A theoretical insight into the reaction mechanisms of a 2,4,6-trinitrotoluene nitroso metabolite with thiols for toxic effects

2,4,6-Trinitrotoluene (TNT) is a class C carcinogen as rated by the Environmental Protection Agency.

Abstract

2,4,6-Trinitrotoluene (TNT) is a class C carcinogen as rated by the Environmental Protection Agency. One of the toxicity mechanisms of TNT is the covalent binding of its metabolites to critical proteins. However, knowledge about their molecular reaction mechanisms is scarce. Herein, we have provided density functional theory (DFT) simulation evidences for the reaction mechanisms of the nitroso metabolite of TNT with the sulfhydryl group of model thiols for the first time. The results show that the solvent-mediated proton-transfer mechanism plays a significant role in the entire process. For the formation of semimercaptal, the mechanism is slightly different from the previous one where the thiolate anion attacks the nitroso group. The rearrangement of semimercaptal needs to be triggered by an acid or hydrated ion (H₃O⁺), which is consistent with the previous assumption. The other pathway, the conversion of semimercaptal to hydroxylamine, has to overcome a higher barrier, although it does not need the participation of an acid or a hydrated ion. In addition, the details on transition states, intermediates and free energy surfaces for three reactions are given, which make up for the lack of experimental knowledge. These conclusions can help to deeply understand the toxic effects of TNT and other nitroaromatic explosives.

Introduction

As one of the most widely used explosives, 2,4,6-trinitrotoluene (TNT) was formerly manufactured on a large scale as a significant component of munitions. However, TNT is a class C carcinogen that causes cataracts, skin lesions, dermatitis, and urinary tract, kidney, and liver tumors.1,2 Its toxicity is mainly attributed to the redox cycling of free radicals and the covalent binding of nitroso and/or hydroxylamine metabolites with critical cellular proteins.3–5 Although the toxic effects of TNT were observed as early as the 1920s,6 the molecular mechanism was not well understood until now. The formation of covalent adducts with proteins (or DNA) is one of the toxic characterizations of several xenobiotics.7–9 Understanding the mechanism for this type of reaction is of universal significance. The main objective of this study is to focus on the reaction mechanism of nitroso metabolites of TNT with proteins.

Liu et al. had previously discovered the covalent-binding of TNT metabolites with proteins many years ago, with a particular emphasis on hemoglobin and hepatic proteins.10,11 Their primary aim was to find a simple means for monitoring the effects of chronic exposure to TNT. Afterwards, Leung et al. further explored the bioactivation of TNT metabolites that led to the covalent-binding of proteins.12 Their study clearly showed that the reaction of the nitroso intermediate of TNT with the sulfhydryl group of proteins results in the formation of protein adducts. Based on the knowledge from previous studies,13,14 they proposed a possible mechanism for the formation of TNT-protein adducts (bold arrows), as shown in Fig. 1. Evidently, in the proposed pathway, there are still deficiencies in detailed information for several significant processes (dotted arrows), such as the rearrangement of SeM to SuA and the conversion of SeM to 4HA. The absence of significant information limits deeper understanding of the toxicity mechanism of TNT, particularly the formation of TNT-protein adducts. In addition, previous studies have proposed a persuasive mechanism for the significant “semimercaptal” (SeM) formation first identified by Eyer et al.,15 that is, a thiolate anion from an acid–base equilibrium of the thiol adds to the nitroso group in a rapid nucleophilic reaction.16 However, Kazanis and McClelland found that both GSH and semimercaptal are predominantly in their neutral forms in the pH range of 6.5–8.17 Therefore, the previously proposed mechanism (attack by the thiolate anion) is not always reasonable, at least in the pH range of 6.5–8.

Fig. 1. Proposed pathways for TNT bioactivation and covalent binding with proteins (bold arrows), showing bioactivation at the 4-position only. The reduction of the nitro group at the 2-position can also form a reactive intermediate. Key: Cys, cysteine; GSH, γ-l-glutamyl-l-cysteinylglycine; SeM, semimercaptal; SuA, the rearranged product of semimercaptal (i.e., sulfinanilide); 4NO, 4-nitroso-2,6-dinitrotoluene; 4HA, 4-hydroxylamino-2,6-dinitrotoluene; 4A, 4-amino-2,6-dinitrotoluene.

