Synthesis and Evaluation of a Photoactive Probe with a Multivalent Carbohydrate for Capturing Carbohydrate–Lectin Interactions

Abstract

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2′-Amino-LNA Functionalized with Galactose Units

“…2-[2-(2-Azidoethoxy)ethoxy]ethoxy-2,3,4,6-tetra- O -acetyl-β- d -galactopyranoside ( 1 ) and 2-[2-(2-azidoethoxy)ethoxy]ethoxy-β- d -galactopyranoside ( 2 ) were synthesized according to procedures in the literature [ 60 , 61 ]. For synthesis of compound 3 we first tried the reduction of azido group 1 into an amino group with 10% Pd/C in different organic solvents (MeOH/EtOH/EtOAc) in an H 2 atmosphere.…”

Effective and transient mapping of protein–protein interactions: Application of novel releasable photoaffinity linkers

“…These methods are based on the conversion of noncovalent interactions between biomolecules into covalent linkages and enable the identification of weak and transient protein–protein interactions frequently found in living cells (He et al, 2017; Peng & Hang, 2015; Winnacker et al, 2009). In addition, bioorthogonal reaction‐based photoaffinity labeling is used to detect interactions in living organisms (Beck et al, 2014; Masaki et al, 2012; Ma et al, 2021; Tsung et al, 2013; Wollrab et al, 2016). However, when photo‐affinity labeling is used to track the interaction partners of low abundance proteins in vivo, large size probes that interfere with the visualization of proteins are often used.…”

Fluorescence “Turn-on” Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein–Glycoprotein Interactions

“…However, in this strategy, depending on the distance from the nucleophilic residues to the protein binding site, different spacer lengths have to be evaluated to find the most suitable fit. 19 In contrast, we envisioned a ligand-directed photoaffinity labeling technique 20 that would be an attractive choice for site-selective assembly of a desired group at the closest location to a lowaffinity carbohydrate-binding pocket.…”

“…To fabricate a useful protein probe, some essential factors need to be considered such as (1) purification of the labeled protein to avoid contamination with nonlabeled proteins and (2) removal of the directing ligand and purification tag to prevent interference with the labeled moiety during PPIs. Recently, we reported a method that used photoactivable and photocleavable probes for labeling proteins 20 and releasing labeled proteins from solid supports. 18 Encouraged by these results, herein, we combined two photosensitive groups in a molecule to design a ligand-directed labeling probe (LLP) for the fabrication of a lectin probe to study protein−glycoprotein interactions.…”