Electrophilic Reactions of Skin-Sensitizing Sultones - ACS Publications

Chem. Res. Toxicol. 2007, 20, 61

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Electrophilic Reactions of Skin-Sensitizing Sultones David W. Roberts,*,† Douglas L. Williams,†,‡,§ and Donald Bethell‡ UnileVer Research Port Sunlight, Quarry Road East, Bebington, Wirral, CH63 3JW, United Kingdom, and Robert Robinson Laboratories, Department of Chemistry, UniVersity of LiVerpool, LiVerpool, L69 3BX, United Kingdom ReceiVed NoVember 21, 2006

Reactions of the strong skin sensitizer hexadec-1-ene-1,3-sultone with sodium hydroxide, sodium methoxide, sodium metabisulfite, sodium butanethiolate, n-butylamine, and aniline have been investigated, and the reaction products have been identified. Most of the nucleophiles studied react by nucleophilic addition (Michael type addition) to the double bond at the 2-position, although in most cases the final products result from further reactions of the initital adducts. The findings are considered together with those reported by Meschkat, Barratt, and Lepoittevin for reactions of hex-1-ene-1,3-sultone and hexane1,3-sultone, and the implications of the two sets of findings for the mechanism of skin-sensitizing action are discussed. It is concluded that nucleophilic attack at the 3-position of the alk-1-ene-1,3-sultones occurs only with those nucleophiles, which either have very low reactivity in nucleophilic addition or are unable to give rise to thermodynamically stable products via initial reaction at the 2-position. It is further concluded that the observed differences in electrophilic reactivity between alk-1-ene-1,3-sultones and alkane-1,3sultones are not large enough to rationalize the differences in skin sensitization properties between the two types of sultone. It is suggested that the differences in specificity arise because the alk-1-ene-1,3sultones act as Michael type electrophiles whereas the alkane sultones act as SN2 electrophiles. It is suggested that the reason for the greater potency of the alk-1-ene-1,3-sultones may be the ability of the initial C2 adducts with protein to undergo further reactions by substitution at C3, leading to cross-linking and consequent perturbation to the protein tertiary structure. Introduction The prediction of skin sensitization potential with minimum animal testing is currently of great importance in the light of new legislation (e.g., the REACH program in the European Union). Research dating back more than 7 decades has established a very strong mechanistic understanding of skin sensitization (1, 2). Skin-sensitizing chemicals are usually electrophilic and are able to react with nucleophilic groups on skin proteins. Thus, the ability to sensitize depends on the ability to bind to carrier protein, which can be modeled by a combination of reactivity and hydrophobicity parameters (3). On the basis of this understanding, in principle, the following approach to nonanimal prediction of skin sensitization potential can be envisaged (4, 5). First, the compound under consideration must be allocated to the appropriate reaction mechanistic applicability domain, depending on the reaction mechanism by which it binds to carrier protein. Second, assuming skin sensitization data exist for other compounds in the same reaction mechanistic applicability domain, a quantitative structureactivity relationship (QSAR) or quantitative mechanistic model (QMM) for that domain may be developed, enabling the sensitization potential to be predicted for the compound of interest. Failing that, it may be possible to estimate the sensitization potential by read-across from data on related compounds. To implement this approach, it is necessary to be able to identify the relevant reaction chemistry of the compound of interest. In some cases, this may be obvious from the chemical * To whom correspondence should be addressed. Present address: School of Pharmacy and Chemistry, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, United Kingdom. E-mail: [email protected]. † Unilever Research Port Sunlight. ‡ University of Liverpool. § Deceased.

structure, and it may be possible to calculate reactivity and hydrophobicity parameters using LFER methods. An example is the recent development and validation of a QMM for the “Schiff base mechanistic applicability domain”, i.e., compounds that can bind to proteins by formation of Schiff base linkages (6). In other cases, the nature of the reaction chemistry may need to be determined experimentally and kinetic studies may be needed to determine reactivity parameters. This is the situation for the compounds discussed here. Alk-1-ene-1,3-sultones 1 and alkane-1,3-sultones 2 of surfactant chain length (total carbon number in the range C12C18) have been recognized for some time as contact allergens (skin sensitizers) (7-9). Although structurally very similar, their skin-sensitizing properties are very different. For surfactant chain length sultones, which have been tested, the alk-1-ene-1,3sultones 1 are classed as very stong sensitizers, capable of inducing sensitization in a high proportion of test animals when applied even at low concentrations, and the alkane-1,3-sultones 2 of surfactant chain length are classed as moderate sensitizers, capable of inducing sensitization in a high proportion of test animals when applied at high concentrations but not when applied at low concentrations (8, 9). Cross-challenge tests reveal a further difference: A set of animals well-sensitized to 1 can be challenged with 2 at a concentration known to elicit response in animals sensitized to 2, and a set of animals well-sensitized to 2 can be challenged with 1 at a concentration known to elicit response in animals sensitized to 1. When these tests were carried out, the results showed complete failure of 1-sensitized animals to respond to challenge with 2 and complete failure of 2-sensitized animals to respond to challenge with 1 (8). These findings of complete lack of cross-reactivity point unambiguously to a clear difference in antigenic specificity.

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62 Chem. Res. Toxicol., Vol. 20, No. 1, 2007

Whereas the alkane sultones 2 can only react by ring-opening substitution at C3 or by attack of the nucleophile at the sulfur atom, for the alkene sultones 1, there is also the possibility of Michael type nucleophilic addition at C2. Here, we present results from work carried out some years ago on the chemistry of alk-1-ene-1,3-sultones 1 and discuss the chemical and biological implications of our findings together with those of Meshkat, Barratt, and Lepoittevin (MBL) for reactions of short chain homologues of 1 and 2 with simple nucleophiles (10) and human serum albumin (HSA) in water (11).

