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Effect of C-Terminal Modification on the Self-Assembly and Hydrogelation of Fluorinated Fmoc-Phe Derivatives Derek M. Ryan, Todd M. Doran, Samuel B. Anderson, and Bradley L. Nilsson* Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, United States
bS Supporting Information ABSTRACT: The development of hydrogels resulting from the self-assembly of low molecular weight (LMW) hydrogelators is a rapidly expanding area of study. Fluorenylmethoxycarbonyl (Fmoc) protected aromatic amino acids derived from phenylalanine (Phe) have been shown to be highly effective LMW hydrogelators. It has been found that side chain functionalization of Fmoc-Phe exerts a significant effect on the self-assembly and hydrogelation behavior of these molecules; fluorinated derivatives, including pentafluorophenylalanine (F5-Phe) and 3-F-phenylalanine (3-F-Phe), spontaneously self-assemble into fibrils that form a hydrogel network upon dissolution into water. In this study, Fmoc-F5-Phe-OH and Fmoc-3-F-Phe-OH were used to characterize the role of the C-terminal carboxylic acid on the self-assembly and hydrogelation of these derivatives. The C-terminal carboxylic acid moieties of Fmoc-F5-Phe-OH and Fmoc-3-F-Phe-OH were converted to C-terminal amide and methyl ester groups in order to perturb the hydrophobicity and hydrogen bond capacity of the C-terminus. Self-assembly and hydrogelation of these derivatives was investigated in comparison to the parent carboxylic acid compounds at neutral and acidic pH. It was found that hydrogelation of the C-terminal acids was highly sensitive to solvent pH, which influences the charge state of the terminal group. Rigid hydrogels form at pH 3.5, but at pH 7 hydrogel rigidity is dramatically weakened. C-terminal esters self-assembled into fibrils only slowly and failed to form hydrogels due to the higher hydrophobicity of these derivatives. C-terminal amide derivatives assembled much more rapidly than the parent carboxylic acids at both acidic and neutral pH, but the resultant hydrogels were unstable to shear stress as a function of the lower water solubility of the amide functionality. Co-assembly of acid and amide functionalized monomers was also explored in order to characterize the properties of hybrid hydrogels; these gels were rigid in unbuffered water but significantly weaker in phosphate buffered saline. These results highlight the complex nature of monomer/ solvent interactions and their ultimate influence on self-assembly and hydrogelation, and provide insight that will facilitate the development of optimal amino acid LMW hydrogelators for gelation of complex buffered media.
’ INTRODUCTION The development of hydrogels formed by the noncovalent self-assembly of simple monomers is a rapidly expanding area of study.1-10 Hydrogels derived from the self-assembly of low molecular weight (LMW) compounds have found use in tissue scaffolding,11 cell culture,12-14 drug delivery,15-18 and wound healing.19 Ideally, hydrogels must exhibit shear responsive behavior, sufficient gel strength, and biocompatibility in order to be practical in these applications.20-22 LMW hydrogelators are attractive due to ease of synthetic preparation and derivatization.23 Hydrogen bonding, van der Waals, hydrophobic, electrostatic, and π-π interactions promote self-assembly of LMW hydrogelators into fibril architectures that subsequently become entangled, resulting in formation of the hydrogel state.2,24 Further investigations into how these physicochemical forces drive selfassembly and gelation will facilitate the design of a new generation of materials. Many of the reported LMW hydrogelators are short peptides designed to adopt conformations that promote self-assembly into entangled fibril networks.9,10,25-36 While some peptide r 2011 American Chemical Society
hydrogel systems are biocompatible,14 shear responsive,20-22 and mechanically robust,37 a substantial drawback of peptide based gels is the high cost of these materials. Small molecule hydrogelators that possess the same advantages as peptide systems are highly attractive due, in part, to the relative low cost of production.23 In addition, recent studies have indicated that appropriate small molecule functionalization can be used to tune the bulk properties of the resulting hydrogels, providing systems that are competitive with modular and tunable peptide-based hydrogels.38-40 Fluorenylmethoxycarbonyl (Fmoc) protected aromatic amino acids have been shown to be highly effective LMW hydrogelators.38-51 It was found that both perfluorination and monohalogenation of the phenyl side chain of Fmoc-Phe resulted in rapid self-assembly and gelation relative to unsubstituted Fmoc-Phe.38,39 It was also observed that polyethyleneglycol Received: December 5, 2010 Revised: February 9, 2011 Published: March 14, 2011 4029
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Figure 1. Structures of C-terminal modified Fmoc-Phe derivatives.
