Synthesis Of Non-Natural Fmoc-Protected Amino Acids To Provide Novel Fluorescent Anion Probes And Their Incorporation Into Synthetic Peptides

Abstract

Cystic fibrosis is a genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR serves to control the gradient of chloride and bicarbonate ions across the cellular membrane of epithelial tissues (e.g., mucosa, intestinal walls, and lungs). Excess chloride on the inside of epithelial cells causes mucus in the lungs to become very thick. This thickening is responsible for a patients' characteristic thick sputum, coughing, and trouble breathing. The thick mucus also creates an ideal environment for opportunistic bacteria like Pseudomonas aeruginosa and Mycobacterium tuberculosis to take residence and proliferate. Very little is known about the transmembrane structure of CFTR, but with current therapies a single mutation can shorten a person's life by about 50%. Many researchers have attempted to bypass the CFTR protein and synthesize anion shuttle transporters or transmembrane pores. The research presented here focuses on the synthesis of anion-responsive amino acids, for eventual incorporation into membrane-spanning peptides. Amino acids 2.1 and 2.2 were successfully synthesized from commercially available Fmoc-Glu-OtBu and Fmoc-Asp-OtBu through the conversion of an acid azide into an isocyanate via a Curtis Rearrangement. An N-substituted-4-amino-naphthalimide was chosen as the chromophore due to its ability to absorb and emit light at a significantly longer wavelength than those produced by naturally occurring amino acids. The fluorescence provides an indirect insight into the binding strength of the novel amino acids, and eventually designer peptides, to various biologically relevant anions. The fluorescence can also be used by a researcher to learn more about a peptides concentration, location, microenvironment, structure, and mechanism of action. The presence of a fluorenylmethyloxycarbonyl (Fmoc) protecting group allows a researcher to use standard solid phase peptide synthesis protocols to produce either a known peptide or any imaginable variation. Fluorescent amino acids are useful tools in grasping a better understanding of protein channelopathies such as cystic fibrosis. One day, the use of synthetic peptide ion channels will be an effective therapy to alleviate the many symptoms and even the causes of death that afflicts the many patients suffering from channelopathies today.