Ana ; Avellón

Jose-Manuel Echevarría

Bernard Weber

Michael Weik

Uwe-Peter Schobel

Wulf R Willems

Wolfram H Gerlich

José M Echevarría

José-Manuel Echevarría

European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants

Keywords: Hepatitis B, Hepatitis B surface antigen, Hepatitis B virus variants, HBsAg mutants, HBsAg testing Hepatitis B, hepatitis B virus surface antigen, hepatitis B virus variants, HBsAg mutants, HBsAg testing

come

 [START_REF] Avellón | Frequency of hepatitis B virus "a" determinant variants in unselected Spanish chronic carriers[END_REF]for full references). (HBV) genome leads to a higher mutation rate and to a broader genetic variability than in most DNA viruses. Random mutations originating in polymerase errors give rise to viral mutants, which may emerge as major viral populations under the pressure of host factors, and some of these mutants display amino acid changes within the major antigenic determinant of the HBV surface antigen (HBsAg). These changes may modify the antigenic properties of the protein and may have impact on relevant issues, such as the immunity to re-infection by the mutant or the sensitivity of diagnostic assays for detecting the mutated form of the antigen [START_REF] Echevarría | Hepatitis B virus genetic diversity[END_REF][START_REF] Weber | Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene[END_REF].

AA residue

Although amino acid changes resulting in decreased binding to specific antibody have been found in many residues of the HBsAg molecule, particular changes in residues 126, 144 and 145 are of special significance, since they may lead to false negative results in diagnostic assays [START_REF] Sl | The analytic sensitivity and mutant detection capability of six hepatitis B surface antigen assays[END_REF][START_REF] Louisirirotchanakul | HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant[END_REF][START_REF] Ly | Sensitivities of four new commercial hepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutant forms[END_REF]Muhlbacher et al., 2008;[START_REF] Scheiblauer | Evaluation of 17 CE-marked HBsAg assays with respect to clinical sensitivity, analytical sensitivity, and hepatitis B virus mutant detection[END_REF][START_REF] Weber | Evaluation of a new automated assay for hepatitis B surface antigen (HBsAg) detection VIDAS HBsAg Ultra[END_REF].

Modifications in the design of an assay can, however, improve significantly its ability to detect these HBsAg mutants [START_REF] Echevarría | Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS(R) HBsAg assay[END_REF][START_REF] Weber | Evaluation of a new automated assay for hepatitis B surface antigen (HBsAg) detection VIDAS HBsAg Ultra[END_REF].

Assessing the success of such changes requires collaborative studies involving, ideally, both clinical samples containing natural variants and samples displaying recombinant mutant proteins, and the performance of such studies has been strongly recommended [START_REF] Gerlich | Diagnostic problems caused by HBsAg mutants-a consensus report of an expert meeting[END_REF].

Enzygnost®

Materials and Methods

Samples containing HBsAg and Pre-S variants The study involved four European laboratories and a total of 201 HBV mutant samples of natural (n=147) or recombinant (n=54) origin (Table 1). The panel spanned mutations predicting amino acid (AA) substitutions between positions 103 to 173 of HBsAg, most involving residues from the major hydrophilic region (AA 124 to 147) that may impair the detection of the antigen [START_REF] Avellón | Frequency of hepatitis B virus "a" determinant variants in unselected Spanish chronic carriers[END_REF][START_REF] Gerlich | Diagnostic problems caused by HBsAg mutants-a consensus report of an expert meeting[END_REF][START_REF] Weber | Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene[END_REF] (Table 2). All samples were aliquoted and stored at -20°C prior to testing.

Multiple freeze/thaw cycles were avoided. Samples were tested undiluted or, in case of limited amounts, diluted as described below. Dilutions were prepared in E6.0 negative control solution for samples tested by Enzygnost or diluted in fetal calf serum, or as recommended by each manufacturer, for samples tested with the remaining assays.

Sixty-five samples of the panel were from Spanish HBV carriers displaying predicted AA substitutions at positions 112 to 145, plus one sample with a predicted stop codon at position 196 [START_REF] Avellón | Frequency of hepatitis B virus "a" determinant variants in unselected Spanish chronic carriers[END_REF]. All the samples were tested at a 1/40 dilution, and again at a 1/10 dilution at least by the E6.0 assay if an absorbance value below the cut-off was obtained. Retesting at a lower dilution by the E5.0 assay was not done when the volume of the sample available was small. The results obtained on undiluted aliquots by the Vitros ® ECi HBsAg and HBsAg ES (Ortho-Clinical Diagnostics, High Wycombe, Buckinghamshire, UK) [START_REF] Echevarría | Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS(R) HBsAg assay[END_REF],

Axsym ® HBsAg V2 (Abbott Diagnostics, Delkenheim, Germany) and the Elecsys ® HBsAg (Roche Diagnostics, Penzberg, Germany) were available for comparison.

Thirty-five samples, supplied by the German National Consulting Laboratory for HBV (NCL), displayed AA changes leading to difficulty in HBsAg detection [START_REF] Gerlich | Diagnostic problems caused by HBsAg mutants-a consensus report of an expert meeting[END_REF][START_REF] Westhoff | Fatal hepatitis B virus reactivation by an escape mutant following rituximab therapy[END_REF]. Samples were tested undiluted or in dilutions ranging from 1/3.16 to 1/25. The results obtained by the Axsym ® HBsAg V2 and by the Prism ® HBsAg (Abbott Diagnostics, Wiesbaden, Germany) were available for comparison.

Forty-seven samples were provided by Laboratoires Réunis Junglister, Luxemburg.

Twenty-two samples were from Luxembourgian HBV carriers [START_REF] Weber | Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene[END_REF]. The remaining 25 were from Nigerian patients with liver disease. Samples were tested in dilutions ranging from 1/2 to 1/100. The results obtained by the Axsym ® HBsAg V2

(Abbott Diagnostics, Delkenheim, Germany), the Elecsys ® HBsAg (Roche Diagnostics, Penzberg, Germany) and by the Vidas ® HBsAg Ultra (Biomérieux, Marcy-l'Etoile, France) were available for comparison.

Fifty-four recombinant HBV mutant samples, comprising HBV genotypes A to D and displaying AA substitutions and insertions between residues 96 to 181 of HBsAg, were München, Germany) were available for comparison.

HBsAg testing

All the samples were tested by the E5.0 and E6.0 assays. The latter test uses sheep polyclonal antibody to HBsAg (anti-HBs) on the solid phase, and a mixture of three monoclonal anti-HBs, targeting different HBsAg epitopes, in the conjugate. Tests were performed following the manufacturer's guidelines and using an automatic processor provided and maintained by the manufacturer. The results were recorded as a signal to cut-off ratio (S/CO). The samples displaying S/CO lower than 1.0 were recorded negative, and the remaining was recorded positive. The samples were not distributed to all participants for testing.

Statistics

Comparison of mean S/CO values was made by the t student test (SPSS software).

Statistical significance was considered for p< 0.005.

Results

S/CO values obtained by the E6.0 assay were always equal to or higher than 1.0. Figure 1 differences in performance favouring the E6.0 assay. Samples in category 2 were also thought to represent significant differences between tests when the S/CO value recorded by one test was at least 1.5 times higher than the value recorded by the other test. No samples placed in the lower right quadrant of the chart were found.

One hundred and seventy-five samples (87.0%) were classified into category 1.

Eighty-one displayed single amino acid substitutions in residues 120,126,129,133,134,140,142,143 or 145, and 41 displayed multiple substitutions involving at least one of these residues. The remaining samples presented either insertions (n=2), substitutions involving the pre-S region only (n=19), or substitutions involving other residues of the HBsAg (n=32). The mean S/CO value found with these samples was significantly higher with the E6.0 assay (51.7, S.D.: 4.8 vs. 39.3, S.D: 3.6; F=16.7, p<0.005).

Sixteen samples (7.9%) were classified into category 3. Ten samples were borderline positive (S/CO <2.0) or negative by the former assay (Table 3a), and one was a natural sample. Multiple amino acid substitutions were recorded in all these ten samples, and eight shared the substitution P120Q.

Finally, 10 samples (4.9%) were classified into category 2 (Table 3b). Six were natural samples, and three of them displayed borderline reactivity by the E6.0 assay (S/CO <2.0) and in some other assays in association with single amino acid changes in the HBsAg (G145R, A184V) or in the pre-S1 protein.

Both natural and recombinant samples with amino acid changes in residues 126, 129, 134, 138, 140, 143 and 148 were strong positive by the E6.0 assay despite the additional occurrence of other relevant changes in some of them. Substitutions in residues 144

were associated with reactivity S/CO <25.0 on recombinant samples when additional changes were present. A single substitution K141E lead to S/CO value of 12.1 in a recombinant sample, but substitutions in residue 142 had no particular effects in absence of additional changes. Amino acid changes in these two residues are uncommon among HBV carriers [Avellon and Echevarria, 2006], and no natural samples with such changes could be tested.

As shown in Table 3b, changes in residues 145 and 184 in natural samples were associated with S/CO values < 2 by the E6.0 assay, similarly to some of the additional assays tested. Single changes in residue 145 had effects both on natural and recombinant samples. The substitution A184V, either alone or in association to other changes, had also effect on the level of reactivity (samples 105 and 109, Table 3b). The pre-S region was studied in 47 samples, and amino acid substitutions were found in 31 samples. The pre-S1 region displayed changes in 14 samples, the pre-S2 region in eight samples, and both regions in nine samples. Twenty samples with pre-S variants displayed a wild-type sequence for HBsAg, and only one (sample 111, table 3b) displayed a borderline S/CO value by the E6.0 assay, paralleling the results obtained with other tests.

Discussion

Warning on the emergence of HBV variants involved potentially in diagnostic failures [START_REF] Gerlich | Diagnostic problems caused by HBsAg mutants-a consensus report of an expert meeting[END_REF] led many companies to modify their assays in order to overcome the problem, and some significant improvements have been noted [START_REF] Echevarría | Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS(R) HBsAg assay[END_REF]. The results of the present study show that the new Enzygnost® HBsAg 6.0 test improves the characteristics of the former with regard to detection of such HBsAg variants.

