Development and Optimization of an Aminooxy Coupling Reaction to Prepare Multivalent Bioconjugates with a Single Noncanonical Amino Acid

Bioconjugates have increasing utility in numerous medical and materials applications; thus, the development of new mechanisms to increase their valency and functional potential has the ability to further their impact. Expansion of the chemical tools used to prepare bioconjugates affords greater flexibility in their preparation and can improve their potency and specificity. This research integrates genetic code expansion methodologies with bioorthogonal reaction development to prepare homogeneous multivalent bioconjugates. Specifically, a novel bioorthogonal reaction has been optimized, reacting an <i>O</i>-alkoxylamine with a 1,3-diyne in the absence of any additional reagents. This reaction has been found to progress to near completion in under 30 min and generate highly stable bioconjugates. Utilizing a cascade sequence involving a bioorthogonal Glaser-Hay coupling, followed by treatment with an aminooxy partner, provides a mechanism to introduce two novel functionalities into proteins. Moreover, the precise control of genetically incorporating an alkynyl amino acid at a specific residue provides a high degree of control over the conjugate structure and activity. This cascade reaction was also optimized to occur in a one-pot fashion, obviating the need for conjugate purification between reactions. Finally, this strategy was employed in producing a highly effective antibody-drug conjugate (ADC) functionalized with monomethyl auristatin E (MMAE) and a fluorescent probe, allowing for monitoring of therapeutic delivery. When tested against HER2+ cells, this trivalent conjugate was specific, potent, and trackable. As this simple proof-of-concept demonstrates, there is limitless potential for the preparation of other therapeutic and diagnostic bioconjugates using this novel approach.