(PEG) functionalization at the C-terminus of these monomers resulted in gel networks that exhibited shear-recovery behavior.40 Our previously described Fmoc-Phe derivative hydrogel systems were prepared in unbuffered water, resulting in gels with pH values ranging from 3.5-3.9. Hydrogel pH was dependent on the pKa of the Fmoc-Phe monomer that was used to form the gel. In order to make these compounds useful for biological applications, efficient self-assembly and hydrogelation must occur at pH 7. Dissolution of monomers at neutral pH will result in an obvious alteration of the bulk carboxylate charge state from partially protonated to completely deprotonated and negatively charged. Based on the observation that incorporation of a PEG moiety at the C-terminus of Fmoc-F5-Phe dramatically affected its self-assembly and rheological properties, we hypothesized that altered charge states and variable C-terminal substitutions would also influence the assembly and gelation of these monomers. In this report, we investigate the effect of perturbation at the Cterminal carboxylic acid on the self-assembly and hydrogelation of Fmoc-F5-Phe and Fmoc-3-F-Phe derivatives. Specifically, we examined the influence of pH and C-terminal amide and ester functionalization on the behavior of these Fmoc-Phe derivatives (Figure 1). It was found that pH and C-terminal functional group manipulation perturbed the self-assembly of these LMW hydrogelators and that the bulk rheological properties of the resulting gels differed from those observed in unbuffered water. These results indicate that the C-terminal carboxylate of halogenated Fmoc-Phe derivatives exerts a strong influence on self-assembly and hydrogelation of these derivatives and that the carboxylate, in addition to the phenyl side chain, provides a convenient handle to tune the properties of the resulting hydrogels.
’ EXPERIMENTAL SECTION Materials and Methods. Fmoc-amino acids and organic solvents were purchased commercially and used without further purification. CH2Cl2 was purified as described in ref 52. Water for gelation experiments was purified using a nanopure filtration system prior to use. Synthetic details and characterization of Fmoc-3-F-Phe-OMe, Fmoc-3F-Phe-NH2, Fmoc-F5-Phe-OMe, and Fmoc-F5-Phe-NH2 are reported in the accompanying Supporting Information. Gelation Conditions. Each of the C-terminal derivatives was dissolved in dimethyl sulfoxide (DMSO; Aldrich) at a concentration of 123 mM. The solutions were then diluted with water to a final amino acid concentration of 2.5 mM in 2% DMSO/H2O (v/v). Co-assembly experiments were conducted by mixing 123 mM DMSO solutions of each monomer in 1:1 ratios prior to dilution with either water or phosphate buffered saline (PBS, pH 7.0) to a final concentration of 2.5 mM total monomer (1.25 mM acid:1.25 mM amide/ester) in 2% DMSO/H2O or 2% DMSO/PBS (v/v). Following dilution, each sample
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was vortexed for 3-5 s and allowed to stand at room temperature. Each sample formed an opaque suspension, which either became optically transparent over the course of 5-30 min or precipitated from solution depending on the identity of the compound under investigation. