Spectroscopic Characterization of the SH2- and Active Site-Directed Peptide Sequences of a Bivalent Src Kinase Inhibitor

Abstract

The spectral properties of the SH2 and active site-directed sequences of the bivalent Src kinase inhibitor Ac-EELL(F₅)Phe-(GABA)₃-pYEEIE-amide (1) have been determined. Ac-pYEEIE-amide (2) and Ac-EELL(F₅)Phe-amide (3), as well as the amino acids phosphotyrosine (pTyr) and pentafluorophenylalanine (F₅)Phe, have been characterized by electronic absorption, fluorescence, and vibrational spectroscopy. Specific and unique marker bands that originate from the phosphate group of pTyr and the fluorinated aromatic ring of (F₅)Phe have been identified, with the latter showing some solvent dependence. Peptide 2 was found to have excitation and emission wavelengths emanating from pTyr at 268 and 295 nm, respectively, whereas peptide 3 displayed excitation and emission peaks attributable to (F₅)Phe at 274 and 315 nm, respectively. Fourier transform infrared (FT-IR) analysis of the amino acid pTyr identified distinct marker bands at approximately 930, 1090, and 1330 cm⁻¹ that could be attributed to the phosphate group. These markers were also observed in the IR spectrum of peptide 2. Likewise, peptide 3 displayed a characteristic C–F stretching mode at 961 cm⁻¹ due to the presence of (F₅)Phe, including two C–F reporting ring modes at 1509 and 1527 cm⁻¹. Identifying and monitoring spectroscopic changes in these marker bands may afford a means to observe the molecular interactions that occur when peptides 1–3 bind to the Src kinase.