The improvement was demonstrated by comparing the mean and individual S/CO values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 that were recorded negative by the former test. Difficulties with current commercial tests for detecting HBsAg variants with substitutions in positions 143 to 145 have been recognised widely [START_REF] Echevarría | Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS(R) HBsAg assay[END_REF][START_REF] Ly | Sensitivities of four new commercial hepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutant forms[END_REF][START_REF] Mühlbacher | Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants[END_REF][START_REF] Weber | Evaluation of a new automated assay for hepatitis B surface antigen (HBsAg) detection VIDAS HBsAg Ultra[END_REF]. Substitutions in residue 120 were associated in the past with false negative results by diagnostic assays [START_REF] Coleman | Immunoassay detection of hepatitis B surface antigen mutants[END_REF], but do not involve such difficulties for the current, modified tests [START_REF] Mühlbacher | Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants[END_REF]. Similar results were obtained with two recombinant samples displaying the substitution Q101H, which correspond to an HBsAg residue placed outside the "a" antigenic determinant. A relative sensitivity reduction in HBsAg testing has been reported recently in association with this substitution, and has been shown to affect a significant proportion of the 70 commercial tests studied [START_REF] Scheiblauer | Performance evaluation of 70 hepatitis B virus (HBV) surface antigen (HBsAg) assays from around the world by a geographically diverse panel with an array of HBV genotypes and HBsAg subtypes[END_REF].

Just ten samples from the present study rendered S/CO values < 25 by the E6.0 assay.

All were, however, positive despite dilution, and all displayed weak reactivity, or negative results, by tests used for comparison. As reported for other tests, these samples displayed often multiple amino acid substitutions [START_REF] Avellón | Frequency of hepatitis B virus "a" determinant variants in unselected Spanish chronic carriers[END_REF] this is a likely explanation for low reactivity by the E6.0 assay of sample 48 [START_REF] Avellón | Frequency of hepatitis B virus "a" determinant variants in unselected Spanish chronic carriers[END_REF]].

This study suggests, in addition, that amino acid changes in the pre-S1 region might also be responsible for low reactivity, since one sample displaying a wild type HBsAg

  within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One-hundred and forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared.A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut-off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centres. transcriptase activity in the replication of the hepatitis B virus

  HBsAg 5.0 (E5.0) is an enzyme-immunoassay (EIA) used widely for the diagnosis of the HBV infection for many years. A novel version of the test (Enzygnost® HBsAg 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany) (E6.0) has been developed recently by the manufacturer, and changes have been introduced in order to improve the detection of HBsAg variants. The aim of the present collaborative study was to assess the HBsAg mutant detection ability of the E6.0 assay in comparison with the former version of the test (E5.0). Clinical samples containing HBsAg variants and supernatants of cells expressing recombinant HBsAg mutants were used in the study.

  Healthcare Diagnostics. The mutants were constructed by sitedirected mutagenesis of wild-type sequences and were expressed in transfected Huh7 cells. All the samples consisted of cell culture supernatants. The HBsAg concentration was not determined. Results obtained by the Monolisa ® HBsAg Ultra (Bio-Rad,

  shows a dispersion chart comparing the S/CO values obtained by the E6.0 and E5.0 assays. Dots arranged on the chart displaying one main and two minor clusters defined by S/CO=25 in both tests (inner lines). Considering that the cut-off value was usually set close to an optical density (OD) value of 0.080 absorbance units in both assays, the S/CO range from 0 to 25.0 would reflect the full range of OD values within the linear range of the tests. Samples placed on the upper right quadrant of the chart (S/CO≥25.0 in both assays) were considered as detected correctly by both methods, and were classified into category 1. Samples placed on the lower left quadrant (S/CO<25.0 in both assays) drew category 2, which included samples displaying false negative results (S/CO<1.0) by either one or both tests. The remaining samples (category 3) placed on the upper left quadrant (S/CO≥25.0 by E6.0, <25.0 by E5.0), and were thought to reflect

  sequence and a single AA change (E54D) in the pre-S1 region was low-reactive by five different tests, including the E5.0 and E6.0 assays. The reduced number of samples available for testing hampered, however, the significance of this finding, which could be due to an unusually low HBsAg concentration in the sample.The results obtained in the present collaborative study show, in conclusion, that modifications introduced in the design of E6.0 have improved significantly the ability of the test to detect HBsAg mutants. After such modifications, the new version displays a high profile of performance in comparison with other assays, and difficulty in detecting HBsAg variants should not be expected with the routine use of the test in diagnostic laboratories or blood transfusion centres.Conflict of interestMichael Weik and Uwe Schobel are employees of Siemens Healthcare Diagnostic Products GmbH. The remaining authors state that no financial or personal relationship exits with other people or organisations that could inappropriately influence their work in regard to the issues of the present report.

Figure 1 .

 1 Figure 1. Dispersion chart of S/CO values. Comparison of Enzygnost HBsAg 6.0 vs. Enzygnost HBsAg 5.0. In black, samples displaying mutations only in the pre-S region.

Table 1 .

 1 Nature, HBV genotype, and number of mutations predicting amino acid changes in HBsAg and/or pre-S proteins with 201 samples used in the study.

				HBsAg variants		Pre-S
			1-3 AA changes	>3 AA changes a	variants only
			Native	Recombinant	Native	Recombinant	Native
	Genotype Subtype	samples	samples	samples	samples	samples Total
		adw2	16	26	5	6	1	54
		adw4q+	1	-	-	-	-	1
	A	ayw1	1	-	-	-	-	1
		ayw2	1	-	-	-	-	1
		?	1	-	-	-	-	1
	B	adw2 ayw1	1 1	8 -	--	--	--	9 1
		adr	8	4	-	-	-	12
	C	ayr	1	-	-	-	-	1
		? b	-	-	1	-	-	1
		ayw2	31	3	3	6	3	46
		ayw3	22	-	1	-	-	23
	D	ayw4	4	1	-	-	-	5
		ayw2/3 c	1	-	-	-	-	1
		? b	6	-	1	-	-	7
	D/E d	ayw? b	1	-	-	-	-	1
		ayw4	9	-	1	-	16	26
	E	ayw2/4 c	1	-	-	-	-	1
		n.a.	2	-	-	-	-	2
	F	adw4q+ adw4q-	2 2	--	1 -	--	--	3 2
	G	adw2	2	-	-	-	-	2
		Total	114	42	13	12	20	201

a : Range: 4-11 changes; b : Unpredictable subtype because of amino acid changes in residues involved in subtype specificity; c : Quasispecies; d : Recombinant strain.

Table 2 .

 2 HBsAg variants in residues involved in difficulties of diagnosis included in the study. Substitutions reported previously in association with such difficulties are highlighted in bold (see

Table 3 .

 3 Nature, S/CO values, and amino acid changes in selected samples. a) Samples testing S/CO ≥25.0 by Enzygnost ® HBsAg 6.0, and border line positive (S/CO <2.0) or negative (bold) (S/CO <1.0) in Enzygnost ® HBsAg 5.0.

			HBsAg reactivity (S/CO) by
			E6.0	E5.0	Other tests
		Sample	(dilution	(dilution	(dilution
	Type	Id	tested)	tested)	tested)	AA changes
					1854 a (1:40)
	Natural	53	55.6 (1:40)	0.83 (1:40)	347 b (1:40) 6.3 c (undil.)	T115N+P120Q+N131K+ D144A
					441 d (1:40)
		215	52.9	0.78	8.3 e	T115R+P120Q+S154L+ E164V+Q181R
		218	56.3	0.99	43.3 e	M133I+F134N+P142S+ T143L+G145R
		219	55.0	1.0	67.8 e	T118V+M133I+F134N+ P142S+T143L+G145R
						V96A+M103I+S114A+
	Recombinant (all samples tested undiluted)	222 225	52.3 53.7	1.2 0.86	1.2 e 0.34 e	T115I+T116N+S117N+ T118R+P120Q+P127T +Q129H+S136Y Q101H+S117N+T118R+ P120Q+S143L
		226	51.3	0.86	0.88 e	S117N+T118R+P120Q
		227	56.3	0.75	1.2 e	Q101H+S117N+T118R+ P120Q
		204	47.4	0.95	2.4 e	P120Q+N131K+G145R
						Y100C+P105R+T118R+
		221	41.4	0.82	0.44 e	P120L+R122K+P142L+
						K160N+L173P
			HBsAg reactivity (S/CO) by
	Type	Sample Id	(dilution E6.0	(dilution E5.0	(dilution Other tests	AA changes
			tested)	tested)	tested)
	Natural	105	17,6 (1:2)	11,2 (1:2)	11,3 a (1:10) 11,2 b (1:10) 23,4 f (1:10)	A184V+ 48T+Q82P+ P94L (preS1)
		154	4,6 (1:10)	3,4 (1:10)	n.a.	M133I+S207N
		111	1,6 (1:2)	1,2 (1:2)	4,6 a (1:10) 1,6 f (1:10) 1,2 b (1:10)	E54D (preS1)
		109	1,5	1,1	6,2 a (1:10)	A184V
			(1:2)	(1:2)	1,2 b (1:10)

b) Samples displaying S/CO <25.0 or negative (bold) results (S/CO <1.0) by Enzygnost ® HBsAg 5.0 and/or Enzygnost ® HBsAg 6.0.

/CO >= 25 BY BOTH METHODS (n =175) S/CO < 25 BY BOTH METHODS ( n= 10). Detailed in Table 3b G145R A184V Pre s1 Enzygnost HBsAg 5.0 Enzygnost HBsAg 5.0 IMPROVEMENT WITH ENZYGNOST HBsAg 6.0 ( n= 16).

	The 10 with lower values in Enzygnost	S
	HBsAg 5.0 are detailed in Table 3a.

