Micellar Catalysis of Nitric Oxide Dissociation from Diazeniumdiolates

Jan 16, 2003 - Diazeniumdiolate reactivity in model membrane systems. Bach T. Dinh , Stacy E. Price , Amr Majul , Mazen El-Hajj , Victor Morozov , Jos...
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Langmuir 2003, 19, 2096-2102

Micellar Catalysis of Nitric Oxide Dissociation from Diazeniumdiolates Stacy E. Price,† Dilara Jappar,† Patricia Lorenzo,† Joseph E. Saavedra,‡ Joseph A. Hrabie,‡ and Keith M. Davies*,† Department of Chemistry, George Mason University, Fairfax, Virginia 22030, and Basic Research Program, SAIC Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702 Received November 4, 2002 The effect of surfactant micelles on the acid-catalyzed dissociation of NO from diazeniumdiolate ions of structure R1R2N[N(O)NO]- has been examined in phosphate-buffered solutions at 37 °C. The reaction behavior of zwitterionic substrates [2, R1 ) R2 ) H2N(CH2)2; 3, R1 ) R2 ) H2N(CH2)3; 4, R1 ) n-Pr, R2 ) H2N(CH2)3; 5, R1 ) H2N(CH2)3, R2 ) H2N(CH2)3NH(CH2)4] and anionic substrates [1, R1 ) R2 ) Et; 6, R1 ) R2 ) n-Pr] has been compared. All but DEA/NO (1) are catalyzed by anionic micelles of sodium dodecyl sulfate (SDS) but are unaffected by the presence of cationic cetyltrimethylammonium bromide or the zwitterionic surfactant 3-(N-dodecyl-N,N-dimethylammonio)-1-propanesulfonate (lauryl sulfobetaine). Catalysis by sodium decylphosphonate micelles has also been demonstrated for 2 (DETA/NO). The surfactantmediated catalysis is discussed in terms of a distribution model with simultaneous reaction in the water and micellar pseudophases. Binding constants (Ks) for diazeniumdiolate association with the surfactant micelles have been obtained, and a comparison of second-order rate constants, k2m and k2w, for their acid-catalyzed dissociation in the micellar and aqueous phases, respectively, has been made. For the zwitterionic polyamine diazeniumdiolates 2-5, the Ks values show good correlation with the number of positively charged nitrogen centers in the substrates, consistent with micellar association between protonated nitrogens in the zwitterions and the anionic headgroups of the micelle. The Coulombic interaction of zwitterionic substrates with SDS micelles is compared with the weak hydrophobic association which was found with the anionic diazeniumdiolate 6.

Introduction Diazeniumdiolates, ions of structure R1R2N[N(O)NO](Figure 1), are widely used nitric oxide generating agents for biomedical research applications because of their ability to release NO spontaneously in physiological fluids.1 They have been used as NO providers in studies exploring a variety of nitric oxide effects including NO’s hemodynamic,2,3 antiplatelet,4 and cytostatic5,6 properties. Although their acid-catalyzed dissociations have been widely studied in buffered aqueous solutions, with the increasing targeting of diazeniumdiolates at specific biological sites and cell types, there is current interest in factors influencing NO release in the more complex environments encountered biologically. Since reaction behavior observed at surfactant interfaces is expected to be more representative of many biological reactions than are reactions studied in dilute aqueous solutions, we have employed a variety of organized surfactant media to assess the effect * To whom correspondence should be addressed. Mailing address: Chemistry Department, George Mason University, 4400 University Drive, Fairfax, VA 22030. Phone: 703-993-1075. Fax: 703-993-1055. E-mail: [email protected]. † George Mason University. ‡ National Cancer Institute at Frederick. (1) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. Methods Enzymol. 1996, 268, 281-293. (2) Saavedra, J. E.; Southan, G. D.; Davies, K. M.; Lundell, A.; Markou, C.; Hanson, S. R.; Adrie, C.; Hurford, W. E.; Zapol, W. M.; Keefer, L. K. J. Med. Chem. 1996, 39, 4361-4365. (3) Diodati, J. G.; Quyyumi, A. A.; Keefer, L. K. J. Cardiovasc. Pharmacol. 1993, 22, 287-292. (4) Diodati, J. G.; Quyyumi, A. A.; Hussain, N.; Keefer, L. K. Thromb. Haemostasis 1993, 70, 654-658. (5) Mooradian, D. L.; Hutsell, T. C.; Keefer, L. K. J. Cardiovasc. Pharmacol. 1995, 25, 674-678. (6) Saavedra, J. E.; Shami, P.; Wang, L. Y.; Davies, K. M.; Booth, M. N.; Citro, M. L.; Keefer, L. K. J. Med. Chem. 2000, 43, 261-269.

