Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2 / 0665 682

Supplement F2: The Chemistry of Amino, Nitroso, Nitro and Related Groups.

Edited by Saul Patai Copyright 1996 John Wiley & Sons, Ltd.

ISBN: 0-471-95171-4

CHAPTER 14

S- Nitroso compounds, formation, reactions and biological activity

D. LYN H. WILLIAMS
Chemistry Department, University of Durham, Durham, UK
Fax: [phone]; e-mail: [e-mail]
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		665
II. S-NITROSOTHIOLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		666
A. Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		666
B. Properties and Chemical Reactions . . . . . . . . . . . . . . . . . . . . . . . . .		669
C. Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		673
III. S-NITROSOTHIOCARBONYL COMPOUNDS . . . . . . . . . . . . . . . . . .		674
A. Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		674
B. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		675
IV. FORMATION AND REACTIONS OF OTHER S-NITROSO
COMPOUNDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		676
A. S-Nitrososulphides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		676
B. S-Nitrososulphinates (Sulphonyl Nitrites) . . . . . . . . . . . . . . . . . . . . .		677
C. S-Nitroso Compounds with Inorganic Anions . . . . . . . . . . . . . . . . . .		678
1.	Nitrosyl thiocyanate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	678
2.	Nitrosyl thiosulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	678
3.	Nitrosyl bisulphite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	679
V. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		680

I. INTRODUCTION

Nitroso compounds in general are quite well-known compounds, readily synthesized and their reactions have been studied. Some are important as intermediates in a number of important industrial processes, e.g. in diazotisation and azo dye formation, in caprolactam synthesis and in paracetamol manufacture. Compounds are very well-known in which the nitroso group is bound to carbon, nitrogen and oxygen sites within molecules. Much less well-known are those in which the nitroso group is bound to sulphur. These include principally S-nitrosothiols (sometimes called thionitrites), RSNO, generally henceforth referred to in this chapter as nitrosothiols. Other species including S-nitrosothiocarbonyl

665

666	D. Lyn H. Williams

compounds are less familiar. Nitrosothiols have not been as widely studied as their oxygen counterparts alkyl nitrites, generally because of their more reduced stability. However, in recent years there has been much interest generated in the chemistry of nitrosothiols following the major discoveries made in the late 1980s and early 1990s concerning the part played by nitric oxide in a number of physiological process in human metabolism. Nitric oxide is believed to be formed naturally in vivo by an enzymatic reaction of L-arginine (leading to L-citrulline production) and also brought about in vivo by the administration of drugs such as glyceryl trinitrate (GTN), which has been used for over a century to treat problems in the blood circulatory system. Nitrosothiols (which have been detected in vivo) are believed to be involved in the nitric oxide saga in two ways: (a) as possible alternative (to GTN) NO-releasing drugs to make up for the deficiencies of spontaneous NO production in some clinical conditions, and (b) as a possible ‘storage’ area for nitric oxide in vivo in the mechanism of NO transfer within the body.

II. S-NITROSOTHIOLS

A. Formation

Nitrosothiols are very easily generated by simple electrophilic nitrosation of thiols1 (equation 1), just as alkyl nitrites are made from alcohols (equation 2), and N-nitrosamines from secondary amines (equation 3). The most convenient reagent is nitrous acid, generated from sodium nitrite and mineral acid in water or in mixed alcohol water solvents. In water the S-nitrosation of thiols is effectively irreversible, contrasting with the corresponding reaction of alcohols. This makes the product separation somewhat easier. The reason for the different behaviour is believed to arise from the differences in basicity (important in the reverse reaction) and nucleophilicity (important in the forward reaction) between the O- and S-sites. The reaction has been studied mechanistically2 and it shows all the characteristics of electrophilic nitrosation (including catalysis by non-basic nucleophiles such as halide ions, thiocyanate ion and thiourea) which are very familiar in N-nitrosation. However, since thiols are not significantly protonated in acid solution S-nitrosation is generally a faster process overall than in N-nitrosation. Some kinetic data (values of k in rate D k[HNO2][HC ][RSH]) are given in Table 1 for the acid catalysed nitrosation of some thiols with nitrous acid. These rate constants tend to approach 7000 dm6 mol 2 s 1 which is believed to be the diffusion controlled limit for the reaction of NOC with the thiols. Similarly, second-order rate constants for the reactions of ClNO, BrNO and ONSCN have been determined for some thiols and are given in Table 2. The by now well-established reactivity trend ClNO > BrNO > ONSCN is evident but the more reactive species are strangely well below the diffusion limit.

