Photoaffinity Labeling with 8-Azidoadenosine and Its Derivatives:  Chemistry of Closed and Opened Adenosine Diazaquinodimethanes

Abstract

The reactive intermediate produced upon photolysis of 8-azidoadenosine was studied by chemical trapping studies, laser flash photolysis with UV-vis and IR detection, and modern computational chemistry. It is concluded that photolysis of 8-azidoadenosine in aqueous solution releases the corresponding singlet nitrene which rapidly tautomerizes to form a closed adenosine diazaquinodimethane in less than 400 fs. A perbenzoylated derivative of 8-azidoadenosine cannot undergo this tautomerization, and instead, it fragments upon photolysis to form an opened adenosine diazaquinodimethane. The singlet nitrene is too short-lived to be observed and, thus, to relax to the lowest triplet state or to become covalently attached to targeted biological macromolecules. The pivotal closed adenosine diazaquinodimethane, the product of nitrene tautomerization, has a lifetime of ca. 1 min or longer in water and in HEPES buffer at ambient temperature. However, this intermediate reacts rapidly with good nucleophiles such as amines, thiols, and phenolates, and significantly more slowly with weak nucleophiles such as alcohols and water. On the basis of these studies, it is clear that the closed adenosine diazaquinodimethane, and not the singlet or triplet nitrene, is the pivotal reactive intermediate involved in photolabeling and cross-linking studies using the 8-azidoadenosine family of photoaffinity labeling reagents.

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

“…To test these hypotheses, we employed the patch clamp technique with excised membrane patches containing CFTR and also photolabeling of CFTR using radioactive nucleotides with a photoactivatable azido (N 3 )-group attached to the adenine ring. The N 3 -group absorbs UV light, resulting in photolysis and formation of a reactive intermediate, which reacts covalently with nearby amino acid residues (38, 39). An advantage of these approaches is that they allow the interactions of nucleotides with CFTR to be studied while CFTR is embedded in the membrane.…”

The ability of triplet nitrenes to abstract hydrogen atoms

“…Singlet nitrene intermediates are useful in applications such as photoaffinity labeling, crosslinking polymers, and modification of surfaces 1–9. The use of singlet nitrenes in these applications is due to their ability to insert efficiently into surrounding chemical bonds 10.…”

The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin

“…37,38 Photo-affinity labeling with azido-ATP and azido-ATP analogs followed by enzymatic digestion and analysis by mass spectrometry have been used to identify ATP interactive regions in several proteins. 39,40 Here, FRET analysis, photo-affinity labeling, and LC/ESI-MS with a novel fluorescent analog of ATP (8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS)), were used to identify the β4-β8 groove on the surface of the α crystallin core domain as the ATP interactive region in αB crystallin.…”