Materials and Methods Caution: Skin contact with alkene sultones must be aVoided. Because they are potent skin sensitizers, they must be handled with care. Hexadec-1-ene-1,3-sultone 1 was prepared from hexadec-1-ene by the method of Roberts, Sztanko, and Williams (12). Reactions of this sultone were carried out in most cases on an approximately 10 mM scale. Work up of reaction product mixtures was typically carried out by the following sequence: removal of solvent and/or excess nucleophile by evaporation, partitioning between aqueous alcohol and petroleum ether to separate ionic products from nonionic products and recovered starting material, and preparative HPLC to separate nonionic products from each other and from recovered starting material. Reaction products were identified by, as appropriate, their proton NMR spectra (in some cases, supplemented by 13C spectra), mass spectra, elemental analysis, and comparison against samples prepared by alternative routes. Details are given in Table 1. Preparation of Alkane-1,3-sultones. Hexadecane-1,3-sultone 2 was isolated by a literature method (13) from the acidic reaction mixture produced by continuous sulfonation of hexadec-1-ene with gaseous sulfur trioxide in a pilot plant short residence time film reactor, under conditions used industrially for olefin sulfonation (14). A batch of the acidic reaction mixture (175 g) was held at room temperature for 15 min in order to maximize the 1,3-sultone content (15, 16) and then added to isopropyl alcohol (500 mL) at room temperature. The precipitate, after recrystallization from isopropyl alcohol, yielded 22.0 g of 2 (R ) C13H27). Butane-1,3sultone 2 was prepared by sulfonation of but-1-ene with sulfur trioxide in dichloroethane as described in the literature (17). Reaction of N,N-Dimethyldodecylamine with Butane-1,3sultone. N,N-Dimethyldodecylamine (11.8 g, 55.5 mmol) and butane-1,3-sultone (7.5 g, 55.1 mmol) were dissolved in ethyl acetate (100 mL), and the solution was heated under reflux for 2.5 h. On standing overnight, a solid precipitated. It was recrystallized from isopropyl alcohol to give a white solid, which was not fully characterized but which was assumed, on the basis of its water solubility and surfactant properties in both neutral and alkaline solution, to be the sulfobetaine N,N-dimethyldodecylammonium)3-methylpropanesulfonate (4.0 g). A further quantity of this product (6.2 g, making a total of 10.2 g, 53% yield) was obtained by heating the remaining reaction solution under reflux for a further 29 h. Further heating after removal of this product yielded no further solid product. Thus, the initial quantity of product obtained after 2.5 h of reaction time corresponds to 39% reaction, from which the rate constant can be estimated as ca. 2 × 10-4 L mol-1 s-1. Reaction of Sodium Thiosulfate with Hexadecane-1,3-sultone. Hexadecane-1,3-sultone (3.04 g, 10 mmol) and sodium thiosulfate pentahydrate (1.89 g, 8 mmol) were added to a mixture of ethanol (12 mL) and water (15 mL), and the mixture was heated with stirring at 70 °C. The initially cloudy reaction mixture became clear

Roberts et al. after ca. 3 h. After a total heating time of 5 h, the reaction mixture was evaporated to dryness on a rotary evaporator. The solid residue, after Soxhlet extraction with acetone to remove excess sultone, was identified as disodium 3-thiosulfato-hexadecane-1-sulfonate monohydrate (2.90 g, 76% yield) (found: C, 40.8; H, 7.4; S, 18.6%. C16H32S3O6Na2‚H2O requires C, 40.0; H, 7.1; S, 20.0%). Reaction Kinetics for Hexadecane-1,3-sultone with n-Butylamine in Dioxan. Hexadecane-1,3-sultone 2 (3.04 g, 0.01 mol) and n-butylamine (2.7 mL) were dissolved in dioxan (total volume, 100 mL). By titration of a 5 mL aliquot with aqueous 0.1 M hydrochloric acid, the initial n-butylamine concentration was determined as 0.24 M. The solution was heated under reflux in a round-bottomed flask equipped with a reflux condenser and a thermometer. The temperature of the solution was 84 °C throughout. Accurately measured aliquots of ca. 5 mL were withdrawn at approximately 2 h intervals by means of a pipet inserted down the top of the condenser, and the samples were titrated with aqueous 0.1 M hydrochloric acid to determine the amount of n-butylamine remaining. The reaction was followed for 12 h, corresponding to approximately 16% reaction. A plot of ln (Tt - 7) against time t where T is the titer of 0.1 M HCl required to neutralize exactly 5 mL of solution (7 is the value of T∞ calculated from the reaction stoichiometry) gave the pseudo first-order rate constant, 3.25 ((0.60) × 10-6 s-1, which divided by the initial butylamine concentration gave 1.35 ((0.25) × 10-5 L mol-1 s-1 as the secondorder rate constant. Kinetics for Deuterium Incorporation in Hexadec-1-ene-1,3sultone with N,N-Dideuterio-n-butylamine. Deuterium oxide (30 g, 1.5 mol) and n-butylamine (10.95 g, 0.15 mol) were mixed, and the mixture was distilled under atmospheric pressure. The fraction boiling at 85 °C was mixed with a further quantity of deuterium oxide (30 g, 1.5 mol), and this mixture was distilled under atmospheric pressure. The fraction boiling at 85 °C was dried over anhydrous magnesium sulfate and shown by NMR to be N,Ndideuterio-n-butylamine. Hexadec-1-ene-1,3-sultone (3.02 g, 10 mmol) and freshly prepared N,N-dideuterio-n-butylamine (4.5 g, 60 mmol) were dissolved in dioxan (100 mL), and the solution was heated at 84 °C. A total of eight samples (5 mL) were removed at measured time intervals (approximately hourly) and evaporated to dryness. The solids thus obtained were examined by proton NMR (using deuteriochloroform as solvent) to measure the relative intensities RI of the multiplets at 6.9 (protons at C2 and C1) and 5.25 ppm (proton at C3). A plot of ln (RI - 1.075) against t gave the psuedo first-order rate constant 1.58 ((0.06) × 10-5 s-1, which, divided by the initial N,N-dideuterio-n-butylamine concentration, gives 2.64 ((0.10) × 10-5 L mol-1 s-1 as the second-order rate constant.

Results Table 2 summarizes the reaction conditions used in our studies with 1 (R ) C13H27) and the products formed. Reaction Pathways. Nucleophilic Addition at C2. For the reactions of sodium methoxide and sodium metabisulphite with 1, the observation of the isolated and characterized addition products 4 and 7 constitutes direct evidence for the addition reaction at C2. The observed reaction products from sodium butanethiolate and n-butylamine with 1 can be rationalized in terms of initial nucleophilic addition at C2 followed by further reactions of the proposed addition products 4 as illustrated in Schemes 1 and 2. Note that for the n-butylamine reaction, because butylamine was removed by evaporation, the product 9 was isolated as such rather than as the corresponding butylammonium salt 9a. Further Reactions with Sodium Methoxide. Although the C2 addition product 4 (X ) OMe) is the initial product in the sodium methoxide reaction, it is not immediately obvious how the final products 5 and 6 arise. Our proposed reaction pathways are shown in Scheme 3. The reactions involve attack of methoxide ion on the hydrogen at C3 (leading to formation of

Sultones and Skin Sensitization

Chem. Res. Toxicol., Vol. 20, No. 1, 2007 63 Table 1. Characterization of Reaction Products

64 Chem. Res. Toxicol., Vol. 20, No. 1, 2007 Table 2. Reactions of 1 with Nucleophiles

Roberts et al. Scheme 1. Proposed Further Reactions of Inital Nucleophilic Addition Product 4 from n-Butanethiolate and 1

Scheme 2. Proposed Further Reactions of Inital Nucleophilic Addition Product 4 from n-Butylamine and 1

