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

## TL;DR

This paper introduces a new chemical method to create complex bioconjugates using a single noncanonical amino acid and bioorthogonal reactions.

## Contribution

A novel aminooxy coupling reaction is developed and optimized for creating multivalent bioconjugates with precise control.

## Key findings

- The aminooxy coupling reaction completes in under 30 minutes and forms stable bioconjugates.
- A one-pot cascade reaction allows introducing two functionalities into proteins without purification steps.
- The method was successfully used to create a potent and trackable antibody-drug conjugate.

## Abstract

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 O-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.