO2-Phe, 4a Fmoc-F5-Phe, 1b Fmoc-Nphe, 2b Fmoc-3FNphe, 3b Fmoc-4-NO2-Nphe, 4b Fmoc-F5-Nphe.

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Figure 2. TEM image of fibrils formed by compounds 1a-4a and microsheets formed by peptoids 1b-4b at 200 µM monomer in 0.17% DMSO/H2O.


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Figure 3. TEM and SEM images of fibrils and crystals formed by compounds 1a-4a and peptoid analogs 1b4b at 4.94 mM in 50% DMSO/H2O (v/v).



Figure 4. Chemical structures and packing architectures of previously reported Fmoc-Phe derivatives that undergo spontaneous transition from hydrogel fibrils to crystalline microtubes.17, 24, 34 A) Fmoc-4-NO2-Phe (3a),17 B) Fmoc-4-CH3-Phe,24 C) Fmoc-4-NO2-Phe/Fmoc-4-CN-Phe.24 82x127mm (300 x 300 DPI)


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Figure 5. Packing of Fmoc-Phe 1a from X-ray diffraction analysis. A) Packing arrangement including DMSO, B) View of packing structure highlighting offset face-to-face aromatic interactions between neighboring side chain phenyl groups and N-terminal Fmoc groups as well as hydrogen bonds (N–H…O) between neighboring carbamate groups.



Figure 6. Extended packing arrangements from X-ray diffraction analysis for crystals of A) Fmoc-Nphe (1b), B) Fmoc-3F-Nphe (2b), C) Fmoc-4-NO2-Phe (3b).


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Figure 7. Significant intermolecular and intramolecular interactions within crystals of Fmoc-Nphe (1b).



Figure 8. A) Extended packing arrangements from X-ray diffraction analysis for crystals of Fmoc-F5-Nphe (4b) with block morphology. B) Intermolecular interactions between Fmoc and pentafluorophenyl groups in neighboring molecules. C) Intermolecular hydrogen bonding between carboxyl groups in neighboring molecules. D) Intermolecular Fmoc-Fmoc and pentafluorophenyl-pentafluorophenyl groups in neighboring molecules. E) Intramolecular face-to-face Fmoc-pentafluorophenyl interactions.


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Figure 9. A) Extended packing arrangements from X-ray diffraction analysis for crystals of Fmoc-F5-Nphe (4b) with needlelike morphology. B) Intermolecular hydrogen bonding between carboxyl groups in neighboring molecules. C) Intermolecular Fmoc-Fmoc and pentafluorophenyl-pentafluorophenyl groups in neighboring molecules. D) Intermolecular pentafluorophenyl C–F...H interactions.



Table of Contents Figure


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Comparison of the Self-Assembly Behavior of Fmoc-Phenylalanine

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