of charged aqueous interfaces and mixed hydrophilic/ hydrophobic environments on the dissociation rates of diazeniumdiolate substrates. We report here details of the catalysis that has been observed in micellar solutions. The use of organized surfactant media to simulate the microenvironment of biological membranes is well documented.7,8 Although it is recognized that such studies are limited in their ability to simulate complex physiological environments, the use of relatively simple surfactant models does provide the means of exploring diazeniumdiolate interactions that may be of relevance to their behavior in biological milieux. A particular application of this information may be in better understanding the nature of lung surfactant interactions. Diazeniumdiolates, when applied intratracheally, have been shown to reduce pulmonary hypertension and improve oxygenation in acute lung injury in pigs.9 Aerosolized DETA/NO (2) has also been administered to a human patient with acute respiratory distress syndrome.10,11 Since aerosolized diazeniumdiolate activity is enhanced by surfactant pretreatment in vivo,9 information on surfactant interactions may help to clarify possible synergistic activity between diazeniumdiolates and surfactant both in vitro and in vivo. In addition to serving as biomimetic media, the polarizing microenvironments that are generated by surfactant assemblies should also provide fundamental information (7) Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982. (8) Jones, M. N.; Chapman, D. Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995. (9) Jacobs, B. R.; Smith, D. J.; Zingarelli, B.; Passerini, D. J.; Ballard, E. T.; Brilli, R. J. Nitric Oxide 2000, 4, 412-422. (10) Lam, C.-F.; Sviri, S.; Ilett, K. F.; Van Heerden, P. V. Expert Opin. Invest. Drugs 2002, 11, 897-909. (11) Lam, C.-F.; Van Heerden, P. V.; Sviri, S.; Roberts, B. L.; Ilett, K. F. Anaesth. Intensive Care 2002, 30, 472-476.

10.1021/la020889s CCC: $25.00 © 2003 American Chemical Society Published on Web 01/16/2003

Catalysis of Diazeniumdiolate Dissociation

Langmuir, Vol. 19, No. 6, 2003 2097 Table 1. Dependence of First-Order Rate Constants for Dissociation of 3, 4, and 5 on SDS Concentration in 0.10 M Phosphate Buffer at pH 7.4 and 37.0 °Ca 3, DPTA/NO

4, PAPA/NO

5, SPER/NO

[SDS] (M)b

104 × kobs (s-1)

[SDS] (M)b

104 × kobs (s-1)

[SDS] (M)b

104 × kobs (s-1)

0 0.0050 0.010 0.020 0.030 0.050 0.100 0.150 0.200

1.39 1.79 2.14 2.69 3.38 4.03 5.12 5.80 6.51

0 0.0050 0.0060 0.0080 0.010 0.100 0.150 0.200

7.02 7.95 8.19 8.71 9.18 19.8 27.1 30.4

0 0.0050 0.020 0.050 0.100 0.150 0.200

4.00 8.37 14.6 15.8 18.2 22.5 20.9

a [Diazeniumdiolate] ) 0.10 mM. b [Micelle]/[Diazeniumdiolate] ) 0.5-20.

Figure 1. Structures of the diazeniumdiolates employed in this study.

on factors influencing NO release rates from this important class of NO donor compounds. Although much progress has been made in understanding mechanistic details of diazeniumdiolate dissociation reactions,12,13 a greater clarification of how the sensitive structure-reactivity relationships displayed by these compounds are tied to electronic distributions in diazeniumdiolate ions, as well as polarizing effects within reaction media, remains a fundamental goal of ongoing diazeniumdiolate studies. Experimental Procedures Materials. Zwitterionic compounds 2-5 were synthesized by treating the appropriate polyamine in CH3CN with NO at 6080 psi for 24 h.14 The product was isolated by filtration, washed with CH3CN and ether, and dried under a vacuum. Compounds 1 and 6 were similarly prepared by the high-pressure reaction of NO with diethylamine and di-n-propylamine, respectively.15 The dialkylammonium salt obtained was converted to the sodium salt using sodium methoxide. Purities were checked by proton NMR (300 MHz, Bruker). The surfactants SDS, sodium dodecyl sulfate (United States Biochemical Corp.), CTAB, cetyltrimethylammonium bromide, and SB-12, 3-(N-dodecyl-N,N-dimethylammonio)-1-propanesulfonate (Sigma) were used as re(12) Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K. J. Am. Chem. Soc. 2001, 123, 5473-5481. (13) Srinivasan, A.; Saavedra, J. E.; Kebede, N.; Nikolaitchik, A. V.; Brady, D. A.; Yourd, E.; Davies, K. M.; Toscano, J. P.; Keefer, L. K. J. Am. Chem. Soc. 2001, 123, 5465-5472. (14) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem. 1993, 58, 1472-1476. (15) Drago, R. S.; Karstetter, B. R. J. Am. Chem. Soc. 1961, 83, 18191822.

