Aminolysis of an N-diazeniumdiolated amidine as an approach to diazeniumdiolated ammonia.

Recent theoretical studies have suggested that the parent diazeniumdiolate ion, H2N-N(O)═NO(-) ("diazeniumdiolated ammonia"), might be stable enough to be isolated and that it could potentially serve as a uniquely advantageous prodrug form of bioactive nitroxyl (HNO). Here, we report on an attempt to isolate its O(2)-benzylated derivative by aminolysis of the C═N bond in PhC(NH2)═N-N(O)═NOBn. The reaction proved remarkably sluggish in comparison to aminolysis of unsubstituted benzamidine, and the desired product could not be isolated, apparently because of base sensitivity of the NH2 group. Consistent with this interpretation, O-benzylhydroxylamine and N2O were recovered from the reaction mixture in high yields, along with N,N'-dibutylbenzamidine. Theoretical calculations rationalize the observed slow aminolysis by demonstrating that the diazeniumdiolate group greatly suppresses the electrophilicity of the adjacent C═N carbon center, rendering attack at that position endothermic. The data provide significant insights into the challenges inherent to the pursuit of diazeniumdiolated ammonia.

Nitric oxide delivery via a permeable balloon catheter inhibits neointimal growth after arterial injury.

BACKGROUND: Neointimal hyperplasia limits the longevity of vascular interventions. Nitric oxide (NO) is well known to inhibit neointimal hyperplasia. However, delivery of NO to the vasculature is challenging. Our study aims to evaluate the efficacy of delivering NO to the site of injury using a permeable balloon catheter. Our hypothesis is that ultra-short duration NO delivery using a permeable balloon catheter will inhibit neointimal hyperplasia. MATERIALS AND METHODS: Ten-week-old male Sprague-Dawley rats underwent carotid artery balloon injury. Groups included: (1) control, (2) injury, (3) injury + periadventitial NO, and (4) injury + endoluminal NO via permeable balloon catheter. The catheter was inflated to 5 atm pressure for 5 min. Arteries were harvested 2 wk following injury. Morphometric assessment for neointimal hyperplasia and immunohistochemical staining for inflammatory markers were performed. RESULTS: Injury increased neointimal hyperplasia compared with control (intima/media area [I/M] ratio 1.07 versus 0.11, respectively, P < 0.001). Periadventitial delivery of NO reduced the I/M area ratio compared with injury alone (55% decrease, P < 0.001). Endoluminal delivery of NO also reduced the I/M area ratio compared with injury alone (65% decrease; P < 0.001). Both endoluminal and periadventitial NO affected the I/M ratio by reducing the intimal area (64% and 46%, respectively, P < 0.001) whereas neither affected the medial area. Periadventitial NO delivery increased lumen area (P < 0.05), whereas endoluminal NO delivery increased circumference (P < 0.05). Periadventitial NO delivery inhibited macrophage intimal infiltration compared with injury alone (P < 0.05). CONCLUSIONS: These data demonstrate that short-duration endoluminal NO delivery via permeable balloon catheters inhibits neointimal hyperplasia following arterial interventions. Endoluminal delivery of NO could become a focus for future clinical interventions.

Nitrogen-bound diazeniumdiolated amidines.

In contrast to amidines bearing ionizable alpha-CH bonds, which react with nitric oxide (NO) to add diazeniumdiolate groups at their alpha-carbons, benzamidine forms an N-bound diazeniumdiolate that can be further derivatized at the other amidine nitrogen and/or the terminal oxygen to form caged NO compounds as potential NO prodrugs.

Insulin enhances the effect of nitric oxide at inhibiting neointimal hyperplasia in a rat model of type 1 diabetes.

Diabetes confers greater restenosis from neointimal hyperplasia following vascular interventions. While localized administration of nitric oxide (NO) is known to inhibit neointimal hyperplasia, the effect of NO in type 1 diabetes is unknown. Thus the aim of this study was to determine the efficacy of NO following arterial injury, with and without exogenous insulin administration. Vascular smooth muscle cells (VSMC) from lean Zucker (LZ) rats were exposed to the NO donor, DETA/NO, following treatment with different glucose and/or insulin concentrations. DETA/NO inhibited VSMC proliferation in a concentration-dependent manner to a greater extent in VSMC exposed to normal-glucose vs. high-glucose environments, and even more effectively in normal-glucose/high-insulin and high-glucose/high-insulin environments. G(0)/G(1) cell cycle arrest and cell death were not responsible for the enhanced efficacy of NO in these environments. Next, type 1 diabetes was induced in LZ rats with streptozotocin. The rat carotid artery injury model was performed. Type 1 diabetic rats experienced no significant reduction in neointimal hyperplasia following arterial injury and treatment with the NO donor PROLI/NO. However, daily administration of insulin to type 1 diabetic rats restored the efficacy of NO at inhibiting neointimal hyperplasia (60% reduction, P < 0.05). In conclusion, these data demonstrate that NO is ineffective at inhibiting neointimal hyperplasia in an uncontrolled rat model of type 1 diabetes; however, insulin administration restores the efficacy of NO at inhibiting neointimal hyperplasia. Thus insulin may play a role in regulating the downstream beneficial effects of NO in the vasculature.

Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome.

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.

Nitric oxide reacts with methoxide.

Despite over a century of reports to the contrary, sodium methoxide has been found to react with nitric oxide (NO). The reaction, whose final organic product is sodium formate, is postulated to occur via an intermediate O-bound diazeniumdiolate [CH3O-N(O)=NO-] that decomposes to formaldehyde and nitrous oxide. Sodium formate forms from the aldehyde via a Cannizzaro reaction. Carboxylate salts have similarly been obtained by exposing sodium benzylate and sodium neopentoxide to NO in dioxane solution. Accordingly, sodium trimethylsilanoate should be considered as a substitute for sodium methoxide as the base used to accomplish the replacement of active hydrogens by the diazeniumdiolate functional group via the Traube reaction.

Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia.

OBJECTIVE: Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia. METHODS: Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group). RESULTS: Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization. CONCLUSIONS: Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.

Diazeniumdiolate reactivity in model membrane systems.

The effect of small unilamellar phospholipid vesicles on the acid-catalyzed dissociation of nitric oxide from diazeniumdiolate ions, R(1)R(2)N[N(O)NO](-), [1: R(1)=H(2)N(CH(2))(3)-, R(2)=H(2)N(CH(2))(3)NH(CH(2))(4)-; 2: R(1)=R(2)=H(2)N(CH(2))(3)-; 3: R(1)=n-butyl-, R(2)=n-butyl-NH2+(CH(2))(6)-; 4: R(1)=R(2)=nPr-] has been examined at pH 7.4 and 37 degrees C. NO release was catalyzed by anionic liposomes (DPPG, DOPG, DMPS, POPS and DOPA) and by mixed phosphatidylglycerol/phosphatidylcholine (DPPG/DPPC and DOPG/DPPC) covesicles, while cationic liposomes derived from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic liposome DMPC did not significantly affect the dissociation rates of the substrates examined. Enhancement of the dissociation rate constant in DPPG liposome media (0.010M phosphate buffer, pH 7.4, 37 degrees C) at 10mM phosphoglycerol levels, ranged from 37 for 1 to 1.2 for the anionic diazeniumdiolate 4, while DOPA effected the greatest rate enhancement, achieving 49-fold rate increases with 1 under similar conditions. The observed catalysis decreases with increase in the bulk concentration of electrolytes in the reaction media. Quantitative analysis of catalytic effects has been obtained through the application of pseudo-phase kinetic models and equilibrium binding constants at different liposome interfaces are compared. The stoichiometry of nitric oxide release from 1 and 2 in DPPG/DPPC liposome media has been obtained through oxyhemoglobin assay. DPPG=1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DOPG=1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DMPS=1,2-dimyristoyl-sn-glycero-3-[phospho-l-serine], POPS=1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine], DOPA=1,2-dioleoyl-sn-glycero-3-phosphate; DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DMPC=1,2-dimyristoyl-sn-glycero-3-phosphocholine, DOTAP=1,2-dioleoyl-3-trimethylammonium-propane.

Beneficial effect of a short-acting NO donor for the prevention of neointimal hyperplasia.

Nitric oxide (NO)-based therapies effectively inhibit neointimal hyperplasia in animal models of arterial injury and bypass grafting, but are not available clinically. We created a simple, effective, locally applied NO-eluting therapy to prevent restenosis after vascular procedures. We investigated the efficacy of perivascular delivery of two distinctly different diazeniumdiolate NO donors, 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO) (short half-life) and diazeniumdiolated poly(acrylonitrile) (PAN/NO) (long half-life), in powder or gel form (30% poloxamer 407), at inhibiting neointimal hyperplasia using the rat carotid artery injury model. Two weeks postinjury, all of the NO-eluting therapies successfully reduced neointimal hyperplasia. However, most dramatically, PROLI/NO powder reduced intimal area by 91.2% (p<0.05) versus injury alone. PROLI/NO powder was noted to reduce the medial area (40.2% vs injury alone, p<0.05), whereas other groups showed no such effect. Three days postinjury, each NO treatment group significantly reduced cellular proliferation. However, inflammatory markers revealed a distinct pattern: PAN/NO groups displayed increased leukocyte infiltration (p<0.05), whereas PROLI/NO groups displayed less macrophage infiltration (p<0.05). In conclusion, perivascular delivery of diazeniumdiolate NO donors in powder or gel form effectively inhibits neointimal hyperplasia. Application of short-acting PROLI/NO powder most effectively inhibited neointimal hyperplasia and inflammation and may represent a simple, clinically applicable NO-eluting therapy to prevent neointimal hyperplasia and restenosis after open vascular interventions.

