2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogs of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

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Abstract
Two new fluorescent nucleotide photoaffinity labels, 3''(2'')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5''-diphosphate (Bz2.epsilon.ADP) and 2''-deoxy-3''-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5''-diphosphate [3''(Bz2)2''d.epsilon.ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N''-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J. A., and Yount, R. G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3''(2'')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., and Yount, R. G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2.epsilon.ADP and 3''(Bz2)-2''d.epsilon.ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the .epsilon.-adenine ring. The degree of stacking was greater with the 2'' isomer than with the 3'' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2.epsilon.ADP and 3''(Bz2)2''d.epsilon.ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3''(2'')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenosine diphosphate (CBH.epsilon.ADP) and 2''-deoxy-3''-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenosine diphosphate [3''(CBH)2''d.epsilon.ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2.epsilon.ADP and 3''(Bz2)2''d.epsilon.ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2.epsilon.ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.