ceived. Solutions of SDP, sodium decylphosphonate, were prepared by treating 1-decylphosphonic acid (Lancaster), in a 0.01 M pH 7.0 phosphate buffer, with sufficient 0.10 M NaOH to return the pH to 7.0. All surfactant solutions were prepared daily prior to their use in rate studies. Kinetic Studies. Rate constants were measured spectrophotometrically by monitoring the decrease in absorbance of the diazeniumdiolate chromophore at 250 nm using a HewlettPackard 8453 Diode Array UV-visible spectrophotometer, as previously described.12 Data obtained with SDS as the surfactant were collected at 37 °C. Because of solubility issues, runs with SDP were made at 50 °C. Buffers of 0.10 M phosphate were prepared by weight from Na2HPO4‚7H2O and NaH2PO4‚H2O. Succinate buffers (0.020 M) were prepared by addition of 1.0 M NaOH to succinic acid solutions. UV Spectral Analysis. Measurement of spectral shifts resulting from protonation of the diazeniumdiolate functional group was carried out as described previously.12 Measurements were made within ca. 5 s of mixing before significant decomposition of the substrate had occurred. NaH2PO4/Na2HPO4 (pH 8.16.0), sodium succinate (pH 5.7-4.2), sodium citrate (pH 4.02.7), and HCl (pH 2.3-1.8) were employed as buffers.

Results Rate Studies with 1 and 2 in Cationic, Zwitterionic, and Anionic Surfactants. Initial studies compared the effect of cationic CTAB, zwitterionic SB-12, and anionic SDS surfactants on the dissociation rate of the anionic diazeniumdiolate, 1 (as a sodium salt), with that of a zwitterionic substrate, 2. CTAB and SB-12 were found to have no effect on the reaction rate of 1 or 2, nor did SDS influence the dissociation rate of 1. When SDS was added to 2, however, the dissociation rate was found to be subject to catalysis by the surfactant at all concentrations employed (2 mM to 0.20 M). These preliminary findings suggested that micellar catalysis resulted from electrostatic interaction of the cationic nitrogen centers in the zwitterion with the negatively charged headgroups of the anionic micelle. To further explore this effect, the dissociation rates of a series of zwitterionic substrates (2-5) were examined as a function of SDS concentration. Tables1-3 show firstorder rate constants, kobs, for dissociation reactions (eq 1)

of diazeniumdiolate ions 2-6 measured as a function of SDS concentration in phosphate-buffered solutions at 37 °C. Typically, the ratios of [micelle]/[diazeniumdiolate]

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Table 2. Dependence of First-Order Rate Constants for Dissociation of 2 on SDS Concentration at 37 °C and pH 7.0 (0.10 M Phosphate Buffer) and pH 5.5 (0.020 M Succinate Buffer)a 2, DETA/NO, pH 7.0

2, DETA/NO, pH 5.5

[SDS] (M)b

105 × kobs (s-1)

[SDS] (M)c

104 × kobs (s-1)

0 0.003 0.007 0.022 0.049 0.079 0.102 0.125 0.151 0.200 0.250

2.03 2.32 2.89 4.60 6.75 8.21 9.55 10.4 11.0 12.1 12.9

0 0.002 0.010 0.02 0.05 0.08 0.10 0.12 0.15

2.00 2.15 27.0 49.7 64.7 64.0 63.0 68.2 57.3

a [Diazeniumdiolate] ) 0.10 mM. b [Micelle]/[Diazeniumdiolate] ) 0.3-25. c [Micelle]/[Diazeniumdiolate] ) 0.2-15.

Table 3. Dependence of First-Order Rate Constants for Dissociation of 6 (DPA/NO) on SDS Concentration in 0.10 M Phosphate Buffer at 37.0 °Ca [SDS] (M)b

103 × kobs (s-1)

[SDS] (M)

103 × kobs (s-1)

0 0.00164 0.00406 0.00649 0.00881 0.0243 0.0292 0.0539

4.45 4.78 4.25 5.28 5.16 5.61 5.90 6.80

0.0742 0.0790 0.0979 0.124 0. 149 0.174 0.200

7.30 7.86 8.19 8.77 9.80 10.1 12.2

a [Diazeniumdiolate] ) 0.10 mM. b [Micelle]/[Diazeniumdiolate] ) 0.16-20.