/CO >= 25 BY BOTH METHODS (n =175) S/CO < 25 BY BOTH METHODS ( n= 10). Detailed in Table 3b G145R A184V Pre s1 IMPROVEMENT WITH ENZYGNOST HBsAg 6.0 ( n= 16).

	The 10 with lower values in Enzygnost	S
	HBsAg 5.0 are detailed in Table 3a.

/CO >= 25 BY BOTH METHODS (n =175) S/CO < 25 BY BOTH METHODS ( n= 10). Detailed in Table 3b IMPROVEMENT WITH ENZYGNOST HBsAg 6.0 ( n= 16).

	The 10 with lower values in Enzygnost HBsAg 5.0 are detailed in Table 3a.	S/CO >= 25 BY BOTH METHODS (n =175)

S/CO < 25 BY BOTH METHODS ( n= 10). Detailed in Table 3b

  low concentration of the antigen. Evaluating the real HBsAg concentration in samples containing variants that may impair binding to capture antibody is, however, a difficult task, and the hypothesis is barely testable in practice. Prior reports suggest, in addition, that difficulty in HBsAg detection in samples containing relevant HBsAg variants are in relation to a major participation of the variant in the quasiespecies viral population, and

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

. A low HBsAg concentration, either alone or in association with relevant amino acid substitutions, could also contribute to weak reactivity or negative results by diagnostic tests. The sample 170, which stems from a patient after antiviral therapy, might display a


Alejandra Fernández-Alonso

Carlos Bravo-Díaz

Solvolyses of 4-methylbenzenediazonium ions in trifluoroethanol/water mixtures. Effects of [TFE], acidity and temperature on the kinetics and mechanism of the reaction

Keywords:

Solvolyses of 4-methylbenzenediazonium ions in trifluoroethanol/water mixtures. Effects of [TFE],

acidity and temperature on the kinetics and mechanism of the reaction.

Reactions of arenediazonium, ArN 2 + , ions are both synthetically useful and mechanistically interesting. They are exploited industrially in the manufacture of pharmaceuticals and dyestuffs among others. New applications are emerging: they are currently widely used as initiators to modify carbon surfaces, to probe the interfacial compositions of colloidal systems and their role in carcinogenic and mutagenic processes is being explored. [1][2][3] Reaction mechanism of arenediazonium ions have been studied by a wide variety of techniques for over a century. New applications continuous to be found in contemporary organic synthesis [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF][START_REF] Canning | [END_REF][6][7][8][9][10][11] , however, few kinetics studies accompany by product analyses. Consequently, mechanistic understanding of the relationships between molecular structure of reactant, solvent, and reaction conditions is less than complete.

Dediazoniations have been studied in a number of solvents, [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF][START_REF] Hegarty | Kinetics and Mechanisms of Reactions Involving Diazonium and Diazo Groups[END_REF][START_REF] Saunders | Aromatic Diazo Compounds[END_REF] and two main mechanisms are reported, heterolytic (1A) and homolytic (1B), Scheme 1. Literature reports [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF] indicate that, in the absence of electron donors and depending on experimental conditions, both homolytic and heterolytic mechanisms occurs in a variety of solvents. Until recently, [3] the initiation step of the homolytic dediazoniation pathway was hypothesized to be an electron transfer from the solvent, but the absence of evidence in for or against, this hypothesis means that the nature of the initiation step remains a matter of debate. [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF] Based on recent solvolytic studies in our laboratory, [START_REF] Pazo-Llorente | [END_REF]15] we concluded that the initiation step is the formation of Z-diazo ether adducts, Ar-N=N-O-R and not an electron transfer. These kinetically controlled products undergo isomerization to the stable E isomer or homolytic bond rupture initiating a radical process, Scheme 2, leading to the formation of different reactivities. [START_REF] Espenson | Chemical Kinetics and Reaction Mechanisms[END_REF] But under our experimental conditions, the solvent ionization is negligible and a potential reaction between ArN 2 + and OH -ions is unlikely, [START_REF] Pazo-Llorente | [END_REF]15,[START_REF] Fernández-Alonso | [END_REF] therefore the formation of an O-adduct (the diazo ether) that initiates a radical process.

The results of our solvolytic studies [START_REF] Pazo-Llorente | [END_REF]15,[START_REF] Fernández-Alonso | [END_REF] permit demonstration the identification of the dual role of ROH molecules as nucleophiles that either (i) solvate the diazonium ions (allowing them to undergo thermal heterolytic decomposition, i.e. decomposing through an ionic mechanism), or (ii) react directly with arenediazonium ions to yield O-coupling adducts in a highly unstable Z configuration, i.e. Z-diazo ethers. The Z-diazo-ethers then undergo homolytic fragmentation initiating radical processes. The kinetics of methanolysis of 4-NO 2and 4-Br-benzenediazonium ions under acidic conditions provides an excellent example of the influence of solvent composition and temperature on the change from a heterolytic mechanism to a homolytic one. [15,[START_REF] Fernández-Alonso | [END_REF] For these compounds, the formation of the diazo ethers is favored on increasing [ROH] by the mass action effect but is inhibited by the medium effect on the equilibrium constant for the formation of the diazo ether, which decreases upon increasing [ROH]. [15,[START_REF] Fernández-Alonso | [END_REF] F o r P e e r R e v i e w

Here we expand our solvolytic studies with a kinetic study of the solvolysis of 4methylbenzenediazonium, 4MBD, ions in 2,2,2-trifluoroethanol, TFE, -water mixtures. The purpose is obtain evidence for or against, the formation of diazo ethers in TFE, by measuring the effects of solvent composition, acidity and temperature on the observed rate constants, k obs , and on the product distributions.

Experimental Section

INSTRUMENTATION

UV-VIS spectra and some kinetic experiments were followed on an Agilent 8453 spectrophotometer equipped with a cell carrier thermostatted by Julabo F12-ED bath and attached to a computer for data storage. Product analysis was carried out on a WATERS HPLC system which included a model W600 pump, a W717 automatic injector, a W2487 dual wavelength detector, and a computer for control and data storage. Products were analyzed on a Nova Pak (Waters) reverse phase column (150 mm length, 3.9 mm internal diameter, and 4 µm particle size) using a mobile phase of 50/50 v/v MeOH/H 2 O containing 10 -4 M HCl. The injection volume was 25 µL in all runs and the UV detector was set at 220 and 280 nm. MATERIALS 4-Methylbenzenediazonium (4MBD) tetrafluoroborate was prepared by an anhydrous method, [18] purified three times from CH 3 CN/cold ether, stored in the dark at low temperatures to minimize its decomposition, and recrystallized periodically. 4 by ignoring the small excess volume of mixed solvents. [19] All aqueous solutions were prepared by using Milli-Q grade water.

METHODS

Beer's law plots (not shown) of 4MBD aqueous solutions up to 2.00 x 10 -4 M were linear (cc.

≥ 0.999). Kinetic data were obtained spectrometrically by monitoring the disappearance of the absorbance of ArN 2 + at an appropriate wavelength. Observed rate constants, k obs , were obtained by fitting the absorbance-time data to the integrated first-order equation ( 1) using a non-linear least squares method. All runs followed first-order kinetics for at least three halflives

t k A A A A obs t - = ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - - ∞ ∞ 0 ln (1)
where A t , A o and A ∞ are the absorbances at any time, at t = 0 and when the reaction is finished (i.e., infinite time). Reactions were initiated by adding an aliquot (< 100 µL) of the ArN 2 + stock solution to the previously thermostated reaction mixture. Duplicate or triplicate experiments gave average deviations less than 10%.

Product yields were obtained by HPLC after dediazoniation was complete.

Preliminary HPLC experiments showed that up to four dediazoniation products may be samples dissolved in MeOH. ArOCH 2 CF 3 is not a commercial product, it was not synthesized, and its yield was not determined. Yields of ArF were determined either because the product was not available in our laboratory. The absence of these products yields produces a systematic error in the total yields. However, this error does not affect the kinetic results because dediazoniation products are formed competitively. This variations in the yields of the ArOH or ArH does not affect the main mechanistic conclusions, that is the reaction with trifluoroethanol goes by a heterolytic pathway unlike aliphatic alcohols, that can be ruled out from the experimental data. [15,[START_REF] Fernández-Alonso | [END_REF]20] Yields of a dediazoniation product, Y, were obtained from the product concentration, ] o , as described elsewhere. [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF]7,21]

Results

1.-Effects of percent TFE on the observed rate constant, k obs , and on the product distribution.

The effects of solvent composition on k obs , was determined by changing the percent TFE in the reaction mixture at a fixed acidity (-log[HCl] = 2). In the absence of TFE, k obs ~ 8

x 10 -4 s -1 at T = 60 ºC, consistent with literature values. [20,22] Values of k obs increase by a factor of almost 3 as % TFE increases from 0 to 98%. At 98%TFE, k obs = 16 x 10 -4 s -1 , close to a previously reported value. [23] At this high acidity (-log[HCl] = 2), the size of the observed rate enhancement is similar to that obtained in solvents such as MeOH or EtOH, [20] but the linear increase in k obs with TFE contrasts with the non-linear variation found in those solvents where exponential variation of k obs with [ROH] were found.

The effect of % TFE on the product distribution was determined by HPLC analysis of the reaction mixture at the end of the reaction, that is, at infinite time. Figure 2 shows that, in the absence of TFE, the major dediazoniation product is ArOH, in keeping with previous results. As expected, ArOH yields decrease upon increasing the percentage of TFE in the reaction mixture and only small amounts of the reduction product ArH were detected in reaction mixtures with TFE content higher than 80% (%ArH < 10%). The ArOH yields at 58% and 85% TFE are very close to those reported in the literature, which have been reported

as 65% and 34% ArOH, respectivele. [START_REF] Canning | [END_REF] 2.-Effects of acidity on k obs and on the product distribution.