Nitric oxide releasing polyurethanes with covalently linked diazeniumdiolated secondary amines.

Two novel strategies for synthesizing stable polyurethanes (PUs) capable of generating bioactive nitric oxide (NO) are described. The methods rely on covalently attaching diazeniumdiolate (N(2)O(2)(-)) groups onto secondary amine nitrogens at various positions within the polymer chain such that, when in contact with water or physiological fluids, only the two molecules of NO available from each diazeniumdiolate moiety are released into the surrounding medium, with potential byproducts remaining covalently bound to the matrix. Extensive analysis of the NO(x)() products released from the polymers was employed to develop appropriate strategies to better stabilize the diazeniumdiolate-based polymer structures. In one approach, diazeniumdiolate groups are attached to secondary amino nitrogens of alkane diamines inserted within the diol chain extender of a PU material. Oxidative loss of NO was minimized by blending the polymer with a biocompatible, relatively nonnucleophilic salt before exposing solutions of the polymer to NO during the diazeniumdiolation step. Fluxes of molecular NO from such materials during immersion in physiological buffer reached levels as high as 19 pmol x cm(-2) x s(-1) with a total recovery of 21 nmol of NO/mg of PU. A second general synthetic strategy involved omega-haloalkylating the urethane nitrogens and then displacing the halide from the resulting polymer with a nucleophilic polyamine to form a PU with pendent amino groups suitable for diazeniumdiolation. Commercially available Pellethane 2363-80AE that was bromobutylated and then reacted with diethylenetriamine and further exposed to gaseous NO proved stable in solid form for several months, but released NO with a total recovery of 17 nmol/mg upon immersion in physiological buffer. This material showed an initial NO flux of 14 pmol x cm(-2) x s(-1) when immersed in pH 7.4 buffer at 37 degrees C, with gradually decreasing but still observable fluxes for up to 6 days.

Carbon-bound diazeniumdiolates from the reaction of nitric oxide with amidines.

[reaction: see text] The enediamine tautomer of a variety of substituted amidine free bases reacts with nitric oxide (NO) to produce compounds containing a carbon-bound diazeniumdiolate [R1R2R3C-N(O)=NO-] functional group (previously called "nitrosohydroxylamines"). The new reaction has been shown to be quite general, although the nature of the products does vary. Amidines containing more than one replaceable hydrogen produce polydiazeniumdiolates as intermolecular salts, while those in which only one diazeniumdiolation can occur provide zwitterionic salts. These diazeniumdiolated amidines are shown to be useful NO donor compounds which undergo very slow spontaneous dissociation on dissolution in pH 7.4 phosphate buffer to produce mixtures of NO and nitrous oxide containing mostly NO. The most advantageous manifestation of the new discovery is the preparation of the monodiazeniumdiolated amidine zwitterions. Reaction of the medically relevant alpha-adrenergic agonists tetrahydrozoline and idazoxan produced monodiazeniumdiolated amidine zwitterions from which NO release was observed for up to 28 days and showed little sign of ending. The reaction should be applicable to a variety of pharmaceutical agents, including NO synthase inhibitors, antitumor agents, and antibacterials.

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.

Mechanistic insight into exclusive nitric oxide recovery from a carbon-bound diazeniumdiolate.

We report that NaON=N(O)-X-N(O)=NONa (1), where X is para-disubstituted benzene, hydrolyzes to 2 mol of nitric oxide (NO) with concurrent production of 1 mol of p-benzoquinone dioxime at physiological pH. The reaction is acid catalyzed, with a rate that slows as the substrate concentration is increased. The results demonstrate that a carbon-bound diazeniumdiolate can be quantitatively hydrolyzed to produce NO as the only gaseous nitrogen-containing product. The data also suggest that N-N bond cleavage is the rate-determining step in NO release, since C-N cleavage followed by dissociation of O=N-N=O to two NO molecules cannot be operative in this case. The finding that this oxime can absorb NO in organic media and regenerate it quantitatively at physiological pHs extends the potential pharmacological implications of the carbon-bound diazeniumdiolates.

Augmentation of intrapericardial nitric oxide level by a prolonged-release nitric oxide donor reduces luminal narrowing after porcine coronary angioplasty.

