Mechanistic Studies of the 5-Iodouracil Chromophore Relevant to Its Use in Nucleoprotein Photo-Cross-Linking

Abstract

The photoreactivity of the 5-iodouracil chromophore was investigated toward understanding photo-cross-linking of nucleic acids bearing the chromophore to functionality in associated proteins. Irradiation of 5-iodouridine (IU) in the presence of a 10-fold excess of N-acetyltyrosine N-ethylamide (1) at 308 nm with a XeCl excimer laser or at 325 nm with a HeCd laser yields uridine (U) and N-acetyl-m-(5-uridinyl)tyrosine N-ethylamide (2) in a 1:2 mole ratio. In the presence of N-acetylphenylalanine N-ethylamide, uridine and analogous ortho, meta, and para regioisomeric adducts (3o, 3m, and 3p) were formed in a similar U to adduct mole ratio. The primary photochemical process leading to products was established as carbon−iodine bond homolysis in the first excited singlet state from a deuterium labeling experiment, photoacoustic calorimetry, and quantum yield measurements. Photoreduction of IU in 2-propanol-d solvent gave U with no deuterium incorporation. Photoacoustic calorimetric measurements established that triplet benzophenone transferred energy to IU with a rate constant of 2 × 109 M-1 s-1. Further, the reaction of IU with 1 to form 2 was sensitized by benzophenone; however, comparison of quantum yields upon direct and sensitized excitation indicated that, at most, only a small portion of the reactions occurred via the triplet state. With direct excitation of IU, quantum yields as a function of the concentration of 1 showed that U and adduct 2 resulted from a common intermediate proposed to be the 5-uridinyl radical. Uridine formation was enhanced by the presence of hydrogen atom donors at the expense of formation of 2. Quantum yields were independent of excitation wavelength in the region 310−330 nm but not the reaction medium. The quantum yield of uridine formation but not adduct formation was approximately an order of magnitude higher in 90% acetonitrile−10% water than in pH 7 water. The results are discussed in terms of high-yield cross-linking of nucleic acids bearing the 5-iodouracil chromophore to associated proteins in light of cocrystal X-ray structural data.