{'fraction_non_alphanumeric': 0.01916376306620209, 'fraction_numerical': 0.0024390243902439024, 'mean_word_length': 5.868421052631579, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Site‐Selective Affinity Labelling of Maltose Binding Protein in Bacterial Cells

Carbohydrates play diverse roles in biological processes through selective binding interactions to their cognate carbohydrate binding protein. This important class of biomolecular interactions is exemplified by the interactions of Siglecs, galectins and selectins, which control many aspects of the immune response. The development of selective probes for carbohydrate binding proteins would provide useful tools to further understand their complex biological roles. Early labelling strategies for lectins focused on photoaffinity techniques. More recently, elegant examples of catalytic affinity-based labelling approaches have been developed. These approaches use a combination of a N,N-dialkyl-4aminopyridine catalyst and a thioester-derivatized fluorophore to facilitate the selective acylation of the lectin of interest. For other classes of ligand binding proteins, selective chemistries involving alternate electrophiles, such as primary tosylates have been developed. Here, we demonstrate that readily synthesised carbohydrate thioester conjugates can be used directly as site-selective labelling agents for the maltose binding protein (MBP) both in vitro and in intact Escherichia coli. Nature uses thioester chemistry extensively for selective acylation reactions. Perhaps the best-known example is from intein-mediated protein splicing, which inspired the development of native chemical ligation, now extensively used in protein synthesis. Thioesters have also shown promise as ligand-directed protein labelling agents. Glutathione transferase has been sitespecifically modified at tyrosine and lysine residues by using glutathione-based thioesters containing aryl substituents. Antiviral compounds that target the HIV nucleocapsid protein include p-mercaptobenzamide thioesters that have been shown to selectively acylate the protein. Recently, the amyloidogenic transthyretin protein was selectively labelled with fluorescent stilbene aryl thioesters at a pKa-perturbed lysine residue, and the Flag-tag peptide has been labelled with a thioester-derived nickel chelator. The labelling construct developed here is composed of a carbohydrate ligand and a fluorescent thioester acyl donor. Maltose binding protein was chosen for evaluation of the labelling reaction. MBP is an E. coli periplasmic protein that binds maltodextrins with micromolar affinity (1–80 mm) and is commonly used as a fusion partner for the expression of poorly behaved proteins. 11] MBP also acts as a convenient purification tag as it displays high affinity for amylose resin. It was hypothesised that recognition of a maltosyl thioester would facilitate delivery of the fluorescent acyl group to a local nucleophilic residue in the MBP binding site, thereby allowing site-selective labelling (Scheme 1).