Efficient synthesis of isotopically pure isotope-coded affinity tagging reagents

Synthesis of an isotopically pure d(8)-ICAT linker, N- [(5,5,6,6,8,8 9,9-H-2)-13-biotinamido-4,7,10-trioxatridecanyl] tert-butyloxy carbamide (12), has been achieved in seven steps with an overall yield of 33%. Conjugation of exchange-inert d(4)-starting materials by classic etherification reaction yielded a pure synthon, carrying eight deuteriums that remained exchange-inert throughout subsequent reactions. This modified synthesis constitutes a significant improvement to the reported syntheses of "heavy" ICAT reagent in terms of expense, yield, and isotopic retention. This synthesis is easily adapted to incorporate additional deuterium atoms and is equally applicable for incorporation of either C-13 and/or O-18. In addition, this synthesis allows for the introduction of different orthogonal functionalities and provides for a high yielding series of differentially encoded ICAT tags.