Free radical induced selenoxide formation in isomeric organoselenium compounds: the effect of chemical structures on antioxidant activity†

Beena G. Singh *^ab, Pavitra Kumar ^ab, P. Phadnis ^c, Michio Iwaoka ^d and K. Indira Priyadarsini *^bc
aRadiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India. E-mail: beenam@barc.gov.in
^bHomi Bhabha National Institute, Anushaktinagar, Mumbai-400092, India. E-mail: kindira@barc.gov.in
^cChemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India
^dDepartment of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan

First published on 26th July 2019

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Introduction

Organoselenium compounds constitute a unique class of antioxidants due to their versatile redox properties. Such compounds exhibit antioxidant properties like free radical scavenging and glutathione peroxidase (GPx) like catalytic activity.^1,2 Studies on several synthetic molecules identified the effect of important structural motifs such as aromaticity, non-bonding interactions, ring size, and nature of functional groups on the GPx like activity.^3–5 Romano et al. have found that the antioxidant activity of aromatic selenocyanates is higher than that of the aliphatic selenocyanates and Rafique et al. have showed that 2-picolylamide-based diselenides having non-bonding interactions showed higher activity.^6,7 Engman et al. have reported that the ring size in cyclic seleno-tocopherols affects their radical trapping ability.⁸ Similarly, Back et al. reported that the GPx activity of a five membered spirodioxaselenanonane was higher than that of a six membered ring.⁹ Davies et al. have reported that the rate constant for the reaction of various free radicals and molecular oxidants with biologically relevant organoselenium compounds is an important parameter.¹⁰

In search of water soluble and low molecular weight antioxidants/radioprotectors, our group has studied the hydroxyl (˙OH) radical reaction with different classes of organoselenium compounds by the pulse radiolysis technique.¹¹ The studies have shown that the initial attack of the ˙OH radical takes place at the selenium centre to form a hydroxyselenouranyl radical adduct (Se∴OH) and the fate of this transient depends on the nature of the functional group (hetero atom) attached to the selenium centre. Of the several compounds screened in our laboratory, trans-3,4-dihydroxyselenolane (DHS), a water-soluble organoselenium compound, has been found to reduce ˙OH radicals, hydroperoxides and peroxynitrite catalytically through the formation of a stable selenoxide, which is reverted back to DHS by thiols.¹² Furthermore, this antioxidant activity was also verified using in vitro and in vivo models.¹³ In contrast, its linear analogue bis(ethan-2-ol)selenide (SeEOH) showed low GPx like activity and was toxic to cells.¹⁴ Therefore, to understand the contrasting behaviour, two structural analogues have been investigated for their reactivity with ROS and the yields of the products formed were estimated. To further complement the observations, quantum chemical calculations have been carried out. The structures of DHS, SeEOH and their selenoxides are given in Scheme 1.

Results

Kinetics of reaction of DHS/SeEOH with different ROS

As reported earlier, SeEOH and DHS reacted with ˙OH radical with bimolecular rate constants of (1.0 ± 0.1) × 10¹⁰ M⁻¹ s⁻¹ and (9.1 ± 0.1) × 10⁹ M⁻¹ s⁻¹, respectively.^12,14 To further understand the antioxidant activity under different conditions, the rate constants for the reaction of these compounds with peroxynitrite, carbonate (CO₃˙⁻) radical and the nitrogen dioxide (NO₂˙) radical were studied. Under physiological conditions peroxynitrite readily reacts with CO₂ to form nitroso-peroxocarbonate which decomposes to form the CO₃˙⁻ radical and nitrogen dioxide NO₂˙ radical, which are important for peroxynitrite induced oxidative stress.^15,16

The reaction between DHS/SeEOH and peroxynitrite was studied by employing competition kinetics using dihydrorhodamine 123 (DHR123) as a reference solute.¹⁵

	(1)

	(2)

The bimolecular rate constant (k₂) was estimated by plotting (F₀/F) − 1 as a function of ((SeEOH or DHS)/DHR123) according to eqn (3):

	(3)

where F₀ and F are the intensities of fluorescence of R123 in the absence and presence of SeEOH/DHS, respectively, and k₁ (8.2 × 10³ M⁻¹ s⁻¹) is the bimolecular rate constant for the reaction of DHR123 with peroxynitrite.^12d The estimated values for k₂ were (9.2 ± 0.1) × 10² M⁻¹ s⁻¹ and (2.2 ± 0.2) × 10³ M⁻¹ s⁻¹ for SeEOH and DHS respectively (Fig. S1, ESI†). The results indicate that DHS showed higher rate constant in neutralizing peroxynitrite as compared to SeEOH.