Figure 2. Plot of kobs versus [SDS] for 2 in 0.10 M phosphate buffer at pH 7.0 and 37 °C.

vary from ca. 0.5 to 20, assuming an aggregation number of 100 for SDS at 37 °C.16 Typical reaction profiles, illustrated in Figure 2 for 2, show kobs values increasing with added SDS and approaching a rate plateau at high surfactant levels, consistent with substrate association with the micellar pseudophase. Table 2 also contains rate data obtained for reaction of 2 in 0.020 M sodium succinate buffer at pH 5.5. The ratesurfactant profile obtained at this pH again shows (16) Kratohvil, J. P. Chem. Phys. Lett. 1979, 60, 238-241.

Figure 3. Plot of kobs versus [SDP] for 2 in 0.10 M phosphate buffer at pH 7.0 and 50 °C.

catalysis by SDS but with rate saturation at lower surfactant concentrations than was apparent in the higher pH solutions. Rate Dependence of 3, 4, and 6 on Solution pH. Rate and equilibrium parameters for the acid-catalyzed dissociation reactions of 3, 4, and 6 have not been previously determined. Details of pH-rate profiles established for the three substrates are shown in Table A of the Supporting Information. All reactions followed excellent first-order behavior over several half-lives and yielded rate data consistent with a single kinetically relevant protonation in the pH ranges studied (5.2-8.0). A nonlinear regression fit of the kobs-[H+] data afforded pKa values of 5.73 ( 0.07, 4.59 ( 0.29, and 5.70 ( 0.06, respectively. These are comparable in magnitude to previous pKa values determined kinetically for secondary amine-derived diazeniumdiolates.12 Micellar Catalysis by Sodium Decylphosphonate. Rate measurements were also obtained for the dissociation of 2 in the presence of SDP micelles in 0.10 M phosphate buffer at pH 7.0. The rate-surfactant profiles obtained with the anionic phosphonate surfactants at 50 °C (Figure 3) were similar to those described above for SDS. Measured rate constants again approached plateau values with increasing [SDP] as the substrate was taken up by the micellar surface. SDS Dependence of Spectrally Determined pKa Values. In addition to any influence that association with the micellar surface might have on the intrinsic diazeniumdiolate dissociation rate, it could also impact on the protonation equilibria that mediate its acid-catalyzed decomposition. Several earlier studies examining the effect of surfactant solutions on acid-base equilibria of indicators have reported surfactant-induced shifts in apparent pKa values of micellar-bound substrates.17-21 To explore the influence of the micellar interphase on protonation equilibria involved in our reactions, we determined the effect of SDS on spectral shifts resulting from protonation of the diazeniumdiolate functional group. Such shifts in (17) Garcia-Soto, J.; Fernandez, M. A. Biochim. Biophys. Acta 1983, 731, 275-281. (18) Romsted, L. S.; Zanette, D. J. Phys. Chem. 1988, 92, 46904698. (19) Romsted, L. S. J. Phys. Chem. 1985, 89, 5107-5118. (20) Fernandez, M. S.; Fromherz, P. J. Phys. Chem. 1977, 81, 17551761. (21) El Seoud, O. A.; Chinelatto, A. M. J. Colloid Interface Sci. 1983, 95, 163-171.

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experimental conditions.

kobs )

Figure 4. Variation of λmax for 3 as a function of solution pH with (b) and without (0) 0.10 M [SDS].

the UV absorption maximum of the diazeniumdiolate chromophore have previously been used to estimate pKa values for diazeniumdiolate substrates.12,22 Figure 4 shows the result of spectral shift measurements conducted on 3. With no SDS present, the absorption maximum changes from 250 nm at pH 7.4 to 229 nm at pH e 3. The midpoint in the transition, observed in plots of λmax versus pH, yielded a pKa value of 3.49. This is in line with values previously determined for the zwitterionic diazeniumdiolates 2 and 5.12 When similar experiments were carried out in the presence of 0.10 M SDS, where the substrate is substantially micellar bound, a significantly higher pKa value of 5.35 was obtained. Experiments conducted with 6, which is only weakly associated with the micellar phase, showed no significant changes in the λmax-pH profile on addition of SDS. Discussion The data obtained for all of the surfactant-catalyzed reactions fit a pseudophase kinetic model (eq 2) in which

the diazeniumdiolate (S) is partitioned between the aqueous and micellar pseudophases, while reacting simultaneously in both.23,24 For such a scheme, the measured first-order rate constants are related to kw and km, the first-order rate constants in the aqueous and surfactant pseudophases, respectively, and to the equilibrium binding constant, Ks, for substrate association with SDS micelles (eq 3). Dn is the concentration of micellized surfactant ([SDS] - cmc), where cmc is the critical micelle concentration under the

kw + kmKs[Dn]

(3)

1 + Ks[Dn]

