pH-Dependent structuring and fluorescence properties of tryptophan-derived polyamidoamino acids

The binding sites of proteins often contain tryptophan residues, whose fluorescent properties may be altered upon ligand binding. Conformational changes within the binding site can result in either fluorescence quenching or enhancement, which may be utilized to quantitatively investigate protein-ligand interactions. Amphoteric fluorescent (L)-, (D)- and (D,L)-tryptophan-deriving polyamidoamino acid (PAAC) homopolymers and their copolymers with glycine and arginine PAAC copolymers were synthesized by the polyaddition of N,N'-methylenebisacrylamide (MBA) with (L)-, (D)- and (D,L)-tryptophan and, respectively, glycine- and (L)-arginine/(L)-tryptophan mixtures. The polymerization reaction was run at 50°C and pH>10 for 7 days under nitrogen atmosphere. All polymers were characterized by NMR spectrometry, UV-Visible spectroscopy, dynamic light scattering (DLS), steady state and time-resolved fluorescence (TRF) spectroscopy. Solubility in aqueous systems at different pH’s was determined by measuring the transmittance at 450 nm. Homopolymers showed similar pH-dependent solubility patterns, with steep solubility drop at pH < 8. Volumetric particle size distribution obtained by DLS analysis showed hydrodynamic radii (Rh) ≈ 100 nm and negligible changes with pH. Fluorescence quantum yield of the MBA-L-tryptophan measured at 10-5 M repeating unit concentration decreased from 6 to 4 % from pH 11 to 8 respectively. Excitation and emission spectra showed maxima at 279 nm and 370 nm respectively, the latter showing a 20 nm blue shift by decreasing pH from 11 to 8. The excited-state lifetimes of the MBA-(L)-tryptophan at the maximum emission wavelength decreased from τ1 = 0.90 ns, τ2 = 4.40 ns to τ1 = 0.81 ns, τ2 = 3.49 ns passing from pH 11 to 8, with a population redistribution in favour of the shortest time. PAAC copolymers were synthesized under the same conditions of the homopolymers. The solubility curves in water of copolymers showed complete solubility for tryptophan content up to 20 % on a molar basis. For tryptophan content up to 30 and 40%, transmittance dropped down, respectively, to 60 % and 5 % in the pH range 2-7. DLS measurements at different pH’s showed average volume particle size 2.5 nm in the range considered. Preliminary TRF spectroscopy data proved that PAAC copolymers were endowed with pH-dependent emission properties. Moreover, significant fluorescence quantum yield was detected at the tryptophan content as low as 5 %. Finally, preliminary circular dichroism measurements showed pH-dependent patterns. In conclusion, tryptophan-based PAAC polymers displayed tunable amphiphilicity, chirality and self-assembly properties.