Self-Assembly and Cytocompatibility of Amino Acid Conjugates Containing a Novel Water-Soluble Aromatic Protecting Group

Abstract

There has been considerable interest in peptides in which the Fmoc (9-fluorenylmethoxycarbonyl) protecting group is retained at the N-terminus, since this bulky aromatic group can drive self-assembly, and Fmoc-peptides are biocompatible and have applications in cell culture biomaterials. Recently, analogues of new amino acids with 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc) protecting groups have been developed for water-based peptide synthesis. Here, we report on the self-assembly and biocompatibility of Smoc-Ala, Smoc-Phe and Smoc-Arg as examples of Smoc conjugates to aliphatic, aromatic, and charged amino acids, respectively. Self-assembly occurs at concentrations above the critical aggregation concentration (CAC). Cryo-TEM imaging and SAXS reveal the presence of nanosheet, nanoribbon or nanotube structures, and spectroscopic methods (ThT fluorescence circular dichroism and FTIR) show the presence of β-sheet secondary structure, although Smoc-Ala solutions contain significant unaggregated monomer content. Smoc shows self-fluorescence, which was used to determine CAC values of the Smoc-amino acids from fluorescence assays. Smoc fluorescence was also exploited in confocal microscopy imaging with fibroblast cells, which revealed its uptake into the cytoplasm. The biocompatibility of these Smoc-amino acids was found to be excellent with zero cytotoxicity (in fact increased metabolism) to fibroblasts at low concentration.

Comparison of the self‐assembly and cytocompatibility of conjugates of Fmoc (9‐fluorenylmethoxycarbonyl) with hydrophobic, aromatic, or charged amino acids

“…the absorbance spectrum in Figure S1). 34 Peaks at these positions were also observed in the UV–vis absorption spectrum for Smoc‐amino acids 28 . The spectra for Fmoc‐Arg and Fmoc‐Phe liquid (pH 8) are qualitatively similar in shape; however, a remarkable ‘chiral inversion’ for Fmoc‐Phe is observed in the CD spectrum for the soft gel at pH 9.…”

“…Fmoc‐Arg forms plate‐like crystals under the pH 3 conditions used to obtain sufficient aqueous solubility. The ordering of Fmoc‐Arg will be driven by Fmoc‐stacking interactions (as for Fmoc‐Phe), but it should be noted that Arg‐Arg interactions with hydrophobic π‐π character are a possible contributor to the intermolecular interactions, as noted for Smoc‐Arg 28 . Fmoc‐Phe forms extended self‐assembled fibril and nanotape structures under the basic pH 8 conditions employed.…”

“…There do not appear to be studies on the bioactivity for the three Fmoc‐aa's that are the focus of the present manuscript. This article complements our recent paper on the self‐assembly and biocompatibility of Smoc‐Ala, Smoc‐Arg, and Smoc‐Phe where Smoc denotes the water‐soluble disulfonated Fmoc analogue 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl 28 . We do not directly compare the results due to the differences in solution conditions used to examine the two systems that arise from the significantly higher hydrophobicity of Fmoc.…”