Diazoacetyl-chymotrypsin

Carbenes, which may be formed photochemically from precursors such as diazo compounds and diazirines, are highly reactive entities containing divalent carbon. The first photogenerated reagent was a carbene, formed by irradiation of diazoacetyl chymotrypsin (Singh et al., 1962). 

In Westheimer s original photoaffinity labeling experiment, diazoacetyl-chymotrypsin was photolysed. Later, Westheimer s group made the improved diazo reagents listed in Table 3.1. 

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). 

The use of PAL in enzyme modifications was first described in 1962, when Westheimer and coworkers reported on the use of a diazoacetyl group to inactivate chymotrypsin [29]. Considerable research on the development of new PAL reagents has taken place ever since [27, 28, 30-36], but only a few photophores, which largely meet the above-mentioned requirements, are being used nowadays in A/BPs. These are aryl azides (first reported use in 1969) [37], diazirines (1973) [38] and benzophenones (1973) [39]. The chemistry of these three photoreactive groups after photolysis as well as their use in recently reported A/BPs will be discussed in Sects. 3.1-3.5. 

Two types of photoactivatable reagents have been employed. The first involves prior covalent attachment of a photoactivatable group to the protein followed by photolysis of the derivatized enzyme. This approach was used in the pioneering studies of Westheimer and his colleagues in which they attached a diazoacetyl group to the reactive serine of chymotrypsin (Singh et al. 1962). 

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