Exploring the Scope of Alkyne-Aminooxy Bioconjugations Employing Noncanonical Amino Acids

Abstract Protein bioconjugates possess widespread utility as potential therapeutics necessitating an expansion of the chemical toolbox used for their production. One particularly promising methodology is the application of noncanonical amino acids (ncAAs) as a route to produce homogeneous and rationally engineered conjugates. Consequently, the development of new ncAAs and bioorthogonal reactions represents a means to further advance the field. Previous work has optimized the reaction of O-aminooxy moieties with an ncAA-introduced diyne functionality to generate multivalent bioconjugates. This work aims to expand this coupling to a monoyne via rationally tuning the reactivity of the monoyne with various substituents in the introduced ncAA. Initial investigations targeted the expansion of this reaction to terminal alkynes, which, while feasible, requires the addition of a CuI alkyne-activating agent. This Cu-dependent coupling progresses to near completion in approximately 2 h at room temperature. In order to eliminate the potentially cytotoxic copper, direct conjugation of the alkyne to an aryl substituent, a bromine substituent, or a combination of both strategies were investigated via the incorporation of three unique ncAAs containing the modification to the alkynyl moiety. Each of these ncAAs were able to conjugate to the O-aminooxy functionality in the absence of any transition metal activation, with the directly conjugated and brominated ncAA proceeding at the fastest rate. The reaction scope was then further expanded via investigation of alternate coupling partners, including N-hydroxylamines and hydrazides. This work ultimately provides a way to systematically modulate alkynyl activity and generates bioconjugates using a novel aminooxy coupling methodology.