Solution acidity has a major effect on the formation and decomposition of diazo ethers intermediates in previous solvolytic studies in MeOH and EtOH. [START_REF] Fernández-Alonso | [END_REF]20] To investigate possible formation of diazo ethers in TFE solutions, we studied the effect of acid strength on k obs and the product distributions at two TFE/H 2 O compositions (35% and 70% TFE). The results in Table 1 show that, at both percentages of TFE, the k obs values increased gradually with increasing the acidity (0.1 < -log[HCl] < 6). The average k obs values, k obs = (11.9 ± 0.9)×10 -4 s - enhancements of 320 fold and stoichiometric amounts of the reduced product were found.

3.-Effect of temperature on k obs for dediazoniation of 4MBD in TFE-H 2 O mixtures.

The temperature effect on k obs was studied at different acidities in two binary mixtures (35 and 70% TFE) at 50ºC and 60ºC, Figure 4. Very similar variations in k obs with acidity were obtained independently of the percentage of TFE in the reaction mixture.

The activation parameters of the reaction were estimated by using the Arrhenius equation and the theory of absolute rates, equation 2, where k B and h stand for the Boltzmann and Plank constants respectively. The results are in Table 2.

RT R T k hk B obs ‡ ‡ H S ) ln( ∆ - ∆ = (2)

DISCUSSION

All runs obeyed first order kinetics for more than three half-lives at any solvent composition.

Upon increasing [TFE], k obs increases modestly, Figure 1 and no significant changes in k obs with acidity were detected, in contrast with results of solvolytic dediazoniations in more nucleophilic solvents such as MeOH or EtOH, where S-shaped variations of k obs with acidity were found, [START_REF] Pazo-Llorente | [END_REF]15,[START_REF] Fernández-Alonso | [END_REF] indicating that formation of diazo ethers of the type Ar-N=N-O-CH 2 -CF 3

does not seem to be important. The low sensitivity of k obs to changes in %TFE is related to its properties as solvent, which combines a lower nucleophilicity, basicity but a higher ionizing power compared to alcohols such as MeOH or EtOH, which makes TFE an excellent solvent to probe nucleophilic solvent assistance and, in general, for solvolytic dediazoniation studies. [5-7, 24, 25] Further evidence supporting the hypothesis comes from the product analyses. Only heterolytic dediazoniation products are obtained in significant yields with increasing %TFE, temperature and acidity, and reduction products like ArH are in low yield. To the contrary, large amounts of the reduction product ArH were obtained upon decreasing the acidity in solvolytic reactions carried out in MeOH or EtOH, for instance, stoichiometric amounts of ArH are found in the ethanolysis of 4MBD at -log[HCl] = 5, meanwhile negligible amounts are found at -log[HCl] = 2. [15,26] Therefore, the absence of significant amounts of reduction products like ArH or biaryls, Ar-Ar indicates that dediazoniation is heterolytic mechanism, Scheme 3.

Rate-limiting nucleophilic attack of TFE on 4MBD would lead to a strong dependence of k obs on solvent composition, which is not observed, Figure 1. The results are consistent with rate determining formation of a highly reactive aryl cation that is trapped by available nucleophiles, Scheme 1. Aryl cations have not been isolated to date in aqueous systems because they are extremely short-lived intermediates (< 0.5 ns [27] ). However, Winkler et al. [28] isolated them in a solid argon matrix and other reports indicate that photolyzed arenediazonium ions generate aryl cations that can be observed as addition products with aromatic compounds. [29] F o r P e e r R e v i e w

Pre-association stepwise mechanisms, Scheme 3, in which the aryl cation has a short but finite lifetime, have been used to describe product distribution in terms of ion-molecule or ion-nucleophile pairs. [22,30] Nucleophilic attack may occur on "free" carbocations, contact ion-molecule pairs or contact ion-solvent pairs. Various research groups have proposed formation of ion-molecule pairs involving ground state arenediazonium ions. Zollinger et al. [31] suggested formation of ion molecule pairs with N 2 at high N 2 pressures. More recently, transient colored complexes between arenediazonium ions and aromatic hydrocarbons have been observed and characterized [32] and solid arenediazonium tetrachlorocuprate(II) complexes [33] have been isolated that decompose to give ArCl in high yields [34] .

Rate constants for nucleophilic attack on carbocations have been reported to be close to the diffusion control limit, ∼ 10 9 -10 13 M -1 s -1 . [29] Aryl cations have lifetimes on the order of nanoseconds and they do not have much time to diffuse away reorganizing their nearest neighbour ions and molecules, similar to the behaviour of than other extremely short lived carbocations. [35] Thus, at low concentration of other components, e.g. anions of salts, arenediazonium ions decompose primarily with solvent in the immediate vicinity of the aryl cation, that is, once the N 2 leaves, one nucleophile molecule in the first coordination shell will form its corresponding dediazoniation product. In other words, to obtain a specific dediazoniation product the precursor nucleophile must be in the solvation shell of the aryl cation prior to N 2 departing. Consistent with this interpretation, Scheme 1, the formation of the aryl cation does involves change transfer into the ring and only a small reorganization of the solvent shell is expected upon formation of the corresponding aryl cation. [36,37] Previous solvolytic dediazoniations in TFE are line with the above conclusions.

Maskill and coworkers [7] investigated the source of fluoride atoms in ArF, concluding that ArF was formed by fluorine abstraction by the presumed aryl cation, in part from the solvent and in part from the tetrafluoroborate counterion within an undissociated ion-pair. They, however, could not identify the nature of the species remaining after fluoride abstraction from TFE or whether fluoride abstraction is concerted or stepwise. [6,7] Analysis of the activation parameters, Table 2, show that both ∆H ‡ and ∆S ‡ values are very similar to those found in water and in short chain alcohols such as MeOH or EtOH. [20] Solvolytic unimolecular reactions can show positive or negative ∆S ‡ values. [START_REF] Pross | Theoretical & Physical Principles of Organic Reactivity[END_REF][START_REF] Maskill | [END_REF] The positive ∆S ‡ values in Table 2 suggest that the transition state has a greater structural freedom than reactants, in line with the reported positive volumes of activation, ∆V ‡ , for a number on dediazoniations in different solvents. [40][41][42][43] On view of Scheme 1, the parent arenediazonium ion and the aryl cation polarize the surrounding solvent molecules to a similar extent, thus the gain of entropy due to the increase in molecular vibrations is not compensated by the loss of entropy due to the restricted motion of the solvating molecules. On the other hand, ∆H ‡ values are relatively high as in many unimolecular reactions, [START_REF] Lowry | Mechanism and Theory in Organic Chemistry[END_REF][START_REF] Maskill | The Physical Basis of Organic Chemistry[END_REF] unlike ∆H ‡ values usually found for bimolecular reactions. In typical S N 2 reactions, [START_REF] Maskill | The Physical Basis of Organic Chemistry[END_REF] activation enthalpies are substantially lower because the bond breaking of old bonds, which requires energy, and bond making, which releases energy, are highly concerted, usually synchronous and of similar energies.

Thus the relatively high ∆H ‡ values for 4MBD in TFE/H 2 O solutions suggest a transition state that has undergone substantial bond breaking with little compensating bond making. Positive ∆S ‡ values, as those found in this work, compensate the large enthalpy term making dediazoniations and ∆G ‡ is relatively small. Thus dediazoniation are often faster than other unimolecular reactions, and consistent with results for a number of dediazoniations, that show a much lower solvent dependence than typical unimolecular reactions. [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF]23,43,[START_REF] Ishida | [END_REF] Finally, it may be worth noting the change in the behaviour of 4MBD ions compared with that of arenediazonium ions bearing electro-withdrawing substituents such as 4-NO 2 or 4-Br-benzenediazonium ions. [15,[START_REF] Fernández-Alonso | [END_REF] Substituent effects on the aromatic ring have a significant effect on the stability and reactivity of arene diazonium ions, but their effects can not be accounted for in terms of the Hammet equation but a dual substituents parameter equation separating the resonance and inductive effects is needed. [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF]47,[START_REF] Zollinger | Opposing Field and Resonance Reaction Parameters in Reactions of Substituted Benzene Derivatives[END_REF] It is well known that 4-alkyl substituents in the aromatic ring make ArN 2 + ions less prone to decomposition through homolytic pathways than those bearing electronwithdrawing substituents [START_REF] Zollinger | Diazo Chemistry I , Aromatic and Heteroaromatic Compounds[END_REF] and this is reflected in the lower values of the equilibrium constant K for the formation of diazo ether of 4MBD compared with those for 4-NO 2 and 4-Br-benzenediazonium ions. [15,[START_REF] Fernández-Alonso | [END_REF] In conclusion, the kinetic and product yield results are not consistent with the formation of diazo ethers with leads to reduction products in TFE. This is in keeping with

  products. The evidence for this mechanism is based on the observed sigmoidal variation in both k obs and product yields with acidity for a number of arenediazonium ions.Product distribution analyses of the reaction mixtures indicate that at relatively high acidities ( -log([HCl] = 2) only heterolytic products are formed, but upon decreasing the acidity, the reduction products become predominant at the expense of the heterolytic ones, suggesting a change in the reaction mechanism. In addition, sigmoideal variations of k obs with acidity are usually observed in reactions of acid-base pairs where both forms are attainable and show

  cresol, ArOH, 4-fluorotoluene, ArF, aryl trifluorotheyl ether, ArOCH 2 CF 3 , and toluene, ArH. Linear (cc. > 0.999) calibration curves for converting chromatographic peak areas, A, into concentrations were obtained for ArOH, and ArH by employing commercial

[

  Analyte] ∞ , and the initial arenediazonium salt concentration, [ArN 2 + ] o , estimated by weight, i.e. Y = 100[Analyte] ∞ / [ArN 2 +

1 (

 1 35% TFE) and k obs = (13.8 ± 0.5)×10 -4 s -1 (70% TFE) are the same as the k obs values at the same %TFE, Figure1.

Figure 3

 3 Figure 3 shows the variation of the product distribution with [HCl]. As shown, the

F o r P e e r R e v i e w 20 Solvent

 20

Figure 1 .Figure 2 .

 12 Figure 1. Influence of %TFE on kobs for dediazoniation decomposition of 4MBD. [4MBD] = 1.2 × 10 -4 M, T = 50ºC and -log[HCl] = 2.

