Long-lived reactive intermediate photogenerated from N-(5-azido-2-nitrobenzoyl)-N '-(D-biotinyl)-1,2-diaminoethane as an affinity reagent to streptavidin

Irradiation of a complex between N-(5-azido-2-nitrobenzoyl)-N '-(D-biotinyl )-1,2-diaminoethane (I) and streptavidin with light of 313 nm led to the co valent attachment of the photobiotin analogue I to the protein. Streptavidi n could also be labelled in the dark with prephotolyzed I. These results in dicate that a long-lived reactive intermediate was formed upon irradiation. Moreover, after cleavage of labelled streptavidin with proteinase K this i ntermediate appears to be covalently attached to the same peptide as the on e obtained by direct photoaffinity labelling. An iminosulfurane II derived from the reaction of biotin sulfur atom with aryl nitrene is responsible fo r the dark-labelling reaction. The photoproduct II converts in an aqueous s olution almost completely into N-(5-amino-2-nitrobenzoyl)-N '-(D-(S-oxo)bio tinyl)-1,2-diaminoethane (the half-life of II is 10 days). (C) 2001 Elsevie r Science B.V. All rights reserved.