Glutathione Peroxidase-Like Activity of Amino-Substituted Water-Soluble Cyclic Selenides: A Shift of the Major Catalytic Cycle in Methanol

Abstract

We previously reported that water-soluble cyclic selenides can mimic the antioxidative function of glutathione peroxidase (GPx) in water through a simple catalytic cycle, in which the selenide (>Se) is oxidized by H₂O₂ to the selenoxide (>Se=O) and the selenoxide is reduced by a thiol back to the selenide. In methanol, however, the GPx-like activity could not be explained by this simple scenario. To look into the reasons for the unusual behaviors in methanol, monoamino-substituted cyclic selenides with a variable ring size were synthesized, and the intermediates of the catalytic cycle were characterized by means of ⁷⁷Se-NMR and LC-MS spectroscopies. In water, it was confirmed that the selenide and the selenoxide mainly contribute to the antioxidative function, though a slight contribution from the dihydroxy selenane (>Se(OH)₂) was also suggested. In methanol, on the other hand, other active species, such as hydroxyselenonium (>Se⁺-OH) and hydroxy perhydroxy selenane (>Se(OH)(OOH)), could be generated to build another catalytic cycle. This over-oxidation would be more feasible for amino-substituted cyclic selenides, probably because the ammonium (NH₃⁺) group would transfer a proton to the selenoxide moiety to produce a hydroxyselenonium species in the absence of an additional proton source. Thus, a shift of the major catalytic cycle in methanol would make the GPx-like antioxidative function of selenides perplexing.

Keywords: antioxidant; enzyme model; glutathione peroxidase; hydroxy perhydroxy selenane; selenide; selenoxide.

Scheme 1

Proposed glutathione peroxidase (GPx)-like catalytic cycle of a selenide. (A) selenide/selenoxide redox pathways mainly observed in water [26,27,28,29]; (B) another catalytic cycle mediated by hydroxyselenonium 3 and hydroxy perhydroxy selenane 4 as a highly active oxidant [32].

Figure 1

Target compounds in this study (5 and 6) and previous selenides (7‒9). Compounds 8 and 9 are racemic compounds.

Scheme 2

Synthesis of monoamino selenides 5 and 6. Reagents and conditions: (i) (1) Boc₂O, Et₃N, 1,4-dioxane/H₂O (5:2), 50 °C, 18 h, (2) NaBH₄, EtOH, reflux, 1 h, (3) MsCl, Et₃N, CH₂Cl₂, 20 h, room temperature (rt); (ii) (1) EtOH, AcCl, reflux, 4 h, (2) Boc₂O, Et₃N, 1,4-dioxane:H₂O = 5:2, 50 °C, 18 h, (3) NaBH₄, EtOH, reflux, 1 h, (4) MsCl, Et₃N, CH₂Cl₂, 20 h, rt; (iii) NaHSe, iPrOH/1,4-dioxane, reflux, 2.5 h for synthesis of 13a from 12a; NaHSe, EtOH/THF, reflux, 3 h for synthesis of 13b from 12b; (iv) HCl, H₂O/EtOH, 35 °C, 20 h. ^a Details of the synthesis are given in Supporting Information.

Figure 2

LC–MS (atmospheric-pressure chemical ionization, APCI+) spectral changes during the oxidation of the selenoxide form of selenide 6 in H₂O at 25 °C. For a‒c, H₂O (100%) was used as an eluent for the LC under a continuous flow at 0.3 mL/min, and 3 μL of the sample solution was injected into the LC and analyzed by the APCI+ mode. Reaction conditions: (a) Selenide 6 (0.038 mmol) and H₂O₂ (0.038 mmol) were mixed in H₂O (800 μL); (b) to a was added H₂O₂ (0.15 mmol); (c): to b was added HCl (0.15 mmol).

Figure 3

GPx-like activity assay in buffer solution and in methanol. (A) Nicotinamide adenine dinucleotide phosphate (NADPH)-coupled GPx assay for selenides 5–9. Reaction conditions were [GSH]₀ = 1.0 mM, [H₂O₂]₀ = 2.5 mM, [NADPH]₀ = 0.3 mM, [glutathione reductase] = 4 units/mL, and [selenide] = 0.2 mM in pH 7.4 phosphate buffer at 25 °C. (B) Percentages of residual dithiothreitol (DTT^red) as a function of reaction time in the oxidation of DTT^red with H₂O₂ in the presence of a selenide catalyst (5–9) in CD₃OD. Reaction conditions were [DTT^red]₀ = [H₂O₂]₀ = 0.14 M and [selenide] = 0.014 M at 25 °C. Data for 7‒9 were quoted from Reference [29].

Figure 4

LC–MS (APCI+) spectral changes during the redox reactions of 7 in MeOH at 25 °C. MeOH (100%) was used as an eluent for the LC. Reaction conditions: (a) selenide 7 (0.038 mmol) in MeOH (800 μL); (b) to a was added H₂O₂ (0.038 mmol).

Scheme 3

Oxidation of a nucleophile (NuH) by dihydroxyselenane 3a.

Scheme 4

A proposed equilibrium between an amino-substituted cyclic selenoxide and the corresponding hydroxyselenonium 3.