Individual values of km and Ks were obtained by a nonlinear regression fit of kobs-[SDS] data to eq 3. Values of kw, the pseudo-first-order rate constant for reaction in the bulk aqueous phase, were taken to be those measured in 0.10 M phosphate-buffered solutions in the absence of SDS. Data for the dissociation reaction of all substrates except 2 were obtained in phosphate buffer at pH 7.4. Due to its particularly long half-life (ca. 20 h) at pH 7.4, measurements on 2 were made in solutions of lower pH (7.0 and 5.5). Of particular interest to our study is the effect of the micellar interphase on the intrinsic reactivity of diazeniumdiolate substrates. For the acid-catalyzed reactions being considered, this requires a comparison of the secondorder rate constant k2w ()kw/[H+]w) for reaction in the water phase with k2m ()km/[H+]m), the rate constant for the acidcatalyzed dissociation of the micellar-bound substrate. This in turn requires separate estimates of [H+]w and [H+]m, the local molarity of hydrogen ions expressed with respect to the volumes of the aqueous and micellar pseudophases, respectively, rather than the total solution volume. This has been achieved through application of the pseudophase ion-exchange (PIE) model (eq 4), devel-

[(

[H+]w kobs )

)

]

k2m [Na+]m + KsKH/Na + k2w V [Na+] w

(1 + Ks[Dn])

(4)

oped by Romsted and others25-27 to interpret the effect of charged interphases on reactions taking place in ionic micelles, microemulsions, and synthetic vesicles. In eq 5, V is the molar volume of the surfactant interphase, generally taken to be the Stern layer (a region encompassing the surfactant headgroups and ca. 70% of their counterions), and KH/Na is an ion-exchange equilibrium constant reflecting competition on the micellar surface between the bound counterions of the surfactant headgroups and other counterions in the aqueous phase. For ionic micelles, the PIE model assumes that the degree of counterion binding, β (i.e., the fraction of the micelle surface covered by counterions), is constant and independent of the nature and concentration of the surfactant. The model has been used successfully to provide quantitative interpretations of micellar effects on a variety of reactions in both the presence and the absence of buffers, although the model fails under conditions of high H+ and OH- concentrations and with strongly hydrophilic counterion surfactants. For the acid-catalyzed reactions in our study, hydrogen ions in the micellar phase are in competition with Na+ ions at the micellar surface through the ion exchange equilibrium represented in eq 5. Since under the experiKH/Na

Na+m + H+w y\z Na+w + H+m

(5)

mental conditions employed the hydrogen ion concentra(22) Arnold, E. V.; Citro, M. L.; Saavedra, E. A.; Davies, K. M.; Keefer, L. K.; Hrabie, J. A. Nitric Oxide 2002, 7, 103-108. (23) Berezin, I. V.; Martinek, K.; Yatsimirski, A. K. Russ. Chem. Rev. 1973, 42, 787-802. (24) Martinek, K.; Yatsimirski, A. K.; Levashov, A. V.; Berezin, I. V. Micellization, Solubilization and Microemulsions; Mittal, K. L., Ed.; Plenum Press: New York, 1977; Vol. 2, p 489.

(25) Bunton, C. A.; Nome, F.; Quina, F. H.; Romsted, L. S. Acc. Chem. Res. 1991, 24, 357-364. (26) Quina, F. H.; Chaimovich, H. J. Phys. Chem. 1979, 83, 18441850. (27) Neves, M. F. S.; Zanette, D.; Quina, F.; Moretti, M. T.; Nome, F. J. Phys. Chem. 1989, 93, 1502-1507.

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protonated diazeniumdiolate following equilibrium protonation of the substrate, S (eqs 6-8). Protonation at the KAH

SH+ y\z S + H+ kAH

Figure 5. Plot of kobs versus Dn for 6 in 0.10 M phosphate buffer at pH 7.4 and 37 °C. The solid curve is calculated with eq 4 using best fit values given in the text. Table 4. Comparison of Rate and Equilibrium Parameters for Micellar-Catalyzed Dissociation of Zwitterionic and Anionic Diazeniumdiolates in Phosphate-Buffered Solutions at 37 °C Ks (M-1)

substrate SPER/NOa DETA/NOb DETA/NOc DPTA/NOa PAPA/NOa DPA/NOa a

5 2 2 3 4 6

88.5 15.4 48.2 14.8 12.1 4.8

k2w (M-1 s-1)

k2m (M-1 s-1)

1.0 × 104 197 126 3.57 × 103 1.72 × 104 1.12 × 105

3.6 × 103 88 56 1.4 × 103 5.80 × 103 3.0 × 104

pH 7.4. b pH 7.0. c pH 5.5.