RSH C HNO2			HC
			! RSNO C H2O				(1)
		HNO2	HC			H2O
ROH	C		!	RONO	C		(2)
R2NH C HNO2			HC
			! R2NNO C H2O				(3)

Other nitrosating agents have been used successfully to synthesize nitrosothiols, notably alkyl nitrites3, nitrosyl chloride4, dinitrogen trioxide5 and dinitrogen tetroxide6. In principle any carrier of NOC would suffice. One of the simplest nitrosothiols, CF3SNO (a red unstable gas), has been made from both nitrosyl chloride and alkyl nitrites in reaction with CF3SH7. Use of these reagents in organic solvents has some advantage over the

14. S-Nitroso compounds, formation, reactions and biological activity

667

TABLE 1. Values of the third-order rate constant

for the acid catalysed nitrosation of thiols with

nitrous acid in water at 25 °C

RSH

k/ dm6 mol 2 s 1

Cysteine methyl ester

213

Cysteine

443

Glutathione

1080

Mercaptosuccinic acid

1334

Thioglycolic acid

2630

Mercaptopropanoic acid

4764

TABLE 2. Second-order rate constants for the reactions of ClNO, BrNO

and ONSCN with thiols in water at 25 °C

k dm3 mol 1 s 1

ClNO

BrNO

ONSCN

Cysteine methyl ester

6

4

2

1.0 ð 106

4.9 ð 104

7.0 ð 102

Cysteine

1.2 ð 106

5.8 ð 105

7.0 ð 103

Glutathione

5.7 ð 10

2.9 ð 104

1.9 ð 10

Mercaptosuccinic acid

2.6 ð 106

7

4

Thioglycolic acid

1.4 ð 10

1.1 ð 105

2.5 ð 10

Mercaptopropanoic acid

4.5 ð 10

nitrous acid method when there are solubility difficulties in water. A well-tried successful procedure6 is to use N2O4 in carbon tetrachloride, hexane, ether or acetonitrile at 10 °C, when reaction is quantitative. Another excellent and more convenient procedure involves the use of t-butyl nitrite in organic solvents such as chloroform or acetonitrile8 where again excellent yields have been reported (equation 4). In mechanistic studies in water it has been shown that nitrosothiols are formed in solution by attack of the thiolate anion (in mildly alkaline solution) with a large range of alkyl nitrites9 (equation 5) and also with N-methyl-N-nitrosotoluene-p-sulphonamide10. In both cases reaction appears to involve simple nucleophilic attack by the thiolate ion at the nitrogen atom of the nitroso group and, as expected, reactions are much facilitated by the presence of electron-withdrawing substituents in the alkyl nitrite. Neither reaction appears to have been much used synthetically. Some rate data are given in Table 3 which give the second-order rate constants k for the reactions of nine alkyl nitrites with three thiolate anions. The rate enhancement by electron-withdrawing groups within the alkyl nitrites is clearly seen. Nitrosothiols can also be formed from disulphides RSSR by photolysis and reaction with nitric oxide, but this does not seem to have much synthetic potential11.

t-BuONO C RSH			CHCl3					(4)
			! RSNO C t-BuOH
RONO + R′S−					RO− + R′SNO			(5)
H +					H +

R′SH ROH

668	D. Lyn H. Williams

TABLE 3. Second-order rate constants for the reaction of alkyl nitrites with the thiolate ions derived from three thiols in water at 25 °C

			kdm3 mol 1 s 1
RONO		Cysteine	N-Acetylcysteine			Thioglycolic acid
Me3CONO	1.7		1.8			4.9
Me2CHONO	11		12			30
EtONO	28		31			75
(Me)2CH(CH2)2ONO	27		30			75
EtO(CH2)2ONO	169		169			417
Cl(CH2)2ONO	1045		1010			2260
Br(CH2)2ONO	1055		1030			2240
I(CH2)2ONO	1060		1020			2260
Cl2CHCH2ONO	1.2 ð 104
Cl3CCH2ONO	Too fast to measure