In order to determine the reaction details for the covalent binding of TNT metabolites with proteins, we chose cysteine (Cys) as the main model compound to investigate the reaction mechanism by Density Functional Theory (DFT) calculations. From Fig. 1, we can see that the generation of SeM is one of the most important processes. However, SeM is only a transient intermediate, and quickly forms the rearranged product (SuA) or the hydroxylamine product (4HA), coupling with the formation of an equivalent amount of the oxidized form of RSH (RSSR) via two parallel pathways. As for the amino product (4A), the formation process is also ambiguous.13 The previous study proposed a pathway,17 however, there is no evidence for the formation of the crucial product, mercaptal. Thus, in this study, we mainly focus on the detailed thermodynamic information for three reaction processes, namely, 4NO to SeM, SeM to SuA, and SeM to 4HA. The bioactivation of TNT catalyzed by enzymes (TNT to 4NO) is also a very important process; we will report the corresponding results in another paper. We believe that the conclusions in this study would not only provide a valuable insight into the toxicity of TNT, but may also help to understand the toxicity of other nitroaromatic compounds.

Computational methods

All species along with the reaction pathways were fully optimized in conjunction with the SMD solvation model18 at the level of M06-2X/6-31G(d,p),19,20 and characterized by a frequency analysis to be minima (no imaginary frequency) or transition states (one and only one imaginary frequency). For all optimizations, the solvent effect was also considered synchronously at the same level of theory. The functional M06-2X and SMD solvation models have been used successfully to study the reactions of explosive systems.21–24 Therefore, we did not testify the reliability of the methods again. Since the reactions generally occur in water, we used the dielectric constant of water at 298.0 K, i.e., ε = 78.4. The energies were then improved by the single-point energy from the calculation of M06-2X/aug-cc-pVDZ levels.25 Zero-point vibrational energies and corrections in enthalpy, entropy and Gibbs free energy were also determined by the calculation of the analytic harmonic vibrational frequencies at the same theory level as that of the geometry optimization. In addition, the intrinsic reaction coordinate (IRC)26 path was traced to check the energy profiles connecting each transition state to the two associated minima of the proposed mechanism. All calculations were carried out with the Gaussian 09 suite of programs,27 and the default parameters of the program were used in all computations. The Gibbs free energies of all reactions were calculated using the standard expression, ΔG = ΔH – T·ΔS, where ΔG is the activation Gibbs free energy, and ΔH and ΔS are the activation enthalpy and entropy, respectively. T is the absolute temperature (298.0 K in this study).

Results and discussion

The formation of semimercaptal

Previous study has shown that the thiol will form a thiolate anion as the nucleophile to attack the nitroso group.15 Hence, we first studied the generation of the thiolate anion in water. Fig. 2a gives the potential energy surface (PES) of the Gibbs free energy for the transformation of cysteine (Cys) to its isomer (Cys′). This reaction needs to overcome a moderate barrier of ΔG^‡ = 36.5 kcal mol^–1 through the transition state TS1. Considering the systematic error of implicit solvent models, the barrier should be less than this value. However, it does not influence the conclusion. IRC calculations further confirm that the final products viaTS1 are a new cysteine (Cys′) and water (H₂O′) by exchanging a hydrogen between the initial Cys and H₂O, and not between the acid–base pair products of Cys^– and H₃O⁺. If the reaction generates Cys^– and H₃O⁺, it would be an endothermic reaction with a high endothermic value of 36.9 kcal mol^–1. Obviously, the pair of products is not stable, and easily return to the reactants if no other intervention factors are present (such as OH^–). Thereby, a mechanism that involves a single thiolate anion (Cys^–) attacking the nitroso group may be no longer tenable in the range of low pH values (e.g., pH = 6.5–8, as mentioned above). With the exception of Cys (numbered as 1), we also checked three other thiols, N-acetylcysteine (2), GSH (3) and 3,4-dichlorobenzenethiol (4), which were previously investigated in the experiments of Leung et al.12 The corresponding structures and thermodynamic data for the transition states are given in Fig. 2b and Table 1, respectively.