5) and at the sulfur atom (leading to formation of 6). We observed only trans stereochemistry for the 4 (X ) OMe) isolated from the reaction mixture. It seems plausible that the minor product 5 is produced mainly if not exclusively from most of the products that are derived from this isomer, although we cannot exclude some involvement of the cis isomer as a transient species. Sodium Hydroxide. For the reaction of sodium hydroxide with 1, we obtained evidence that 2-hydroxyhexadecane-1,3sultone 4 (X ) OH), which was not detected as a reaction product, is not an intermediate on the pathway to the observed reaction product 3. The lower homologue 4 (R ) C3H7, X ) OH) was prepared in low yield by dissolving sodium 2-epoxyhexane-1-sulfonate (made by treatment of sodium hex-2-ene sulfonate in water with aqueous 40% peracetic acid) in aqueous 30% sulfuric acid and then extracting with ethyl acetate. Upon treatment with either dilute aqueous acid or base, the 2epoxysulfonate was regenerated immediately. Treatment of sodium 2-epoxyhexadecane-1-sulfonate with sodium hydroxide

under the conditions used for reaction of 1 with for sodium hydroxide did not give 3, the reaction product having an infrared spectrum consistent with sodium 2,3-dihydroxyhexadecane-1sulfonate: absorption at 3540 (OH stretch) but no absorption at 1700-1710 (CdO stretch). Bearing in mind that hydroxide ion is a very hard nucleophile and is strongly solvated in aqueous solution, we rationalize the formation of 3 in terms of initial attack at the sulfonyl sulfur atom of the sultone, as shown in Scheme 4. In the first step, hydroxide ion attacks reversibly at sulfur. An analogous reaction can also occur with methoxide ion. In the second step, the anionic adduct 1a is deprotonated to give the dianionic species 1b. With methoxide, the analogue of 1a cannot be deprotonated to give 1b. This can explain why methoxide does not give products derived from initial attack at sulfur. The charged oxygen atoms of the dianionic species 1b are now sufficiently basic to attack the hydrogen at C3, giving rise to 1c, the notional hydroxide ion adduct of an alk-2-ene1,3-sultone. Compound 1c can then undergo ring cleavage leading to the observed product. As a variant on this pathway, we can envisage 1a reacting with hydroxide to give 1c directly in a concerted pathway avoiding free 1b, as shown in Scheme 4. Aniline. For the reaction of aniline with 1, the product 10 is consistent with either nucleophilic substitution at C3 or re-


Chem. Res. Toxicol., Vol. 20, No. 1, 2007 65 Table 3. Deuterium Exchange Experiments

Scheme 3. Reaction of Sodium Methoxide with 1

Scheme 4. Reaction of Sodium Hydroxide with 1

Scheme 5. Rearrangement Route to C3 Substitution Products from 1

arrangement of the C2 addition product as shown in Scheme 5, for X ) -NHPh. Further evidence was obtained from deuterium exchange experiments, in which 1 was treated with deuterated solvents and deuterated nucleophiles, and the recovered 1 was examined by proton NMR to measure changes in the relative intensities of the multiplets at 5.25 (proton at C3) and 6.9 ppm (protons at C1 and C2). Reversible addition of nucleophile at C2 leads to deuterium incorporation at C1 and a consequent decrease in the relative intensity of the multiplet at 6.9 ppm. Table 3 summarizes the findings. The deuterium exchange results provide evidence for addition at C2 by the nucleophiles methoxide, butanethiolate, and n-butylamine but not by aniline. The failure of N,N-dideuterioaniline to give any deuterium incorporation at C1 shows that

reagent/solvent

conditions

D exchange at C1

D2O/MeOD D2O/MeOD, pH 10.4 MeOD MeONa/MeOD BuSNa/BuSD BuND2 BuND2/dioxan

reflux, 90 min reflux, 7 h 65 °C, 3 h 20 °C, 10 min 80 °C, 10-20 min 80 °C, 1 h 84 °C

PhND2

100 °C, 2 h

20% 75% complete complete complete complete k ) 2.64 ((0.10) × 10-5 L mol-1 s-1 none

aniline does not add at C2 and indicates that the reaction product 10 arises by substitution at C3 and not via the pathway of Scheme 5. Kinetics with n-Butylamine. The rate constant for deuterium incorporation in 1 with 0.6 M deuterated n-butylamine in dioxan at 84 °C is 2.64 ((0.10) × 10-5 L mol-1 s-1. For comparison, we determined the rate constant for the ring-opening nucleophilic substitution reaction of hexadecane-1,3-sultone 2 (R ) C13H27) with n-butylamine in dioxan at 84 °C. This rate constant is 1.35 ((0.25) × 10-5 L mol-1 s-1. Thus, the forward rate of the reversible addition of n-butylamine to 1 is only about two times greater than the overall rate of product formation from 2 with n-butylamine under the same conditions. It follows that in terms of rates of final adduct formation, 1 is not substantially more reactive than 2 toward n-butylamine and could indeed be less reactive. Mechanistic Principles. Overall, our results indicate that apart from hydroxide and aniline, all of the nucleophiles studied give reaction products derived from attack on 1 at C2. It is clear from Table 3 that this nucleophilic addition reaction is reversible. In several cases, the final reaction products result from further reactions of the C2 adducts. Only aniline reacts with 1 by nucleophilic substitution at C3. We note that methoxide ion, a very hard nucleophile (18), appears to be one of the more reactive of the nucleophiles studied and attacks 1 at the “softer” reaction center, C2, to give the addition product, 4. In contrast, aniline, which is considered borderline on the hard/soft spectrum (18), reacts with 1 exclusively at the “harder” reaction center, C3. Clearly, HSAB principles play little part in the choice between attack at C2 and attack at C3. This may reflect the lateness of the transition state for the addition reaction as compared with that for the substitution reaction. The addition reaction proceeds via a high-energy intermediate whereas the substitution at C3 proceeds directly to stable products. It therefore follows from the Hammond postulate (18) that in the addition reaction transition state the charge on the nucleophile becomes less negative/more positive to a much greater extent than in the substitution transition state. Thus, the ability of the nucleophile to accommodate this change, i.e., the basicity of the nucleophile, affects reactivity in the addition reaction more than it affects reactivity in the substitution reaction. Our interpretation is that ring-opening nucleophilic substitution at C3 in 1 is disfavored, relative to substitution at C3 in alkane-1,3-sultones 2 and relative to nucleophilic addition at C2 in 1. This is consistent with the principles put forward by Bordwell et al. (19) to account for the effects of methyl substitution on rates of alkane-1,3-sultone solvolysis and later applied by two of us to sultone isomerization reactions (15). The basic concept underlying these principles is that in ringopening reactions the two atoms between which bond heterolysis occurs cannot separate linearly as in open chain compounds. Instead, separation of these atoms occurs by rotation about the bonds of the ring, and the effects of substituents can be analyzed in terms of the restriction they provide to such rotations. In


