J. Chem. Soc., Perkin Trans. 2, 1999, 1989–1993 1989 This journal is © The Royal Society of Chemistry 1999 Reactivity of nitrogen nucleophiles towards S-nitrosopenicillamine Andrew P. Munro and D. Lyn H. Williams * Department of Chemistry, Durham University, South Road, Durham, UK DH1 3LE Received (in Cambridge, UK) 21st July 1999, Accepted 20th August 1999 We report the results of a kinetic study of the reactions of a number of nitrogen nucleophiles with the nitrosothiol S-nitrosopenicillamine (SPEN).The range of nucleophiles includes primary, secondary and tertiary aliphatic amines, together with hydrazine, hydroxylamine, azide ion, ammonia, semicarbazide, thiomorpholine and S-methylcysteine. Secondary amines form N-nitrosamines quantitatively. As expected, reaction occurs via the free base forms of the nucleophiles and consequently most of the reactions take place readily only at relatively high pH. Experiments were carried out with [nucleophile] [RSNO], and for many reactions, plots of the .rst order rate constant vs.[nucleophile] were linear. For ammonia and the primary amines, however, this plot tended to level o. at high [nucleophile] and an explanation is o.ered involving the reversible formation of an inactive RSNO–amine complex, for which there is spectral evidence, in parallel with the main reaction. For the secondary amines there is a reasonably good Brönsted plot with a ß value of ~0.2. The much greater reactivities of S-methylcysteine and thiomorpholine, compared to those of primary amines and morpholine respectively are consistent with initial attack at the sulfur atom, followed by an internal rearrangement.Over the whole range of nucleophiles studied there is a reasonable correlation with the Ritchie N parameter, and not with the Pearson n scale. Comparisons are made with the corresponding reactions of alkyl nitrites and N-methyl-N-nitrosotoluene-p-sulfonamide (MNTS). There is currently much interest in the chemistry of S-nitrosothiols (or thionitrites) RSNO, in connection with aspects of the now well-known1 ‘nitric oxide story’.They are under active consideration as nitric oxide donors for possible medical use to make up for any de.ciencies in the natural in vivo synthesis of NO from .-arginine, and they are currently believed to be involved in the storage and transport of NO within the body.2 S-Nitrosoglutathione (GSNO) is presently used medically to inhibit platelet aggregation during some operations,3 and both GSNO and S-nitroso-N-acetylpenicillamine (SNAP), are stable solids which have been, and continue to be, much used in a range of in vivo and in vitro experiments as NO generators. Less stable (generally non-isolable in the pure state) RSNOs are sometimes used for the same purpose, when they are generated in solution by thiol nitrosation 4 and used as such in situ.Clearly the mechanism of NO release is important.We have shown5 that apart from the thermal reaction (which is usually very slow at room temperature) and the photochemical pathway, reaction can occur in aqueous solution at pH 7.4, generating the disul.de and initially nitric oxide. This occurs in a Cu-catalysed process, in which the catalyst is generated from Cu2 and RS, which are often present in bu.ered solutions of RSNOs at trace impurity levels. In aerated aqueous solutions the nitric oxide is rapidly and quantitatively converted to nitrite anion, at the concentrations used.There is a large structure– reactivity dependence, which has been rationalised. It has been further shown that ascorbate (at low concentration) will also act as a reducing agent, and that Cu can also be generated, by thiolate reduction from Cu2 bound to peptides and proteins.6 Another important reaction of RSNOs is that with thiolate ion (eqn. (1)), which leads to a transnitrosation, forming a new RSNO RS RS RSNO (1) S-nitrosothiol if the R groups are di.erent.This exchange process has been known qualitatively for some time,7 but more recent work has measured the equilibrium and rate constants for the process.8 Kinetic studies over a range of pH values have shown, as expected, that the reactive species is the thiolate ion, and that mechanistically the reaction can be regarded as a nucleophilic substitution at the nitroso nitrogen atom,9 or alternatively as an electrophilic S-nitrosation by RSNO.At this stage there is no evidence in favour of (or against) the involvement of a reaction intermediate. Since there is a wide range of reactivity of RSNOs towards NO generation,10 it is possible that NO can be stored in vivo as a ‘stable’ RSNO, which could release NO by .rst undergoing a transnitrosation to give a more unstable RSNO. It is known for example that S-nitrosoproteins are much more stable than is S-nitrosocysteine itself to loss of NO.11 Another example of reactions where RSNOs can act as electrophilic nitrosating species is to be found in their reactions with ascorbate.As previously mentioned, it has been demonstrated that ascorbate can act as a reducing agent (at quite low concentration, typically 1 × 104 mol dm3) to generate Cu, which brings about RSNO decomposition. However, in the presence of EDTA, which cuts out any metal ion promoted reactions, and at higher concentrations, ascorbate brings about the decomposition of RSNOs generating NO and the corresponding thiol 12 (eqn.