# Unsaturated Phosphorus Electrophiles to Probe Protein Tyrosine Phosphatases

## TL;DR

Researchers developed new chemical probes to selectively study protein tyrosine phosphatases, which are important drug targets.

## Contribution

Introduces ethynyl-substituted aryl phosphonamidic and phosphonic acids as selective, low-reactivity probes for tyrosine phosphatases.

## Key findings

- The probes selectively target the catalytic cysteine residue of phosphatases without off-target reactivity.
- Phosphonic acid probes enriched the target phosphatase in human cell lysates during proteomics experiments.
- The strategy enables precise profiling of phosphatase interactions with high specificity.

## Abstract

Protein tyrosine phosphatases (PTPs) represent an important pharmacological target and subject of study. Although a number of broad‐spectrum electrophilic, phosphotyrosine‐mimicking probes have been developed to covalently capture the catalytic site of these enzymes, there is still a high demand for PTP probes with high target selectivity that are accessible in a synthetically straightforward way. Unsaturated phosphorus (V) (P(V)) compounds have recently emerged as powerful cysteine‐selective bioconjugation reagents (P5‐labeling). Herein, we introduce ethynyl‐substituted aryl phosphonamidic and phosphonic acids as phosphotyrosine mimics, which serve as active‐site‐directed, covalent probes for tyrosine phosphatases. We show that these P(V) electrophiles can be readily incorporated into a peptide sequence, allowing proximity‐enabled reactivity and selective targeting of the catalytic cysteine residue of an interacting phosphatase, as exemplified for PTP1B, a protein tyrosine phosphatase that acts as a key negative regulator of insulin signaling. Both ethynyl phosphonamidic acid and ethynyl phosphonic acid show no reactivity towards nontarget cysteine residues, though the phosphonamidic acid probe was notably less reactive toward its intended target. Proteomics experiments in human cell lysates demonstrated that the phosphonic acid probe selectively enriches its interacting phosphatase in the human proteome. Our study highlights a versatile strategy to obtain remarkably precise peptide‐based PTP probes, thereby enabling the characterization of phosphatase interactions with high specificity.

Charged aryl‐ethynyl phosphonamidic and phosphonic acids are introduced as low‐reactivity electrophiles for peptide‐based activity probes that enable selective, target‐specific profiling of protein tyrosine phosphatases. The probes show no off‐target cysteine reactivity and engage only the interacting phosphatase in global proteomic analysis.