Photoaffinity tags

Diaziridines and diazirines were first prepared by Abendroth and Henrich <1959AGE283>, Schmitz <1959AGE127>, and Paulsen <1960AGE781> at the turn of the 1960s. Nearly 50 years later, both diaziridines and diazirines have seen tremendous utility in synthetic chemistry and biochemistry. In particular, diazirines have been widely used as photochemical and thermal precursors of carbenes in their primary role as photoaffinity tags. Unlike diazo compounds, diazirines are reasonably stable and have been used extensively in both photolysis and thermolysis. 

The C5 position of dU has been used to attach a variety of labels or reporter groups, in particular fluorophores ° (see section 3.5). The pyrene-modified analogue (39) has been used to detect RNA bulge conformations in the HIV-TAR RNA sequence where the fluorescence is greatly enhanced,and as a donor for the red emitter nile red (40) when the two dyes are adjacent in duplex DNA the result is white light that is emitted upon excitation of (39). A zinc-porphyrin complex has been added to C5 of dU for use in electron transfer. Norbornene has been attached via an alkyne linker for post-synthetic modification with nitrile oxides in a copper-free Click reaction, " and various dienes have been attached for Diels-Alder tagging of DNA. The photoaffinity tag (41) has been incorporated into DNA adjacent to a damaged DNA base (8-oxo-dG or thymine dimer) such that when exposed to repair systems, the repair enzyme is trapped by the diazirine for mass spectroscopic characterisation. ... 

When the Sanger sequencing is performed in conjunction with mass spectrometry, the gel separation and detection of the radiolabeled or photoaffinity-tagged... 

Radiolabeled photoaffinity ligands, in which the radio-tag is an integral part of the photo-phore, are used to simplify the analysis of the ligand-acceptor conjugate. 92 Identification of the photo-insertion site requires that both the photophore and the radio-tag will remain bound to the fragmented acceptor. To this end, introduction of Phe(3,5-3H2,4-NH2) offers an efficient and supposedly facile solution to this problem. 

In cases where it has proved impractical to assay for activity at each step, protein purification has been aided by tagging a fraction of the molecules with a photoaffinity reagent (e.g. the lactose carrier of Escherichia coli Newman et al., 1981 and the /J-adrenergic receptor of frog erythrocytes Shorr et al., 1982). 

Identification of Molecular Target To isolate the binding protein of pladienolide, [ H]-labeled (3), fluorescence-tagged (4) and photoaffinity/biotin (PB)-tagged (5) pladienolide B were synthesized and treated with HeLa cells [7], The [ H] probe... 

Identification of the phenylamide receptor is hampered by loss of its ability to bind phenylamides during purification. To overcome this a photoaffinity probe for the phenylamide receptor has been synthesized. The biological properties of this probe allow its use to tag the components of the enzyme complex involved in binding. 

Mixed isotope photoaffinity reagents have been developed to facilitate target identification by mass spectrometry. To demonstrate the approach, cyclosporine was both biotinylated and functionalised with a mixed isotope pho-toactivatable benzophenone probe (Figure 1.8). Irradiation of the probe in the presence of a mixture of proteins led to selective cross-linking to cyclophilin A. The biotin tag enabled easy separation of the cross-linked protein from other proteins in the mixture. Subsequently, after tryptic digestion, the mixed isotope tag facilitated the identification, by mass spectrometry, of the peptides that were cross-linked to the functionalised probe. 

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