These methods are known to be nitration agents for phenolic compounds [19,20]. The reaction of AgNO₃ with BF₃·OEt₂ in acetonitrile and in a nitrogen atmosphere at room temperature resulted in yields of 72%–75%. Employing the reaction with Al(H₂PO₄)₃, the solid acid [21] with concentrated nitric acid at room temperature resulted in yields of 56%–61%. The use of this reagent provides the possibility of the catalytic oxidative coupling of thiols under mild conditions. The disulfides produced are isolated with excellent purity and good yields (Figure 1).

Nutritional analyses revealed that thiosulfonates 2 and 3 presented a toxic effect when ingested by larvae. The thiosulfonates 2 and 3, when incorporated in an artificial diet at 0.2% and 0.1%, respectively, reduced ECI and ECD and increased AD and metabolic cost (CM) for A. kuehniella larvae, when compared with the control. Thiosulfonate 2 significantly decreased both ECI and ECD by 21% and 45%, respectively, and AD and CM were increased by 6.5% and 5.5%, respectively, when compared with larvae of A. kuehniella that were reared on control diets. Thiosulfonate 3 decreased both ECI and ECD by 45% and 52%, respectively, and AD and CM were increased by 14% and 7%, respectively, when compared with larvae of A. kuehniella that were reared on control diets.

The AD value for larvae of A. kuehniella, in the present study, was increased throughout the feeding period of the experiment; this finding suggests that, during this treatment, the food remained for a greater time in the insect’s gut to allow the detoxification of the thiosulfonates. A greater AD would help to meet the increased demand for nutrients [22,23] and compensate for the deficiency in food stuff conversion (reduction in ECI and ECD), perhaps by diverting energy from biomass production to detoxification [20]. This behavior has also been observed by others [4,24]. A drop in ECI indicates that more food is being metabolized for energy and less is being converted to body mass, i.e., growth of the insect [14]. ECD also decreases as the proportion of digested food metabolized for energy increases [25]. We suggest that the reduction in ECD is likely to result from a reduction in the efficiency to convert foodstuffs into growth, perhaps by a diversion of energy from the production of biomass into detoxification of thiosulfonates increase in costs. Results show that there were problems in the conversion of the food assimilated, suggesting that during this treatment, the diet remained in the gut of the insect for a longer time to allow detoxifying of the effects caused by the thiosulfonates 2 and 3[3]. There was probably a greater expenditure of energy from the diet to be used in the process of degradation of thiosulfonates present and also the maintenance of the vital processes of the insect.

Studies were undertaken to evaluate the action of these thiosulfonates on the tryptic activity of the larvae of A. kuehniella, utilizing BAPNA as a synthetic substrate. Figure 3 shows the effects of different doses of thiosulfonates 2 and 3 (1 μg) on the in vitro activity of digestive proteinases from fourth instar larvae. The thiosulfonates 2 and 3 altered the tryptic activity of these proteinases by 20% and 28%, respectively.

Larvae reared on artificial diets containing thiosulfonates 2 (0.2%) and 3 (0.1%) were observed for trypsin proteinase activity. The enzyme assay showed that larvae fed on thiosulfonates 2 and 3 resulted in low levels of trypsin activity in the gut and in fecal material (Figure 4a,b). Tryptic activities in the midgets of fourth instar A. kuehniella larvae reared on artificial diets containing thiosulfonates 2 and 3 were altered by 59% and 41%, respectively (Figure 4a), and the tryptic activities in the feces were altered by 47% and 26%, respectively (Figure 4b), when compared with those of larvae on control diets. The decrease in the trypsin activities of feces of thiosulfonate-fed larvae suggest that thiosulfonates did not cause the rupture of the peritrophic membrane of A. kuehniella.

These thiosulfonate compounds presented a toxic effect on A. kuehniella larvae as they can block the trypsin enzyme involved in this insect’s digestion. In addition, all trypsin proteinase activities in thiosulfonate-fed larvae were sensitive to thiosulfonates, indicating that no novel proteolytic form resistant to thiosulfonates was induced in larvae reared on a diet containing these compounds. Similar results were observed with a Kunitz-type inhibitor (APTI) of 21 KDa isolated from the seeds of Adenanthera pavonina, the seed proteinase inhibitors act as a part of the plant defensive system against pest via inhibition of their proteolytic enzymes [3,26].

The TLCK (tosyl-l-lysine chloromethyl ketone) is an irreversible inhibitor of trypsin and has a sulfonyl group in its molecular structure. This compound binds covalently to a serine at the active site of trypsin, inhibiting its enzymatic action; the sulfonyl group does not participate directly in this connection [27]. Regarding the tested arylthiosulfonates, we suggest that they interact with trypsin but not by a covalent bond as it was observed that only a decreasing of trypitic activity, which might be due to hydrogen bonding to hydrophilic amino acids in the active site of this enzyme [27]. This would explain the greater toxicity of thiosulfonate 3 compared to thiosulfonate 2, since the latter compound has methoxyl groups.

