Conjugation of Two Alkyne Molecules at High Efficiency Under Physiological Conditions

The present technology describes a double-conjugation method in which two copper(l)-catalyzed azide-alkyne cycloadditions (CuAAC) reactions occur sequentially in a single reaction mixture without an intervening deprotection step or purification of intermediates. The sequential chemoselective ligation reactions are enabled by the different reactivities of chelating and non-chelating azido groups included in an unsymmetrical bisazide. These linkers can be used in combinatorial chemistry to cross-ligate alkyne molecules, in the modification of ethynyl-functionalized surface chemoselectively, and in extension, in the conjugation of three biomolecular fragments.

This method affords an excellent regioselectivity while preserving the fast kinetics and large substrate scope of the CuAAC reaction. In addition to the reduced workload comparing to currently available bifunctional linkers, the disclosed products are relatively easy to prepare. The substrate scope is broad, and the chemistry proceeds well under a vast array of conditions, including physiological conditions.