tion in the micellar phase is expected to be much less than the concentration of bound Na+ counterions, we estimate [Na+]m/[Na+]w assuming [Na+]m ) βDn and [Na+]w ) (1 β)Dn + cmc + [Na+]buffer.28 Reported literature values of β ) 0.7, V ) 0.25 M-1, and KH/Na ) 1 were used in fitting our data.18,28 Critical micelle concentration values of 1.4 and 0.83 mM have been reported for SDS in 0.10 and 0.20 M NaCl solutions, respectively, at 25 °C.29 A value of 1.0 mM was assumed under our experimental conditions of 0.10 M phosphate buffer at 37 °C. With these assumptions and by use of Ks values obtained initially from fitting the data to eq 3, rate-surfactant profiles for the substrates 2-6 were fitted to eq 4 to provide best fit values for k2m. The value of k2w was calculated from kobs/[H+] for reaction in phosphate buffer in the absence of SDS. Typical fits are shown in Figure 5 for 6. A summary of the k2m, k2w, and Ks values obtained for 2-6 is shown in Table 4. The fit of the kobsd-[SDS] data is relatively insensitive to the values selected for KH/Na and cmc but shows a greater dependence on the value used for V, the volume of the interfacial region. Although polar solutes are generally expected to be located largely in the Stern layer, micellar phase reactant concentrations have, in some instances, been calculated in terms of the total volume of the micelle which is approximately twice that of the Stern layer. Mechanism of Diazeniumdiolate Dissociation in Micellar Solution. Diazeniumdiolate dissociation in phosphate-buffered solution at physiological pH has been shown to proceed through unimolecular decay of the (28) Ruzza, A. A.; Rosania, M.; Walter, K.; Nome, F.; Zanette, D. J. Phys. Chem. 1992, 96, 1463-1467. (29) Jones, M. N.; Chapman, D. Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995; p 68 and references therein.

(6)

SH+ 98 R2NH + 2NO

(7)

kobs ) kAH[H+]/([H+] + KAH)

(8)

parent amine (R2N-) nitrogen has been shown to be responsible for triggering the decomposition of the N2O2group with release of NO under these conditions.12 A similar mechanism is assumed in the phosphate-buffered aqueous phase of the micellar solutions. Since the catalysis by anionic surfactants is believed to arise from the concentration of the diazeniumdiolate substrate and hydrogen ions in the Stern layer, a similar acid-catalyzed decomposion mechanism is assumed there also. The calculated k2m values for micellar-mediated dissociations of diazeniumdiolate substrates are not greatly different from k2w values obtained in the aqueous phase (k2m/k2w ) 0.27-0.45), although the extent to which binding of the diazeniumdiolate substrates at the surfactant interface contributes to the differences in k2m and k2w is uncertain. The substrates with the greatest hydrophobic component in their micellar binding, 4 and 6, show the lowest k2m/k2w ratios, and it is possible that penetration of their alkyl groups into the micellar interior does produce a small rate inhibition. It is also likely that after loss of NO, the amine product at the interface is partitioned into the lipophilic interior of the micelle through its small unprotonated fraction. However, calculated k2m values that are 2-3-fold smaller than k2w has been a common finding in many previous studies of micellar-catalyzed organic reactions. In light of the uncertainty in the value assigned to V, particularly for more hydrophobic substrates, our calculated k2m values for micellar-mediated dissociations of NO cannot be considered substantially different from those occurring in the aquous phase, particularly since bulk water and surfactant media are, in any event, expected to produce some differences in kinetic behavior. Some unimolecular reactions that are sensitive to solvent polarity have shown very different rate constants, although diazeniumdiolate dissociation reactions do not appear to exhibit this behavior. Micellar Binding of Diazeniumdiolate Substrates. Of particular note is the fact that the data in Table 4 show a good correlation between the number of positively charged nitrogen centers in zwitterionic substrates and the magnitude of their binding constants. Values range from 88.5 M-1 for compound 5 with three cationic nitrogens to 12.1 M-1 for 4 with a single protonated amino group. The ionic association of zwitterionic substrates with SDS micelles is perhaps not surprising in light of the many examples of Coulombic interactions involving cationic nitrogen centers in polyamines. The polyammonium ions derived from spermine, the parent polyamine of 5, are known to have high affinity for the polyanionic nucleic acids at physiological pHs. Rate reductions observed in reactions between spermine and phosphoimidazolide analogues of guanosine triphosphate are also believed to be due to Coulombic association of polyamines protonated at the nitrogen sites.30 The binding of local anesthetics such as tetracaine to axonal nerve membranes also involves Coulombic association of protonated secondary (30) Kanavarioti, A.; Baird, E. E.; Smith, P. J. J. Org. Chem. 1995, 60, 4873-4883.