Early workers isolated nitrosothiols of varying degrees of stability, including EtSNO4, Ph3CSNO12 and Me3CSNO13. However, in 1978 Field and coworkers14 prepared the nitrosothiol derived from N-acetyl-D,L-penicillamine (SNAP) which is indefinitely stable in the solid form as deep green crystals with red reflections. SNAP (1), because of its solid state stability, has been much used as the typical nitrosothiol in a large number of biological experiments. Similarly, the derivative from glutathione (2) is now widely available as a stable solid15. The mounting interest in the biological activity of nitrosothiols

Me	SNO			O	SNO
Me						H
		HO2 C				N	CO2 H
					N
HN	CO2 H
					H
Ac		NH2				O
(1)					(2)
Me	SNO	Me		SNO	Me	SNO
Me		Me			Me
	NH3 +Cl −			NHAc	HN	CO2 H
					CHO
	(3)		(4)		(5)
	Me	Me	O	CO2 H
	ONS			N	SNO
		NHAc		H Me	Me
			(6)

14. S-Nitroso compounds, formation, reactions and biological activity

669

has resulted in the isolation of a large number of stable nitrosothiols including 316, 416, 517 and the S,S0 -dinitrosothiol 617.

B. Properties and Chemical Reactions

The stable nitrosothiols are coloured either green or red. In general the tertiary structures (e.g. SNAP) are green. There is a general UV absorption band in the range 330 350 nm with extinction coefficients about 103 mol 1 dm3 cm 1, which has been used to monitor reactions of nitrosothiols by spectrophotometry in kinetic studies. The UV-visible spectra have been analysed and the electronic transitions assigned18. The infrared spectra of some nitrosothiols have also been analysed7 and the stretching (1480 1530 cm 1) and bending frequencies of the NO group identified, as has the C S vibration in the 600 730 cm 1 region. Both 1H and 13C NMR spectra of nitrosothiols have also been examined16. There is a significant downfield shift of the ˛-protons upon nitrosation of thiols and there is a similar shift of the ˛-carbon resonances, which makes the techniques useful in showing whether S-nitrosation has occurred.

The molecular structure of SNAP has been obtained by X-ray crystallography14, and is shown in 7. The C S bond is rather long, but other features are as expected.

100.4°

°

1.214 A

C N

S O

°

1.841 A

°

1.771 A 113.2°

(7)

Since the discovery that nitric oxide is crucially involved in a range of physiological processes and indeed that it is synthesized in vivo from L-arginine (for review articles see References 19 22), there has been intense interest in a range of compounds which might act as NO donors. Consequently, the most studied reaction of nitrosothiols is that where decomposition to nitric oxide occurs.

Nitrosothiols decompose photochemically and thermally to give the corresponding disulphides and nitric oxide18,23,14,24 (equation 6). In most cases the nitric oxide has not been identified as the primary product but rather as its oxidized form, nitrogen dioxide.

	h		C
2RSNO	!	RSSR		2NO	6

The reaction in water at pH 7.4 has been much studied since the discovery of the importance of nitric oxide. The products are as for the thermal and photochemical reactions, except that the final product is nitrite ion. This is to be expected since nitric oxide in aerated water at pH 7.4 also yields quantitatively nitrite ion25, by it is believed the series of equations 7 9, which involves oxidation to nitrogen dioxide, further reaction to give dinitrogen trioxide which, in mildly alkaline solution, is hydrolysed to nitrite ion. Under anaerobic conditions it is possible to detect nitric oxide directly from the decomposition of nitrosothiols using a NO-probe electrode system26. Solutions of nitrosothiols both in

670	D. Lyn H. Williams

water26 and in organic solvents6,14 will nitrosate, e.g. amines, probably via NO loss, oxidation and N2O3 formation. In the absence of oxygen no nitrosation product is detected. Aryl halides are generated in excellent yields from nitrosothiols and arylamines at ambient temperatures in the presence of anhydrous Cu(II) halides in acetonitrile (equation 10)11. This probably involves an initial NO group transfer, probably indirectly via NO formation, although no mechanistic studies have been carried out.

2NO C O2 D 2NO2

(7)

NO2 C NO D N2O3

(8)

N2O3 C 2OH D 2NO2

C H2O

(9)

CuX2

ArNH2

C

RSNO

!

