Photoactivatable

Radioiodinated derivatives have been prepared to define more closely the target site of a-conotoxins on the acetylcholine receptor (R. Myers, unpublished data). In membrane preparations from Torpedo electroplax, photoactivatable azidosalicylate derivatives of a-conotoxin GIA preferentially label the p and 7 subunits of the acetylcholine receptor. However, when the photoactivatable derivative is cross-linked to detergent solubilized acetylcholine receptor (AChR), only the 7 subunit is labeled. Since snake a-neurotoxins mainly bind to the a subunits of AChR and a-conotoxins compete directly with a-bungarotoxin, the cross-linking results above are both intriguing and problematic. 

Verga, D. Richter, S. N. Palumbo, M. Gandolfi, R. Freccero, M. Bipyridyl ligands as photoactivatable mono- and bis-alkylating agents capable of DNA cross-linking. Org. Biomol. Chem. 2007, 5, 233-235. 

Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297 1873-1877... 

Henderson JN, Gepshtein R, Heenan JR, Kallio K, Huppert D, Remington SJ (2009) Structure and mechanism of the photoactivatable green fluorescent protein. J Am Chem Soc 131 4176 1177... 

Verkhusha, V. V. and Sorkin, A. (2005). Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem. Biol. 12,... 

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24, 461-5. 

Visible fluorescent proteins from jellyfish and corals (VFPs) la. Spectral variants lb. Dual-VFP or VFP-luciferase constructs for sensing ions, pH, covalent modification,... lc. Photoactivatable, photoconvertible, 238 27 + ... 

SADP, N-succinimidyl-(4-azidophenyl)l,3 -dithiopropionate, is a photoreactive heterobifunctional crosslinker that is cleavable by treatment with a disulfide reducing agent (Thermo Fisher). The crosslinker contains an amine-reactive NHS ester and a photoactivatable phenyl azide group, providing specific, directed coupling at one end and nonselective insertion capability at the other end. 

Baenzigen J.U., and Fiete, D. (1982) Photoactivatable glycopeptide reagents for site-specific labeling of lectins./. Biol. Chem. 257, 4421M425. 

Denney, J.B., and Blobel, G. (1984) 1251-Labeled cross-linking reagent that is hydrophilic, photoactivat-able, and cleavable through an azo linkage. Proc. Natl. Acad. Sci. USA 81, 5286-5290. 

Goodfellow, V.S., Settineri, M., and Lawton, R.G. (1989) p-Nitrophenyl 3-diazopyruvate and diazopyru-vamides, a new family of photoactivatable cross-linking bioprobes. Biochemistry 28, 6346. 

Ji, T.H., and Ji, I. (1982) Macromolecular photoaffinity labeling with radioactive photoactivatable heterobifunctional reagents. Anal. Biocbem. 121, 286-289. 

Prossnitz, E., Nikaido, K., Ulbrich, S.J., and Ames, G.F. (1988) Formaldehyde and photoactivatable cross-linking of the periplasmic binding protein to a membrane component of the histidine transport system of Salmonella typhimurium. J. Biol. Chem. 263, 17917-17920. 

Yan, M., Cai, S.X., Wybourne, M.N., and Keana, J.F.W. (1994) N-Hydroxysuccinimide ester functionalized perfluorophenyl azides as novel photoactivatable heterobifunctional cross-linking reagents. The covalent immobilization of biomolecules to polymer surfaces. Bioconjugate Chem. 5, 151-157. 

Alternatively, arene displacement can also be photo- rather than thermally-induced. In this respect, we studied the photoactivation of the dinuclear ruthenium-arene complex [ RuCl (rj6-indane) 2(p-2,3-dpp)]2+ (2,3-dpp, 2,3-bis(2-pyridyl)pyrazine) (21). The thermal reactivity of this compound is limited to the stepwise double aquation (which shows biexponential kinetics), but irradiation of the sample results in photoinduced loss of the arene. This photoactivation pathway produces ruthenium species that are more active than their ruthenium-arene precursors (Fig. 18). At the same time, free indane fluoresces 40 times more strongly than bound indane, opening up possibilities to use the arene as a fluorescent marker for imaging purposes. The photoactivation pathway is different from those previously discussed for photoactivated Pt(IV) diazido complexes, as it involves photosubstitution rather than photoreduction. Importantly, the photoactivation mechanism is independent of oxygen (see Section II on photoactivatable platinum drugs) (83). 

The major requirements for photoactivatable amino acids are that they should exhibit high optical purity and contain appropriate protecting groups for solid-phase synthesis there is also a particular advantage if they contain the radiolabel. While p-azido-phenylalanines can easily be labeled with 125I and... 

Fig. 4. Frequently used photoactivatable bifunctional cross-linker reagents...

The aryl azido derivatives of [32P]NAD+ were employed to site-specifically incorporate photoactivatable ADP-riboses to transducin at Cys-347 by means of pertussis toxin, and the conjugation was followed by irradiation. Photocrosslinking revealed three major bands after irradiation which corresponded to a-y(47 kDa), a-a (83 kDa),and a-a-a (105kDa) subunit contact domains [88]. 

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

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