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and tertiary amino groups in anesthetics to regions of high negative surface charge near sodium channels in the nerve membrane.17 In addition to the Coulombic interaction with the sulfate headgroups, the data in Table 4 also provide evidence for a hydrophobic component in the micellar binding of diazeniumdiolate substrates. The weak binding constant of the anionic diazeniumdiolate 6 is due exclusively to hydrophobic interaction of its propyl groups with the micellar interior, although the smaller ethyl groups in the analogous compound 1 were unable to effect a comparable association. The similar binding constants obtained for the zwitterions 4 and 3, despite the one less nitrogen site in the former, also presumably arise from some hydrophobic component to the binding of 4 through its propyl group. Reaction of 2 in Succinate Buffer at pH 5.5. The pseudophase ion-exchange model was also applied to rate data obtained for reaction of 2 in 0.020 M succinate buffer at pH 5.5. Treatment of the data with eq 4 yields Ks ) 48.2 M-1, k2m ) 56 M-1 s-1, and k2m/k2w ) 0.44. The same values were employed for the parameters V, KH/Na, and cmc in succinate buffer at pH 5.5 as had been employed in phosphate buffer at pH 7.0. The best fit of the data to eq 4 is shown in the Supporting Information. The value of 56 M-1 s-1 obtained for k2m is in reasonable agreement with the value of 88 M-1 s-1 estimated from data obtained at pH 7.0, considering the many assumptions of the model and the change in buffer medium and concentration. The larger Ks value of 48.2 M-1 obtained for binding of 2 to SDS micelles at pH 5.5, compared to the value of 15.4 M-1 at pH 7.0, is consistent with the more extensive protonation of nitrogen sites at the lower pH, particularly that of the nitrogen carrying the N2O2- functional group that triggers the release of NO. The pKa value of 5.9, determined kinetically for protonation of 2 at this site,12 predicts that it should be substantially more protonated at pH 5.5 (73%) than at pH 7.0 (8%), thereby contributing to the larger binding constant at the lower pH. Catalysis by Sodium Decylphosphonate. The ratesurfactant profile determined for 2 at pH 7.0 in phosphatebuffered sodium decylphosphonate solutions at 50 °C resembles that obtained with SDS at 37 °C. Measured rate constants for diazeniumdiolate dissociation increase with SDP concentration and approach a limiting value at high [SDP]. Treatment of the data with eq 4, using a cmc value reported for sodium dodecyl phosphate micelles,28 yielded a Ks value of 9.1 M-1 for 2 in 0.050 M succinatebuffered SDP at 50 °C compared to the value of 15.6 M-1 obtained in 0.10 M phosphate-buffered SDS at 37 °C. Critical micelle concentration values of 1-2 mM gave a better fit to the rate data, although much lower precision was evident with SDP micelles at all cmc values employed. In several previous studies of acid-base and rate behavior in sodium monoalkyl phosphate surfactants, no significant differences were found from those observed in SDS.18,28 From the limited study we have carried out with 3 in SDP, comparable binding affinities also appear to exist for diazeniumdiolates at sulfate and phosphonate micellar surfaces. It appears that the headgroups of micellized SDP surfactants resemble those of alkyl phosphates and exist primarily as monoanions under our experimental conditions.31 Micellar Effects on Diazeniumdiolate Acid-Base Equilibria. Many earlier studies have reported shifts in the apparent acidity constants of micellized substrates.