ArX

C

N2

C

RSSR

C

R2S3

(10)

3

CN

CH

Kinetic studies of the reaction of nitrosothiols in water at pH 7.4 have been reported and reveal a large range of different rate forms and half-lives. However, in 1993 it was realized27 that reaction is brought about by catalytic quantities of Cu2C . There is a sufficient concentration of Cu2C in many samples of distilled water, and particularly buffer components, to allow reaction to occur. This accounts for the wildly erratic behaviour reported in the literature. When the concentration of Cu2C is carefully controlled sensible results emerge. There is a range of [Cu2C ] for most of the nitrosothiols studied for which the second-order rate equation (equation 11) applies. Values of the second-order rate constant k vary with the structure of the nitrosothiol in such a way which suggests that the copper needs to be bidentately linked (a) with the N of the NO group (or the S of the SNO group) and (b) with another electron-donating system such as a nitrogen atom or negatively charged oxygen atom at positions within the molecule which allow a six-membered ring to be formed26. Both N-acetylation and the addition of a methylene group (which would give a 7-membered ring) resulted in sharp rate reductions. Very little reaction was discernible in the absence of function (b), e.g. for t-BuSNO. When Cu2C was rigorously excluded by complexation with EDTA, then again the reaction rate was reduced to a negligible value, even for reaction of the ‘normally very reactive’ nitrosothiols such as nitrosocysteine.

Rate D k[RSNO][Cu2C ]

11

Outside a given [Cu2C ], zero-order behaviour (at high [Cu2C ]) and the presence of an induction period (at low [Cu2C ]) raised the possibility that the actual catalyst is CuC and not Cu2C , formed by reduction of the latter by thiolate ion, a well-known reaction28 (equation 12). This was confirmed by the use of a specific CuC -chelating agent neocuproine (8) which resulted in a sharp rate reduction, leading eventually to a complete suppression of the reaction. The spectrum of the CuC 8 complex was observed from the reaction mixture. Further, the increased reactivity brought about by the addition of a thiol species supported this suggestion. At low concentrations of added thiol there was a sharp increase in the rate constant for the reaction of SNAP, whereas at higher concentrations there was a gentle reduction leading eventually to a stabilization effect (see Figure 1). The initial catalysis represents an increase in rate of CuC formation (equation 12) whereas the subsequent rate reduction is believed to arise by complexation of Cu2C by thiolate (a well-known reaction29). This accounts for the contrasting literature reports, which state that in some cases added thiol increases the rate of decomposition whereas in other cases a stabilization effect is claimed. The outline reaction scheme is given in equations 13 15. Intermediate X1 is probably RSCuC and X2 is probably a structure similar to those given

14. S-Nitroso compounds, formation, reactions and biological activity

671

kobs (s−1)

0.0025

0.02

0.015

0.01

0.005

0

0

100

200

300

400

500

600

700

800

900

1000

106 [NAP]added (mol dm−3)

FIGURE 1. Rate constant for decomposition of S-nitroso-N-acetylpenicillamine in the presence of added N-acetylpenicillamine (NAP)

in 9 and 10 for two different nitrosothiols. It is likely that here CuC is also coordinated to two water molecules. Details of how NO is released from 9 and 10 are not yet clear, and it is possible that coordination is at the sulphur atom rather than the nitroso nitrogen atom.

Cu2C C RS

! CuC C RSž

! RSSR

(12)

Cu2C

C

RS

!

X1

!

RSž

C

CuC

(13)

Cu2C

RSNO

C

CuC

!

X2

!

RS

C

C

NO

(14)

RSNO C RSž

! RSSR C NO

(15)

or 2RSž

!

RSSR

H2 C

S

H2 C

S

H2 C

N

O

C

N O

N

N

NH2

Cu+

O

−

O

Cu+

Me

Me

(8)

(9)

(10)

Nitrosothiols are also readily decomposed by mercuric ion to the corresponding coordinated thiols and nitrous acid (in acid solution) as in equation 16. This reaction has been used as the basis of an analytical procedure for thiol determination30. Mechanistic

672	D. Lyn H. Williams

studies have shown31 that this is quite a different reaction to the Cu2C catalysed reaction since (a) the Hg2C reactions are generally much faster, (b) the reactions with Hg2C are stoichiometric rather than catalytic, (c) no trace of NO was detected when the reaction was carried out anaerobically and (d) there is very little structure reactivity dependence which is so evident for the Cu2C reaction. All the evidence suggests that [RS(Hg)NO]2C is first formed and then undergoes nucleophilic attack by water at the nitroso nitrogen atom to release nitrous acid. A similar reaction can be achieved with an acid catalyst (equation 17) but only at fairly high acid concentration (¾2M H2SO4) and only in the presence of a nitrous acid trap (such as sulphamic acid) which ensures the irreversibility of the process32.

