Photoaffinity probe

ABPP is only applicable to targets that possess a nucleophilic active-site residue (Ser, Cys, Lys) susceptible to covalent labeling by an electrophile. When this is lacking, an alternative is to add a photoaffinity group to an inhibitor scaffold so that a covalent adduct with the target can be created by exposure to UV light. 

If the probe must also possess a reporter group for detection and/or enrichment of labeled targets, it can be functionalized with an 

The sustained attractiveness of photolabeling is apparent from its prominence in studies of y-secretase, an intramembrane protease that contributes to forming amyloid-p peptides and is a major target in Alzheimer s disease [60-62]. y-Secretase is a complex of at least four different polypeptides, and is difficult to engage with high-resolution structural methods. However, in a case of this kind that involves a known target, immunodetection of proteins can often specify the target of y-secretase inhibitor photoaffinity probes such as 19, and proteomic mass spectrometry is not needed. 

Peptidic photoprobes can be based on the photoreactive amino acid p-benzoyl-L-phenylalanine inserted into a peptide in place of a natural aromatic residue by peptide synthesis [65] or by manipulation of the genetic code [66]. The use of p-benzoyl-L-phenylalanine for this purpose is not new, but the nature of peptide probes naturally offers opportunities for the location of linkage sites by proteomic analysis [67]. 

Finally, GTP is bound by many proteins, and diazirine-bearing thiol-cleavable GTP analogs (e.g., 23) have been proposed as probes to detect such proteins. By this method, H-Ras was successfully labeled in a nuclear extract of cultured human cells [70]. 

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Figure 2 Double-stranded oligonucleotide photoprobes that simulate modified DNA and intended to cross-link to DNA-binding proteins. (A) Probe modeling interstrand cross-linking by cisplatin Source From Ref. [63], with permission from the American Chemical Society via the Rightslink service (license number 2458870278307 granted June 30, 2010). The benzophenone probe prior to reaction with DNA is shown in the lower part of the panel. (B) Photoaffinity probe for bacterial DNA repair proteins. TT is a simulated thymine dimer intended to be recognized as a site of damage in DNA, and T (two instances) is the diazirine thymine derivative T Source From Ref. [64], with permission from Wiley.

Drafler, F.L., and Marinetti, G.V. (1977) Synthesis of a photoaffinity probe for the b-adrenergic receptor. Biochem. Biophys. Res. Comm. 79, 1. 

Fig. 7. Photoaffinity probes for studying G-protein coupled receptors... 

Several non-peptidic CCK photoaffinity probes were synthesized by Darrow et al. [65]. Benzophenone and diazirine photophores were linked to 1,5-benzo-... 

Fig. 8. Photoaffinity probes for studying light-induced and chemosensory signaling pathways... 

A different set of photoaffinity probes (29, Fig. 12) was designed and synthesized by Atkinson et al. The photosensitive 4-azido-2,3,5,6-tetrafluorobenzyloxy group was placed at the terminus of the phytyl side chain [112]. 

Fig. 13. Photoaffinity probes for studying transcription factors, inositol-polyphosphate binding proteins, and biopolymers containing an SH2 domain...

Fig. 14. Photoaffinity probes studying biomolecular targets in protein prenylation...

Fig. 15. Miscellaneous photoaffinity probes for studying interactions in tyrosine kinase, calmodulin and nitric oxide related signaling...

Fig. 16. Photoaffinity probes for studying signaling pathways in immunomodulation...

Fig. 17. A photoaffinity probe for integrin signaling and photoactivatable membrane probes...

Antonovic, L., Hodek, P., Smrcek, S., Novak, P., Sulc, M., and Strobel, H. W. (1999) Heterobifunctional photoaffinity probes for cytochrome P450 2B. Arch. Biochem. Biophys. 370, 208-215. 

The area of covalent glycosidase inhibitors has recently been highlighted in an excellent review that also includes compounds containing such reporter groups as photolabile functional groups as photoaffinity probes, and others.161... 

Arenediazonium salts substituted with electron-donating groups are good candidates for photoaffinity probes.111191 Photoactivation of these salts leads to the corresponding aryl cations (Scheme 10), which can be stabilized by electron-donating groups at the 2- and 4-posi t ions J11191... 

Carbene generation from photolysis of diazirine compounds leads to efficient insertion into C—H or N—H bonds and also causes addition reactions with points of unsaturation within target molecules. Diazirine-containing photoaffinity probes have... 

Chong, P. C., and Hodges, R. S. (1982). Photochemical cross-linking between rabbit skeletal troponin and alpha-tropomyosin. Attachment of the photoaffinity probe N-(4-azidobenzoyl-[2-3H]glycyl)-S-(2-thiopyridyl)-cysteine to cysteine 190 of alpha-tropomyosin. / Biol. Chem. 257, 9152-9160. 

The synthesis of the photoaffinity probe, NAZA-FL, lc, will be described elsewhere. The effects of this acyclic modifier on mitochondrial activities were assayed as previously described (5,6). The results are summarized in the Table. The effects, and the effective concentration range, of compounds lc and La are similar in the dark. With the photolabile modifier,< T(c, illumination with strong light resulted in an irreversible preferential inhibition of the energy-dependent proton movements this effect was not relieved by phosphatidylcholine vesicles. Hence, the modifier became covalently bonded in the vicinity of its implantation. Additional experiments were performed with phosphate-washed (12) membrane preparations (ATPase-enriched inner membrane frac-... 

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