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded on an AVIV 202 circular dichroism spectrometer. Gel samples were prepared by diluting concentrated MeOH or DMSO solutions of the Fmoc amino acids (123 mM) into water or PBS to final concentrations of 2.5 mM Fmoc amino acid in 2% MeOH/H2O or 2%DMSO/ H2O (v/v). Co-assembly mixtures were similarly prepared by mixing (123 mM) MeOH solutions in 1:1 ratios prior to dilution with water or PBS. The solutions were then immediately transferred into a 0.1 mm path length quartz cuvette, and gelation was allowed to proceed in the cuvette. Spectra of the transparent gels were collected at 25 °C from 350 to 190 nm with a 1.0 nm step, 1.0 nm bandwidth, and 6 s averaging time per step. NMR Spectroscopy. 19F NMR spectra were collected on a Br€uker AMX-300 MHz NMR instrument. Solutions of 1.23 mM Fmoc-3-FPhe-OH in 2% DMSO/PBS v/v and 1.23 mM Fmoc-3-F-Phe-OH in DMSO were prepared in NMR tubes fitted with an internal capillary containing 7.41 mM Fmoc-4-CF3-Phe as a standard. The reported values for soluble monomer remaining in solution were determined from the difference between the integrated signals of the monomeric Fmoc-3-F-Phe-OH (DMSO solution) and assembled Fmoc-3-F-PheOH (2% DMSO/PBS) relative to the Fmoc-4-CF3-Phe standard. Solutions of 1.23 mM Fmoc-F5-Phe-OH in 2% DMSO/PBS v/v and 1.23 mM in DMSO were prepared in NMR tubes fitted with an internal capillary containing 24 mM Fmoc-3-F-Phe-OH as an integrative standard. The reported values for soluble monomer remaining in solution were calculated from the difference between integrated values of the monomeric Fmoc-3-F-Phe-OH (DMSO solution) and assembled Fmoc-3-F-Phe-OH (2% DMSO/PBS). Co-assembly of mixtures of Fmoc-3-F-Phe-OH/Fmoc-3-F-Phe-NH2 and Fmoc-F5-Phe-OH/FmocF5-Phe-OH were prepared in NMR tubes fitted with Fmoc-4-CF3-PheOH and Fmoc-3-F-Phe-OH standards, respectively, and monomer remaining in solution after self-assembly was determined as described previously. Transmission Electron Microscopy (TEM). Images were obtained with a Hitachi 7650 transmission electron microscope with an accelerating voltage of 80 kV. Samples of gel were applied directly onto 200 mesh carbon coated copper grids and allowed to stand for 30-45 s. Excess gel was carefully removed by capillary action (filter paper), and the grids were then immediately stained with uranyl acetate (20 μL for 45 s). Excess stain was removed by capillary action, and the grids were allowed to air-dry for 10-15 min. Rheology. Rheological measurements were conducted on a TA Instruments AR-G2 rheometer operating in oscillatory mode, using a 20 mm parallel plate geometry equipped with a solvent trap filled with silicon oil to prevent evaporation. DMSO solutions of Fmoc amino acid were diluted into water and immediately applied to the stage (1.4 mm gap) and covered with the solvent trap in order to prevent sample evaporation. Following application of the sample to the stage, a dynamic time sweep was immediately performed at 25 °C for 35-45 min with an angular frequency of 6.283 rad s-1 and 0.2% strain. A dynamic frequency sweep was performed immediately following the time sweep experiment from 0.1-50 rad s-1 with 0.2% strain at 25 °C. All reported values for G0 and G00 are an average of at least three runs. All measurements were performed in the linear viscoelastic region for each gel.
’ RESULTS AND DISCUSSION The Effect of Charge and C-Terminal Functionalization on Self-Assembly and Hydrogelation. The propensity of a small
molecule to efficiently self-assemble is driven by the balance between monomer solubility and hydrophobicity.24,53,54 If a 4030
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Table 1. Self-Assembly Behavior of Fmoc-Phe Derivatives in Water/DMSOa fibril
clarification time compound
pHb
(min)c
appearanced
diameter
Fmoc-3-F-Phe-OH Fmoc-F5-Phe-OH
3.5 3.5
10-15 5-10
TG TG
21 ( 3 9(2
Fmoc-3-F-Phe-NH2
7.3