(2)). As yet the exact form of 2RSNO Ascorbate .. 2RS 2NO Dehydroascorbic acid 2H (2) the nucleophile has not been properly established, but since the reactions were carried out at pH 7.4, it is likely that the nucleophile is the monoanion of ascorbic acid. Similarly it has been shown recently that S-nitrosothiols react with hydrogen peroxide (via the anion) generating initially the peroxynitrite anion, stable at high pH, but which isomerises to nitrate anion at lower pH.13 We thought it to be of some interest to determine the range of nucleophiles which would undergo electrophilic nitrosation by RSNOs, thus establishing the possible generality of the reaction.There are literature reports 14 that amines can be nitrosated in this way, but there has not been a proper mechanistic study. It is possible that amine nitrosation could occur by RSNO decomposition (by the copper catalysed route) to give NO,1990 J.Chem. Soc., Perkin Trans. 2, 1999, 1989–1993 which by oxidation and further reaction with NO would give N2O3, which could readily nitrosate any amine group present. Indeed we showed this to be the case 10 with the reaction of SNAP with N-methylaniline, where in the presence of air, the N-nitrosamine was formed essentially quantitatively, whereas when oxygen was rigorously excluded, very little N-nitrosamine was formed. The copper catalysed pathway is readily blocked by EDTA addition. Electrophilic nitrosation by other compounds containing the -NO group such as alkyl nitrites and a range of N–NO compounds is well known, and has signi.cant synthetic advantages over the more usual nitrous acid nitrosation, in that generally it can be carried out under basic conditions, and also in a wider range of solvents.Detailed mechanistic studies of reactions of alkyl nitrites 15 and of N-nitrososulfonamides16 have been published, and there are some results on the reactions of N-nitrosamines. 17 We hoped to compare the results of RSNO reactions with nitrogen nucleophiles with these literature data. We found generally that the reactions of amines with RSNO species were quite slow, so drawing on our experience with reactions of several RSNOs18 with thiolate ion, we elected to work initially with one substrate, S-nitrosopenicillamine (SPEN) 1. This compound was signi.cantly more reactive than the other nitrosothiols used, towards thiolate ion at high [thiolate ion], when a di.erent reaction, leading principally to ammonia formation, occurs, but where the rate limiting step is believed to involve thiolate attack at a nitrosothiol.Although SPEN has never been isolated in the pure state, it is easy to work with a solution generated by S-nitrosation of penicillamine, which is su.ciently stable as a stock solution in mildly acidic solution for the purpose. Results and discussion (a) Reactions of secondary aliphatic amines A range of secondary aliphatic amines reacted with SPEN (generated in situ from equimolar quantities of penicillamine and nitrous acid) in water at pH values near to the pKa of the protonated amine.Reactions were followed by noting the disappearance of the absorbance at 340 nm due to SPEN. Final spectral scans all showed the complete disappearance of the 340 nm band and the appearance of absorbances in the 230–240 nm region consistent with N-nitrosamine formation.Spectral measurements in this range showed that nitrosamine formation is essentially quantitative. All kinetic experiments were carried out with [amine] [SPEN], and in the presence of EDTA to prevent SPEN decomposition to NO via a copper-catalysed reaction. Good .rst-order kinetics were always found and the .rst-order rate constant (kO) was linearly dependent on the total stoichiometric amine concentration, [Amine]T, as shown in Fig. 1 for both the reactions of pyrrolidine (a) and piperidine (b) at a constant pH. This establishes experimentally the rate eqn. (3). For both of these amines, values of kO were also Rate = k[SPEN][Amine]T = kO[SPEN] (3) obtained over a range of pH values in the region of the pKa value of the protonated amine. The experimental results for