Carbohydrate‐Directed Labeling Probes for UV‐Independent Chemoproteomic Profiling of Carbohydrate–Protein Interactions

ABSTRACT Weak and transient carbohydrate–protein interactions hinder the detection of carbohydrate‐binding proteins (CBPs) in native settings. Here, we introduce carbohydrate‐directed labeling probes (CDLPs), modular ligand‐directed covalent probes composed of a glycan ligand, an acyl‐transfer electrophile, and a distal azide handle. Glycan binding positions the electrophile for proximity‐enhanced, glycan‐releasing acyl transfer, leaving an azide‐bearing acyl tag on the protein. Benchmark CDLPs capture carbohydrate–protein interactions with monovalent K D values extending into the millimolar range. In Daudi B cells, a 6′‐sialyllactose CDLP preferentially captures CD22 (Siglec‐2), demonstrating glycan‐dependent labeling in live cells. Quantitative chemoproteomics using 6′‐sialyllactose‐ and lactose‐CDLPs yields complementary competition‐sensitive CDLP‐capture profiles (6′SL‐CDLP: 95; Lac‐CDLP: 113; 21 shared), including canonical CBPs, glycan‐processing proteins, and candidate proteins lacking prior glycan‐related annotations. CDLPs provide an accessible, UV‐independent route to durable covalent readouts for targeted validation and proteome‐scale candidate discovery.