Fluorescent ε-ATP analogues for probing physicochemical properties of proteins. Synthesis, biochemical evaluation, and sensitivity to properties of the medium

Despite the significance of the elucidation of proteins' physicochemical parameters to understand various molecular phenomena, direct methods for measuring these parameters are not readily available. Here, we propose the use of 8-[p-amino-Ph]-ε-ATP, 3b, as a fluorescent probe for the elucidation of physicochemical parameters of binding sites in certain proteins. We synthesized novel fluorescent nucleotide analogues based on an extension of the ε-ATP scaffold. These analogues bear a primary or tertiary p-amino-phenyl moiety on the etheno-bridge. We explored the recognition of the fluorescent analogues by the target proteins: P2Y ₁-receptor (P2Y ₁-R) and NTPDase1. Based on the high affinity to the P2Y ₁-R (EC ₅₀ 100 nM), 3b proved a suitable probe for the investigation of this receptor. Next, we elucidated the dependencies of the absorption and emission spectra of 3b on environmental parameters, for establishing correlation equations. These equations will help determine the properties of the ATP-binding site from the spectral data of the protein-bound 3b. For this purpose, the sensitivity of the probe to acidity, dielectricity, H-bonding, viscosity, and to correlation between these parameters was determined. Thus, the pH-dependence of 3b emission intensity is bell shaped. At pH 2.8 the quantum yield (φ) is enhanced 150-fold, as compared to neutral pH. The basic nitrogen atoms of 3b were assigned and pK _a values were determined. A linear relationship was found between log φ and log viscosity, however, emission maxima (λ _max) remained constant. A linear relationship was found between both φ and λ _max and dielectricity, as measured in protic or aprotic solvents of comparable viscosity. pK _a-like values were measured in acid-titrated alcohols with varying dielectricity but comparable viscosity, or with varying viscosity but comparable dielectricity. An inverse relationship and a linear relationship were found between the pK _a values of 3b and the medium dielectricity and viscosity, respectively. These correlations help the calibration of properties of a protein ATP-binding site.