Abstract
We report the synthesis and testing of a series of "caged" nitric oxide compounds that are stable indefinitely in oxygen-containing solutions until photolyzed by ultraviolet irradiation, whereupon they release nitric oxide (NO) with quantum yields of delta 8% for 3a (CNO-1) and delta 2% for compounds 3b-e (CNO2-5). After a flash, NO release is complete within 5 ms, so that precise temporal control of NO release is possible. NO donor 3d (CNO-4) includes two carboxylate negative charges at physiological pH, which reduce membrane permeability and enable photolytic generation of NO to be selectively confined to either extracellular or intracellular compartments. Esterification of these carboxyls with acetoxymethyl groups produces 3e (CNO-5), which is membrane-permeant and intracellularly hydrolyzable. Therefore, large populations of intact cells can be conveniently intracellularly loaded with "caged" NO donor 3d by incubation with 3e (CNO-5). The biological efficacy of these NO donors and their absolute dependence on UV-irradiation was demonstrated by inhibition of thrombin-stimulated platelet aggregation. Extracellular hemoglobin blocked the effects of NO generated outside but not inside platelets, verifying the sidedness of the NO donors and the limited spatial range of NO action. These molecules should permit precise spatial, temporal, and concentration control of NO release for investigation of its important biological functions.
Highlights
From the Howard Hughes Medical Institute and Departmentsof Pharmacology and Chemistry, University of California aStan Diego, fill this need, we report photochemically triggered NO donors that can be tailored either to permeate cell membranes or to remain excluded from cells or to becomspeecifically trapped in their intracellular spaces.Theserelatively small molecules are
The formation of NO from compounds 3a-e was determined spectrophotometricallyusing the well knownNO trap deoxyhemoglobin,Hb (22,23).As shown in Fig. 2, irradiation of 3b at 365 n m in the presence of Hb converted the absorbance spectrum of Hb to the spectrum characteristic of nitrosylhemoglobin (HbNO) (24)
Ru(NO)C13 was very effectiveat producingHbNO upon photolysis; howeveirt, failed to inhibit platelet aggregation when tested in thesame way and at the same concentrations as 3b, 3d, and 3e (Table 11)
Summary
NO donors release NO relatively slowly, a property that can be desirablein some applications. The precipitate was washed with cold water, collected,and dried under vacuum to give 3 mg (7.3 p o l ) of the free acid This material was suspended in 200 111 of dry CHZClz,and 30 plof diisopropylethylamine and 20 pl (200 m o l ) of bromomethyl acetate were added. The recorded change in light transmission relative to that obtained with the same concentration of thrombin in the absence of any NO donor was used to calculate values for percent inhibition of platelet aggregation. In experiments where oxyhemoglobin was used as an extracellular NO trap, the HbOz was added producing a final concentration of 20 p~ just prior to the photolysis of the NO donor.Control experiments involving photolysisof the platelet suspension with and without hemoglobin in the absence of NO donors causedless than 5% inhibition relative to unphotolyzed controls. Incubation of the platelets with the NO donors in the darfkor 10minunder the experimental conditions described abovaelso resulted in less than 5% inhibition of thrombin-inducedplatelet aggregation
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