Ligand photoaffinity

While none of these neutrophil receptors has yet been sequenced, partial purification of the formyl peptide receptors has been reported (16). The formyl peptide receptor is particularly attractive because of the extensive structure-activity studies on peptide ligands by Freer and coworkers (17,18). The receptor is a trans-membrane glycoprotein with apparent molecular weight of 60,000 based on proteolysis (19) and photoaffinity (20) labeling studies. 

J. Lehmann and U. P. Weitzel, Synthesis and application of a-D-mannosyl clusters as photoaffinity ligands for mannose-binding proteins Concanavalin A as a model receptor, Carbohydr. Res., 294 (1996) 65-94. 

In the classical photoaffinity experiment a tissue homogenate is solubilized by standard detergents and incubated with the radiolabeled photoprobe for non-covalent binding prior to irradiation. By illumination at the excitation wavelength a covalent linkage is established between the ligand and receptor around the binding site as shown in Scheme 2. 

It has been found that the catalytic activity of PKC is enhanced by a lipid component of the cell membrane, namely phosphatidylserine. This activity is further stimulated by sn-1,2-diacylglycerol. Oleic acid also activates the enzyme in the presence of 1,2-diacylglycerol, and thus it is presumed to mimic phosphatidylserine. In order to identify that modulating binding site for oleic acid on PKC, a photoaffinity analogue was devised. A carbene generating photophore, diazirine was placed in the apolar terminus of the unsaturated fatty acid ligand (30, Fig. 12). The synthesis and the photochemical activation properties were reported by Ruhmann and Wentrup [113]. 

In another recent example, Hashimoto reported photoaffinity experiments on retinoic acid receptors (RAR). Retinoic acid plays a critical role in cell proliferation and differentiation. RARs belong to the superfamily of nuclear/ thyroid hormone receptors. They consist of six transmembrane domains (A-F) which is a general feature of these receptors. The A/B domains have an autonomous transactivation function while the C-domain contains the Zn-finger, which binds to DNA. The large E-domain participates in ligand binding, dimerization, and ligand dependent transactivation. Finally, D- and F-domains help the orientation and stabilization of the E-domain. 

Vaughan, R. A. (1995) Photoaffinity-labeled ligand binding domains on dopamine transporters identified by peptide mapping. Mol. Pharmacol. 47,956-964. 

Vaughan, R. A., Gaffaney, J. D., Lever, J. R., Reith, M. E., and Dutta, A. K. (2001) Dual incorporation of photoaffinity ligands on dopamine transporters implicates proximity of labeled domains. Mol. Pharmacol. 59,1157-1164. 

Gartner, C. A. (2003) Photoaffinity ligands in the study of cytochrome p450 active site structure. Curr. Med. Chem. 10, 671-689. 

Scheme 2 Photoaffinity Labeling and Identification of a Ligand-Contact Site...

Radiolabeled photoaffinity ligands, in which the radio-tag is an integral part of the photo-phore, are used to simplify the analysis of the ligand-acceptor conjugate. 92 Identification of the photo-insertion site requires that both the photophore and the radio-tag will remain bound to the fragmented acceptor. To this end, introduction of Phe(3,5-3H2,4-NH2) offers an efficient and supposedly facile solution to this problem. 

In summary, the inherent chemical susceptibility of the arenediazonium-based photoaffinity ligands does not make them a popular tool for routine photo-cross-linking studies. Examples of arenediazonium-based photophores that have been incoporated into peptides are given in Table 3. 

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