5-Azido-2-aminopyridine, a New Nitrene/Nitrenium Ion Photoaffinity Labeling Agent That Exhibits Reversible Intersystem Crossing between Singlet and Triplet Nitrenes

Abstract

The photochemistry of a new photoaffinity labeling (PAL) agent, 5-azido-2-(N,N-diethylamino)pyridine, was studied in aprotic and protic solvents using femtosecond-to-microsecond transient absorption and product analysis, in conjunction with ab initio multiconfigurational and multireference quantum chemical calculations. The excited singlet S1 state is spectroscopically dark, whereas photoexcitation to higher-lying singlet excited S2 and S3 states drives the photochemical reaction toward a barrierless ultrafast relaxation path via two conical intersections to S1, where N2 elimination leads to the formation of the closed-shell singlet nitrene. The singlet nitrene undergoes intersystem crossing (ISC) to the triplet nitrene in aprotic and protic solvents as well as protonation to form the nitrenium ion. The ISC rate constants in aprotic solvents increase with solvent polarity, displaying a "direct" gap effect, whereas an "inverse" gap effect is observed in protic solvents. Transient absorption actinometry experiments suggest that a solvent-dependent fraction from 20% to 50% of nitrenium ions is generated on a time scale of a few tens of picoseconds. The closed-shell singlet and triplet nitrene are separated by a small energy gap in protic solvents. As a result, the unreactive triplet state nitrene undergoes delayed, thermally activated reverse ISC to reform the reactive closed-shell singlet nitrene, which subsequently protonates, forming the remaining fraction of nitrenium ions. The product studies demonstrate that the resulting nitrenium ion stabilized by the electron-donating 4-amino group yields the final cross-linked product with high, almost quantitative efficiency. The enhanced PAL function of this new azide with respect to the widely applied 4-amino-3-nitrophenyl azide is discussed.

Deuterium‐ and Tritium‐Labelled Compounds: Applications in the Life Sciences

“…[298] Them ethod is used to investigate plasma proteins [299] and lipidic biomembranes, [300] and can also be used for activitybased proteomics [301] to study enzyme activities and protein complexes.Photoaffinity probes typically involve three functionalities:1 )a ligand (affinity unit) for reversible and specific binding to the active site of the target macromolecule; 2) ap hotoreactive group (e.g., aryl azides,d iazirines,o r benzophenones) for covalent target binding upon light activation and generation of highly reactive species;a nd 3) ar eporter tag (typically aradiolabel, afluorescent dye,or an affinity tag, such as biotin) for isolation/detection of the resulting probe-protein adducts (Scheme 28). [302] In contrast to all other identification tags,atritium label can be introduced without altering the chemical structure of the ligand and thus preserves the affinity towards at arget receptor. [303] Additionally,radiolabels can be easily quantified with high sensitivity and low background interference and can also be used for in vivo activity assessment in living cells.…”

Photochemical Reactions in the Synthesis of Protein–Drug Conjugates

“…Under acidic conditions protonation of the 1 ArN species gives the nitrenium ions . However, with unsubstituted 1 ArN species, protonation by water proceeds at such a slow rate that it does not compete efficiently with the rearrangement reaction.…”

Deuterium‐ und tritiummarkierte Verbindungen: Anwendungen in den modernen Biowissenschaften

“…[296] [ 3 H]-Digoxin wurde z. [302] Im Gegensatz zu allen anderen Identifizierungs-Tags kann eine Tritiummarkierung ohne Veränderung der chemischen Struktur des Liganden eingeführt werden und erhält dadurch die Affinitätg egenüber dem Zielrezeptor aufrecht. [297] Photoaffinitätsmarkierung (PAL) ist eine weitere leistungsfähige biochemische Methode zur Untersuchung von Ligand-Protein-Interaktionen, die oftmals in der Wirkstoffsuche fürd ie Isolierung und Identifizierung unbekannter molekularer Zielstrukturen, zur Untersuchung von Interaktionen mit Off-Target-Strukturen und zur Strukturaufklärung von Bindungsstellen herangezogen wird.…”