particular, restriction to rotation about the C1-C2 bond in 1,3sultones has a large effect on the reaction rate. Thus, for example, 2,2-dimethylpropane-1,3-sultone is less reactive than propane-1,3-sultone (2, R ) H) to hydrolyis by water by a factor of 285 (19). Dividing this factor by 9.5 to allow for the contribution of the neopentyl effect [neopentyl benzene sulfonate is less reactive than n-propyl benzene sulfonate toward hydrolysis by a factor of 9.5 (19)] gives a factor of 30 as the rotational hindrance effect of two methyl groups on C2 of propane-1,3-sultone. This factor is of the same order of magnitude as that which normally applies for allylic activation in SN2 reactions: For example, in reactions of primary chlorides with potassium iodide in acetone at 50 °C, the rate constant for allyl chloride is 72 times that for n-propyl chloride (20). Bearing in mind that the rotational restriction effect of the double bond in alkene sultones is likely to be even greater than that of two methyl groups on C2 of propane-1,3-sultone, we conclude that for alkene sultones, allylic activation of the C3 position toward nucleophilic substitution is largely negated and is probably even outweighed by the severe restriction to rotation about the C1C2 double bond acting as a barrier to heterolysis of the bond between the C3 and the sulfonate oxygen atom. Therefore, reaction of 1 at C3 is to be expected only with those nucleophiles that either, like aniline (21, 22), have very low reactivity in nucleophilic addition or that are unable to give rise to thermodynamically stable products via initial reaction at C2. The low reactivity of aniline in nucleophilic addition probably reflects its low basicity and the lateness of the transition state. Hydroxide ion is the only nucleophile to give a product derived from initial attack on 1 at sulfur. The reason why methoxide does not do so is probably that there is no low energy pathway from the initial adduct analogous to 1a (Scheme 4). Comparison with Findings of MBL (10, 11). We now consider the findings of MBL in the light of the above. We note that whereas we chose to work with the C16 homologue (1, R ) C13H27), because of its known strong sensitization potential (7-9) and its relevance as a potential contaminant in consumer products (3), MBL worked with the C6 alkene sultone (1, R ) C3H7). The C6 alkene sultone is a less potent sensitizer and not relevant in the context of potential human exposure, but it is water-soluble and is therefore convenient for their studies on binding to the water-soluble protein HSA. They also studied the C6 alkane-1,3 sultone (2, R ) C3H7). To the best of our knowledge, no sensitization studies on 2, R ) C3H7, have been reported, but it would be expected to be less potent than its C16 homologue. It is not relevant in terms of potential human exposure, but for MBL’s studies, it too has the advantage of being water-soluble. All reactions reported by MBL were done at ambient temperature with C6 sultones 1 and 2, with 13C at positions 2 or 3. Reactions were followed by 13C NMR. Table 4 summarizes their results with model nucleophiles. Overall, the results shown in Table 4 are analogous to our own shown in Table 2 and are consistent with our mechanistic interpretation of our findings. In particular, for propanethiolate and n-butylamine, MBL observed the same or homologous products as we did, and in the case of n-butylamine, their observation of the C2 addition product 4, X ) NHBu, seems to confirm our interpretation that 4, X ) NHBu, although we did not detect it, is the precursor of 9. For the 3-substituted cis-alk-1-ene sulfonates 10, which MBL observe as reaction products with phenolate, imidazole, and diethylamine, it is not possible on the available evidence to select unequivocally from three possibilities: (i) substitution at C3

Roberts et al. Table 4. MBL Findings for Reactions of 1 with Nucleophiles

occurring more readily than addition at C2; (ii) addition at C2 occurring more readily than substitution at C3, but the latter reaction prevailing due to thermodynamic instability and lack of further reaction pathways for the addition product; and (iii) formation of 3-substituted cis-alk-1-ene sulfonates 10 from the addition products 4 as per Scheme 5. We note that imidazole is the weakest base studied by MBL, the pKa value of its conjugate acid (6.95) being intermediate between those for the conjugate acids of aniline (4.63), for which we have shown that (i) above applies and n-butylamine (10.77), which we propose reacts by nucleophilic addition at C2. On that basis, it seems reasonable to interpret MBL’s observation of both 4 and 10 as reaction products of 1 with imidazole in terms of substitution at C3 and addition at C2 occurring competitively. Phenolate, which gives the cis-alk-1-ene sulfonate 10, X ) OPh exclusively, is substantially more basic (the pKa of its conjugate acid is 9.89) than imidazole and not much less basic than n-butylamine; therefore, for phenolate, it seems likely that (ii) or (iii) above applies. Of these two possibilities, (ii) seems the more likely since, for (iii), formation of 10 from 4 (X ) OPh), the internal nucleophile would be the ether oxygen, which is only feebly nucleophilic. Diethylamine is very similar in its basicity to n-butylamine (pKa values of the conjugate acids are 10.49 and 10.77, respectively). We note also from Table 4 that the solvent effects are very similar in magnitude for the two nucleophiles, suggesting that although the final products are very different,

Sultones and Skin Sensitization Scheme 6. MBL Proposed Reaction Pathway for 1 with n-Butylamine

the rate-determining steps are probably similar. We therefore consider it likely that, for diethylamine, (iii) above applies. We note that the aziridinium ion analogue of 9, with two ethyl substituents on the nitrogen, is more sterically hindered than the butylamine-derived analogue 9 and, hence, may be thermodynamically unstable. Reaction of 1 with n-Butylamine: Addition at C2 or Substitution at C3? Although we interpret our and MBL’s results for reactions of 1 with n-butylamine in terms of nucleophilic addition at C2, the MBL interpretation of their results is different. They argue that the main initial reaction in water and deuteriochloroform is substitution at C3 to give the adduct 10 (X ) NHBu), which reacts rapidly by intramolecular addition to give the aziridine 9 (Scheme 6). They argue that direct C2 addition only occurs as a slower side reaction (except in neat butylamine). The MBL arguments in support of this reaction pathway are that the cis/trans ratio in aziridine 9a (1/1) does not correspond with that of the aminosultone 4 (predominantly trans). If 9 is formed from 4, the stereochemistry should be unchanged. Furthermore, the reaction product (main component 4) from reaction with neat butylamine does not generate 9a when dissolved in water. The proposed intermediate 10 (X ) NHBu) is not detected in any of the experiments. Although we cannot definitively rule it out, the reaction pathway of Scheme 6 seems to us rather unlikely, for two reasons. First, it implies that the double bond in 10 (X ) NHBu) is more reactive toward intramolecular nucleophilic addition than the double bond in 1 toward intermolecular nucleophilic addition, i.e., in Scheme 6, k2 > k1 [BuNH2]. This seems inconsistent with the very much lower electronegativity of the anionic sulfonate group as compared with the covalent sulfonate ester group. Nucleophilic addition to olefinic ionic sulfonates has been reported but only under forcing conditions. Thus, the salt PhCHdCHSO3Na requires heating with aqueous ethylamine at 160 °C for 40 h to give the adduct in 51% yield (22). Even allowing for an intramolecular enhancement effect, typically of the order of a factor of 15 (23), these figures seem incompatible with formation of 9 from 10 at 20 °C. Second, the solvent effects reported by MBL are not consistent with their proposed reaction scheme. The reasoning, based on the Hammond postulate and the Hughes Ingold rules (18), is as follows. Suppose that, as MBL propose, the reaction scheme is as shown in Scheme 6. Then, because 10 is not observed as a reaction product, its formation, with rate constant k1, must be the rate-determining step in formation of 9. The transition state for this step will be partly zwitterionic in character, the nitrogen of the attacking butylamine entity bearing approximately half a positive charge and the incipient sulfonate group bearing approximately half a negative charge. For the addition reaction, with rate constant k3, the transition state will be similar in energy and structure to the intermediate 4a, with

