Tyrosine-derived stimuli responsive, fluorescent amino acids

Abstract

A series of fluorescent unnatural amino acids (UAAs) bearing stilbene and meta-phenylenevinylene (m-PPV) backbone have been synthesized and their optical properties were studied. These novel UAAs were derived from protected diiodo-l-tyrosine using palladium-catalyzed Heck couplings with a series of styrene analogs. Unlike the other fluorescent UAAs, whose emissions are restricted to a narrow range of wavelengths, these new amino acids display the emission peaks at broad range wavelengths (from 400-800 nm); including NIR with QY of 4% in HEPES buffer. The incorporation of both pyridine and phenol functional groups leads to distinct red, green, and blue (RGB) emission, in its basic, acidic and neutral states, respectively. More importantly, these amino acids showed reversible pH and redox response showing their promise as stimuli responsive fluorescent probes. To further demonstrate the utility of these UAAs in peptide synthesis, one of the amino acids was incorporated into a cell penetrating peptide (CPP) sequence through standard solid phase peptide synthesis. Resultant CPP was treated with two different cell lines and the internalization was monitored by confocal fluorescence microscopy.

Fig. 1. Fluorescent UAAs derived from tyrosine/phenylalanine by extending the π-conjugation of aromatic side chain.

Scheme 1. Synthesis of fluorescent unnatural amino acids 4a–f and 5a–g starting from tyrosine.

Fig. 2. Absorption and emission spectra of mono-styryl tyrosine analogs 4a–f in DMSO: UV-vis spectra (A) before the addition (B) after the addition of NaOH; (C) a clear red-shift in emission spectra is due to the extended conjugation of the phenolate ion; (D) PL spectra; before (solid line) and after (dotted lines) the addition of NaOH.

Fig. 3. Absorption (left) and emission (right) properties of compounds 5a–f in DMSO; left inset: overlapped absorption spectra of 5a, 5b, 5e; right inset: emission colors of fluorescent amino acids in DMSO that covers the visible spectrum from violet to red.

Fig. 4. Emission spectra of 5f in neutral (blue), acidic (green) and basic (red) environment showing the RGB emission.

Fig. 5. pH (left) and redox (right) sensitivity of compound 5b. Emission spectra were recorded using 50 μM and 10 μM solutions, respectively.

Fig. 6. Absorption and emission spectra (left) of NIR emission of deprotonated 5g; c = 100 μM dissolved in HEPES buffer (pH 7.3). Shown in inset is quenching of NIR emission upon acidification (pH 2) due to the protonation of phenolate; a schematic representation (right) showing the origin of NIR emission through ICT mechanism.

Scheme 2. Solid phase synthesis of peptide 1.

Fig. 7. Internalization of peptide 1 by HeLa (left) and NIH 3T3 (right) cells visualized by confocal scanning microscope.