Fmoc-4-(Boc-aminomethyl)-L-phenylalanine

A practical synthesis of the novel highly fluorinated amino acid Fmoc-perfluoro-tert-butyl tyrosine was developed. The sequence proceeds in two steps from commercially available Fmoc-4-NH2-phenylalanine via diazotization followed by diazonium coupling reaction with perfluoro-tert-butanol. In peptides, perfluoro-tert-butyl tyrosine was detected in 30 s by NMR spectroscopy at 500 nM peptide concentration due to nine chemically equivalent fluorines that are a sharp singlet by 19F NMR. Perfluoro-tert-butyl ether has an estimated σp Hammett substituent constant of +0.30.

Supramolecular hydrogels formed by self-assembly of low molecular weight (LMW) compounds have been identified as promising materials for applications in tissue engineering and regenerative medicine. In many cases, the relationship between the chemical structure of the gelator and the emergent hydrogel properties is poorly understood. As a result, empirical screening strategies instead of rational design approaches are often relied upon to tune the emergent properties of the gels. Herein, we describe a novel strategy to identify improved phenylalanine (Phe) derived gelators using a focused empirical approach. Fluorenylmethoxycarbonyl (Fmoc) protected Phe derivatives are a privileged class of gelators that spontaneously self-assemble into fibrils that entangle to form a hydrogel network upon dissolution into water. However, the Fmoc group has been shown to have toxicity drawbacks for potential biological applications, requiring the identification of new N-terminal modifications that promote efficient self-assembly but lack the shortcomings of the Fmoc group. We previously discovered that fibrils in Fmoc-p-nitrophenylalanine (Fmoc-4-NO2-Phe) hydrogels transition to crystalline microtubes after several hours by a mechanism that involves the hierarchical assembly and fusion of the hydrogel fibrils. We hypothesized that this hierarchical crystallization behavior could form the basis of a screening approach to identify alternative N-terminal functional groups to replace Fmoc in Phe-derived LMW gelators. Specifically, screening N-terminal modifying groups for 4-NO2-Phe that stabilize the hydrogel state by preventing subsequent hierarchical crystallization would facilitate empirical identification of functional Fmoc replacements. To test this approach, we screened a small series of 4-NO2-Phe derivatives with various N-terminal modifying groups to determine if any provided stable LMW supramolecular hydrogels. All but one of the 4-NO2-Phe derivatives assembled into crystalline forms. Only the 1-naphthaleneacetic acid (1-Nap) 4-NO2-Phe derivative self-assembled into a stable hydrogel network. Additional Phe derivatives were modified by N-terminal 1-Nap groups to confirm the general potential of 1-Nap as a suitable replacement for Fmoc, and all derivatives formed stable hydrogels under similar conditions to their Fmoc-Phe counterparts. These results illustrate the potential of this approach to identify next-generation Phe-derived LMW gelators with improved emergent properties.

Molecular hydrogels formed by Fmoc-short peptides have been demonstrated to be a class of promising scaffolds/carrier for in vitro cell cultures/drug delivery. In this paper, we firstly studied the gelation property of Fmoc-halogenated phenylalanine and found that the halogenated compounds had better gelation properties than the Fmoc-phenylalanine in aqueous solutions. The most efficient gelator is Fmoc-4-fluoro-phenylalanine, which can gel PBS buffer solution at the minimum gelation concentration of 0.15 wt%. All of the hydrogels formed by halogenated or non-halogenated Fmoc-phenylalanine were characterized by SEM and fluorescence spectrometer. But unfortunately, they were not suitable for NIH 3T3 cell culture. Based on these information and the fact that arginine-glycine-aspartic acid (RGD) peptide could promote cells adhesion and division, we then synthesized a Fmoc-peptide (Fmoc-fFfFGRGD) based on the best gelator of 4-fluoro-phenylalanine (fF) and the cell adhesion peptide of RGD. We observed the formation of molecular hydrogels from Fmoc-fFfFGRGD and the hydrogels could promote NIH 3T3 cell adhesion and proliferation efficiently. This study provides useful information about the gelation property of peptides containing halogenated phenylalanine and the hydrogels reported in this paper had potentials to be used as materials for tissue engineering and drug delivery.