Chem. Res. Toxicol., Vol. 20, No. 1, 2007 67 Scheme 7. Reactions of Alkane-1,3-sultones 2

the nitrogen atom bearing almost a full positive charge and C1 bearing almost a full negative charge. Although we do not have detailed information on charge distributions in the two transition states, we can be confident that the transition state for the addition reaction at C2 is more polar than the transition state for the substitution reaction at C3. Both transition states will be stabilized, relative to the uncharged reactants, by polar solvents, but the stabilizing effect will be greater for the more fully zwitterionic transition state corresponding to k3. Thus, on changing from a less polar solvent (butylamine) to a more polar solvent (water), k3 should increase more than k1, and the ratio of 4 to 9 in the reaction product mixture should increase. This is the converse of what MBL observed: Compound 4 was the major product when the solvent was butylamine but was not observed when the solvent was water. Our interpretation is as shown in Scheme 2. The equilibrium composition of the reaction products will depend on the polarity of the medium and on the acidity/basicity of the medium. The polarity of the products can be summarized as follows: Compound 9a is the most polar, and compound 4 is least polar. This explains why in water and deuteriochloroform with excess butylamine, 9a is the major product. When the reaction is done with butylamine alone, the medium is much less polar, and 4 is more favored. When the product mixture from the neat butylamine reaction is isolated and put into water, there is no butylamine or other base present to drive the equilibrium over to 9a, so the composition remains at the equilibrium position between 4 and 9. If, as we suggest, the product composition is determined more by thermodynamics rather than kinetics, the MBL observations regarding steroechemistry are easily rationalized. For the adducts 4, the trans isomer should clearly be the more stable, since nonbonding interactions between the substituents on C2 and C3 are lower than in the cis isomer. For 9 and 9a (R ) C3H7), there are four configurations to consider as follows: propyl, butyl, and sulfomethyl groups all on the same side of the ring (clearly, the least stable and likely to be strongly disfavored); propyl and sulfomethyl mutually cis with butyl trans to them; and propyl and sulfomethyl mutually trans with the butyl group cis to one and trans to the other (two isomers). The latter two should not differ much in their nonbonding interactions; therefore, a cis/trans ratio (as regards the propyl and sulfomethyl groups) close to 1/1 is not surprising. Reactivity Comparison between Alk-1-ene-1,3-sultones 1 and Alkane-1,3-sultones 2. Reactions of alkane-1,3-sultones 2 with nucleophiles give substitution products 12, sometimes accompanied by hydrolysis products 13 and elimination products 14 (Scheme 7). As a broad generalization, our findings do not reveal large differences between 1 and 2 in overall rates of electrophilic reactions. As mentioned earlier, the rate constants for reactions of 1 and 2 with n-butylamine in dioxan are similar. On the basis of our own findings and those of others (17, 24), reactions of other nucleophiles with 2 require conditions broadly similar to

68 Chem. Res. Toxicol., Vol. 20, No. 1, 2007 Table 5. Reactivity of Alkane-1,3-sultones 2 to Various Nucleophiles

a The reaction conditions appear to have been selected somewhat arbitrarily and were probably more severe than necessary. b This reaction was carreid out with hexadecane-1,4-sultone 15. Alkane-1,4-sultones react similarly to alkane-1,3-sultones but more slowly.

those found necessary for reactions of alk-1-ene-1,3-sultones 1. Some examples are shown in Table 5. The comparison can be extended by considering the findings reported by MBL. MBL investigated reactions of 2, mostly in water, with the same nucleophiles used in their studies with 1, under the same conditions (10). For n-butylamine, diethylamine, and imidazole, the hydrolysis product 13, accompanied by smaller amounts of the elimination product 14, was formed to a greater extent than the substitution product 12. For sodium phenolate, the substitution and hydrolyis reactions occurred to about the same extent, and in a competitive reaction with a mixture of 2 and 1, it was found that 2 reacts faster than 1 with sodium phenolate. With sodium propanethiolate, the substitution reaction was found to be faster than the hydrolysis reaction; 12 (X ) SPr) is the major reaction product . Thus, the order of nucleophilic reactivity toward C3 in 2 is PrSNa > PhONa > BuNH2, Et2NH, and imidazole. We note

Roberts et al.

that this reactivity order is not consistent with the concept that reactivity toward sultones is strongly governed by the HSAB principle and that C3 in 1,3-sultones is a hard reaction center. MBL also report findings from reactions of 1 and 2 (R ) C3H7 in both cases) with HSA in water (11). The HSA molecule contains 18 tyrosine units and 59 lysine units, although at the pH of the MBL experiments (8.1), most of the latter are protonated and unavailable for reaction. The ionized tyrosine unit is modeled by sodium phenolate, and the unprotonated lysine unit is modeled by n-butylamine. MBL found that when aqueous HSA was incubated with excess 1, covalent binding occurred to one of the lysine units and at least four of the tyrosine units, while 2 reacted with the tyrosine units but not with the available lysine unit. Although MBL do not give kinetic data, it is possible from the information given to make order of magnitude estimates of rate constants for the reactions of 1 and 2 (R ) C3H7) with several nucleophiles. Table 6 summarizes the rate constants estimated from the MBL data; how the rate constants are estimated is described in detail in the Appendix. In agreement with our general observations for reactions in organic solvents, for the reactions in water investigated by MBL, the differences in reactivity between 1 and 2 do not appear to be large, except for the case of hydrolysis. In most cases, 1 appears to be somewhat more reactive than 2. The converse applies when phenolate is the nucleophile, and this is consistent with the formation of the product 10 (X ) OPh) by SN2 attack of phenolate ion at C3; this position in 1 is deactivated relative to the C3 position in 2. For hydrolysis, which presumably proceeds by nucleophilic attack of water at C3, the greater reactivity of 2 as compared to 1 is again attributable to deactivation of the C3 position in 1. The MBL findings are supported by unpublished work from our laboratory on hydrolysis of long chain sultones in aqueous anionic surfactant micelles. The reaction rates are independent of hydroxide ion concentration, indicating that water is the nucleophile, and at 95 °C, the alkane-1,3-sultones 2 are about 24 times as reactive as the alkene-1,3-sultones 1. Comparing against the factor of ca. 250 derived from the MBL data at 20 °C, we can estimate that the difference in activation energy for hydrolysis of 1 and 2 is about 6.6 kcal/mol. The fact that MBL observed no coupling between 2 (R ) C3H7) and the lysine unit of HSA (0.12 mmol/l) can be attributed more to 2’s high reactivity toward hydrolysis than to any lack of reactivity toward the amino group. Thus, the only significant difference observed by MBL between 1 and 2 (R ) C3H7) in their reactions with HSA is not relevant to the water-insoluble skin sensitizers of interest, 1 and 2 with R in the range C9H19 to C13H27. Protein Binding and Skin Sensitization. At first sight, it seems reasonable to attribute the difference in sensitization potential between 1 and 2 (R in the range C9H19 to C13H27) to the differences observed by MBL in the reactions of 1 and 2 (R ) C3H7) with HSA in water, in particular the failure of 2 (R

Table 6. Estimated Rate Constants for Reactions of Sultones 1 and 2 nucleophile

k (1) L mol-1 s-1

BuNH2 in water BuNH2 in CDCl3 Et2NH in water Et2NH in CDCl3 PrSNa in water

>2 × 10-3 2 × 10-3 4 × 10-5 could be >6 × 10-3 2 × 10-5 1 × 10-6 5.5 × 10-7 s-1

PhONa in water imidazole in water hydrolysis, pH 8.1

k (2) L mol-1 s-1

k(1)/k(2)