The rate constant for the reaction of DHS with the CO₃˙⁻ radical was reported to be (1.2 ± 0.2) × 10⁹ M⁻¹ s⁻¹.¹² In the present study, SeEOH was tested for its ability to scavenge CO₃˙⁻ and NO₂˙ radicals using the pulse radiolysis technique. The kinetics between SeEOH and the CO₃˙⁻ radical was estimated by following the characteristic decay of CO₃˙⁻ at ∼600 nm (ε_600nm = 1680 M⁻¹ cm⁻¹) in the presence of varying concentration of SeEOH. Fig. S2 (ESI†) shows the absorption–time plot of the CO₃˙⁻ radical in the absence and presence of 10 μM SeEOH. In the absence of SeEOH, the CO₃˙⁻ radical decayed by second order kinetics with a 2k/εl value of (5.7 ± 0.1) × 10⁷ s⁻¹ at pH 7.4. On addition of 10 μM SeEOH, the CO₃˙⁻ radical decayed very fast and the reaction was found to follow pseudo-first order kinetics (k_obs). The bimolecular rate constant was estimated from the slope of the linear plot of k_obs at different concentrations of SeEOH (10–100 μM), and was found to be (6.5 ± 0.3) × 10⁸ M⁻¹ s⁻¹. Both DHS and SeEOH did not show any reaction with the NO₂˙ radical, which could be due to the unfavourable redox potential of these compounds.

To further understand the mechanism of electron transfer in these compounds, the nature of the transients produced during these ROS reactions was investigated as discussed below.

Transient studies

The reaction of SeEOH and DHS with the ˙OH radical formed two centered three electron bonded dimer radical cations (Se∴Se)⁺, which decayed by following second order kinetics with 2k/εl of (2.7 ± 0.2) × 10⁵ s⁻¹ and (3.9 ± 0.3) × 10⁵ s⁻¹ for DHS and SeEOH, respectively. Using the reported ε value for the (Se∴Se)⁺ radical of DHS, and presuming a similar value for SeEOH, the 2k value for DHS and SeEOH was estimated to be (1.6 ± 0.1) × 10⁹ and (2.3 ± 0.2) × 10⁹ M⁻¹ s⁻¹, respectively. This indicates that the (Se∴Se)⁺ radical of DHS is more stable than that of SeEOH. In the case of SeEOH along with the (Se∴Se)⁺ radical, a species absorbing at 320 nm was attributed to a carbon centred radical of the type α-(hydroxyl ethyl) seleno methyl radical (HOCH₂CH₂SeCH₂˙) or α-reducing radical (–SeC˙). Such radicals are formed by rearrangement of the (Se∴OH) radical at the α-position to the selenium centre followed by elimination of water and formaldehyde (HCHO) molecules via the Barton reaction.¹⁷ In the case of DHS, no indication for α-reducing radical formation was observed.

The relative yield of the radical cations, estimated by the ABTS˙^−18,19 reaction, was found to be 1.8 times higher for DHS than SeEOH. As reported earlier, the (Se∴Se)⁺ radical undergoes radical–radical disproportionation to form selenoxides, as estimated below.

Product analysis by HPLC

Selenoxides and formaldehyde products formed during the reaction of ˙OH radicals with SeEOH/DHS were identified and quantified by HPLC measurements.

Discussion

In the present study two isomeric compounds, DHS and SeEOH, were investigated to understand the effect of small structural alteration on their ROS scavenging activity. DHS and SeEOH were examined for their ability to scavenge ROS like ˙OH and CO₃˙⁻ radicals and peroxynitrite. The rate constants for the ˙OH radical reaction for both SeEOH and DHS were comparable. However the rate constant with peroxynitrite was two times more for DHS as compared to SeEOH. To explain this significantly different activity by a minor change in the structure, a detailed mechanism of the radical reaction with DHS/SeEOH was studied. The overall reaction of SeEOH and DHS is depicted in Scheme 2.

On reaction with ˙OH radical, both the compounds were converted to their corresponding selenoxide, where DHS produced a significantly higher amount of selenoxide than SeEOH.

The experimental results were complemented by calculating the energetics determined by quantum chemical calculations. The HOMO energy of DHS is higher than that of SeEOH indicating the easier oxidation of DHS. The reaction of initial addition of the ˙OH radical on the selenium atom to form a (Se∴OH) adduct is endothermic in SeEOH (ΔE = +3.48 kcal mol⁻¹) but exothermic in DHS (ΔE = −17.29 kcal mol⁻¹). This (Se∴OH) adduct generally decays to form (Se∴Se)⁺. The dimerization process was found to be more exothermic in DHS as compared to SeEOH. Additionally, the stability of the dimer radical cation of DHS is due to the direct interaction between selenium and oxygen of DHS and this prevents deprotonation to form a carbon centred radical. Thus a higher HOMO value coupled with higher stability of the intermediate (Se∴Se)⁺ results in a higher yield of selenoxide formation and thereby resulting in better antioxidant activity of DHS as compared to SeEOH. The selenoxide being reversible in the presence of thiols imparts higher antioxidant activity in DHS than in SeEOH against ROS.