Fig. 2. (a) The free energy profiles for the deprotonation reaction of the thiol Cys (kcal mol^–1). The energies are relative to the initial reactants of Cys + H₂O and are mass balanced. (b) The molecular structures for the transition states (TSs). For TSs, the silver, blue, red, yellow and cyan spheres (or rods) represent H, N, O, S and C, respectively.

Table 1. The activation (ΔG^‡) and reaction (ΔG‡R) Gibbs free energies for the deprotonation reactions of four thiols (kcal mol^–1). ΔΔG‡R = ΔG‡R – ΔG^‡.

	ΔG‡	ΔG‡R	ΔΔG‡R
1	36.5	36.9	0.4
2	37.5	37.0	–0.5
3	35.1	37.0	1.9
4	31.5	28.9	–2.6

As shown in Fig. 2, the molecular structures of TS2–TS4 are very similar to that of TS1. The hydrogen of the sulfhydryl group transfers to H₂O and forms H₃O⁺, in which two of the H atoms are at an almost equal distance from the S atom in Cys^–. The data (ΔG‡R and ΔG‡R) in Table 1 clearly shows that the products of the acid–base pairs are still unstable, such as Cys^–/H₃O⁺. For 4, it has a slight barrier of 2.6 kcal mol^–1 for the reverse reaction. Hence, we infer that the acid–base pairs of all thiols should be unstable, at least in neutral conditions (pH ∼7). It may be the reason why the neutral forms of GSH and semimercaptal were found in the range of pH 6.5–8.17 Of course, if the alkali of sodium hydrate (e.g. NaOH) is added, as done in previous experiments,12,17 it can advance the formation of thiolate anions.

Then, we explored an alternative mechanism for the formation of SeM. We built a trimer model including 4NO (a nitroso metabolite of TNT), Cys and H₂O, considering the participation of water in a hydrogen transfer reaction. In fact, a proton-transfer mechanism mediated by solvent molecules, especially the water molecule, was prevalent.28–32 The corresponding PES of the Gibbs free energy and the structure of the transition states are shown in Fig. 3. We can see that the new reaction pathway containing H₂O passes through a transition state TS5 with a barrier of 34.2 kcal mol^–1 and an exothermicity of 6.6 kcal mol^–1. This barrier is slightly less than that of a dimer model with Cys and H₂O (36.5 kcal mol^–1 in Fig. 2a). Significantly, the IRC results show that TS5 directly connects the trimer complex and the semimercaptal product (SeM1), not their ion pairs. If the products are the ions SeM1^– and H₃O⁺, the barrier (ΔΔG‡R) of the reverse reaction is only 1.5 kcal mol^–1, as shown in Table 2. It again explains the reason why this experiment only found the neutral forms of GSH and semimercaptal.17

Fig. 3. (a) The free energy profiles for the formation of SeM1 (kcal mol^–1), which are relative to the initial reactants of Cys + H₂O + 4NO and are mass balanced, and the structure of TS5, (b) the molecular structures of TS6–TS8. For all TSs, the silver, blue, red, yellow and cyan spheres (or rods) represent H, N, O, S and C, respectively. The labelled distance is given in Å.

Table 2. The activation (ΔG^‡) and reaction (ΔG‡R) Gibbs free energies for the formation of four semimercaptals (kcal mol^–1). ΔΔG‡R = ΔG‡R – ΔG^‡.

	ΔG‡	ΔG‡R	ΔΔG‡R
1	34.2	–6.6	–1.5
2	31.1	–6.5	2.5
3	34.3	–6.2	–2.0
4	24.5	–3.9	8.4

For the above result, the participation of H₂O plays a crucial role. From Fig. 3, we can clearly see that TS5 forms a six-membered loop structure including water (H₂O), nitroso (NO) and a sulfhydryl group (SH). The IRC shows a complete process in which the hydrogen of the SH group first transfers to its neighbouring H₂O and forms the hydroxonium ion (H₃O⁺) because the distance between S and H is 1.90 Å (broken bond), but that between O and H is 1.06 Å (bonding) in TS5 (Fig. 3). It is similar to the situation without the addition of 4NO shown in Fig. 2. The difference occurs in the subsequent step where the oxygen of the NO group forms a hydrogen bond with the hydrogen of H₂O. Meanwhile, the hydrogen bond between H₃O⁺ and NO promoted the transfer of H from H₃O⁺ towards the O of the NO group. If there was no nitroso group, then the H of H₃O⁺ would return to the thiolate anion. However, the added nitroso group accepts the transferred hydrogen, which provides a chance for the thiolate anion (RS^–) and starts a nucleophilic attack on the neighbouring N atom. The distance of N···S is shortened to 2.61 Å in TS5 (Fig. 3). It finally forms the N–S covalent bond.