8-12 × 10-5

>20

8-70 × 10-6

>30

>4 × 10-4 2 × 10-4 1.4 × 10-4 s-1

could be >10 g0.1 0.1 0.004


Chem. Res. Toxicol., Vol. 20, No. 1, 2007 69

Table 7. Differences in Electrophilic Reactivity and Skin Sensitization Protential

a

sensitizers

relative reactivitya

alkane-1,3-sultones 2 and -1,4-sultones 15 (3, 8) C12H25SO3Me 16 and MeSO3C12H25 17 (28, 29) 1 and 2

2 more reactive, by factor of ca. 10 16 more reactive, by factor of ca. 30 1 unlikely to be more reactive by a factor >100; less than five times more reactive in nonpolar media

relative sensitization potentialb 2 more potent, by factor of ca. 10 16 more potent, by factor of ca. 30 1 more potent by factor of ca. 104 (3)

cross-reactivity mutually cross-reactive mutually cross-reactive complete lack of cross-reactivity (8)

Toward n-butylamine. b Relative concentrations required to induce the same degree of sensitization, calculated from RAI correlations.

) C3H7) to react with the available lysine unit. On this basis, to explain the higher sensitization potential of alk-1-ene-1,3sultones 1, it has to be argued that coupling of allergen to tyrosine units does not make protein antigenic to the same extent that coupling to lysine does. However, on closer consideration, this explanation is seen to have several deficiencies. First, as discussed above, the failure of 2 (R ) C3H7) to react with the available lysine unit of aqueous HSA is not relevant to the water-insoluble skin sensitizers of interest. Second, the argument that coupling of allergen to tyrosine units does not make protein antigenic to the same extent that coupling to lysine does is an ad hoc assumption for which there is no obvious theoretical justification and for which there is no experimental evidence. Third, this explanation rationalizes the difference in sensitization potential by trying to explain the low sensitization potential of alkane sultones, rather than the high sensitization potential of alkene sultones. However, in comparison to other SN2 electrophiles such as alkyl alkane sulfonates, which have been shown to react with lysine units in HSA (25), the alkane sultones 2 are not anomalously weak sensitizers. It is the very high sensitization potential of the alkene sultones 1 that is anomalous. The location and the nature of the protein or proteins whose modification by low molecular weight allergens leads to skin sensitization are not known. The possibilities, discussed in some detail by Smith and Hotchkiss (26), include binding of the allergen to extracellular proteins, with subsequent recognition by cell surface molecules on antigen presenting cells; binding of allergen to membrane-bound proteins on antigen presenting cells; and penetration of allergen to the interior of the antigen presenting cell before binding to intracellular protein. At one extreme, any protein that the allergen happens to encounter could react and subsequently be processed leading to presentation by antigen-presenting cells. At the other extreme, there could be specific proteins in specific locations whose function is to react with xenobiotic electrophiles so as to produce an immune response. In any event, it is well-recognized that for many classes of electrophilic chemicals, including saturated and unsaturated sultones, skin sensitization potential is an increasing function of hydrophobicity (27), suggesting strongly that the relevant protein or proteins are in a lipid environment, such as a cell membrane (3, 8). This concept is a key feature of the RAI model for QSARs in skin sensitization. It is therefore appropriate to consider how the relative reactivities listed in Table 4 could vary according to the reaction medium. The Hughes-Ingold principles for solvent effects on reaction rates (18) predict that the reactions of both sultones with primary amines such as n-butylamine or lysine units of protein) will be faster in aqueous solution than in a nonpolar medium. Some idea of the magnitude of such solvent effects can be gained from the MBL data on reactions of n-butylamine and diethylamine with 1: Changing from water to chloroform reduces the

rate constants by a factor of at least 3000. This is consistent with the generation from neutral reagents of transition states having substantial zwitterionic character. On the other hand, the reactions of the sultones with charged nucleophiles such as phenolate and thiolate ions, or ionized tyrosine units and cysteine units of proteins, will be faster in a nonpolar medium than in water. For a given nucleophile, whether the saturated or the unsaturated sultones will be more sensitive to solvent effects could be predicted if the rate-determining step in the reaction of the latter were known. For example, if the rate-determining step in reaction with phenolate ion or ionized tyrosine units with 1 is substitution at position 3, then the relative reactivities of 1 and 2 to these nucleophiles should not be greatly influenced by the nature of the medium. If the rate-determining step in reaction of butylamine or lysine units with 1 is addition at position 2, then the reactivity of 1 would be predicted to be reduced more than that of 2 by changing the reaction medium from water to a less polar solvent [cf. our finding of k(1)/k(2) < 2 for reaction with n-butylamine in aqueous dioxan]. Why Alk-1-ene-1,3-sultones and Alkane-1,3-sultones Differ in Their Skin Sensitization Properties. Overall, the reactivities of the alkene sultones 1 and the alkane-1,3-sultones 2 toward nucleophiles have not been shown to be greatly different. Of the nuceleophiles representing those commonly found in proteins, the thiolate ions are clearly the most reactive, toward both alkene sultones 1 and alkane sultones 2. Thus, cysteine units, if present in the proteins whose modification leads to skin sensitization, are the most likely targets for both sultones. To put the reactivity differences between 1 and 2 into context, we compare them in Table 7 with those for other pairs of structurally similar skin-sensitizing electrophiles for which skin sensitization data are available (3, 8, 28, 29). Clearly, the evidence does not support the concept that the differences in skin sensitization properties between 1 and 2 can be attributed to their different reactivities toward protein nucleophiles. The unsaturated sultones 1 are much more potent sensitizers, in comparison to the saturated sultones 2, than would be expected purely on the basis of their observed reactivity toward any of the nucleophiles studied by us or by MBL. The key difference between the two types of sultones is that the unsaturated sultones 1 react predominantly as Michael type electrophiles, and the saturated sultones 2 react as SN2 electrophiles. Thus, they belong to different reaction mechanistic applicability domains. When a saturated sultone 2 reacts with carrier protein by the SN2 mechanism, an anionic sulfoalkyl group becomes covalently bound to the protein, whereas when an alk-2-ene-1,3-sultone 1 reacts by nucleophilic addition, the group that becomes covalently bound to carrier protein is a nonionic sultone entity. If the reaction stops at this point, the difference in specificity is obvious: The two groups bound to carrier protein are sufficiently different that a T-cell receptor able to bind strongly to


the anionic sulfoalkyl group (i.e., recognize it as an antigenic determinant) is unlikely to recognize the nonionic sultone entity and vice versa. Furthermore, the noncovalent interactions of these groups with other parts of the carrier protein molecule will be different. Thus, conformational changes in the tertiary structure of the carrier protein, which can be recognized as antigenic determinants by T-cells, will be different. Similar arguments apply if, as is the case with most of the simple nucleophiles studied here, the initially formed nonionic sultone entity derived from 1 undergoes further reactions by nucleophilic attack at C3: This will result in a cross-linked structure, again with conformational changes to the carrier protein’s tertiary structure different from those produced by the saturated sultone 2. Thus, the difference in specificity can be rationalized. The large difference in sensitization potency may result from the cross-linking in the case of the alk-1-ene-1,3-sultones. Other cases are known where cross-linking agents are significantly more potent than would be expected based on their reactivity relative to other compounds reacting by the same mechanism. For example, mouse local lymph node assay (LLNA) skin sensitization potency of aldehydes and ketones, which can react via Schiff base formation, is well-correlated with reactivity and hydrophobicity parameters, but the cross-linking agents formaldehyde and glutaraldehyde are, respectively, 10 times and 1000 times as potent as predicted from the correlation established with noncross-linking homologues (6, 30). Why cross-linking should increase sensitization potency is not known. Our interpretation is that cross-linking produces more extensive changes in the carrier protein tertiary structure, resulting in a larger number of different antigenic determinants and consequently proliferation of a larger number of T-cell clones. This concept will be developed more fully elsewhere.