Figure 3 .Figure 4 .

 34 Figure 3. Influence of [HCl] on the product distribution for the decomposition of 4MBD in A) 35% TFE-H 2 O, B) 70% TFE-H 2 O. ■ 4MBOH, • 4MBH, [4MBD] = 1.7 × 10 -4 M, T = 50ºC.

Table 1 .

 1 Influence of [HCl] on k obs for the decomposition of 4MBD in two selected TFE/H 2 O reaction mixtures. [4MBD] = 1.2 × 10 -4 M, T = 50ºC.

	previous solvolytic studies showing that heterolytic dediazoniations tend to occurs in solvents
	of low nucleophilicity such as H 2 O or fluorinated alcohols such as TFE, but that both ionic
	and homolytic dediazoniations occur in non-fluorinated alcohols of higher nucleophilicity
	such as MeOH and EtOH or DMSO. Our results are completely consistent with the
	heterolytic D N + A N mechanism shown in Scheme 3, that is rate-limiting formation of an
	extremely reactive aryl cation that is trapped by any nucleophile available in its immediate
	vicinity.
	AKNOWLEDGEMENT
	We would like to thank Prof. H. Maskill for helpful discussions. Financial support from the
	following institutions is acknowledged: Xunta de Galicia (PGDIT06PXIB314249PR),
	Ministerio de Educación y Ciencia (CTQ2006-13969-BQU), Fondo Europeo para el
	Desarrollo Regional (FEDER) and Universidad de Vigo.

Scheme 1. Simplified dediazoniation mechanisms. (A) Heterolytic; (B) homolytic. Scheme 2. Proposed competitive heterolytic (HET) and radical mechanisms (HOM) for the reaction of arenediazonium ions with alcohols under acidic conditions.

Table 2 .

 2 Estimated activation parameters for the solvolyses of 4MBD in 35% and 70% TFE-H 2 O reaction mixtures. For comparison, literature values in other solvents are included.

	T∆S ‡	∆G ‡
	kJ mol -1	kJ mol -1

http://mc.manuscriptcentral.com/poc Journal of Physical Organic Chemistry


Howard ; Maskill

Juan Crugeiras

William Mcfarlane

Ana Rios

Miguel A Rios

Iain D Menneer

William Macfarlane

interconversion of conformational isomers of N nitroso dimethylhydroxylamine

Keywords: nitrosation, NMR kinetics dialkylhydroxylamines, nitrosation, nitrosohydroxylamines, NMR kinetics, conformational isomers, computational chemistry

  L'archive ouverte pluridisciplinaire

Introduction

There are many known nitrosating agents, e.g. nitrosyl compounds (halides, thiocyanate, and acetate), nitrogen oxides (nitric oxide, dinitrogen trioxide, and dinitrogen tetroxide), the nitrosonium ion (NO + ), and nitroso sulphur compounds, but by far the most common reagent is the undissociated weak acid, nitrous acid (HNO 2 ). Nitrosation of organic compounds has been studied since the earliest days of systematic organic chemistry, [1,2,3] the first report being by Behrend and König who prepared N-nitroso-dibenzylhydroxylamine by treating N,Odibenzylhydroxylamine (1 in Fig. 1) with nitrous acid. [1] Catalysis of nitrosation by nucleophiles has also been investigated. [4] Nitrous acid (pK a = 3.3) is readily formed in aqueous solution by adding a nitrite salt (typically sodium nitrite) to an aqueous solution of a strong acid. [5] Under these conditions, it exchanges oxygen with the water, a reaction which can be exploited when oxygen isotope labelling is desired in the nitroso group. [6] Under acidic conditions, nitrous acid has been reported to be in equilibrium with its protonated form, (H 2 NO 2 ) + , [4] its anhydride (N 2 O 3 ), [7,8] and the nitrosonium ion (NO + ), [9] all of which are potential nitrosating agents; in addition, it undergoes decomposition to nitric oxide and nitric acid. There have been many wide-ranging discussions of nitrosation of amino compounds (including hydroxylamine) notably by Ridd and Stedman and their colleagues, [9][10][11][12] and the field has been comprehensively reviewed by Williams. [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] Particularly relevant to our current investigation is the effect of acidity upon the rate of nitrosation of O-alkylhydroxylamines by nitrous acid reported by Stedman. [10,11] We reported recently an investigation of the mechanism of the acid-catalysed decomposition of a series of N-nitroso-dialkylhydroxylamines. [START_REF] Maskill | [END_REF]15] We now report the kinetics and mechanism of nitrosation of N,O-dialkylhydroxylamines (1-3) in acidic 1:1 (v/v) aqueous methanol and of N,O-dimethylhydroxylamine (4) in aqueous acid without the methanol cosolvent. We also report an investigation of the conformational properties of N-nitrosodimethylhydroxylamine (5) by NMR spectroscopy and theoretical methods; dynamic, thermodynamic, and structural parameters have been determined.

Methods and Results

Kinetics of nitrosation of N,O-dibenzylhydroxylamine (1), tetrahydro-1,2-oxazine (2), and Nbenzyl,O-methylhydroxylamine (3). Of the range of N-nitroso-dialkylhydroxylamines we had prepared for solvolytic studies, [15] the formations of three by nitrosation of the parent dialkylhydroxylamines (N,O-dibenzylhydroxylamine (1), tetrahydro-1,2-oxazine (2), and Nbenzyl,O-methylhydroxylamine (3)) were selected as representative for mechanistic investigation and comparison with literature results. All were available as the substituted hydroxylammonium chlorides which, given their pK a values (typically ≈5), [5,11] remain very largely in their protonated forms in aqueous acidic solution. In each case, therefore, the total initial concentration of all hydroxylamine species was taken to be equal to the initial concentration of the hydroxylammonium chloride and represented henceforth as

[RNH 2 OR' + ] 0 . And since [RNH 2 OR' + ] 0 >> [NaNO 2 ] 0 , it will (like [H 3 O + ] 0 ) remain
approximately constant throughout the reaction. However, a high level of methanol in water (1:1, v/v) was needed to achieve the concentrations of hydroxylammonium chlorides that were required to ensure they were in large excess over the initial concentrations of nitrous acid. Similarly, as [H 3 O + ] 0 >> [NaNO 2 ] 0 and pK a = 3.3 for HNO 2 , [5] the initial concentration of nitrous acid, [HNO 2 ] 0 , was taken to be equal to the initial concentration of the sodium nitrite, [NaNO 2 ] 0 , (and it decreased to zero as the reaction went to completion).

The UV range 300-400 nm was monitored and the crown-shaped absorbance attributed to nitrous acid (the limiting reactant) was seen to decrease by a clean first-order rate law (even though the overall changes in absorbances were small) as broad absorbances due to the nitroso-products increased. As seen in Table 1, the range in reactivity for compounds 1-3 is modest, as anticipated from the earlier work by Stedman. [11] Compounds 1 and 2 were then shown to be first order in hydroxylammonium by using different initial excess concentrations of RNH 2 OR' + Cl -in the usual way (results not shown), [START_REF] Maskill | The physical basis of organic chemistry[END_REF] and 3 was studied at different initial excess perchloric acid concentrations (Table 2). Results in Tables 1 and2 indicate that precision and reproducibility are satisfactory. Although nitrosation is first order in both hydroxylammonium and nitrous acid, Table 2 and Fig. 2 indicate that increasing initial hydronium ion concentrations lead to lower pseudo-first-order rate constants for 3, so a complex rate law is implicated. 2 and Fig. 2)

were not as anticipated from results reported earlier by Stedman who detected minimal dependence of k obs upon [H 3 O + ] 0 for a series of O-alkylhydroxylamines in water. [11] Consequently, we reinvestigated the nitrosation of the simpler N,O-dialkylhydroxylamine (4) in aqueous acid without methanol as cosolvent. The reaction was monitored by following the increase in absorbance due to the formation of the N-nitroso product (5), and results for different starting concentrations of sodium nitrite in the range (0.05-5.0)x10 -3 mol dm -3 are shown in Table 3. As seen, the value of k obs increases slightly with increasing [NaNO 2 ] 0 ; however, the absorbance-time data deviated from first-order behaviour at higher values of [NaNO 2 ] 0 , especially at the higher acidity. This effect had been noted earlier by Stedman for nitrosation of the parent hydroxylamine and attributed to competing nitrosation by N 2 O 3 . [12] Consequently, our further experiments were all carried out at [NaNO 2 ] 0 = 5x10 -5 mol dm -3 .

Figure 3 shows the effect of [4H + ] 0 (the initial concentration of the hydroxylammonium ion) upon k obs at [H 3 O + ] 0 = 0.05 mol dm -3 ; the results appear to be in accord with a rate law comprising first and second order terms in [4H + ] 0 , i.e. rate = k obs [HNO 2 ] with k obs given by eqn 1, and a plot of k obs / [4H + ] 0 versus [4H + ] 0 was indeed linear with a = (2.75 ± 0.06)x10 -2 dm 3 mol -1 s -1 and b = (2.80 ± 0.02) dm 6 mol -2 s -1 (Fig. 4).

k obs = a [4H + ] 0 + b [4H + ] 0 2 (1)
However, since the initial concentration of the hydroxylammonium is equal to that of

chloride, b [4H + ] 0 2 = b [4H + ] 0 [Cl -]
, so the second term in eqn 1 could be due to nucleophile catalysis by Cl -as previously reported by Stedman for halides and SCN -in the nitrosation of several hydroxylamines by nitrous acid. [11] To test this, Cl -was replaced by perchlorate using Amberlyte IRA-410 anionic resin. Although 100% exchange was not achieved, the value of b in eqn 1 was much reduced whereas a was unchanged. In addition, Fig. 5 shows the effect of increasing concentrations of NaCl upon k obs for nitrosation of 4 at [H 3 O + ] 0 = 0.05 mol dm -3 and [4H + ] 0 = 0.01 mol dm -3 . The intercept divided by [4H + ] 0 gave the result a = 4.5 (± 2.1)x10 -2 dm 3 mol -1 s -1 which, although of low precision, compares favourably with the result given above, (2.75± 0.02)x10 -2 dm 3 mol -1 s -1 . The slope divided by [4H + ] 0 gave the result b = 2.56 (± 0.07) dm 6 mol -2 s -1 in fair agreement with the value given above, 2.80 (± 0.02) dm 6 mol -2 s -1 .