piperidine are given in Table 1. In both cases, plots of kO vs. pH are S-shaped with an in.exion point in the vicinity of the pKa value, con.rming that reaction occurs via the free base form of the amine as expected.This means that kO and the acidity should be related by eqn. (4), where k2 is the bimolecular rate kO = k2Ka[Amine]T/(Ka [H]) (4) constant for reaction between SPEN and the free base form of the amine. Plots of (kO)1 against [H] should then be linear with a positive slope and intercept. A typical plot is shown in Fig. 2 for the reaction of piperidine. The data for pyrrolidine gave a similar straight line plot.Values of the pKa of the protonated form of the amines found from the slopes and intercepts of these lines were determined as 11.15 (piperidine) and 11.55 (pyrrolidine), which agree well with the respective literature values 19 of 11.12 and 11.30. These plots also gave values of 1.34 × 103 dm3 mol1 s1 (piperidine) and 7.00 × 103 dm3 mol1 s1 (pyrrolidine) for k2, the bimolecular rate constant for reaction of the free amine with SPEN. The reactions of eight secondary amines with SPEN were followed kinetically.Each individual experiment gave good .rst-order behaviour, and plots of kO against [Amine]T were all linear. Typical experimental plots (for piperidine and pyrrolidine) are shown in Fig. 1. The other amines behaved similarly Fig. 1 Plots of kO vs. total amine concentration for the reaction of SPEN with (a) pyrrolidine at pH 11.57 and (b) piperidine at pH 11.21. Fig. 2 Plot of (kO)1 vs. [H] for the reaction of SPEN with piperidine. Table 1 Values of kO for the reaction of SPEN with piperidine (0.2 mol dm3) as a function of pH pH kO/105 s1 10.58 10.73 10.87 11.00 11.21 11.39 11.56 11.74 11.92 12.02 12.10 5.8 7.2 9.1 10.6 13.9 16.6 19.8 22.3 23.0 24.2 25.3J. Chem.Soc., Perkin Trans. 2, 1999, 1989–1993 1991 except that in some cases there was a small positive intercept at [Amine]T = 0, which will be discussed later. Values of k2 obtained from the slopes and from eqn. (4) are listed in Table 2.The value for pyrrolidine is duplicated, within the experimental error, when reaction is carried out anaerobically, demonstrating that dissolved oxygen plays no part in these reactions. Small positive intercepts for the kO against [Amine]T plots are evident for the slower reacting amines, notably for proline, piperazine and morpholine, and can reasonably be ascribed to the alkaline hydrolysis pathway, where the nucleophile is the hydroxide ion. This explanation was suggested 20 to account for similar small intercepts observed in the nitrosation of amines brought about in alkaline solution by N-methyl-N-nitrosotoluene- p-sulfonamide (MNTS).To check this possibility, we examined the kinetics of the alkaline hydrolysis of SPEN independently. Over the range of [OH] 0.06–0.20 mol dm3, .rst order behaviour occurred and the plot of kO against [OH] is linear with a very small intercept, which could arise from the spontaneous thermal decomposition of SPEN (see Fig. 3). The expected ‘nitrogen’ product in the hydrolysis reaction is the nitrite anion, which was determined in the .nal reaction solution only at ~50% yield. This suggests that in addition to attack of OH at the nitrogen atom, some reaction also occurs possibly by way of initial attack at the sulfur atom, which could lead to nitrous oxide formation, notionally by release of NO. Reactions of nucleophiles with MNTS show this ambident feature,21 for while amines and other nitrogen nucleophiles attack the nitroso-nitrogen atom (leading to amine nitrosation), the ‘harder’ nucleophiles OH and OEt attack at the sulfur atom leading to diazomethane formation.There is an acceptable agreement between the k2 values obtained for piperidine and pyrrolidine from the variation of kO with [Amine]T (Table 2) and those obtained from the reciprocal plot (Fig. 2). Qualitatively there is a clear link between the reactivity of the secondary amines and their basicity, and there Fig. 3 Plot of kO vs. [OH] for the alkaline hydrolysis of SPEN. Table 2 Values of k2 from eqn. (4) for the reaction of SPEN with a range of secondary amines Amine k2/dm3 mol1 s1 Pyrrolidine Piperidine .,.