Appendix Estimation of Rate Constants from MBL Data. The reactions reported by MBL were carried out under pseudo firstorder conditions, with the nucleophiles in 10 or 30 M excess over the sultones. Although fully quantitative data are not given for the changes in composition of reaction mixtures, rough estimates of rate constants can be made as follows. 1. With n-Butylamine and Diethylamine. MBL report that in water, with excess n-butylamine at 0.45 mol/L, reaction of 1 was complete after 1 h. Bearing in mind that analysis is by 13C NMR, we can interpret “complete reaction” as beyond 4 halflives (6% of the original sultone would remain after 4 halflives). Thus, k(1, BuNH2, H2O) > 2 × 10-3 L mol-1 s-1. The same estimate applies for diethylamine with 1. For 1 in deuteriochloroform, MBL report that most of the sultone remains after 30 days, from which we can infer that the half-life is greater than 30 days. Thus, k(1, BuNH2, CDCl3) < 6 × 10-7 L mol-1 s-1. The same estimate applies for diethylamine with 1. For the reaction of n-butylamine (0.45 mol/L) with 2 in water, MBL report that after 11 h some 2 is still present, although most of it has reacted, and that the reaction is complete after 35 h. From this, we infer that 11 h corresponds to between two half-lives (75% reaction) and three half-lives (87.5% reaction). Thus, k(2, BuNH2, H2O) lies between 8 × 10-7 and 10 × 10-7 L mol-1 s-1. For the reaction of diethylamine (0.45 mol/L) with 2 in water, MBL report that the main product results from hydrolysis. Comparing with the finding for sodium phenolate (0.15 mol/L) with 2 in water (see below), we can conclude that the pseudo first-order rate constant for reaction of 2 with 0.45 M diethyl-

Roberts et al.

amine is less than the pseudo first-order rate constant for reaction of 2 with 0.15 M sodium phenolate. This gives an upper limit for the rate constant. Since, however, MBL state that the substitution product was observed and bearing in mind the detection limits in 13C NMR, we assume that at least 10% of the sultone reacts with the diethylamine. This gives a lower limit for the rate constant. Thus, we estimate the rate constant to be in the range 8 × 10-6 to 7 × 10-5 L mol-1 s-1. 2. With Sodium Phenolate in Water. From the data reported by MBL for reaction of a mixture of 1 and 2 with aqueous sodium phenolate (0.15 mol/L), we estimate rate constants as follows. In the 9 h 13C NMR spectrum of Figure 9 of ref 5, the signal due to 2 is clearly visible but minor as compared to those of the substitution product product and the hydrolysis product (formed in approximately equal amounts). We take the extent of reaction as 3 half-lives (from the NMR spectrum, it is unlikely to be as low as 2 or as high as 4 half-lives). On this basis and taking into account that the rate constant for disappearance of 2 is the sum of the rate constants for hydrolysis and reaction of phenolate ion, we get k(2, PhONa, H2O), ca. 2 × 10-4 L mol-1 s-1. For 1, the 7 day or 10 day (7 is given in the text, but 10 is given in the figure) 13C NMR spectrum of Figure 9 of ref 5 shows the signal due to 1 clearly visible but minor as compared to those of the substitution product (dominant) and the hydrolysis product (minor). Assuming 3 half-lives, we get k(1, PhONa, H2O), ca. 2 × 10-5 L mol-1 s-1. Thus, the alkane sultone 2 is about 10 times as reactive as the alkene sultone 1 towards sodium phenolate in water (and, by implication, towards ionized tyrosine units of HSA in water). This approximate estimate is unlikely to be out by much more than a factor of 2. 3. With Sodium Propanethiolate in Water. MBL report that for 1 with sodium propanethiolate (0.15mol/L) in water, the reaction was “rapid”, whereas the reaction with sodium phenolate in water was “much slower”. If we interpret “rapid” as “similar to the reaction of 1 with aqueous butylamine”, which MBL also describe as rapid and which they report to be complete after 1 h, we arrive at >6 × 10-3 L mol-1 s-1 for k (1, PrSNa, H2O). However, in the absence of any information as to the reaction time, a more conservative estimate can be made by interpreting “much slower” as indicating that sodium propanethiolate reacts at least twice as fast as sodium phenolate. On that basis, the conservative conclusion is that k (1, PrSNa, H2O) > 4 × 10-5 L mol-1 s-1. For reaction of 2 with sodium propanethiolate (0.15 mol/L) in water, MBL report that the substitution reaction was faster than the hydrolysis reaction, with the substitution product being the major product. By comparison with the information for reaction of sodium phenolate with 2 (similar amounts of hydrolysis and substitution products) and assuming at least twice as much substitution product as hydrolysis product in the case of sodium propanethiolate, we estimate that k (2, PrSNa, H2O) > 4 × 10-4 L mol-1 s-1. 4. With Imidazole in Water. MBL report that for 1 with imidazole (0.45 mol/L) in water, “some starting material was still present after 40 days”. Assuming 3 half-lives, we estimate k(1, imidazole, H2O), ca. 1 × 10-6 L mol-1 s-1. 5. Hydrolyis at pH 8.1. From the data reported by MBL for hydrolysis of a mixture of 1 and 2 with pH 8.1 buffer in the presence of HSA, we estimate rate constants as follows. After 4 h, the signal of 2 has, according to the text, almost disappeared, although in Figure 10 of ref 2 it is clearly visible. Assuming 3


half-lives, we estimate kobs (2, hydrolysis, pH 8.1 buffer), ca. 1.4 × 10-4 s-1. After 24 h, 1 is, according to the text “almost intact”, and from the spectrum reproduced in Figure 10 of ref 2, we see that the signal corresponding to the hydrolysis product is just visible. Assuming 5% reaction, we arrive at a figure of 0.002 h-1 for the rate constant. We can check this figure against the observation that after 34 days “some unsaturated sultone was still present”. From the spectrum reproduced in Figure 10 of ref 2, we infer the extent of reaction to be between 2 and 3 half-lives, corresponding to a rate constant values between 0.0017 and 0.0025 h-1 (note that the small difference between these figures illustrates the relatively small error in under- or overestimating the number of half lives). We thus arrive at kobs (1, hydrolysis, pH 8.1 buffer), ca. 5.5 × 10-7 s-1. The saturated sultone 2 is about 250 times more reactive than the unsaturated sultone 1 towards hydrolysis in aqueous solution. Note Added after Print Publication: Because of an e-mail transmission error, many of the author-submitted corrections were inadvertently not made in the version posted on the Web January 16, 2007 (ASAP) and published in the January 2007 issue (Vol. 20, No. 1, pp 61-71). The correct electronic version of the paper was published on March 19, 2007, and an Addition and Correction appears in the March 2007 issue (Vol. 20, No. 3).

