Improved synthesis and iodination of a cleavable photoactivated probe

J. B. Denny and G. Blobel (1984, Proc, Natl. Acad. Sci. USA 81, 5286-5290) have described the synthesis of a water-soluble novel heterobifunctional 125I-labeled photocrosslinking reagent, N-[4-(p-azido-m-[125I]iodophenylazo)benzoyl]-3-aminopropionyl-N'- oxysulfosuccinimide, which contains a cleavable internal azo bond. We report several modifications of their synthesis which greatly increase the yield of a synthetic intermediate, N-[4-(p-aminophenylazo)benzoyl]-3-aminopropionic acid (compound VII). Evidence is presented that direct iodination of compound VII with chloramine-T produces low yields of 125I-labeled compound VII in a reaction which is difficult to control. Alternatively, the yield of 125I-labeled reagent can be greatly improved if the iodination is performed following the derivatization of N-[4-(p-axidophenylazo)benzoyl]-3-aminopropionyl-N'-oxysulfosuc cinimide (compound IX) to a ligand such as dextran. We have demonstrated the transfer of 125I label from the reagent when derivatized to amino-dextran to proteins in solution after photolysis and cleavage of this 125I-labeled reagent.