RSNO C Hg2C C H2O D RSHgC C HNO2 C HC	(16)
HC
RSNO C H2O ! RSH C HNO2 (removed)	(17)

Nitrosothiols can also be reduced with sodium borohydride, leading (with SNAP14) to the disulphide formation (equation 18).

NaBH4
ONSCMe2CH(NHAc)CO2H ! SCMe2CH(NHAc)CO2H]2	18

Oxidation is also a known reaction; examples are known where the reagents are fuming nitric acid33 or N2O434, the product being in each case the corresponding thionitrate (equation 19).

HNO3
RSNO ! RSNO2	19

An important reaction of nitrosothiols is the exchange reaction of the nitroso group with another thiol, i.e. a transnitrosation. This has been demonstrated on a number of occasions6,35. Often, the final product is not the new nitrosothiol but its decomposition product, the disulphide. All three possible disulphides, for example, have been identified in the product mixture of the reaction of nitrosoglutathione (GSNO) with cysteine (equation 20). It is, however, possible to identify spectrophotometrically the primary products of transnitrosation36,37. Kinetic studies37 have shown quite clearly that the reaction involves attack by the thiolate anion, probably in a direct reaction, and is an example of nucleophilic substitution at the nitroso nitrogen atom. The process (equation 21) is thus very similar to that reported in Section II.A for the corresponding reaction of the thiolate anions with alkyl nitrites (equation 5). It seems likely that other powerful nucleophiles will react in a similar fashion, but there appear to be no reports in the literature.

RSNO C R0 SH ! RSSR C R0 SSR C R0 SSR0								(20)
RSNO + R′S−					RS− + R′SNO			(21)
H +					H +

R′SH RSH

14. S-Nitroso compounds, formation, reactions and biological activity

673

C. Biological Activity

Since 1986, a remarkable number of discoveries have been made concerning the physiological actions of nitric oxide. A large number of review articles have appeared (typically References 19 21) and a vast amount of research is being undertaken in this area. It is now known that nitric oxide is synthesized in vivo from L-arginine and controls, among other functions vasodilation in the blood circulatory system. Administered drugs such as glyceryl trinitrate (GTN, by far the most widely used) and other organic nitrates also generate nitric oxide in vivo and are effective as a treatment for angina and other circulatory problems. There is a problem with glyceryl trinitrate in that it quickly induces a tolerance in some patients, so that there is a need for other NO-releasing compounds which can be used medically. Nitrosothiols present an obvious alternative solution.

There is no doubt that many nitrosothiols effect vasodilation38 and also have a powerful inhibition effect on platelet aggregation39. Among those tested and shown to have significant activity are nitrosocysteine, SNAP, GSNO and nitrosocaptopril. Use is now made of these properties clinically by way of the administration of nitrosoglutathione (GSNO)

(a) during coronary angioplasty40 to inhibit platelet aggregation and (b) to treat a form of pre-eclampsia in pregnant women41. Details of the mode of action are not known, but a major factor is believed to be the ability to inhibit platelet aggregation at concentration levels which do not lower blood pressure, in contrast to some other NO-donors.

It has been argued that the so-called Endothelium Derived Relaxing Factor is in fact a nitrosothiol42 and not nitric oxide itself. This, however, is not the generally held view at the present time. Nonetheless, it is quite likely that nitrosothiols are involved at some stage and the bulk of the nitric oxide in the blood43 and in other tissues such as the lung44 is found primarily in the form of nitrosothiol derivatives of proteins and peptides, notably GSNO. The anti-platelet aggregation effects of nitrosoproteins may involve lower molecular weight nitrosothiols following the known (equation 21) transnitrosation between nitrosothiols and thiols45.