References (1) Lepoittevin, J.-P., Basketter, D. A., Goosens, A., and Karlberg, A.T., Eds. Allergic Contact Dermatitis. The Molecular Basis, Springer, Heidelberg. (2) Roberts, D. W., Aptula, O. A., and Patlewicz, G. Electrophilic chemistry related to skin sensitization. Reaction mechanistic applicability domain classification for a published data set of 106 chemicals tested in the mouse local lymph node assay. Chem. Res. Toxicol. 2007, 20, 44-60. (3) Roberts, D. W., and Williams, D. L. (1982) The derivation of quantitative correlations between skin sensitisation and physicochemical parameters for alkylating agents and their application to experimental data for sultones. J. Theor. Biol. 99, 807-825. (4) Aptula, A. O., and Roberts, D. W. (2006) Mechanistic applicability domains for non-animal based toxicological endpoints. General principles, and application to reactive toxicity. Chem. Res. Toxicol. 19 (8), 1097-1105. (5) Roberts, D. W., Aptula, A. O., Cronin, M. T. D., Hulzebos, E., and Patlewicz, G. (2006) Global (Q)SARs for skin sensitizations Assessment against OECD principles. SAR QSAR, in press. (6) Aptula, A. O., Roberts, D. W., and Patlewicz, G. (2006) Mechanistic applicability domains for non-animal based toxicological endpoints. QSAR analysis of the Schiff Base applicability domain for skin sensitization. Chem. Res. Toxicol. 19 (9), 1228-1233. (7) Ritz, H. L., Connor, D. S., and Sauter, E. D. (1975) Contact sensitization of guinea pigs with unsaturated and halogenated sultones. Contact Dermatitis 1, 349-358. (8) Goodwin, B. F. J., Roberts, D. W., Williams, D. L., and Johnson, A. W. (1983) Skin sensitisation potential of saturated and unsaturated sultones. In Immunotoxicology (Gibson, G. G., Hubbard, R., and Parke,

Chem. Res. Toxicol., Vol. 20, No. 1, 2007 71 D. V., Eds.) pp 443-448, Academic Press, London. (9) Roberts, D. W., and Williams, D. L. (1983) Sultones as by-products in anionic surfactants. Tenside Deterg. 20, 109-111. (10) Meschkat, E., Barratt, M. D., and Lepoittevin, J.-P. (2001) Studies of the chemical selectivity of hapten, reactivity and skin sensitization potency. 1. Synthesis and studies on the reactivity towards model nucleophiles of the 13C-labeled skin sensitizers hex-1-ene- and hexane1,3-sultones. Chem. Res. Toxicol. 14, 110-117. (11) Meschkat, E., Barratt, M. D., and Lepoittevin, J.-P. (2001) Studies of the chemical selectivity of hapten, reactivity and skin sensitization potency. 2. Studies of the covalent binding of the 13C-labeled skin sensitizers 2-[13C]- and 3-[13C]-hex-1-ene- and 3-[13C]-hexane-1,3sultones to human serum albumin. Chem. Res. Toxicol. 14, 118-126. (12) Roberts, D. W., Sztanko, S., and Williams, D. L. (1981) An improved route to alk-l-ene 1,3 sultones, involving sulphonation of R-olefins by means of a sulphur trioxide/dioxan complex. Tenside Deterg. 18, 113-116. (13) Trowbridge, J. R., and Sundby, B. (1972) Canadian Patent CA 894,830. (14) Roberts, D. W. (1998) Sulfonation technology for anionic surfactant manufacture. Org. Proc. Res. DeV. 2, 194-202. (15) Roberts, D. W., and Williams, D. L. (1990) Formation of sultones in olefin sulphonation. J. Am. Oil Chem. Soc. 67, 1020-1027. (16) Roberts, D. W. (1997) Kinetics and mechanism in olefin sulphonation. RiV. Ital. Sostanze Grasse 74, 567-570. (17) Blaser, B. (1965) U.S. Patent 3,164,608. (18) Isaacs, N. (1995) Physical Organic Chemistry, 2nd ed., Longman, Harlow, United Kingdom. (19) Bordwell, F. G., Osborne, C. E., and Chapman, R. D. (1959) The hydrolysis of sultones. The effect of methyl groups on the rates of ring-opening solvolysis. J. Am. Chem. Soc. 81, 2698-2705. (20) Hine, J. (1962) Physical Organic Chemistry, 2nd ed., International Student Edition, McGraw-Hill, New York and Kogakusha, Tokyo. (21) Jones, J. B., and Young, J. M. (1966) Carcinogenicity of lactones. I. The reaction of 4-methylbuteno- and 4-methylbutano-γ-lactones with primary amines. Can. J. Chem. 44, 1059-1068. (22) Meyle, E., Keller, E., and Otto, H.-H. (1985) Darstellung und eigenschaften von 2,3-disubstituirten 1,2-thiazetidin-1,1-dioxiden. Liebigs Ann. Chem. 802-812. (23) Kirby, A. J. (1980) Effective molarities for intramolecular reactions. AdV. Phys. Org. Chem. 17, 183-278. (24) Tamai, I., Yokoi, K., and Ichikawa, C. (1971) German Patent GE 2,164,179. (25) Lepoittevin, J. P., and Benezra, C. (1992) 13C-Enriched methyl alkanesulfonates: New lipophilic methylating agents for the identification of nucleophilic amino acids of proteins by NMR. Tetrahedron Lett. 33, 3875-3878. (26) Smith, C. K., and Hotchkiss, S. A. M. (2001) Allergic Contact Dermatitis. Chemical and Metabolic Mechanisms, pp 210-214, Tayor and Francis, London, United Kingdom. (27) Barratt, M. D., Basketter, D. J., and Roberts, D. W. (1997) Structureactivity relationships for contact hypersensitivity. In Allergic Contact Dermatitis. The Molecular Basis (Lepoittevin, J.-P., Basketter, D. A., Goosens, A., and Karlberg, A.-T., Eds.) pp 129-154, Springer, Heidelberg. (28) Roberts, D. W., and Basketter, D. A. (1990) A quantitative structureactivity/dose relationship for contact allergenic potential of alkyl group transfer agents. Contact Dermatitis 23, 331-335. (29) Roberts, D. W., and Basketter, D. A. (1997) Further evaluation of the quantitative structure-activity relationship for skin-sensitizing alkyl transfer agents. Contact Dermatitis 37, 107-112. (30) Aptula, A. O., Patlewicz, G., and Roberts, D. W. (2005) Skin sensitization: Reaction mechanistic applicability domains for structureactivity relationships. Chem. Res. Toxicol. 18, 1420-1426.

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