Thiol (7 mmol) was dissolved in anhydrous acetonitrile (23 mL) in a round-bottom flask under a nitrogen atmosphere, before adding AgNO₃ (7.7 mmol) and BF₃·OEt₂ (0.8 mL) slowly to the solution. The reaction mixture was stirred for 17 h at room temperature. The reaction solution was then quenched with ice water, and the pH was increased to 7 by the careful addition of sodium bicarbonate saturation solution. The resulting solution was filtered and the filtrate was extracted with ethyl acetate, the organic layer was washed with distilled water and brine. The organic layer was dried over magnesium sulfate and the solvent was evaporated at reduced pressure; the resulting material was purified by flash chromatography using hexane-ethyl acetate (8:2) as eluent.

The Al(H₂PO₄)₃ catalyst was elaborated by addition of 85% H₃PO₄ (16 mL) in neutral Al₂O₃ (39 mmol) in a porcelain crucible. The mixture was heated at 220 °C in a muffle for 2 h. The mixture was then placed in a vacuum desiccator and cooled to room temperature. The catalyst, thus prepared, was then stored in sealed sample vial.

To thiol (32 mmol) was added Al(H₂PO₄)₃ (0.16 mmol) and concentrated HNO₃ (3 mL). The reaction mixture was stirred for 24 h at room temperature. The mixture was transferred to a dropping funnel, and ethyl acetate was added. The pH was increased to 7 by the careful addition of sodium bicarbonate saturation solution. The organic layer was washed with distilled water, and brine, and dried over magnesium sulfate. The solvent was evaporated at reduced pressure, and the resulting material was purified by flash chromatography using hexane-ethyl acetate (8:2) as eluent.

White solid; mp 36–38 °C, lit. 35–37 °C [29]. ¹H NMR (300 MHz, CDCl₃) δ 7.34 (m, 4H), 7.45 (m, 3H), 7.56 (m, 3H).¹³C NMR (CDCl₃) δ 127.5 (CH), 127.8 (C), 128.7 (CH), 129.4 (CH), 131.3 (CH), 133.5 (CH), 162.1 (C), 163.5 (CH), 142.9 (C). MS m/z (%): 250 [M⁺] (32); 141 (33); 125 (100); 109 (36); 77 (76).

White solid; mp 77–78 °C, lit. 77–78 °C [30]. ¹H NMR (300 MHz, CDCl₃) δ 2.63 (s, 3H), 2.41 (s, 3H), 7.13 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H). ¹³C NMR (CDCl₃) δ 21.4 (CH₃), 21.6 (CH₃), 124.5 (C), 127.5 (CH), 129.3 (CH), 130.1 (CH), 136.4 (CH), 140.4 (C), 142.0 (C), 144.5 (C). MS m/z (%): 278 [M⁺] (30); 155 (20); 139 (100); 124 (32); 123 (34); 91 (87).

White solid; mp 84 °C, lit. 84–85 °C [31]. ¹H NMR (300 MHz, CDCl₃) δ 3.81 (s, 3H), 3.85 (s, 3H), 6.83 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H). ¹³C NMR (CDCl₃) δ 55.4 (CH₃), 55.6 (CH₃), 113.7 (CH), 114.8 (CH), 118.8 (C), 129.8 (CH), 134.8 (C), 138.3 (CH), 162.1 (C), 163.4 (C). MS m/z (%): 310 [M⁺] (16); 171 (13); 155 (63); 140 (56); 139 (100); 125 (35).

Fourth instar larvae were cold-immobilized and dissected in cold 250 mM NaCl. The midguts were surgically removed from the larvae using tweezers. Only actively feeding larvae with guts that were filled with food were used. The gut portion taken was posterior to the proventriculus and anterior to the malpighian tubules. After removing all extraneous tissue and freeing the lumen of its contents by rinsing in 250 mM NaCl, the midgut tissues were homogenized in cold distilled water in a hand-held Potter-Elvehjem homogenizer immersed in ice. Midgut homogenates were centrifuged at 17,000× g for 20 min at 4 °C and the supernatants were collected in a known volume of appropriate buffer and used immediately as enzyme sources for enzymatic assays, and when necessary were stored at −20 °C.

Feces of the caterpillars were collected during the experiment and frozen (−20 °C). When necessary, they were macerated, homogenized in 200 mM Tris-HCl buffer (Tris-Hydroxymethylaminomethane), pH = 8.5, centrifuged at 20,000× g for 30 min at 4 °C and supernatants were used for in vitro enzymatic assays.

Trypsin-like enzymes of whole midgut extracts and fecal samples from A. kuehniella larvae, fed on diets containing thiosulfonates (2 and 3) and control diets were assayed using the synthetic substrate, N-benzoyl-dl-arginine p-nitroanilide (BAPNA), as described by Erlanger [32]. The linearity of the relationship between the changes in absorbance with time was checked to ensure that substrate concentrations were not limiting. Substrate and enzyme controls were run to ensure the validity of sample absorbance readings.