# Unlocking the Silent Proteome: Chemoselective Asn/Gln Activation for Multidimensional Protein Diversification

## TL;DR

This paper introduces a new method to chemically modify asparagine and glutamine in proteins, enabling diverse applications in biochemistry and drug development.

## Contribution

A novel strategy for chemoselective activation of Asn/Gln residues using bioorthogonal nitrile handles and carbometalation.

## Key findings

- The method enables selective modification of Asn and Gln residues in native peptides and proteins.
- It allows for the synthesis of functional antibody-fluorophore conjugates and fluorosequencing of Asn residues.
- The approach expands the chemical space of biomolecules and identifies previously silent Asn/Gln sites.

## Abstract

Amides are ubiquitous
in pharmaceuticals, natural products, and
biomolecules, owing to their exceptional stability and hydrogen-bonding
capacity. Among the amino acids, asparagine (Asn) and glutamine (Gln)
contain neutral primary amide side chains and constitute over 8% of
the human proteome. Despite their abundance, these residues have remained
largely inaccessible to selective chemical modification due to their
low intrinsic reactivity and the propensity of proteinogenic side
chains to poison transition-metal catalysts via chelation. Here, we
report a general strategy that converts the primary amides of Asn
and Gln into bioorthogonal nitrile handles, which can be further diversified
through carbometalation with aryl boronic acids to yield aryl ketone
products. This transformation proceeds with exceptional chemoselectivity,
enabling the modification of native peptides and proteins. We demonstrate
its broad utility in the synthesis of unnatural amino acids, late-stage
diversification of peptides, fluorosequencing of Asn residues, and
site-selective protein modification, culminating in the synthesis
of a functional antibody-fluorophore conjugate. The versatility and
selectivity of this approach expand the accessible chemical space
of biomolecules and provide a powerful route for uncovering previously
uncharacterized Asn/Gln sites within the chemically silent proteome.