To check the universality of this model, we also calculated the PES for the reactions of three other thiols (i.e., 2, 3 and 4, respectively) in water. The data on the Gibbs free energies and transition states are given in Table 2 and Fig. 3, respectively. TS6, TS7 and TS8 for the other three thiols also form six-membered loop structures (Fig. 3b), and the corresponding bond lengths are also similar to that of TS5. The direct production of semimercaptal makes these reactions slightly exothermic, as shown in Table 2. The barrier of the reaction of 4 with 4NO is the lowest; 24.5 kcal mol^–1 in water. This is in agreement with the previous experimental findings,12 in which Leung et al. found that 4 can form the most amount of covalent adducts. Comparing the data in Tables 1 and 2, we can see that the presence of the solvent makes the barriers for the trimer reactions decline slightly in comparison with those of the dimer reactions, except for obtaining stable semimercaptal products. It clarifies that the essence of this reaction is the cleavage of the S–H bond. The solvent only promotes the proton-transfer, causing the direct formation of semimercaptal, and opposes the process of producing RS^–/ArN(OH)⁺ pairs. It is a new mechanism that is different from the previous one, where the thiolate anion attacks the nitroso group.16 Although our primary research focus is the nitroso metabolite of TNT, we believe that the new mechanism is also applicable to other nitroso compounds.

Rearrangement of semimercaptal to sulfinanilide

The SeM is an unstable intermediate, it prefers rearrangement and produces the isomer sulfinanilide (SuA). Up to now, the mechanism of this reaction was still elusive. Kazanis and McClelland17 proposed a mechanism whereby the first rate-limiting step involved the dissociative cleavage of the hydroxyl group, either in an unassisted manner where the hydroxide ion itself was the leaving group or with catalysis by buffer acids or H⁺, in which H₂O was the product. Herein, we provide a more detailed mechanism for the rearrangement of SeM to SuA. Since the above results showed that the first reaction step has no obvious difference in the four thiols (1–4), we only used the Cys (i.e.1) as a representative. First, we checked the leaving reaction of the OH group and the results are given in Fig. 4. The corresponding SeM1 needs to overcome a high barrier of 48.7 kcal mol^–1 to leave the hydroxyl ion. IRC confirms that the product is the isomer of SeM1, SeM1_i (Fig. 4), not the ion pairs, SeM1⁺/OH^– (with an energy penalty of 42.4 kcal mol^–1). Therefore, the hydroxyl ion as the leaving group is not preferred thermodynamically. It also supports the key finding of the previous study that the O in the S O group is derived from the solvent, not from the O of the starting NO group.17

Fig. 4. The free energy profiles for the transfer reaction of the hydroxyl group in SeM1 from N to S atoms (kcal mol^–1), and the molecular structures of SeM1, SeM1_i and TS9. For TS9, the silver, blue, red, yellow and cyan spheres (or rods) represent H, N, O, S and C, respectively.

