Affinity Labeling with 4-Azidophthalimide (AzPI): Relation between Labeling Rate and Fluorescence Intensity

Abstract

We recently developed 4-azidophthalimide (AzPI) as a compact fluorogenic photoreactive tag that can be attached to ligands to achieve selective fluorescence labeling of target proteins even in the presence of a large excess of non-target proteins. To further establish the utility of the AzPI tag, we focused here on streptavidin labeling with biotin-AzPI conjugates, and evaluated the relation between the amount of covalently labeled streptavidin (labeling rate) and fluorescence intensity. The labeling rate was proportional to the fluorescence intensity under standardized photo-irradiation conditions. Prolongation of the photo-irradiation time led to a marked increase in the labeling rate, but this was accompanied by a gradual decrease in the fluorescence intensity, which appeared to be due at least in part to photo-induced degradation of the target streptavidin. These findings should be helpful for achieving sensitive fluorescence detection of target proteins by using the AzPI tag.Key words photoaffinity labeling; azidophthalimide; target identification; fluorescence; chemiluminescence In general, affinity labeling utilizes chemical probes composed of a bioactive small molecule (i.e. ligand), a reactive group (e.g. photoreactive group) and a tracer unit (e.g. fluorescent group), and is considered a reliable target identification method.1-8) However, design and synthesis of highly functionalized chemical probes are often time-consuming. 9-11)To address this issue, we recently presented a simple design strategy for chemical probes bearing only a small alkoxy nitrobenzoxadiazole (O-NBD, 180 Da), 12) 2,3-dichloromaleimide (diCMI, 164 Da) 13) or 4-azidophthalimide (AzPI, 189 Da) 14) tag, each of which can serve as a combined reactive group and tracer unit. The AzPI tag is particularly useful for identifying target proteins of bioactive small molecules because it generates highly reactive nitrene species under photo-irradiation conditions and is capable of forming a covalent bond with various amino acids. In previous studies, 14) we confirmed that target proteins can be selectively fluorescence-labeled with ligand-AzPI conjugates even in the presence of a large excess of non-target proteins. Encouraged by these results, we have further investigated the utility of the AzPI tag by exploring the relation between the amount of covalently labeled target protein (labeling rate) and the fluorescence intensity. Results and DiscussionEvaluation Design and Probe Synthesis Biotin and streptavidin were selected as a model ligand and its target protein, because the labeling rate can be easily evaluated by using streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate, which can quantitatively visualize biotinylated proteins (Fig. 1). Based on the reported X-ray co-crystal structure of biotin bound to streptavidin, 15) chemical probes 1a-d with a variety of linker lengths were designed (Fig. 2). These probes were synthesized from biotin in three steps as depicted in Chart 1. Relation between Labeling Rate and FluorescentInte...

Recent advances in identifying protein targets in drug discovery

“…ACT showed selective and efficient reaction with proximal nucleophiles on target proteins under UV irradiation (Herner et al, 2016). In contrast, AzPI exhibited fluorescence after UV irradiation, enabling covalent fluorescence labeling (Chiba et al, 2017).…”

3-Benzoylquinoxalinone as a photoaffinity labelling derivative with fluorogenic properties allowing reaction monitoring under “no-wash” conditions

“…An alternative, yet underexplored approach, consists in using fluorogenic photoreactive functions which are able to both photocrosslink and unveil a fluorescent scaffold in a single-step process. This approach has been combined with diazo-, 8 azide- [8][9][10][11] and diazirine-based photoreactive functions. [12][13] This strategy was extended to the photoreactive diaryltetrazole function, which has shown to preferentially react with aspartic acid and glutamic acid residues of proteins through the formation of a nitrile imine intermediate.…”