Photoprobes

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.

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]. 

Horney, M.J. et al. (2001) Synthesis and characterization of insulin-like growth factor (IGF)-l photoprobes selective for the IgG-binding proteins (IGFBPs) photoaffinity labeling of the IGF-binding domain on IGFBP-2./. Biol. Chem. 276(4), 2880-2889. 

Pandurangi, R.S. et al. (1997b) Chemistry of bifunctional photoprobes Part 1. Perfluoro azido functionalized phosphorus hydrazides as novel photoreactive heterobifunctional chelating agents High efficiency nitrene insertion on model solvents and proteins./. Org. Chem. 62(9), 2798-2807. 

Pandurangi, R.S. et al. (1998) Chemistry of bifunctional photoprobes Part 4. Synthesis of the chromoge-nic, cleavable, water soluble and heterobifunctional (N-Methyl amino perfluoroaryl azide benzamido)-ethyl-l,3-dithiopropionyl sulfosuccinimide An efficient protein cross-linking agent. Bioorg. Chem. 26(4), 201-212. 

The potential of photoprobes is increasingly utilized as a remotely controllable tool in the drug discovery process. The difference between the dark state stability and light sensitivity allows the separation of two actions ... 

The design of the photoprobe is based on structure-activity relationship (SAR) studies if available. Ideally the photoprobe should be bioactive over the same range as its parent compound. The next step is to synthesize the bioactive photoprobe in radiolabeled form. Similarly, non-radioactive labels (primarily biotin) can also be attached via a linker arm [18]. 

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. 

Miller reported several monomeric, photolabile CCK agonists and antagonists. The photoreactive residue (L-Bpa) was placed at the N-terminal and a fluorescent reporter group was also linked to it. The CCK receptor in the study was expressed on Chinese hamster ovary-CCKR cells. To identify the labeled domains on the receptor capillary electrophoresis was used with laser induced fluorescence detection. Separate regions were labeled with the two photoprobes, one labeled the first extracellular loop (96-121), while the other probe labeled a fragment in the second extracellular loop (174-195). 

Non-peptidic tachykinine antagonists were converted to photoprobe ligands by Ward. First, a piperidine derivative, CP-99,994 (Glaxo) was appended with a diazirine photophore (6, Fig. 7) to study SP (NK1) receptors [74]. A similar modification on a neurokinin A antagonist, SR 48968 (Sanofi) produced a photoligand (5, Fig. 7) in order to investigate NK2 receptor proteins [75]. 

Prostaglandin receptors, including prostacyclin (PGI2), are all G-protein coupled receptors, which activate cAMP cyclase, resulting in cAMP. PGI2 receptors were identified in mouse mastocytoma P-815 cells (43 kDa) by a photolabile analogue, [15-3H]-19-(3-azidophenyl)-20-norisocarbacyclin (21, Fig. 10). The photoprobe also labeled a 45-kDa band in membranes of procine platelets [98]. 

Finally a French group developed a novel taxoid photoprobe [111], which allowed the first identification of an amino-acid sequence in the a-subunit (281-304) together with expected labeling in the /1-subunit (217-229). 

The molecular elements of that pathway were mapped with photoaffinity labeling by different investigators. Farnesyltransferase contains a and heterodimer subunits, and binds to both protein and farnesyl diphosphate. The main recognition elements for the protein is the C-terminal CAAX motif. Coleman et al. attached two benzophenones to the recognition sequence and the resulting photoprobe (38, Fig. 14) specifically labeled both subunits [125]. 

Katzenellenbogen studied several steroid receptors [21,144]. Estrogen receptor is one of the most frequently investigated receptors by PAL. Hexestrol is a non-steroidal agonist, which was modified with a diazirine photophore via a sulfide linkage (52, Fig. 18). The photoprobe efficiently (30%) labeled the... 

A couple of extensive reviews on the chemistry of benzophenone photoprobes and their applications in biochemistry have been published over the last decade.13 6 ... 

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