-Proline Sarcosine Piperazine 4-Hydroxyproline Morpholine N-Methylaniline 7.2 × 103 7.0 × 103 a 7.9 × 103 b 1.7 × 103 1.4 × 103 a 5.8 × 104 1.3 × 103 3.5 × 104 c 6.1 × 104 3.8 × 104 Too slow to measure a From the reciprocal plot (kO)1 vs. [H]. b From a reaction carried out anaerobically.c Corrected for the statistical factor. is a reasonable Brönsted plot with a ß value of ~0.2. This is signi.cantly smaller than the value of 0.6 found for the corresponding reactions of secondary amines with MNTS 21 suggesting that charge transfer in the transition state of the RSNO reactions is less extensive, possibly re.ecting the powerful electron withdrawing e.ect of the sulfonyl group in MNTS. Less basic amines such as N-methylaniline (NMA) react much too slowly for a meaningful rate constant to be obtained.This means that an earlier report of nitrosamine formation 10 from NMA and a nitrosothiol, carried out without removal of all trace amounts of Cu2 ions, was correctly interpreted in terms of prior NO formation followed by oxidation and hydrolysis giving nitrous acid which e.ects N-nitrosation, rather than as a direct nitrosation reaction by the nitrosothiol. (b) Reactions of other nitrogen nucleophiles Primary aliphatic amines behave very much like their secondary counterparts, particularly at relatively low concentrations of amine and k2 values were obtained in the same way.Values obtained are given in Table 3. Again reaction was shown to occur via the free base form by the linearity of a plot of (kO)1 vs. [H], and satisfactory agreement was found between the derived and literature pKa values. Reactivity was comparable with that of the secondary amines of similar basicity.Other nitrogen nucleophiles (of lower basicity), azide ion, and hydrazine reacted much more rapidly than did the amines: hydroxylamine also reacted, but at a rate comparable with those of the primary amines. Reaction also occurred with tertiary amines e.g. trimethylamine and quinuclidine, as is the case with nitrous acid nitrosation,22 but reactions were quite slow and were not examined in detail. We did .nd one signi.cant di.erence between the behaviour of the primary and secondary amines, in that for the former the plots of kO against [Amine]T showed a very pronounced tendency to curvature leading to a levelling o.at high [Amine]T, which was not present for any of the secondary amines studied. An example is shown in Fig. 4 for the reaction of propylamine at pH 10.9. All of the primary amines behaved similarly, but there was no obvious curvature in the plots for the reactions of azide ion, hydrazine and hydroxylamine. For the primary amines therefore values of k2 were obtained at lower concentrations of amine (below ~0.09 mol dm3) where the inequality [Amine]T [SPEN] still was valid.The products were not characterised; it is very likely that a primary aliphatic diazonium ion is formed, as in the nitrous acid reaction, leading to a range of products of deamination. There was, as expected, no absorbance formation in the 230–240 nm range corresponding to the formation of a stable nitrosamine. The curvature of the k2 vs.[Amine]T plot was even more pronounced in the reaction of ammonia with SPEN, which did proceed readily (with kO values in the region of 1 × 103 s1), Table 3 Values of k2 for reaction of SPEN with a range of nitrogen nucleophiles Nucleophile k2/dm3 mol1 s1 Propylamine Ethylamine Ethylenediamine Quinuclidine Trimethylamine Hydrazine Azide ion Methoxyamine Hydroxylamine Semicarbazide Thiomorpholine S-Methylcysteine 7.8 × 104 4.9 × 104 1.8 × 104 a 7.1 × 104 3.6 × 104 3.3 × 102 a 1.1 × 102 a 1.5 × 105 9.9 × 104 4.4 × 105 1.5 × 102 3.8 × 102 a Corrected for the statistical factor.1992 J.Chem. Soc., Perkin Trans. 2, 1999, 1989�C1993 which precluded the determination of a k2 value. Curved plots of this type could arise if an inactive complex between the amine and the nitrosothiol is formed rapidly as indicated in Scheme 1. The general expression for kO from such a scheme is RSNO RNH2 [RSNORNH2] rapid K RSNO RNH2 ¡ú RS RN2 slow k2 Scheme 1 given by eqn.(5), which predicts a constant value for kO at high kO = k2Ka[RNH2]T/(Ka [H] KKa[RNH2]T) (5) [RNH2]T if K[RNH2]T 1. Support for this idea comes from the observation of a signicant change in the initial spectrum of SPEN in the 340 nm range at high [RNH2]T. This is shown in Fig. 5 for the interaction of ammonia with SPEN at pH 9.55. A possible structure for this inactive complex could be that shown in Fig. 6, where there are two H-bonding interactions involving the O- and S-atoms of RSNO in a six-membered ring structure.This is possible for primary amines and ammonia, but not for secondary amines, in keeping with the experimental ndings. (c) Ambident nucleophiles There are two examples in Table 2 where there is evidence that SPEN eects nitrosation initially at a sulfur site, which is then followed by an internal rearrangement of the nitroso group to the nitrogen atom. The rate constant for the reaction with thiomorpholine is 40 times greater than that for morpholine, and the value for S-methylcysteine is ~100 times larger than the values for simple primary amines: a direct comparison with e.g.alanine is not possible since, unusually, the