Anion binding by fluorescent Fmoc-protected amino acids

Two non-natural amino acids with fluorescent urea side-chains were prepared from Fmoc-protected aspartic and glutamic acids. In acetonitrile solution, the emission of the Asp derivative is strongly quenched by HCO₃⁻  or H₂PO₄⁻  (K ≥ 10⁴ M^− 1) but not by less-basic Cl⁻  or NO₃⁻ . Solutions containing excess bicarbonate ion appear peach-colored, with λ_abs at 394 and 495 nm ascribed to the anion complex and urea-deprotonated sensor, respectively. Corresponding fluorescence bands are observed at 475 and 579 nm. Dihydrogenphosphate is not sufficiently basic to remove H⁺ from the ground state of the fluorophore. However, deprotonation of the excited state occurs in the presence of>1 equiv of H₂PO₄⁻  (λ_em = 578 nm). According to ¹H NMR in DMSO-d₆, recognition of H₂PO₄⁻  occurs at the urea N–H groups and the amino acid backbone N–H. DFT techniques further predict that the backbone C = O group accepts an H-bond from the anion. The Glu derivative has lower affinity for anions; the additional CH₂ group in its side-chain apparently sets the backbone N–H and C = O too far from the urea to contribute significantly to binding. To demonstrate suitability for standard Fmoc-based solid-phase peptide synthesis, the Asp derivative was incorporated into a 12-residue peptide.