High Efficiency Photolabeling of Human Serum Albumin and Human .gamma.-Globulin with [14C]Methyl 4-Azido-2,3,5,6-tetrafluorobenzoate

Abstract

The efficiency of photolabeling of HSA and IgG with [14C]methyl 4-azido-2,3,5,6-tetrafluorobenzoate has been studied using size exclusion chromatography in conjunction with liquid scintillation counting. Labeling efficiencies of 78% for HSA and 82% for IgG have been determined. The extent of bond insertion into proteins exceeds the C-H insertion efficiency in cyclohexane with less wastage into anilinium and azo side products. These results suggest that the photoprobe accesses hydrophobic regions of both proteins prior to photolysis.

Chemistry of Bifunctional Photoprobes

“…Preassociation of the lipophilic perfluoroarylazido labeling agent with hydrophobic regions of the aggregates could account for the large increase in insertion yields. This observation is similar to the enhancement of insertion yield when nitrenes are generated near to hydrophobic parts of proteins (17). Selective insertion at the secondary amine position is favored because of the lower nucleophilicity of the protonated primary amine of lisinopril at pH 7.…”

“…Photochemical modification requires that photogenerated precursors exhibit a high efficiency for CH or NH bond insertion rather than hydrogen abstraction, which leads to reduced byproducts. Experiments with model solvents (e.g., cyclohexane or diethyl amine) demonstrate that insertion efficiency is enhanced by the presence of electron withdrawing groups on the para position to azide group (16)(17)(18)(19). Perfluoroazidobenzonitrile 1 was shown to exhibit high CH insertion yield on cyclohexane (20).…”

“…Positions which can be modified without significantly decreasing the inhibitor binding can be subsequently derivatized with bifunctional chelating agents to carry radiometals or photoaffinity probes. The modifications described here, carried out with derivatives of perfluoroarylazides, extend the potential for covalent incorporation of heterobifunctional radioimaging agents, developed in our laboratory (15)(16)(17)(18)(19)(20), to lisinopril. The same modification procedure can be used for preparation of photoaffinity labeling probes which are useful for mapping the binding sites of enzymes and receptors (21 ).…”

Chemistry of Bifunctional Photoprobes

“…Study of the fundamental photochemistry of perfluoroaryl azides by Keana et al (10,11) and the development of high efficiency heterobifunctional perfluoroaryl azide based chelating agents for covalent attachment to proteins and antibodies in our laboratory (12)(13)(14)(15)(16) and by others (17,18) suggest that perfluoroaryl azides are potentially good candidates as cross-linking agents. The promising nature of perfluoroaryl azides as cross-linkers stems from the fact that singlet perfluoroaryl nitrene rearranges more slowly to didehydroazipines, allowing the reactive intermediate to react with even unactivated CH bonds by formal insertion (19)(20)(21)(22)(23).…”

“…The promising nature of perfluoroaryl azides as cross-linkers stems from the fact that singlet perfluoroaryl nitrene rearranges more slowly to didehydroazipines, allowing the reactive intermediate to react with even unactivated CH bonds by formal insertion (19)(20)(21)(22)(23). Recently, we have shown from flash photolysis studies that the bifunctional nitrenes carrying electron withdrawing groups (amide and ester groups) exhibited a longer lifetime for singlet nitrenes (Ͼ250 ns), leading to the highest CH insertion in model solvents (1) and in proteins (12,15,16). Such observations prompted us to design and report, here, a first example of the synthesis of a chromogenic, cleavable, water soluble, and heterobifunctional sulfosuccinimidyl (N-methylamino perfluoroazido benzamido)ethyl-1,3Ј-dithiopropionate 7 (Scheme 1).…”