A New Strategy for Neurochemical Photodelivery: Metal-Ligand Heterolytic Cleavage Leonardo Zayat, Cecilia Calero, Pablo Albore ´ s, Luis Baraldo,* and Roberto Etchenique* Departamento de Quı ´mica Inorga ´ nica, INQUIMAE, Facultad de Ciencias Exactas y Naturales, UniVersidad de Buenos Aires, Ciudad UniVersitaria Pabello ´ n 2 AR1428EHA Buenos Aires, Argentina Received July 28, 2002 ; E-mail: rober@qi.fcen.uba.ar; baraldo@qi.fcen.uba.ar Over the past few years, the uncaging of biomolecules using phototriggers has been established as a promising technique in biological sciences, especially in the field of neurophysiology. 1 In most cases, the communication between neurons is achieved by means of the release and detection of molecules called neurotransmitters. 2 Thus, the controlled delivery of either these molecules, their analogues, or other substances that promote a neuronal response is a powerful tool for the determination of circuitry in a neuronal tissue. A number of biomolecules, including neurocompounds, have been caged to produce molecules that can be phototriggered. 3 The basic approach involves the modification of the molecule by introducing a group that can be cleaved by the absorption of light, thus releasing the bioactive compound. This strategy requires breaking a σ covalent bond, which involves the use of UV (∼300 nm) photons. This type of radiation may induce cellular damage and requires expensive optical equipment. To lower the energy needed for the biomolecule release, the use of coordination metal compounds in which the biomolecule acts as a ligand is an interesting possibility. There are many known complexes that undergo heterolytic photocleavage using low-energy light. 4 Although some chelate compounds have been used as caged metal ions (Ca 2+ , La 3+ , etc.), 5 the release of the radical NO from nitrosyl complexes 6 is the sole example of the use of the photochemical properties of coordination compounds for this purposes. In this work, we present a new strategy for designing photo- triggers based in transition-metal complexes in which the bioactive compound is released using visible light pulses. The photorelease of 4-aminopyridine (4AP), a widely used neurocompound that blocks certain K + channels, 7 promoting depolarization and increas- ing neuron activity, is achieved from a ruthenium polypyridyl complex. Its synthesis, characterization, and biological activity are presented in this work, and some inherent advantages of this new approach are discussed. [Ru(bpy) 2 (4AP) 2 ]Cl 2 (bpy ) 2,2′ bipyridine) was obtained by the reaction of Ru(bpy) 2 Cl 2 in water with excess of 4AP and purified by recrystalization (One hundred and fifty-nine milligrams of Ru- (bpy) 2 Cl 2 were suspended in 7 mL of water at 85 °C under N 2 . After dissolution, 66 mg of 4AP was added, and the solution was heated for 20 min. The compound was precipitated by addition of NH 4 PF 6 , washed, and dried. The red solid was dissolved in acetone and reprecipitated with tetraethylammonium chloride. Yield: 79%.) UV-vis spectra in water were obtained with a HP 8453 diode array spectrophotometer. RMN 1 H spectra were done using a Bruker 500 MHz equipment. CV measurements were performed with a PAR 273A potentiostat. The irradiation of the samples was effected by means of a pulsed Xe lamp, (pulse energy ∼0.5 J), with a low- pass filter at 480 nm. Irradiation using a 473-nm DPSS laser gave similar results. The compound [Ru(bpy) 2 (4AP) 2 ]Cl 2 (RU4AP 2 ) is very soluble in water and stable in the dark, while undergoing decomposition under irradiation with visible light in its MLCT band, centered at 489 nm. (In CH 3 CN solution, the absorption band is red-shifted to 492 nm, consistently with the lower polarity of the solvent, despite a previous characterization that reported 450 nm. However, light exposure of CH 3 CN solutions produced a yellow compound with absorption maximum at 450 nm that possibly corresponds to the previously misinterpreted assignments for this compound. 8 This photoproduct is probably the complex [Ru(bpy) 2 (4AP)(CH 3 CN)] 2+ .) Several ruthenium polypyridyl complexes present this behavior. 9 Although at pH 7 the spectrum of the irradiated complex is very similar to that of the original complex, a diminished shoulder at 470 nm becomes evident. To determine the nature of the photo- reaction, NMR spectra were taken before and after irradiation with visible light. Figure 1 shows the signal assigned to the meta hydrogens RU4AP 2 (m1). After irradiation this signal decreases, and two new ones appear at lower fields: one corresponding to the free ligand (m3), and the other to the aquo-4AP complex (m2), indicating photorelease of the 4AP. These two latter signals integrated for 0.30 and 0.27 of the initial signal, which corresponds to photoreaction of 60%. The redox potential of the couple Ru III /Ru II for RU4AP 2 measured in water is E ) 0.76 V vs Ag/AgCl, which is consistent with the higher basicity of 4AP compared with that of pyridine. Thus, the redox and the photochemistry of this compound is in total agreement with previous results corresponding to the Ru(bpy) 2 XY family, X and Y being monodentate ligands. 10 The photoactivity of these compounds has been explained in terms of a reaction pathway that involves the transition between the MLCT state to a lower-energy d-d state, which promotes ligand release. There is a direct correspondence between the energy of the MLCT transition and the quantum yield of the photoreaction. 9 Figure 1. Partial 1 H NMR spectra of RU4AP2, showing the signals corresponding to the 4AP meta hydrogens. m1: in [Ru(bpy)2(4AP)2] 2+ m2: in [Ru(bpy)2(H2O)(4AP)] 2+ , and m3: in free ligand 4AP. Published on Web 01/01/2003 882 9 J. AM. CHEM. SOC. 2003, 125, 882-883 10.1021/ja0278943 CCC: $25.00 © 2003 American Chemical Society