Finally in this context, Fig. 6 shows the effect of [H 3 O + ] 0 upon k obs for nitrosation of 4 at [4H + Cl -] 0 = 0.05 mol dm -3 in water; clearly, the effect is only small up to 0.9 mol dm -3 in agreement with previous observations by Stedman for related compounds, [11] and quite different from what we had found for 3 in aqueous methanol.

Determination of thermodynamic and kinetic parameters for the conformational isomerisation of N-nitroso-dimethylhydroxylamine (5) by NMR spectroscopy and related computational methods. N-Nitrosamines and N-nitroso-hydroxylamines have been investigated in the past by 1 H and 15 N NMR spectroscopy, [START_REF] Karabatsos | [END_REF] and conformational isomerism of several N-nitroso-dialkylhydroxylamines was reported by Axenrod et al. [18] We have carried out new 1 H measurements on 5 at 500 MHz and -50 o C, and isotropic NMR parameters were calculated using B3LYP/6-311++G(d,p) in Gaussian03 with the Gauge-Invariant Atomic Orbital (GIAO) scheme and geometries optimised at the B3LYP/6-311++G(d,p) level. [START_REF] Foresman | Exploring Chemistry with Electronic Structure Methods[END_REF] Identical calculations were also carried out on tetramethylsilane and the results used as references to calculate proton and carbon chemical shifts. As seen in Table 4, the computed and new experimental 1 H chemical shifts for cis-and trans-5 are in good agreement with each other and with earlier experimental values. We have also measured [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] C NMR spectra of 5 at 125.77 MHz and 23 o C in CDCl 3 (assignments were confirmed by twodimensional HSQC experiments) for comparison with our computed results in the gas (Table 4).

In order to determine the thermodynamic and kinetic parameters for the conformational isomerisation of compound 5, Scheme 1, 1 H NMR spectra were recorded at 500. 16 As the temperature is raised, the signals broaden as they begin to coalesce due to interconversion of the conformational isomers becoming faster compared with the time-scale of the NMR process; spectra in all solvents at the highest temperatures comprise only two broad signals (one for -O-CH 3 and one for >N-CH 3 ). At each temperature, a first-order exchange rate constant, k ex , was determined by line-shape analysis and simulation of the spectrum. The simulations of the 1 H NMR spectra were performed using Program gNMR, [START_REF] Program Gnmr By | [END_REF] MHz showed similar line broadening and coalescence behaviour fully consistent with the results from the 1 H spectra, although the signal-to-noise ratios attainable in the times available were insufficient to achieve comparable precision in spectral simulations.

Each exchange rate constant corresponds to a rate of conformational equilibration and is the sum of the rate constants for forward and reverse processes (k ex = k f + k r ) shown in Scheme 1. [START_REF] Maskill | The physical basis of organic chemistry[END_REF] From these and the ratios of the rate constants from the equilibrium constants, k f and k r can be separately determined at each temperature. The enthalpies and entropies of activation for k f and k r processes in the various solvents were then obtained from plots using the Eyring equation: ln(k/T) = 23.73 -(∆H ‡ /8.31T) + (∆S ‡ /8.31); in all cases, the least squares fits to straight lines had R 2 > 0.994. and results for the k f process are included in Table 5.

Results for ∆H o (= ∆H f ‡ -∆H r ‡ ) and ∆S o (= ∆S f ‡ -∆S r ‡
) were the same as results obtained by the more usual van't Hoff method from values of K at different temperatures shown (with experimental values for ∆G o 298 ) in Table 6.

Computational structural investigation on N-nitroso-dimethylhydroxylamine (5). Structures were optimized using the B3LYP method with a 6-31G(d) basis set using the Gaussian03

program. [START_REF] Foresman | Exploring Chemistry with Electronic Structure Methods[END_REF] For geometry optimizations in chloroform, the Polarized Continuum Model (PCM) approximation was used (TABS = 300.0, ALPHA = 1.21, TSNUM = 70, RADII = PAULING). Vibrational frequency calculations were performed on all stationary points at the B3LYP level to characterise them as minima (all real frequencies) or as transition structures (one imaginary frequency). Stable cis and trans conformational isomers, and two transition structures (TS1 and TS2) for their interconversion, were identified (Fig. 8). TS2 was found to be less stable than TS1, which was confirmed by results of higher level calculations (QCISD/6-311+G(d,p) and B3LYP/6-311++G(d,p)), and is not considered further. Key 7.

Zero-point vibrational energies, entropies, and thermal corrections to enthalpy and Gibbs free-energy values were obtained from the unscaled harmonic frequencies and moments of inertia by standard methods. [START_REF] Mcquarrie | Statistical Mechanics[END_REF] These led to the computed thermodynamic parameters for the conformational isomerisation cis-5 ⇌ trans-5 included in Table 6 (along with NMR experimental values), and computed activation parameters are compared with NMR experimental results in Table 5.

Discussion

Mechanism of nitrosation of N,O-dibenzylhydroxylamine (1), tetrahydro-1,2-oxazine (2), and N-benzyl,O-methylhydroxylamine (3) in aqueous methanol. In aqueous acidic solution, nitrite anions will be essentially fully protonated (pK a = 3.3 for HNO 2 ) [5] and it has been previously assumed that the HNO 2 so formed will be further protonated on the hydroxyl but to only a small degree, eqn 2. [4] On this assumption, a kinetic scheme involving ONOH 2 + as a reactive intermediate nitrosating agent may be derived (see later). However, the pK a of ONOH 2 + has recently been reported to be 1.7, [START_REF] Riordan | [END_REF] in which case its concentration would be appreciable in aqueous acidic solutions of nitrites (even though it had eluded previous attempts at detection), and the steady state approximation may not be applied to it. This pK a value requires that replacement of a hydrogen in H 2 O by a nitroso group is both strongly acid-enhancing (pK a = 15.7 for H 2 O and 3.3 for HONO) and appreciably base-enhancing (pK a = -1.74 for H 3 O + and +1.7 for ONOH 2 + ). In contrast, our recent computational studies indicate that ONOH 2 + is not only a stronger acid than H 3 O + , but that it is also very unstable with respect to dissociation into NO + and water, eqn 3. [23] Consequently, we combine eqns 2 and 3 into eqn 4 and, following earlier workers, [9] propose NO + as the nitrosating agent additional to nitrous acid in dilute aqueous solutions of nitrite salts (but see later).

HONO + + H 2 O (2) H 2 ONO + H 3 O + (3) + H 2 O H 2 ONO + NO + HONO + ; K NO (4) + 2 H 2 O H 3 O + NO +
Hydroxylammonium ions (pK a ≈ 5) are the conjugate acids of moderately strong bases, [5,11] so will be deprotonated in aqueous acids to only a very small degree; however, the free base will be the most reactive nucleophile present even though its concentration is very low. We

F
k obs = . . K a1 [RNHOR'] 0 ( k 1 + k 2 . K NO . [H 3 O + ] ) . (K a1 [H 3 O ]) + + (1 K NO . [H 3 O + ]) + (5) (6) = k obs [RNHOR'] 0 K a1 . k 1 [H 3 O ] + + k 2 . K NO
Equation 6 predicts a linear relationship between k obs and the reciprocal of [H 3 O + ] with a finite intercept. The plot is shown in Fig. 9, which indicates that the experimental results obtained for 3 are indeed compatible with the mechanism in Scheme 2. Moreover, the two second order rate constants k 1 and k 2 may be determined from the gradient and intercept of the plot, respectively, with values for K a1 and K NO .

Using [RNHOR'] 0 = 0.075 mol dm -3 , K a1 = 3.2 x 10 -5 mol dm -3 , [START_REF] Mcquarrie | Statistical Mechanics[END_REF] and K NO = 3.5 x 10 -7 dm 3 mol -1 , [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] we obtain k 1 = 1.4 x 10 2 dm 3 mol -1 s -1 and k 2 = 5.9 x 10 9 dm 3 mol -1 s -1 . We conclude that reaction between dialkylhydroxylamines and nitrosonium ion is diffusion controlled, and the lower overall reactivity of 3 at higher values of [H 3 O + ] is because the increasing concentrations of NO + do not compensate for the decreasing concentrations of the free hydroxylamine.

Mechanism of nitrosation of N,O-dimethylhydroxylamine (4) in aqueous acid. We saw above in the nitrosation of 3H + Cl -in aqueous methanol that k obs decreased with increasing [H 3 O + ] (Fig. 2) and that this is due to the k 1 term in eqn 6 corresponding to reaction of the decreasing concentrations of the free base with HNO 2 (the k 1 route in Scheme 2). This was not observed in the reaction of 4H + Cl -in water; in fact k obs increased slightly with increasing [H 3 O + ] (Fig. 6), the anticipated result on the basis of earlier findings by Stedman for related compounds.

k obs = [RNHOR'] 0 . K a1 . (k 2 . K NO + k 3 . K ClNO .[Cl ]) (8)
_ (7) k obs = (K a1 + [H 3 O + ]) _ [RNHOR'] 0 . K a1 . [H 3 O + ] . (k 2 . K NO + k 3 . K ClNO .[Cl ])
This complex rate law may be approximated to eqn 8 since K a1 << [H 3 O + ] (see below).

