Development of bifunctional photoactivatable benzophenone probes and their application to glycoside substrates

Abstract

Photoaffinity labeling is used to covalently attach ligands to macromolecules to determine their spatial arrangement and structure. Benzophenone (BP) groups are widely used for covalent photoaffinity labeling and for probing protein interactions. We developed bifunctional BP photoactivatable derivatives using three different general chemical approaches. In addition to the photoaffinity reactivity of the BP, these derivatives contain an additional group: A radioactive tracer for biological studies, or an N-ethylmaleimide group as an additional crosslinker, or a biotin group to be used during purification and characterization of probe-protein complexes using the high-affinity biotin-avidin interaction. A model series of photoaffinity labeling probes was synthesized based on the arbutin ligand. These compounds can be used as probes to study the arbutin binding site of microbial beta-glucoside transporters by photolabeling residues in its vicinity. The second functionality provides additional options for studying proteins and binding sites. The probes were developed using different methodologies: (i) a diazotation reaction; (ii) protecting group methodology; and (iii) solid-phase synthesis. These procedures are general and provide a simple and versatile approach for synthesizing bifunctional BP ligands, as demonstrated here on arbutin.

Binding is not enough: Flexibility is needed for photocrosslinking of Lck kinase by benzophenone photoligands

“…Since benzophenone is a reversible photophore (i.e., benzophenone can be photoactivated multiple times until it undergoes photochemical reactions), it is generally believed that photolabeling by benzophenone reflects target-binding better than many other photocrosslinking agents, such as diazirine and azide; since diazirine and azide can be photoactivated only once, they are more susceptible to many factors that affects the kinetics of photochemical reaction, such as conformational flexibility. Many studies of benzophenone photoprobes describe preliminary biological data, such as K d , K i , and/or IC 50 , to show that the synthesized compounds bind to their intended targets 30–34. However, an important question that has not been addressed in existing literature is whether binding affinity is predictive of successful photolabeling experiments.…”

Recent Advances in Target Characterization and Identification by Photoaffinity Probes

“…The advantages of benzophenones include their stability in most organic solvents and their compatibility with several synthetic strategies. However, the major drawbacks of benzophenone- derived probes are that their bulkiness could hinder the binding with their targets and their normal requirement for long irradiation times, during which non-specific labeling can occur [1,7,8].…”

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3

“…[4] Importantly,t he selected photophore and the reporter tag should not significantly impair binding. [7] Photoactivated benzophenones are The CXCR3 receptor,ac lass AG protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. [5,6] Each photophore has its own advantages and disadvantages.…”

“…Diazirines and benzophenones are activated at al onger wavelength (~360 nm), which prevents protein damage contrastingt oa zides, which need to be irradiated at as horter wavelength (~250 nm). [7] Photoactivated benzophenones are The CXCR3 receptor,ac lass AG protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3a ntagonists have been proposedt ob e beneficial for the treatment of aw ide range of disorders including but not limited to inflammatorya nd autoimmune diseases.…”