β Sheets Not Required: Combined Experimental and Computational Studies of Self-Assembly and Gelation of the Ester-Containing Analogue of an Fmoc-Dipeptide Hydrogelator

Abstract

In our work toward developing ester-containing self-assembling peptides as soft biomaterials, we have found that a fluorenylmethoxycarbonyl (Fmoc)-conjugated alanine-lactic acid (Ala-Lac) sequence self-assembles into nanostructures that gel in water. This process occurs despite Fmoc-Ala-Lac’s inability to interact with other Fmoc-Ala-Lac molecules via β-sheet-like amide–amide hydrogen bonding, a condition previously thought to be crucial to the self-assembly of Fmoc-conjugated peptides. Experimental comparisons of Fmoc-Ala-Lac to its self-assembling peptide sequence analogue Fmoc-Ala-Ala using a variety of microscopic, spectroscopic, and bulk characterization techniques demonstrate distinct features of the two systems and show that while angstrom-scale self-assembled structures are similar, their nanometer-scale size and morphological properties diverge and give rise to different bulk mechanical properties. Molecular dynamics simulations were performed to gain more insight into the differences between the two systems. An analysis of the hydrogen-bonding and solvent-surface interface properties of the simulated fibrils revealed that Fmoc-Ala-Lac fibrils are stronger and less hydrophilic than Fmoc-Ala-Ala fibrils. We propose that this difference in fibril amphiphilicity gives rise to differences in the higher-order assembly of fibrils into nanostructures seen in TEM. Importantly, we confirm experimentally that β-sheet-type hydrogen bonding is not crucial to the self-assembly of short, conjugated peptides, and we demonstrate computationally that the amide bond in such systems may act mainly to mediate the solvation of the self-assembled single fibrils and therefore regulate a more extensive higher-order aggregation of fibrils. This work provides a basic understanding for future research in designing highly degradable self-assembling materials with peptide-like bioactivity for biomedical applications.

Understanding the self-assembly of Fmoc–phenylalanine to hydrogel formation

“…[15][16][17] Apart from peptides, fluorenylmethyloxycarbonyl (Fmoc) protected amino acids have also been very effective hydrogelators. [18][19][20][21] These hydrogels have comparable physical and mechanical properties to those formed from longer peptides and covalently cross-linked polymers. 18,[22][23][24] For the first time, the formation of hydrogels from Fmoc-protected dipeptides, Fmoc-Leu-Asp was described.…”

Expanding the Functional Scope of the Fmoc‐Diphenylalanine Hydrogelator by Introducing a Rigidifying and Chemically Active Urea Backbone Modification

“…Yet, to our knowledge, its backbone modification has been attempted only twice by modifying the Fmoc‐FF amide group into N ‐benzyl glycine (Nphe) or ester. These modifications resulted in decreased hydrogen bonding, which led, in turn, to decreased rigidity of the formed hydrogel, as compared to the Fmoc‐FF under the same conditions . Accordingly, it would be expected that increasing the hydrogen bonding capability of the hydrogelator by the incorporation of a urea moiety would enhance the rigidity of its hydrogels.…”

Structural determinants in a library of low molecular weight gelators

“…38,39 Similar values have been found elsewhere for Fmoc-dipeptides, and have been ascribed to either distances between ß-sheets or distances between the Fmoc groups. 32,[40][41][42] The interstrand separation in Å, indicated by the strongest meridional signals, are generally grouped by the identity of the naphthalene group since HNap-AA and H-Nap-AV show a meridional repeat of 4.68 -4.70 Å whilst the peptides containing Br or CN have shorter repeats closer to 4.55 Å (Table S3). Reflection positions, relative intensity and axial alignment of reflections for each fXRD pattern must arise from the molecular packing of the dipeptides in the self-assembled fibres.…”

“…Reflections at approximately 26 Å have been found for Fmoc-dipeptides and ascribed to the fibre widths. 32,40,42 This data has been used to build a model for the assembly, although it is not clear to us that this model can be directly translated to our systems. Further, the reflections with the greatest relative intensity are matched in Br-Nap and CN-Nap systems.…”

“…For related Fmoc-dipeptide systems, it has been suggested that the driving force is assembly of the aromatic rings. 32,[40][41][42] Related work on a system where one amino acid was replaced with lactic acid showed that the formation of beta-sheets is not a pre-requisite for gelation (and also makes the point that interpretation of IR data is difficult for such short peptides). 40 In general, dipeptides such as those described here form micellar aggregates at high pH (i.e.…”