Fmoc-(S)-3-Amino-2-benzylpropanoic acid

Here we report the first large-scale synthesis of Fmoc-(S)-2-amino-6,6,6-trifluorohexanoic acid via asymmetric alkylation of chiral Ni(II)-complex of glycine Schiff base with CF3(CH2)3I. The synthesis was performed on over 100 g scale and can be recommended as the most advanced procedure for reliable preparation of large amounts of enantiomerically pure Fmoc-(S)-2-amino-6,6,6-trifluorohexanoic acid for protein engineering and drug design. Chiral auxiliary used in this protocol can be >90 % recovered and reused.

We have previously improved the bioactivity of PEG5k-FTS2 system by incorporating disulfide bond (PEG5k-S-S-FTS2) to facilitate the release of farnesyl thiosalicylic acid (FTS).1 Later, fluorenylmethyloxycarbonyl (Fmoc) moiety has been introduced to PEG5k-FTS2 system (PEG5k-Fmoc-FTS2) in order to enhance drug loading capacity (DLC) and formulation stability.2 In this study, we have brought in both disulfide linkage and Fmoc group to PEG5k-FTS2 to form a simple PEG5k-Fmoc-S-S-FTS2 micellar system. PEG5k-Fmoc-S-S-FTS2 conjugate formed filamentous micelles with a ~10-fold decrease in critical micellar concentration (CMC). Compared with PEG5k-Fmoc-FTS2, our novel system exhibited further strengthened DLC and colloidal stability. More FTS was freed from PEG5k-Fmoc-S-S-FTS2 in treated tumor cells compared to PEG5k-Fmoc-FTS2, which was correlated to an increased cytotoxicity of our new carrier in these cancer cells. After loading Paclitaxel (PTX) into PEG5k-Fmoc-S-S-FTS2 micelles, it showed more potent efficiency in inhibition of tumor cell proliferation than Taxol and PTX-loaded PEG5k-Fmoc-FTS2. PTX release kinetics of PTX/PEG5k-Fmoc-S-S-FTS2 was much slower than that of Taxol and PTX/PEG5k-Fmoc-FTS2 in normal release medium. In contrast, in glutathione (GSH)-containing medium, PTX in PEG5k-Fmoc-S-S-FTS2 micelles revealed faster and more complete release. Pharmacokinetics and tissue distribution study showed that our PEG5k-Fmoc-S-S-FTS2 system maintained PTX in circulation for a longer time and delivered more PTX to tumor sites with less accumulation in major organs. Finally, PTX-loaded PEG5k-Fmoc-S-S-FTS2 micelles resulted in a superior therapeutic effect in vivo compared to Taxol and PTX formulated in PEG5k-Fmoc-FTS2 micelles.

This paper describes the application of dynamic combinatorial libraries (DCL) towards the discovery of self-assembling nanostructures based on aromatic peptide derivatives and the continuous enzymatic exchange of amino acid sequences. Ultimately, the most thermodynamically stable self-assembling structures will dominate the system. In this respect, a library of precursor components, based on N-fluorenyl-9-methoxycarbonyl (Fmoc)-amino acids (serine, S and threonine, T) and nucleophiles (leucine, L-; phenylalanine, F-; tyrosine, Y-; valine, V-; glycine, G-; alanine, A-OMe amino-acid esters) were investigated to produce Fmoc-dipeptide esters, denoted Fmoc-XY-OMe. Upon exposure to a protease (thermolysin), which catalyses peptide bond formation and hydrolysis under aqueous conditions at pH 8, dynamic libraries of self-assembling gelator species were generated. Depending on the molecular composition of the precursors present in the library different behaviours were observed. Single components, Fmoc-SF-OMe and Fmoc-TF-OMe, dominated over time in Fmoc-S/(L+F+Y+V+G+A)-OMe and Fmoc-T/ (L+F+Y+V+G+A)-OMe libraries. This represented > 80% of all peptide formed suggesting that a single component molecular structure dominates in these systems. In a competition experiment between Fmoc-(S+T)/F-OMe, conversions to each peptide corresponded directly with ratios of starting materials, implying that a bi-component nanostructure, where Fmoc-TF-OMe and Fmoc-SF-OMe are incorporated equally favourably, was formed. Several techniques including HPLC, LCMS and fluorescence spectroscopy were used to characterize library composition and molecular interactions within the self-selecting libraries. Fluorescence spectroscopy analysis suggests that the most stable peptide nanostructures show significant pi-pi intermolecular electronic communication. Overall, the paper demonstrates a novel evolution-based approach with self-selection and amplification of supramolecular peptide nanostructures from a complex mixture of amino acid precursors.