Nucleophilic reactivity of thiolate, hydroxide and phenolate ions towards a model O-arylated diazeniumdiolate prodrug in aqueous and cationic surfactant media.

The kinetics of aromatic nucleophilic substitution of the nitric oxide generating diazeniumdiolate ion, DEA/NO, by thiols, (L-glutathione, L-cysteine, DL-homocysteine, 1-propanethiol, 2-mercaptoethanol and sodium thioglycolate) from the prodrug, DNP-DEA/NO, has been examined in aqueous solution and in solutions of cationic DOTAP vesicles. Second-order rate constants in buffered aqueous solutions (k(RS(-) ) = 3.48 - 30.9 M(-1)s(-1); 30 °C) gave a linear Brønsted plot (ß(nuc) = 0.414 ± 0.068) consistent with rate-limiting S(N)Ar nucleophilic attack by thiolate ions. Cationic DOTAP vesicles catalyze the thiolysis reactions with rate enhancements between 11 and 486-fold in Tris-HCl buffered solutions at pH 7.4. The maximum rate increase was obtained with thioglycolate ion. Thiolysis data are compared to data for nucleophilic displacement by phenolate (k(PhO(-) ) = 0.114 M(-1)s(-1)) and hydroxide (k(OH(-) ) = 1.82 × 10(-2) M(-1)s(-1), 37 °C) ions. The base hydrolysis reaction is accelerated by CTAB micelles and DODAC vesicles with vesicles being ca 3-fold more effective as catalysts. Analysis of the data using pseudophase ion-exchange formalism implies that the rate enhancement of the thiolysis and base hydrolysis reactions is due primarily to reactant concentration in the surfactant pseudophase.

Thiol activation of a model O2-aryl diazeniumdiolate prodrug in phospholipid vesicle media.

Thiolysis of the model diazeniumdiolate prodrug, O2-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DNP-DEA/NO), by glutathione (GSH), cysteine (CYSH) and 1-heptanethiol (heptylmercaptan, HM) has been examined in anionic (DOPG), neutral (DPPC, DOPE) and cationic (DOTAP) vesicle media and in glycine buffered aqueous solutions. DOTAP vesicles accelerate the bimolecular reaction with glutathione, cysteine and 1-heptanethiol by factors of 81, 8.2 and 4630, respectively, while reaction is inhibited 5- to 10-fold in the presence of neutral and anionic vesicles. The intrinsic nucleophilicity of the thiols has been compared through the second-order rate constants, 22.9, 5.24 and 43.1M(-1)s(-1), for nucleophilic attack on 1 by GS(-), CYS(-) and M(-), respectively, obtained in buffered aqueous media. Analysis of the catalysis by DOTAP vesicles, using pseudophase ion-exchange formalism, suggests that the rate increase is due to reactant concentration in the bilayer and interfacial region coupled with enhanced dissociation of the thiol at the vesicle surface. Some contribution from enhanced nucleophilic reactivity at the vesicle interface may also contribute to the greater catalysis by HM. Inhibition of the thiolysis reaction by phospholipid liposomes is attributed to repulsion of the thiolate anions by the negatively charged acyl phosphate of the lipid head group. DOPG=1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DOPE=1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOTAP=1,2-dioleoyl-3-trimethylammonium-propane.

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.