Then, we explored the other pathway (see Fig. 5), in which the initial step is when the hydronium ion (H₃O⁺) is used to capture the OH^– group. The generated cationic intermediate IN1 can further react with a solvent water molecule at the site of the S atom. Subsequently, the formed isomer SeM1_i could generate the final rearrangement product, sulfinanilide (SuA), via the two H₂O molecules-mediated proton-transfer. For the first step of the OH^– cleavage viaTS10, as shown in Fig. 5, it needs to overcome an energy barrier of 19.1 kcal mol^–1. The formation of IN1 also brings an exothermicity of 15.1 kcal mol^–1. From IN1 to SeM1_i, there are three water molecules that are involved in the reaction. Among them, a H₂O molecule with a “hard nucleophile” prefers to use the O atom to attack the S atom with a “hard electrophile” because the charge of the S atom obviously increases (0.29 to 0.70 Mulliken charge) after OH^– leaves.33 The sequence from IN1 + 3H₂O to SeM1_i + H₂O + H₃O⁺ proceeds with a barrier of 21.3 kcal mol^–1, and is exothermic by 10.8 kcal mol^–1. We also investigated the reaction with the participation of two water molecules. Unfortunately, no transition states could be found. Probably, the third H₂O molecule is indispensable. After the production of the isomer SeM1_i, it easily transforms into the final product SuAvia the transition state of TS12 with a relatively low barrier of 14.8 kcal mol^–1, and gives an exothermicity of 11.8 kcal mol^–1. For TS12, it also forms a loop-structure like those of TS5–TS8 (Fig. 3), which can promote the proton-transfer by a water bridge.

Fig. 5. The free energy profiles for the rearrangement reaction of SeM1 to SuA1 (kcal mol^–1), which is mass balanced, and the molecular structures of the key intermediates, TSs and products. For all TSs, the silver, blue, red, yellow and cyan spheres (or rods) represent H, N, O, S and C, respectively. The labelled distance is given in Å.

The conversion of semimercaptal to hydroxylamine

Previous study has inferred that the formation of N-arylhydroxylamine corresponds to the mechanism of RS^– attacking the S of semimercaptal,17 where the solvent simultaneously transfers a proton to N. The transition state would be described to exhibit an ArN^–(OH) character, with the proton transfer lagging behind the breaking of S–N bond. However, we still think that it should be a concerted process, whereby the solvent (H₂O) mediates the proton-transfer from the thiol (RSH) to N and the formed RS^– attacks the S under neutral conditions because the thiol (RSH) is unlikely to form RS^– without the assistance of alkaline reagents (Fig. 2). We further investigated our proposed concerted reaction mechanism in detail. The results are given in Fig. 6.

Fig. 6. The free energy profiles for the conversion of semimercaptal (SeM1) to hydroxylamine (4HA) (kcal mol^–1), and the molecular structure of TS13 and product CSSC. For TS13, the silver, blue, red, yellow and cyan spheres (or rods) represent H, N, O, S and C, respectively. The labelled distance is given in Å.

Herein, we take the reaction of SeM1 with Cys as an example. From the above analysis, SeM1 results from the water-mediated proton-transfer reaction between 4NO and Cys. After that, when the second Cys comes closer to SeM1, the system will proceed with another water-mediated proton-transfer reaction, and generates a hydroxylamine product 4HA and an oxidized product CSSC, as shown in Fig. 6. This reaction needs the participation of two water molecules. From the structure of TS13, we know that Cys first donates a hydrogen to the oxygen atom of the neighbouring water (the distance of S···H is 2.22 Å). Then, the other water molecule transfers a proton to the nitrogen atom of SeM1 (the distance of N···H is 1.58 Å). Simultaneously, the N–S bond of SeM1 is elongated from 1.72 Å to 1.91 Å, which results in the adjacent positioning of two S atoms (the distance of S···S is 2.64 Å). Interestingly, the transition state TS13 forms an eight-membered loop structure with two water molecules. It is this ring structure that promotes the proton-transfer process. We also tried to find the transition state via a six-membered loop structure with one water molecule, but were unsuccessful. It is slightly different to the formation of semimercaptal (Fig. 3). Specifically, water molecules participate in the conversion of semimercaptal (SeM1) to hydroxylamine (4HA), which supports previous speculations.15 The difference is that the mechanism described in this study is a concerted reaction, not an asynchronous procedure.

Comparison

To entirely view the reaction of the nitroso metabolite (4NO) of TNT with the thiols, all the corresponding potential energy surfaces (PES) for the above three reaction pathways (the formation of semimercaptal, the rearrangement of semimercaptal to sulfinanilide, and the conversion of semimercaptal to the hydroxylamine product) are summarized in Fig. 7.

Fig. 7. The summarized ΔG potential energy surfaces for the metabolite of TNT (4NO) with the model thiols. The corresponding compounds represented by the notations can be seen in the above figures.