kinetics for that reaction were not straightforward. It is quite clear however that Fig. 4 Plot of kO vs. total amine concentration for the reaction of SPEN with propylamine. Fig. 5 Spectra of (a) SPEN (2.5 ¡Á 10�C4 mol dm3) alone and (b) with added ammonia (0.2 mol dm3) at pH 9.55.the presence of the sulfur atom results in an increased reactivity, suggesting that reaction occurs initially at sulfur, a more nucleophilic site. This contrasts with the results obtained for the reactions of MNTS,16 where the kinetic results indicate that the nitrogen site is more nucleophilic than the sulfur atom. This suggests that MNTS and SPEN possess dierent discriminating properties. Details of the reaction of S-nitrosothiols with a range of sulfur nucleophiles will be published shortly,23 when this point will be discussed further.Comparable ¡®additional reactivity¡� has been described for the nitrosation of S-methylcysteine, 24 thioproline 25 and thiomorpholine 26 by nitrous acid itself, and the results were also interpreted in terms of initial attack at the sulfur atom followed by internal rearrangement of the NO group to the amino nitrogen atom, but these results refer to reaction in an acid medium, where the amino group is substantially protonated, and so provides no direct comparison for the relative nucleophilicities of N- and S- sites.Correlation of the kinetic results Although there is a reasonable correlation between log k2 and the pKa values for the family of secondary amines, there is no discernible link when all of the nitrogen nucleophiles are included. Similarly there is no correlation with the Pearson nucleophilicity parameter n27 for the nucleophiles in this work for which values are available (for example both hydrazine and azide ion are substantially more reactive than are pyrrolidine and piperidine, even though their n values would predict otherwise).There is however a reasonable correlation between log k2 and the Ritchie N parameter 28 as shown in Fig. 7. The slope of the drawn line is 0.8, a little lower than the predicted value of 1.0 and the one point for hydrazine lies well above the line. These results parallel almost exactly those reported in an extensive study of nucleophilic reactivity for attack at the nitrogen atom of the nitroso group in the nitrososulfonamide MNTS.16,20,21 Here also there was a correlation with N but not with n.This was interpreted using the ideas of Hoz 29 and Shaik 30 in terms of a Klopman frontier-controlled reaction 31 in which the transition state has substantial diradical nature. From the general parallel between the MNTS (and an alkyl nitrite) and SPEN reactions it appears that direct nitrosation Fig. 6 Possible structure of a complex between RSNO and a secondary amine or ammonia (R = H). Fig. 7 Plot of log k2 vs. the Ritchie N parameter for the reactions of SPEN with a range of nucleophiles. (1) Semicarbazide, (2) methoxyamine, (3) hydroxylamine, (4) morpholine, (5) ethylamine, (6) piperazine, (7) piperidine, (8) hydrazine and (9) azide.J. Chem. Soc., Perkin Trans. 2, 1999, 1989–1993 1993 reactions e.ected by S-nitrosothiols can be characterised in the same way.Experimental SPEN was generated in mildly acid solution by the reaction of equimolar quantities (around 1 × 102 mol dm3) of sodium nitrite and penicillamine. This stock solution was stable for many hours and a fresh batch was made up daily. Reaction solutions were prepared with [SPEN] typically 1 × 103 mol dm3 and the nucleophile typically in the range 0.002–0.200 mol dm3. The pH was adjusted by the addition of perchloric acid or sodium hydroxide solution, and the amine/amine salt acted as a bu.er.All other materials used were commercial samples of the highest purity available. Kinetic measurements were carried out in aqueous solution containing EDTA (1 × 10–4 mol dm3) at 25 C at 340 nm in a conventional spectrophotometer following the disappearance of the absorbance due to the nitrosothiol. Good .rst order behaviour was found throughout and rate constants were obtained using the Enz.tter program and were generally reproducible to ±3%.Nitrite analysis was done by the conventional diazotisation–azo dye procedure, following pH adjustment to 7. Acknowledgements We thank the EPSRC for the award of a research studentship to APM and Dr M. R. Crampton and Dr S. Amado for constructive discussions. References 1 P. L. Feldman, O. W. Gri.th and D. J. Stuehr, Chem. Eng. News, 1993, Dec. 20, 26. 2 J. S. Stamler, O. Jaraki, J. Osborne, D. I. Simon, J. 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Hoz, Acc. Chem. Res., 1993, 26, 69. 30 S. S. Shaik, J. Org. Chem., 1987, 52, 1563. 31 G. Klopman, J. Am. Chem. Soc., 1968, 90, 223. Paper 9/05901F