New nitrosothiols are being synthesized and tested for activity. These include structures 1 6 in Section II.A as well as those derived from cysteine residues within proteins. One example of a S-nitrosocysteine within a polypeptide46 is remarkably stable in the solid form, contrasting with the marked instability of S-nitrosocysteine itself. The S,S0- dinitrosodithiol 11 has been shown to have platelet aggregation inhibition properties of the same order of magnitude as GTN and vasodilation properties somewhat less than those possessed by GTN17.

Me	Me	O	CO2 H
ONS		N	SNO
		H	Me Me
	NHAc

(11)

Nitrosothiols thus appear to have the necessary properties for clinical use as an alternative for GTN. There does not appear to be the same problem regarding tolerance47. Much testing experiments in vivo remain to be undertaken.

Little is known about the mode of action of nitrosothiols regarding their biological properties. There appears to be no direct connection between their reactivities towards nitric oxide loss and biological activity38,48. However, the experiments which have been carried out in these studies relating to rates of nitric oxide formation do not recognize

674	D. Lyn H. Williams

the vital part played by Cu2C in these reactions and the quoted results may not represent a true reactivity sequence. It has been suggested49 that some of the biological activities result from NOC loss from RSNO, not in a unimolecular process (which would not make good chemical sense) but in the transnitrosation process discussed earlier in Section II.B (equation 21).

One important result, however, does point to the fact that nitric oxide release from nitrosothiols is a necessary reaction for there to be biological activity50. In a study using nitrosocysteine and GSNO it was shown that the specific copper(I) chelators neocuproine

(8) and the structurally related bathocuproine sulphonate reduce the biological activity of both nitrosothiols. This indicates that copper(I) is required for biological activity, which ties in with the in vitro experiments described in Section II.B. The realization that copper plays such an important part in nitrosothiol decomposition accounts for the widely differing decomposition rates quoted in the literature, e.g. the half-life of nitrosocysteine has been reported as 15 min and also variously between 4 and 83 seconds. One of the factors important here, apart from the level of the [Cu2C ] present adventitiously, is the nature and concentration of the buffers used, since many of these, particularly those containing carboxylic acid groups, will themselves bind Cu2C and hence reduce the catalytic activity. It is clear that there is a long way to go before the biological activities of nitrosothiols (and of nitric oxide) are fully understood at the molecular level.

III. S-NITROSOTHIOCARBONYL COMPOUNDS

A. Formation

The sulphur atom of a thiocarbonyl compound is a powerful nucleophilic centre. Examples are to be found in the S-alkylation of thioamides and in the reaction of thioureas to give isothiuronium salts from alkyl halides. In a conventional SN 2 reaction thiourea is approximately as powerful a nucleophile as is iodide ion in polar solvents as measured by the Pearson nucleophilicity parameter51. It is no surprise therefore that thiourea reacts with nitrous acid to give initially the S-nitrososulphonium ion (equation 22). This generates a red or yellow colour in solution, which is fairly characteristic of S-nitroso species. No salts have been isolated and the ion decomposes fairly readily in solution. In fact Werner52 showed that thiourea can undergo two reactions with nitrous acid, one leading to thiocyanate ion and nitrogen products (equation 23) and the other to the disulphide cation (equation 24). These findings can be rationalized in terms of N-nitrosation (at low acidities) leading to nitrogen formation, and S-nitrosation (at higher acidities) leading to the disulphide cation.

(NH2)2CS C HNO2	HC	+
	!	(NH2)2CSNO C H2O	(22)
(NH2)2CS C HNO2	D SCN C N2 C HC C 2H2O		(23)
		+ +
2(NH2)2CS C 2HNO2 C 2HC D (NH2)2 CSSC(NH2)2 C 2NO C 2H2O			(24)

The equilibrium constant for S-nitrososulphonium ion formation (equation 25) has been measured spectrophotometrically as 5000 dm6 mol 2 at 25 °C in water53. This is a much larger figure than for the corresponding reactions leading to nitrosyl chloride, bromide and thiocyanate formation, reflecting the greater nucleophilicity of thiourea. The same effect is evident in the analysis of the rate constant for the nitrosation of thiourea by nitrous acid (equation 26). The value of the third-order rate constant k is 6900 dm6 mol 2 s 1 at 25 °C, which is very close to that found for a large number of reactive aromatic amines