For the formation of semimercaptal from the reactions of 4NO with different compounds with a sulfhydryl group, there is a moderate free energy activation barrier of 34.2 kcal mol^–1 for cysteine (1), as shown in Fig. 7. The reaction with GSH (3) has a very similar barrier (34.3 kcal mol^–1). The lowest barrier is of 24.5 kcal mol^–1 for the reaction of 4NO with 3,4-dichlorobenzenethiol (4), which illustrates that this reaction occurs easily compared with those of the other three thiols. The result is also supported by the findings of Leung et al.12 The generated semimercaptal (SeM1 as an example) can rearrange into the isomer sulfinanilide (SuA1, the S O bond is a marked characteristic). For this unknown and complex process, we initially proposed two possible paths for the production of the other isomer SeM1_i. However, in the first pathway, the direct transfer of OH (Fig. 4) is easily ruled out because of the high barrier of 48.7 kcal mol^–1. The result is also consistent with the ¹⁸O-tracer experiment that shows the S O oxygen is derived from the solvent.15,16 The other pathway follows the catalysis of the hydronium ion (H₃O⁺), as proposed by Kazanis and McClelland,17 which captures the S–OH hydroxyl group and produces ArN⁺SR and H₂O. This reaction needs to overcome a barrier of 19.1 kcal mol^–1 through TS10, which is far less than that of the first route. The following reactions include the participation of two water molecules, which donate a hydroxyl group to ArN⁺SR simultaneously, to produce a new H₃O⁺, in which the highest barrier is 21.3 kcal mol^–1. Obviously, the second pathway is preferred thermodynamically. In addition, the conversion from semimercaptal to hydroxylamine is a competing pathway. However, it has a relatively high barrier of 38.3 kcal mol^–1, which substantially decreases the competitive edge for this route. It is worthwhile to note that the above possible mechanism for the rearrangement of semimercaptal needs acidic conditions or H₃O⁺, however, the conversion of semimercaptal to hydroxylamine does not.

Conclusions

In conclusion, considering the importance of the toxicity mechanisms of TNT and its metabolites, we have provided detailed evidences at the molecular level for the formation of semimercaptal, which is produced from the reactions of the nitroso metabolites of TNT with four thiols (cysteine, N-acetylcysteine, GSH, 3,4-dichlorobenzenethiol), which are important constituents of proteins, and the subsequent transformation, respectively. Firstly, we found that the water-mediated proton-transfer mechanism plays a significant role in several key processes, such as the formation of semimercaptal and the conversion of semimercaptal to hydroxylamine. The essence of water participation in the reactions is that a water molecule directly interacting with the nitroso and sulfhydryl group can stabilize the RS^–/H₃O⁺ ion pair and act as a proton transporter by forming a complex with a six (or eight)-membered loop structure. Otherwise, the stable thiolate anion cannot be directly formed. Since the products of the RS^–/H₃O⁺ ion pairs generally have higher free energy than the transition states, the reverse reactions are more thermodynamically favorable. Thus, the key for water-mediated proton-transfer reactions depends on the ability of thiols to donate the proton. The result can be easily obtained by comparing the ΔG^‡ data of Tables 1 and 2. Secondly, the rearrangement of semimercaptal needs the hydrated ions to trigger the first-step reaction with a low barrier of 19.1 kcal mol^–1. The subsequent reaction where a water molecule provides a hydroxyl group to the N atom with a positive charge has the highest barrier of 21.3 kcal mol^–1, and is the rate-limiting step. In the final step, the proton-transfer reaction involving two water molecules participates in the reactions again and produces the rearranged product with a S O group. To the best of our knowledge, this is the first molecular mechanism showing the rearrangement of semimercaptal. Finally, the molecular mechanism for the conversion of semimercaptal to hydroxylamine is clearly provided, which is also the water-mediated proton-transfer reaction. It has a relatively high barrier of 38.3 kcal mol^–1. The advantage of this competing reaction is that it does not require acidic conditions. This study shows the first clear molecular reaction mechanism for the nitroso metabolites of TNT binding covalently with proteins. Since the covalent binding of TNT metabolites to critical proteins is of significance to TNT toxicity, we hope that the results of this study can help to understand the toxicity of TNT in depth, and apply in further treatments.

Conflicts of interest

There are no conflicts to declare.