Experimental

The organoselenium compounds studied were synthesized as per the reported method.^14a,c 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid-sodium salt) (ABTS²⁻), DHR123, diphenylpicrylhydrazine (DNPH), dithiothreitol reduced (DTT) and oxidized dithiothreitol (DTT_ox) were purchased from Sigma Aldrich and were of >95% purity. All the other chemicals and reagents were of “Analar” grade and used as such. The solutions were freshly prepared for each experiment in nanopure water with a conductivity of 0.1 μS cm⁻¹, obtained from a Millipore water purification system. The pH of the solutions was adjusted using monosodium phosphate (NaH₂PO₄), disodium phosphate (Na₂HPO₄·2H₂O) and perchloric acid (HClO₄).

Peroxynitrite was prepared by the ozonolysis of 0.1 M sodium azide solution containing 0.1 M NaOH.²³ For all experiments stock peroxynitrite solutions were diluted using 10 mM NaOH and during the experiments, the pH was maintained at 7.4 by using 70 mM phosphate buffer. The concentration of peroxynitrite was estimated by measuring the absorbance at 302 nm (ε_302nm = 1705 M⁻¹ cm⁻¹) on a UV-Visible spectrophotometer (Jasco V-639).

Pulse radiolysis studies were carried using a 7 MeV electron beam with 100 ns pulse width and the details of the instrument are reported. An average dose of 9 Gy as estimated by using a thiocyanate dosimeter (aerated aqueous solution of 10 mM KSCN, G_ε475nm = 2.59 × 10⁻⁴ m² J⁻¹) was used for all experiments.²⁴ Solutions were saturated with N₂O to monitor the ˙OH radical reaction (G_˙OH = 0.6 μmole J⁻¹). The carbonate (CO₃˙⁻) radical was generated on pulse radiolyzing N₂O saturated solution containing sodium carbonate (0.1 M) at pH 7. The product formed on reaction of the ˙OH radical with the organoselenium compound was quantified by the HPLC technique. For this, N₂O saturated aqueous solutions of 10 mM SeEOH were radiolysed using the ⁶⁰Co γ-source with a dose rate of 40 Gy min⁻¹. The absorbed dose was set in a way that not more than 10% of the compound undergoes radiolysis. Selenoxide estimation was performed as per the reported method. In brief, 180 μl of radiolysed samples were treated with 20 μl DTT (10 mM) and incubated for 5 minutes. 20 μl of the mixture was injected into the C-18 reverse phase column. The different compounds were eluted using an acetonitrile:water (5:95) mixture as the mobile phase and absorption peaks were detected at 240 nm. The yield of formaldehyde formed during radiolysis of the organoselenium compound was estimated by derivatizing the aldehyde with DNPH. The radiated solutions were mixed with 1 mM DNPH dissolved in 10 mM HCl and stirred for 5 minutes. 20 μl of the resulting solutions were injected into the C-18 reverse phase column. Acetonitrile:water (60:40) was used as the mobile phase and the detector was set at 345 nm.

Reaction of the organoselenium compounds with peroxynitrite was studied by competition kinetics using dihydrorhodamine (DHR)123 as standard. The fluorescence measurements were done on a Hitachi F-4500 fluorescence spectrophotometer with excitation and emission wavelengths of 510 nm and 536 nm respectively. Peroxynitrite solution (5 μM) was added to DHR123 (10 μM) containing 0.1 mM DTPA in 70 mM phosphate buffer (pH 7.5) in the absence and presence of the organoselenium compound (10–100 μM).

The structures of the transients were optimized in vacuo by regressive variation in the starting geometry at the B3LYP/6-31++G(d,p) level (the Becke nonlocal model and Lee–Yang–Parr nonlocal correlation functionals). The geometries obtained were checked by frequency calculations. The global minimum structures were then further optimized in water at the B3LYP/6-31+G(d,p) level in water using the PCM-SMD model. Geometry optimization and frequency calculations were performed by adopting the GAMESS suite of programs on a PC-based LINUX cluster platform.²⁵ The absorption maximum wavelength was calculated in water by using the UCIS model at the B3LYP;6-31-G(d,p) level using Gaussian 09. The transient's geometries and molecular orbitals were visualized using chemissian V4.38 software.

Conclusions

Two isomeric organoselenium compounds, a cyclic compound, DHS, and a linear compound, SeEOH, were studied for their antioxidant ROS scavenging activity, where DHS, a cyclic compound, was found to be much better than its linear isomer SeEOH. The enhanced activities in DHS have been attributed to the strain induced in the cyclic structure that increased the HOMO energy level and easy oxidation. Furthermore, the cyclic structure assists in stabilizing the selenium centred dimer radical cations which favour formation of stable selenoxides, which can be reversed by thiols. Thus the cyclic structural feature of DHS can be used as a molecular design for developing new organoselenium compounds with enhanced antioxidant activity.

Conflicts of interest

The authors declare that there is no conflict of interest.

Acknowledgements

The authors acknowledge the LINAC team, RPCD, BARC and Computer Division, BARC, for the support for radiolysis and computing facility.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj02227a

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