Photoaffinity reagent

Chowdhry Westheimer described many of the strategies and applications of photoaffinity labeling to the investigation of biological systems. (See Azido Photoaffinity Reagents) To provide an illustrative case, we describe... 

AZIDO PHOTOAFFINITY REAGENTS 3 -(p-Azido-m-iodophenylbutyryl)-2, 5 -dideoxyadenosine,... 

AZIDO PHOTOAFFINITY REAGENTS 2-Azido-NAD as G-protein structure probe, AZIDO PHOTOAFFINITY REAGENTS... 

AZIDO PHOTOAFFINITY REAGENTS AZIDO PHOTOAFFINITY REAGENTS 5-Azido-UDPglucose,... 

FOURIER TRANSFORM IR/PHOTOACOUS-TIC SPECTROSCOPY TO ASSESS SECONDARY STRUCTURE PHOTOAFFINITY LABELING AZIDO PHOTOAFFINITY REAGENTS AFFINITY LABELING Photobleaching,... 

A photoaffinity reagent (Fig. 1.1.) is a ligand that is chemically inert but conceals a highly reactive intermediate that is unmasked by irradiation with... 

Finally, these properties permit the use of photoaffinity reagents in experiments in which kinetic phenomena are examined, and it has proved possible to extend the resolution to the millisecond time-scale. 

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). 

Further examples of a, 3-unsaturated ketones include molecules such as steroid derivatives that were not originally designed as photoaffinity reagents. Polyunsaturated compounds that may be photolysed efficiently at wavelengths above 300 nm are preferred (e.g. Fig. 2.6 Dureetal., 1980 Nordeen et al., 1981 Sadler and Mailer, 1982). 

Aryl halides have been used with moderate success as photoaffinity reagents and they react in a process initiated by homolytic fission at the carbon-halogen bond (Sharma and Kharash, 1968, Grimshaw and de Silva, 1981). 

Goeldner, Hirth and colleagues have presented evidence that the efficiency of labeling by certain photoaffinity reagents is improved in the environment provided by the receptor. They define a photosuicide inhibitor as a ligand analog of an enzyme or a receptor, the photodecomposition of which is selectively induced by the intrinsic physico-chemical properties of an active site (Goeldner et al., 1982). 

Aryl azides which were introduced as photoaffinity reagents by Fleet et al. (1969) are now the most commonly used photoactivatable reagents. By early 1976 there were almost one hundred examples of their use (Bayley and Knowles, 1977) and today it would be impracticable to list a complete bibliography. The ease of synthesis of aryl azides, and not their other desirable properties, is probably responsible for their great popularity. 

The major intermolecular reaction of triplet aryl nitrenes in solution is hydrogen atom abstaction to form primary amines. For a photoaffinity reagent bound to a receptor, this would result in a failure to couple. However, it is possible that the intramolecular photochemistry of aryl azides is more relevant, and here numerous examples of insertion by triplets have been noted. Presumably, these are two step processes hydrogen atom abstraction, followed by radical coupling (cf. Figs. 2.1 and 2.3). 

Ketenes are highly reactive electrophiles but not nearly so indiscriminate as carbenes. When the properties of diazoacetyl photoaffinity reagents were evaluated the Wolff rearrangement was found to be a major problem accounting for 30 to 60% of the products arising from O-esters and 100% of the products fromS-esters. For instance, diazoacetyl-chymotrypsin gave rise to O-carboxymethyl serine formed by the attack of water on the ketene (Shafer et al., 1966) (Fig. 3.10). 

Just as we inquired for diazo compounds and azides, we may ask why certain diazirines are not used as photoaffinity reagents and what prospects there are for new diazirine reagents. 

Photoaffinity reagents of low molecular weight can be made by total synthesis or by attaching photoactivatable groups to preexisting ligands. For macromolecules, such as polypeptides, only the latter strategy is feasible. 

Fig. 3.26. The interaction of macromolecular photoaffinity reagents with a receptor, a The natural ligand binds tightly to a receptor on a membrane (or in solution), b A ligand modified with a bifunctional reagent close to its site of interaction with the receptor may not bind well. c If the ligand is modified with a short-armed reagent at an alternative site it may not label the receptor but, d if a long-armed reagent is used neighboring or irrelevant polypeptides may...

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