Photoaffinity labeling nitrenes

Aryl nitrenes have been studied extensively over the last decades and are used in several industrial processes such as microlithography. Aryl nitrenes have also been used in photoaffinity labeling bioorganic molecules. The pursuit for organic magnetic material has sparked renewed interested in nitrene intermediates, which are ideal candidates for magnetic material because of their high spin properties. ... 

Photochemically reactive molecules have often been used as labels for specific sites in proteins and nucleic acids. Psoralen derivatives serve as relatively nonspecific photochemically activated crosslinking agents for DNA and double-stranded RNA.195 Aryl azides are converted by light to aryl nitrenes, which react in a variety of ways including insertion into C-H bonds (Eq. 23-27).200 201 In some cases UV irradiation can be used to join natural substrates to enzymes or hormones to receptors. For example, progesterone, testosterone, and other steroids have been used for direct photoaffinity labeling of their receptors.202 Synthetic benzophenones have also been used widely as photoactivated probes.203... 

Another general approach is the use of photoaffinity labels.9-11 A compound that is stable in the absence of light but that is activated by photolysis is reversibly bound to an enzyme and photolyzed. The usual reagents are diazo compounds that when photolyzed give highly reactive carbenes, or azides that give highly reactive nitrenes ... 

In the literature of photoaffinity labeling, much ado is made about the half-lifes of carbenes, nitrenes and other reactive intermediates. It is often implied that the half-life has a fixed value for each intermediate, but it is of course a function of the temperature and environment. 

The reaction products from aryl azides in photoaffinity labeling experiments have not been fully characterized. Indeed the reactions of aryl nitrenes in solution remain a topic of current research. 

Nitrenes, the nitrogen analogs of carbenes have been introduced as a reactive group in photoaffinity labels for antibodies by Fleet et al. (1969). Nitrenes tend to be more selective in their reactions and possibly will provide fewer products than carbenes (but see also 6.2.3). The types of reaction that they can undergo (eq. 6.2, Knowles 1972) include insertions into C-H bonds to yield secondary amines, cycloadditions to double bonds to form cyclic 3-member imines and... 

These authors have emphasized that the best way to distinguish between these two possible mechanisms of labeling is by the addition of scavengers of carbenes, nitrenes or whatever the chemical nature of the activated form of the photoaffinity label is. A true photoaffinity label (i.e. one which reacts irreversibly with the same protein molecule to which it was bound at the instant of photolysis) will not be inhibited by added scavengers, whereas a pseudo photoaffinity label (i.e. one which accomplishes derivatization via a reactive intermediate which has been generated in solution) will be. In their studies with nitrenes, Ruoho et al. (1973) found that 10 M p-aminobenzoic acid was an effective scavenger. It should be emphasized that the two possible mechan-... 

Studies of the photodecomposition of azido derivatives of PCB have attracted interest because of their potential use as photolabels in intracellular distribution studies.Polyfluorinated aryl azides are also of current interest as new reagents for photoaffinity labelling. The major products of photolysis of methyl 4-azidotetrafluorobenzoate in cyclohexane or diethylamine, for example, arise by insertion, a property which enhances its use in labelling. Singlet pentafluorophenyl nitrene, formed in an analogous fashion on irradiation of pentafluorophenyl azide, can be trapped by toluene as the insertion products (79) and (80). °... 

Bayley, H. and Staros, J., Photoaffinity labeling and related techniques, in Azides and Nitrenes Reactivity and Utility, Scriven, E.F.V., Ed., Academic Press, New York, 1984, pp. 434-490. 

Despite the fact that they can be much more reactive than nitrenes, carbene derivatives are less frequently cited in reports on photoaffinity labels. Two such derivatives of thyroid hormone have been described in which the amino group of the alanine side chain of 3,5,3 -triiodo-L-thyronine or 3,5,3, 5 -tetraiodo-L-thyronine was derivatized with 2-diazo-3,3,3-trifluoropropionate (246). Irradiation at 254 nm of these compounds with several cell lines yielded low incorporation of reagent (0.5-13.5%) into two forms of thyroid hormone receptor proteins. Low incorporation was also characteristic of early reports of photoaffinity labeling (e.g., 247) in which 0 -ethyl-2-diazomalonyl-cAMP and A, C)2. (ji(ethyl-2-diazomalonyl)-cAMP were used to generate carbenes which modify rabbit muscle phosphofructokinase. Similarly, only 3-14% of the potential sites of lactate dehydrogenase were labeled by the carbene derived from 3-(3//-diazirino)pyridine adenine dinucleotide (248). 

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