BACKGROUND: Nitric oxide (NO) is a potent vasodilator and antiplatelet agent that suppresses vascular smooth muscle cell proliferation. Hypothesizing that generating NO in the pericardial space would reduce luminal narrowing after coronary angioplasty without affecting systemic hemodynamics, we have determined the effect of a novel NO donor on vascular healing after balloon overstretch. METHODS AND RESULTS: Diazeniumdiolated bovine serum albumin (D-BSA; molecular weight 74 kDa, half-life for NO release 20 days) was radioiodinated and found by intravital gamma-imaging to have a longer residence time in pig pericardium than a low-molecular-weight (0.5 kDa) analogue (22 versus 4.6 hours, respectively). Intrapericardial injection of D-BSA immediately before 30% overstretch of normal left anterior descending and left circumflex coronary arteries dose dependently reduced the intimal/medial area ratio by up to 50% relative to controls treated with underivatized albumin when measured 2 weeks after intervention. Positive remodeling was also noted, which increased luminal area relative to control. CONCLUSIONS: Perivascular exposure of coronary arteries to NO via intrapericardial D-BSA administration reduced flow-restricting lesion development after angioplasty in pigs without causing significant systemic effects. The data suggest that intrapericardial delivery of NO donors for which NO release rates and pericardial residence times are matched and optimized might be a beneficial adjunct to coronary angioplasty.

A nitric oxide-releasing polydiazeniumdiolate derived from acetonitrile.

Acetonitrile, frequently used as a solvent in reactions of nitric oxide (NO) with amines and other nucleophiles to introduce the [N(O)NO](-) (diazeniumdiolate) functional group, has itself been shown to react with NO in the presence of strong base to yield methane trisdiazeniumdiolate (1), presumably via an intermediate trisdiazeniumdiolated imidate. Aqueous hydrolysis of 1 does not follow simple first-order kinetics and produces mixtures of NO and nitrous oxide in ratios that vary with solution pH. [reaction: see text]

Qualitative thin-layer and high-performance liquid chromatographic analysis of 1-substituted diazen-1-ium-1,2-diolates on aminopropyl-bonded silica gel.

High-performance liquid (HPLC) and thin-layer (TLC) chromatographic methods for the detection and quantification of diazeniumdiolates are described. The HPLC determinations were made using a Rocket Platinum NH2 column (7 x 53 mm, 100-A pore size, 3-microm particle size), under isocratic conditions with mixtures of acetonitrile, methanol, and water containing 0.1% diethylamine (v/v), at a flow rate of 2.5 ml/min, a column temperature of 22 degrees C, and UV detection at 250 nm. The TLC determinations were similarly made using Merck NH2 F254S precoated glass plates (approximately 2 x 5 cm, 5-microm particle size, 0.2-mm layer thickness) with mixtures of acetonitrile, methanol, and water containing 0.1% diethylamine (v/v). Preexisting traces of carcinogenic nitrosamines were detected in some samples of diazeniumdiolates. The methods provide a more efficient means of characterizing the purity of diazeniumdiolate samples prepared for use in biomedical applications than older procedures which rely on differential absorbance measurements at 250 and 350 nm, respectively.

Piperazine as a Linker for Incorporating the Nitric Oxide-Releasing Diazeniumdiolate Group into Other Biomedically Relevant Functional Molecules.

Synthetic procedures have been devised to exploit the bifunctional amine piperazine (pip) as a linker capable of attaching the nitric oxide (NO)-releasing diazeniumdiolate functional group [N(O)NO]- to a diverse selection of biomedically useful molecules. One of the amino groups bears the diazeniumdiolate, which may be substituted on oxygen as necessary to control its dissociation to NO, while the other is used to provide a site suitable for covalent bonding to the molecule requiring NO donor capability. N,N'-Disubstituted piperazines of the structure R-pip-N(O)[Formula: see text]NOE were prepared either by using the nucleophilic character of the amino group or by converting it into an electrophilic moiety for reaction with nucleophilic centers in the molecules to be derivatized. Examples are reported in which E = CH3 and the R groups are bound to the N'-nitrogen: via amide linkages to the carboxyl groups of the drug ibuprofen and the amino acid derivative N-acetylmethionine; through a urea grouping to the ε-amino group of a protected lysine; via a carbamate linkage to poly(ethylene glycol); and by replacing the NH2 nitrogens of nicotinamide and adenosine. Synthesis of analogues in which E = vinyl has been facilitated by introduction of BrCH2CH2OSO2Cl as a novel, efficient bromoethylating agent. Spontaneous NO releasers in the diazeniumdiolated piperazine series include both a fluorescent anion of half-life 5.5 min in which E = Na and R = dansyl and "MOM-PIPERAZI/NO" (E = CH3OCH2, R = H), whose half-life for NO release was estimated as 17 days. The latter agent has made possible the conversion of poly(vinyl chloride) and phosphatidylethanolamine to NO-releasing derivatives. This chemistry should allow introduction of diazeniumdiolate groups into a wide variety of natural products, drugs, polymers, and other molecules whose activities could be beneficially combined with the ability to generate NO for biomedical applications.