Acid-base indicators, in particular, have been used widely to examine the interfacial properties of surfactant micelles and develop quantitative treatments of acid-base equilibria at a variety of charged aqueous interfaces including those of micelles,17-21 microemulsions,32 and vesicles.33 Apparent pKa shifts, typically 1-2 units, for micellarbound indicators, have been attributed to a variety of factors. In terms of the PIE model, surfactant-induced equilibrium changes are expected to reflect the increased substrate and hydrogen ion concentrations resulting from their partitioning into the much smaller volume of the Stern layer.18 Changes in intrinsic weak acid behavior at the lower dielectric and surface potential found at the micellar interface17,20 and specific stabilizing or destabilizing interactions between weak acid components and the surfactant headgroups21 are also expected to contribute. Added salts can further influence pKa values through their displacement of H+ ions at the micellar surface.19 The negative ∆pKa ()pKa(water) - pKa(micelle)) we have observed for 3 in 0.10 M SDS is typical of equilibrium changes found with anionic surfactants. The SDS-induced increase in the apparent pKa of the trialkylammonium nitrogen of the local anesthetic tetracaine has been related to the lower dielectric and the surface electric potential of the anionic micellar surface.17 The higher pKa of bromocresol green in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles has been explained in terms of electrostatic destabilization of the negatively charged conjugate base of the indicator by the anionic headgroups of the micelle.21 Since the spectral shift in λmax employed for the determination of 3 has been assigned to protonation of oxygen sites in the diazeniumdiolate functional group, our result could be rationalized in similar terms. Since this protonation is expected to initiate a second acidcatalyzed dissociation path for 3 in the lower pH solutions, the higher pKa value in SDS solutions would be expected to enhance the rate of NO dissociation from diazeniumdiolate substrates. An additional SDS-induced shift in the pKa of the protonated nitrogen centers in 3, which initiates the major NO dissociation path at pH 7.4, is also possible although it is not detectable spectrally. The situation is expected to be even more complex for zwitterions such as 3, however, due to the possible interdependence of the different protonation equilibria involved. Just as micelles can change the apparent pKa of a bound substrate, they can also disrupt buffer equilibria and influence the solution pH by preferentially binding one component of a buffer system. This is expected where one or both buffer components are of opposite charge to that of the surfactant headgroup and with organic buffers which can associate strongly with micellar interiors. Since the active buffering species in both the phosphate and succinate buffer systems are hydrophilic, and anionic under the conditions employed in our study, they are expected to reside almost exclusively in the aqueous phase and have negligible interaction with the micellar surface. They therefore represent the best situation for maintaining constant pH of the aqueous phase in the presence of different surfactant concentrations. Consistent with this, measured pH values of micellar solutions in our studies were not significantly different from those obtained in the absence of surfactant. These findings were in contrast to the experiments we carried out initially in SDS solutions with organic buffers which showed considerable differences (>0.5 units) in pH.

(31) Arakawa, J.; Pethica, B. A. J. Colloid Interface Sci. 1980, 75, 441-450.

(32) Mackay, R. A. Adv. Colloid Interface Sci. 1981, 15, 131-156. (33) Fernandez, M. S. Biochem. Biophys. Acta 1981, 646, 23-26.

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Conclusions We have shown that the acid-catalyzed dissociation of nitric oxide from diazeniumdiolates is subject to catalysis by anionic surfactants with the catalytic activity arising from an increase in the local concentration of the reaction partners, diazeniumdiolate substrate and hydrogen ions, at the charged micellar interface. Micellar association occurs principally through Coulombic interaction of positively charged nitrogen centers and anionic headgroups of the micelle and, to a lesser extent, through hydrophobic association of alkyl chains with the micellar interior. There appear to be no significant differences in diazeniumdiolate binding to anionic sulfate and phosphonate micellar surfaces. Diazeniumdiolate dissociation rates are unaffected by cationic CTAB or zwitterionic sulfobetaine surfactants. This is presumably due to the expulsion of hydrogen ions from the Stern layer as a result of their repulsion by the cationic micellar surfaces. The lack of any rate inhibition in CTAB solutions suggests that there is insufficient attraction between the cationic micellar surface and the [N(O)NO]- functional group to significantly partition the diazeniumdiolate substrate away from the aqueous phase. This may be a consequence of the delocalization of the negative charge density of the [N(O)NO]- group over the different heteroatoms in the ion. Strong solvation of the functional group by water molecules could produce a similar result. Although analogies have been made between rate enhancements at micelles and biomembranes, it is recognized that surfactant aggregates can only crudely model catalysis in biological systems. The micellar interface and the active sites of proteins and other lipophilic membrane components are expected to be very different with respect

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to their interactions with substrate molecules. Proximity effects are likely to be less important at protein interfaces where more specific recognition of substrates is generally found. Acid-catalyzed reactions at enzyme surfaces also frequently involve general acid catalysis rather than catalysis by hydronium ions. Notwithstanding these considerations, our findings do provide important information on the nature of the interactions expected between diazeniumdiolate substrates and charged aqueous interfaces. This information will be of relevance to diazeniumdiolates employed as NO donors in biological milieux. A particular application may be in the choice of aerosolized diazeniumdiolates administered for the treatment of acute lung injury after surfactant pretreatment in vitro. Our findings also speak to the robust nature of the diazeniumdiolate functional group and its ability to resist changes in its intrinsic dissociation rate when subjected to potentially polarizing reaction environments. Acknowledgment. Support of this work by the National Institutes of Health (Grant Number R15 GM61560-01) is gratefully acknowledged. This publication has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400. Supporting Information Available: The pH dependence of the first-order rate constants for dissociation of 3, 4, and 6 and a plot of kobs versus Dn for 2 in 0.020 M succinate buffer at pH 5.5, showing the fit of the data to eq 4. This information is available free of charge via the Internet at http://pubs.acs.org. LA020889S