Synthesis and self-alkylation of isotope-coded affinity tag reagents.

A pair of ICAT reagents, N-(13-iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)biotinamide (8d, ICAT-d(8)) and N-(13-iodoacetamido-4,7,10-trioxa-tridecanyl)biotinamide (8c, ICAT-d(0)), and an alternative pair of ICAT reagents, N-(10-iodoacetamido-2,5,5,6,6,9-hexadeutero-4,7-dioxa-decanyl)biotinamide (8b, s-ICAT-d(6)) and N-(10-iodoacetamido-4,7-dioxa-decanyl)biotinamide (8a, s-ICAT-d(0)), were successfully synthesized. A mixture of sodium borohydride and cobalt(II) chloride reduced the intermediate dinitrile to the diamine without loss of the deuterium labels, which occurred when Raney nickel was the reducing agent. The problem caused by unsymmetrical biotinylation of the intermediate diamine was solved by using the solid-phase method in which one end of the diamine was attached to a chlorotrityl chloride resin, followed by biotinylation of the resin-bound amine. The self-alkylation of ICAT reagents that accounted for their instability and their limitations in the applications was also studied.