Equation 8 predicts a linear plot of k obs versus total chloride concentration, [Cl -] T , as is observed in Fig. 5. From the gradient of the plot (2.56x10 -2 dm 3 mol -1 s -1 ), [4H + ] 0 = 0.01 mol dm -3 , and literature values for K a1 (1.78 x 10 -5 mol dm -3 ), [24] K NO (3.5 x 10 -7 dm 3 mol -1 ), [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] and K ClNO (1.1 x 10 -3 dm 6 mol -2 ), [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] we calculate k 3 = 1.3 x 10 8 dm 3 mol -1 s -1 at 25 o C which compares well with the result of 1.5 x 10 7 dm 3 mol -1 s -1 at 0 o C reported by Stedman. [11] From the intercept in Fig. 5 (5 x 10 -4 s -1 ) and the values for K NO and K a1 given above, [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF]24] we obtain k 2 = 8 x 10 9 dm 3 mol -1 s -1 , i.e. diffusion control, in excellent agreement with the result obtained for 3 (see above). The mechanism of Scheme 3, therefore, accounts for the experimental results for nitrosation of compound 4 under the experimental conditions we used.

Comparison of the nitrosation of compounds 1-3 and 4. Nitrosation of compounds 1-3 was carried out in 1:1 (v/v) aqueous methanol; compound 4 was nitrosated in aqueous solution without the methanol; all substrates were used as their hydrochloride salts (RNH 2 OR' + Cl -).

For 3 in aqueous methanol, we have shown that a mechanism involving HNO 2 and NO + as the effective nitrosating agents satisfactorily accommodates the experimental findings, and HNO 2 is more than 10 7 times less reactive than NO + which reacts under diffusion control.

Significantly, there was no evidence of catalysis by the counter-ion of the substrate, Cl -. In contrast, a second-order term in [RNH 2 OR' + Cl -] implicates Cl -as a nucleophile catalyst for nitrosation of 4 in water. Under these conditions, both ClNO (formed in the catalytic route) and NO + react with 4 close to or at the diffusion limit, and the much less reactive HNO 2 cannot compete as a nitrosating agent. Apart from the relatively minor differences in structure between 3 and 4, the only obvious difference between the two reactions is the presence of methanol as cosolvent for 3. Consequently, we have to conclude that the Base strength of nitrous acid. In Scheme 2 and the associated text, we invoked NO + as the reactive nitrosating agent (in addition to HNO 2 ) rather than its precursor, ONOH 2 + , eqns 2-4.

This was based upon a computational investigation of the protonation of HONO. [23] In the event that ONOH 2 + is trapped by RNHOR' faster than it fragments to NO + and H 2 O, eqn 9, and ONOH 2 + rather than NO + is the reactive nitrosating agent, a mechanism analogous to that in Scheme 2 can be readily devised. With the usual approximations, the experimental rate constant is then given by eqn 10, rather than eqn 6, where K a2 is the equilibrium constant for the reverse of eqn 2 above, i.e. the acid dissociation constant of protonated nitrous acid. If we assume that ONOH 2 + is comparable with ClNO as a nitrosating agent, [START_REF] We Thank Drs | McClelland for helpful discussions on this issue[END_REF] i.e. k 2 ' ~ 10 8 dm 3 mol -1 s -1 , we calculate pK a2 ~ -5. Whilst this is an approximate result, we suggest it is more credible than the value of 1.7 recently reported. [START_REF] Riordan | [END_REF] k obs = . The agreement between the experimental and simulated NMR spectra shown in Fig. 7 is satisfactory indicating that the derived rate constants are reliable. Computed and experimental activation parameters for the cis-5 → trans-5 conversion are compared in Table 5. As with the enthalpies and entropies of reaction, agreement amongst the experimental results in the several solvents, and with the computed values, is within the probable experimental error of the NMR results. Again, we see no computational prediction of a solvent effect upon the conformational interconversion and none is found. This is perhaps surprising in view of the computed greater dipolarity of the ground state forms compared with the transition structure for their interconversion (see above and Table 7).

K a1 [RNHOR'] 0 k 1 + k 2 ' [H 3 O + ] K a2 (10)
Structure of N-nitroso-dimethylhydroxylamine (5). The interconversion of cis and trans conformational isomers of compound 5 is shown in Scheme 4 along with their alternative resonance structures. We note that the degree of planarity at the central N and the dihedral angles in cis and trans forms will reflect the contribution of the alternative resonance canonicals, i.e. the degree of delocalisation. We also note that internal rotation about the central N-N bond which interconverts cis and trans forms of 5 reduces the degree of delocalisation, which will be close to zero in the transition structures when the dihedral angle is about 90 o .

The N-N bonds in the two stable conformers are very similar in length (Table 7), and shorter than a normal N-N bond, [27] i.e. as expected for partial double bond character indicated in Scheme 4. In contrast, the N-N bond in TS1 is appreciably longer and corresponds to no discernable resonance interaction in the transition structure. Correspondingly, the nitroso N-O bond in TS1 is normal for a double bond between O and N whereas the very similar N-O bonds in cis-5 and trans-5 are longer than normal indicating partial single bond character. [27] In accordance with these findings, the sums of angles at N1 shown in Table 7 indicate (i) a high degree of pyramidality in TS1 but only little in cis-5 and trans-5, (ii) dihedral angles in cis-5 and trans-5 only about 15 o out of planarity, and (iii) appreciably greater dipolarity in cis-5 and trans-5 than in TS1.

Experimental

Representative kinetics measurement of the nitrosation of N-benzyl-O-methylhydroxylamine

(3). Aqueous perchloric acid (5.0 cm 3 , 0.4 mol dm -3 ) was mixed with methanolic N-benzyl- O-methylhydroxylammonium chloride (5.0 cm 3 , 0.30 mol dm -3 ) to give solution A (10 cm 3 ) which is 0.2 mol dm -3 in perchloric acid and 0.15 mol dm -3 in N-benzyl-Omethylhydroxylammonium chloride in 1:1 aqueous methanol.

Sodium nitrite (13.8 mg) was dissolved in 1:1 methanol:water (25 cm 3 ) to give solution B which is 8.0 x 10 -3 mol dm -3 in NaNO 2 . Typically, 1 cm 3 of each of the solutions A and B was injected into a 1 cm pathlength quartz UV cell whereupon the data acquisition was started at a wavelength in the range 320-330 nm (corresponding to the wavelength of greatest change in absorbance as the reaction proceeds) in the usual manner. [START_REF] Maskill | The physical basis of organic chemistry[END_REF] (The initial concentrations in the 1:1 methanol:water in the cell were [HClO 4 ] = 0.1 mol dm -3 ; [hydroxylammonium salt] 0 = 0.075 mol dm -3 , and [NaNO 2 ] 0 = 4.0 x 10 -3 mol dm -3 .)

The absorbance-time data were fitted to the exponential form of the first-order rate law equation to determine the pseudo first order rate constant.

NMR spectroscopic measurements of the conformational equilibration of N-Nitroso-N,Odimethylhydroxylamine (5). Pulsed FT 1 H (500.16 MHz) NMR spectra were obtained from samples containing ca. 5 % of 5 in 5 mm o.d. tubes on a JEOL Lambda spectrometer using 70º flip angles and a digital resolution in the transformed spectra of 0.15 Hz per point.

Temperature calibration was done using the 1 H spectra of samples of neat MeOH and 1,2ethanediol. The number of transients recorded depended upon the amount of line-broadening at the different temperatures and was adjusted to give a satisfactory signal-to-noise ratio in each case. Any exponential window applied to the FID prior to transformation was adjusted to ensure the resulting additional line-broadening was insignificant in each case. [START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] b In each case, the wavelength at which the decreasing absorbance change was greatest was selected.

K NO = [NO + ] [H 3 O + ] [HNO 2 ] , mechanistic rate = . ( k 1 + k 2 . K NO . [H 3 O + ]) . K a1 [RNHOR'] 0 [HNO 2 ] T . . ( 1 + K NO . [H 3 O + ]) (K a1 + [H 3 O + ]) F o r P e e r
Table 2: Effect of initial hydronium ion concentration on kinetics of nitrosation of N-benzyl,O-methylhydroxylamine (3) under pseudo-first-order conditions, 1:1 (v/v) aqueous methanol, 25.0 o C. [a] [HClO 4 ] 0 /mol dm -3 10 3 k obs /s -1 10 5 Standard Error 0.10 [b,c] 8.25 7.52 0.25 [b] 6.42 5.06 0.50 [b] 5.59 20.9

1.00 [d] 5.28 4.92 a For all cells, [RNH 2 OR' + ] 0 = 0.075 mol dm -3 ;

b [NaNO 2 ] 0 = 4.0x10 -3 mol dm -3 ; c repeat of experiment 3 in Table 1 using different solutions and spectrophotometer, etc.; ). [a] [H 3 O + ] 0 = 0.10 mol dm -3 [H 3 O + ] 0 = 0.010 mol dm -3 10 3 [NaNO 2 ] 0 /mol dm -3 10 3 k obs /s -1 10 3 [NaNO 2 ] 0 /mol dm -3 10 3 k obs /s -1 0.05 [b] 8.58 0.05 [b] 8.35 0.5 [c] 9.35 0.5 [c] 12.7 5.0 [d] 14.0 5.0 [d] 24.0 a [MeNH 2 OMe + Cl -] 0 = 5x10 -2 mol dm - a The cis and trans short-hand descriptors here indicate the relationship between the oxygens bonded to the respective N atoms in the approximately planar forms (see Schemes 1 and 4 and text); earlier authors have used cis and trans to describe the relationships between the >N-CH 3 and the O of the nitroso. [18] b -30 o C, 60 MHz; [18] c present work, -50 °C, 500 MHz;

4

 4 Kinetics of nitrosation of N,O-dimethylhydroxylamine (4) in aqueous acid. Our results for the effect of acidity upon the rate of nitrosation of 3 in aqueous methanol (Table

  MHz at different temperatures in deuteriochloroform (263-328 K), methanol-d 4 (263-328 K), toluened 8 (238-373 K), and dimethyl sulfoxide-d 6 (295-336 K; this relatively high lower temperature is enforced by the melting point of DMSO). At the lowest temperatures in each solvent, we see two sharp singlets (>NCH 3 and -OCH 3 ) for each of the two conformational isomers of CH 3 N(NO)OCH 3 (see Fig. 7 for the spectrum in CDCl 3 at 253 K). Integration of the signals gives the relative proportions of the conformational isomers and hence the equilibrium constant at the measured temperature, which is the ratio of forward and reverse rate constants for the conformational equilibration (K = k f /k r ).

  for the variation of the proportions of cis-and trans-5 with temperature, and also for the temperature dependence of the various chemical shifts which was particularly marked in the case of solutions in toluene. These latter effects can be attributed to reversible association of the solvent and solute and the large anisotropic diamagnetic susceptibility of toluene. In CDCl 3 and methanol, the chemical shifts of the >N-Me and -O-Me resonances of the more abundant cis-5 were quite close, and their assignments were confirmed by two-dimensional EXSY experiments at 5 °C which related them to the corresponding widely separated resonances in the less abundant trans isomer. Representative experimental and simulated spectra in deuteriochloroform at specified temperatures, along with calculated exchange rate constants, are shown in Fig. 7. The 13 C NMR spectra at 125

  B3LYP/6-31G(d)) and dipole moments for cis-5, trans-5, and TS1 in the gas phase are given in Table

  rule out any appreciable reaction between the free base 4 and HNO 2 under these reaction conditions. However, like Stedman, we observed catalysis by chloride so propose the mechanism in Scheme 3 for 4 with two principal reaction channels. If the mechanism of Scheme 3 is correct, k obs of the experimental rate law (rate = k obs [HNO 2 ]) is related to the parameters in Scheme 3 by eqn 7.

  affects the relative magnitudes of the various equilibrium and rate constants sufficiently to determine the subtle balance between the competing nitrosation routes for 1-3 on the one hand and 4 on the other. There are insufficient data, however, either in the literature or from our own work, to allow extrapolation and accurate prediction of likely results in aqueous methanol solutions of other compositions.

2 +

 2 Conformational isomerisation of N-nitroso-dimethylhydroxylamine (5) by temperaturedependent1 H NMR spectroscopy and computational methods. Computed and experimental thermodynamic parameters for cis-5 ⇌ trans-5 are given in Table6. Agreement amongst the experimental results in the several solvents and the computed results is within the probable experimental error of the NMR results. The computed results indicate no major effect of solvation by chloroform upon the equilibrium and none is found experimentally in non-polar, H-bonding, and dipolar aprotic solvents employed. Equilibrium favours the cis isomer but there is still about 20% of the less stable trans form present at 25 o C. Resolution of the free energy of reaction into enthalpy and entropy terms indicates no huge compensating effects or differences amongst the solvents: the entropy change for the cis to trans conversion is positive so compensates to only a small degree for the small positive enthalpy change associated with the conversion.

15 Figure 1 :Figure 2 : 16 Figure 3 :

 1512163 Figure 1: Hydroxylamines 1-4 and N-nitroso,dimethylhydroxylamine, 5

Figure 4 :

 4 Figure 4: Plot of k obs /[4H + ] 0 versus [4H + ] 0 for nitrosation of 4 in aqueous solution at [H 3 O + ] 0 = 0.05 mol dm -3 , [NaNO 2 ] 0 = 5x10 -5 mol dm -3 , 25 o C, I = 1.0 (NaClO 4 ).

Figure 5 :

 5 Figure 5: The effect of increasing concentrations of NaCl upon k obs for the nitrosation of 4 at [H 3 O + ] 0 = 0.05 mol dm -3 , [4H + ] 0 = 0.01 mol dm -3 , [NaNO 2 ] 0 = 5x10 -5 mol dm -3 , 25 o C, I = 1.0 (NaClO 4 ).

Figure 6 :

 6 Figure 6: The effect of [H 3 O + ] 0 upon k obs for nitrosation of 4 in water at [4H + ] 0 = 0.05 mol dm -3 , [NaNO 2 ] 0 = 5x10 -5 mol dm -3 , 25 o C, I = 1.0 (NaClO 4 ).

Figure 7 :

 7 Figure 7: Comparison of experimental and simulated NMR spectra for N-nitrosodimethylhydroxylamine (5) in CDCl 3 . (Temperatures are given below experimental spectra and corresponding calculated exchange rate constants are given below the simulated spectra.)

Figure 8 :

 8 Figure 8: Gas phase optimized structures (B3LYP/6-31G(d)) for the cis and trans conformational isomers of CH 3 N(NO)OCH 3 (5) and two transition structures (TS1 and TS2) for their interconversion

Figure 9 : 21 Scheme 1 :

 9211 Figure 9: Plot of pseudo-first-order rate constants against the reciprocal of the initial hydronium ion concentrations for nitrosation of N-benzyl-O-methylhydroxylamine (3) in 1:1 (v/v) aqueous methanol at 25.0 °C. ([NaNO 2 ] 0 = 4x10 -3 mol dm -3 ; [RNH 2 OR' + ] 0 = 0.075 mol dm -3 ; gradient = 3.30x10 -4 mol dm -3 s -1 (standard error = 1.4x10 -5 ); intercept = 4.98x10 -3 s -1(standard error = 7.6x10 -5 ); correlation coefficient > 0.99).

25 Table 3 :

 253 d [NaNO 2 ] 0 = 3.87x10 -3 mol dm -3 . Dependence of k obs for nitrosation of 4 on [NaNO 2 ] 0 in aqueous acidic solution at 25 o C, I = 1.0 mol dm -3 (NaClO 4

  , the mechanism shown in Scheme 2 with two parallel reaction channels. A third possible channel involving catalysis by chloride to give ClNO can be ruled out by the finding that there is no kinetic term second-order in [RNH 2 OR' + Cl -] 0 for the nitrosation of 3 as was observed for the nitrosation of 4 (see later). The rate law derived from the mechanism in Scheme 2 for compounds 1-3 in aqueous methanol is included in Scheme 2.The experimental rate law for loss of nitrous acid is pseudo-first order, i.e. rate = k obs [HNO 2 ], so, if the mechanism of Scheme 2 is correct and assuming [HNO 2 ] ≈ [HNO 2 ] T , k obs is related to the parameters in Scheme 2 and eqn 4 by eqn 5.As [H 3 O + ] >> K a1 and K NO . [H 3 O + ] << 1 (see below),

	propose, therefore
	o r
	P
	e
	e r
	R
	e
	v i e
	w
	8

Table 4

 4 regarding cis and trans nomenclature.) Scheme 2. Proposed mechanism and derived rate law for nitrosation of 1-3 by nitrous acid and nitrosonium ion in aqueous methanol.

		CH 3	N N	OCH 3 O	k f k r	CH 3 O	N N	OCH 3	K = k f /k r
			cis-5					trans-5
		RNH 2 OR' +	+ H 2 O		RNHOR' +	H 3 O +	; K a1
		RNHOR' + HONO	k 1	RN	NO OR'	+ H 2 O
		RNHOR' +	NO +	k 2 H 2 O	RN	NO OR'	+	+ H 3 O
	mechanistic rate = k 1 [RNHOR'] [HNO 2 ] + k 2 [RNHOR'][NO ] +
	Substituting in this equation using
	[RNHOR'] 0 = [RNH 2 OR'] + [RNHOR'] , [HNO 2 ] T = [HNO 2 ] + [NO + ] ,
	K a1 =	[RNHOR'] [H 3 O + ] [RNH 2 OR'] +	, and (from eqn 4)

+ we obtain

Table 1 :

 1 Proposed mechanism and derived rate law for nitrosation of 4 in aqueous perchloric acid containing sodium nitrite with catalysis by chloride. Electronic structures and interconversion of conformational isomers of N-nitrosodimethylhydroxylamine(5). (See footnote[a] to Table4regarding cis and trans Kinetics of nitrosation of N,O-dibenzylhydroxylamine (1), tetrahydro-1,2-oxazine (2), and N-benzyl,O-methylhydroxylamine (3) under pseudo-first-order conditions in 1:1 (v/v) aqueous methanol containing perchloric acid, 25.0 o C.[a]

	R
	e
	v i e
	w

Table 4 :

 4 Experimental and computed 1 H and[START_REF] Williams | Nitrosation Reactions and the Chemistry of Nitric Oxide[END_REF] C NMR chemical shifts (ppm relative to TMS) for cis-and trans-N-nitroso-dimethylhydroxylamines (5) in CDCl 3 .[a]

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

3

; b monitored at 240 nm; c monitored at 250 nm; d monitored at 330 nm.

Table 5 :

 5 present work, 23 o C, 125.77 MHz. Computed and NMR experimental activation parameters for the conformational

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

d present work, gas phase; e

Table 6 :

 6 Computed and NMR experimental thermodynamic parameters for the conformational isomerisation cis-5 ⇌ trans-5.

		B3LYP	B3LYP	NMR	NMR	NMR	NMR
		(gas)	(CHCl 3 )	(CDCl 3 )	(toluene)	(DMSO-d 6 ) [a]	(CD 3 OD)
	∆H o /kJ mol -1	3.9	4.3	4.1	4.0	5.7	5.1
	∆S o /J K -1 mol -1	8.1	8.3	2.8	2.4	7.6	5.4
	∆G o 298 /kJ mol -1	1.5	1.8	3.3	3.3	3.4	3.5
	(equil. constant)	(0.55)	(0.48)	(0.26)	(0.26)	(0.25)	(0.24)
	a Limited temperature range.

Table 7 :

 7 Key geometric parameters (B3LYP/6-31G(d)) and dipole moments for cis-5, trans-5, and the transition structure TS1 for their interconversion in the gas phase.[a]

	Parameter	cis-5	TS1	trans-5
	d(N-N)/Å	1.351	1.571	1.347
	d(N=O)/Å	1.220	1.183	1.225
	Φ(O-N-N-O)/ o	-15.3	-119.2	165.8
	µ/D	3.4	1.3	3.6
	∑θ/ o at N1	350.3	306.7	351.8

a calculated results in chloroform are only slightly different.

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Acknowledgements

We thank the Centro de Supercomputacion de Galicia (CESGA) for computing facilities, and the EPSRC (UK) for partial funding of NMR facilities at Newcastle.