Integrating Diphenyl Diselenide and Its Mehg + Detoxificant Mechanism on a Conceptual DFT Framework

: Methylmercury (MeHg + ) is an important environmental contaminant and its toxicity is associated with its interaction with selenium (e.g., selenol groups of selenoproteins or HSe − , which is the pivotal metabolite for Se incorporation into selenoproteins). We hypothesized that (PhSe) 2 mediated MeHg + detoxification could be indirectly altered by its open or closed conformation. The two conformations of (PhSe) 2 were located on the potential energy surface (PES) computed at ZORA-OPBE-D3(BJ)/ZORA-def2-TZVP level of theory. HPLC analysis indicated that (PhSe) 2 did not react with MeHg + , but its reduced intermediate formed a stable complex with MeHg + . The nudged elastic band (NEB) method revealed conformational changes from closed to open state with an H − (2 electrons) transfer from NaBH 4 , forming a reactant complex-like transition state (TS). The UV-Vis spectrophotometer used in combination with the time-dependent density functional theory (TD-DFT) indicated that the signal of (PhSe) 2 at 239 nm was possibly the open conformer’s signal with oscillator strength 0.1 and a π → π * electron transfer character. The experimental band gap energy of (PhSe) 2 at 5.20 eV matched to the excitation energy of the open conformation. The local softness (S − ) on the selenium atoms almost doubles, as state changes from closed to open. The theoretical results have indicated that the open conformation of (PhSe) 2 is likely the one that reacts with NaBH 4 to form the PhSeH, which can react with MeHg + .


Introduction
In the aquatic food web, methylmercury (MeHg + ) can be biomagnified and can reach toxic levels in the edible muscle of predatory or piscivorous fish [1,2]. The frequent consumption of predatory fish can result in MeHg + intoxication [2]. The toxicity of the soft electrophilic MeHg + is mediated by inactivation of proteins containing soft nucleophilic sites (e.g., thiol-and selenol-containing proteins) [3][4][5][6][7]. MeHg + has an extremely high affinity for -SH and -SeH groups [5,8]. Experimental and theoretical studies have indicated that the affinity of MeHg + for -SeH is greater than for -SH groups [5]. Selenium (Se) is an essential element for vertebrates as part of the selenol group present in the selenocysteine residues found in selenoproteins. In a previous study, we demonstrated that diphenyl diselenide (PhSe)2 decreased the deposition of Hg in mice treated with MeHg + . A decreased mercury burden in liver, kidney, cerebrum and cerebellum of mouse was reported in mice treated with (PhSe)2 [3,4,9]. We have hypothesized that (PhSe)2 could be reduced to its selenol intermediate PhSeH, which formed a complex with MeHg + (i.e., PhSeHgMe) [5,9,10]. The reduction of (PhSe)2 and its eventual reaction with methylmercury have several intricate and interesting parts, as reactions may eventually depend on the conformation assumed by (PhSe)2 in the reacting medium [11][12][13].
(PhSe)2 presents two conformations in its ground state, referred to as the closed and open conformation (Scheme 1) [12,14]. The reduction of (PhSe)2 using NaBH4 involves a hydride transfer via a likely single-step mechanism [15], Scheme 2. It has been reported [16] that the hydride transfer process is strongly affected by solvent, with the open state acting as the hydride acceptor from the NaBH4 donor. A likely mechanism is shown in Scheme 2. The nudged elastic band (NEB) method [17] was used to locate the relevant points on the PES, i.e., the minima and transition states. TD-DFT [18] calculations were performed at ZORA-CAM-B3LYP/zora-def2-TZVP to compute the excitation energies and interpret the experimental spectrum. Then, we used the conceptual density functional theory (c-DFT), [19] which involves the use of DFT, electron density to unravel the reactivity of chemical systems. Particularly, the Fukui function f(r) was computed. This function is the second derivative of energy (E) at a constant external potential, derived by perturbing the chemical system from N to N + 1 and N to N − 1 [20]. This function indicates the regioselectivity [21], suggesting a region on a molecule where there will be either a nucleophilic or electrophilic attack.

Experimental
A stock solution of 10 mM was prepared by dissolving 19.6 mg of (PhSe)2 in 6.28 mL of 70% Acetonitrile, from which 5 µL ((PhSe)2 final concentration of 50 µM) was used in the reaction, same concentration applied to NaBH4, DTT and MeHg. The UV absorption spectrum of (PhSe)2 was recorded in the region of 220-450 nm with 70% acetonitrile as the solvent using a UV-1800 Shimadzu spectrophotometer (Shimadzu, Kyoto, Japan). Analysis of methylmercury reaction with (PhSe)2 was performed using a Shimadzu SPD-20A UV/V is Detector (Shimadzu, Kyoto, Japan). CBM-20A communication bus module and DGU-20A5 Degasser prominence high-performance liquid chromatography (HPLC) controlled by the LCSolution software system version 1.22 SP1. Detection was monitored at 239 nm UV wavelength. The separation was achieved on a VerticalTM VertiSep GES C18 HPLC column (4.6 × 150 mm). The mobile phase used for the analysis of the reaction was at 70% acetonitrile-0.5% phosphoric acid aqueous solution (70:30, v/v) with a flow rate of 0.8 mL/min. An injection volume of 50 µL was used. The retention time was approximately 12.5 min for (PhSe)2. The peak areas were used for quantification. The estimated void volume is 1.75 mL and at 0.8 mL/min flow rate gives 2.19 min.

Computational Methods
All Density Functional Theory (DFT) calculations were done with ORCA 4.1.2 [22,23]. Geometry optimizations and vibrational frequencies were performed at ZORA-OPBE-D3(BJ)/ZORA-def2-TZVP [24,25] level of theory. The Zeroth-order regular approximation (ZORA) was used in order to include scalar relativistic effects due to the presence of selenium atom [26] as previously benchmarked [11]. In addition, the effect of dispersion was included using Grimme's approximation (D3(BJ)) [27,28]. The FMO (frontier molecular orbitals) energies and related parameters were obtained at ZORA-OPBE-D3(BJ)/ZORA-def2-TZVP level. The NEB analysis was carried out at ZORA-OPBE-D3(BJ)/ZORA-def2-TZVP level. The chemical reactivity descriptors used are the local softness(s), global hardness (η) and global softness (S), defined as: η = (IE − EA)/2 and S = 1/(IE − EA) where IE is the Ionization Energy which corresponds to the HOMO energy taken with a negative sign and EA is the electron affinity which corresponds to the LUMO energy taken with a negative sign. The local softness is (s) = S (Global Softness) × f(r) where the Fukui function f(r) [20,29,30] is the second derivative of E at a constant external potential. The dual descriptors f(2)(r) > 0 indicate preferable sites for nucleophilic attack and f(2)(r) < 0 shows sites for electrophilic attack. The experimental band gap energy was obtained by calculating the energy using the Planck-Einstein relationship E(eV) = hc/λ. The pkCSM server [31] (http://biosig.unimelb.edu.au/pkcsm/prediction) was used in predicting the total clearance (CLtot) of (PhSe)2, MeHg + and MeHgSePh [32].

Results
A reactant-like transition state (TS) geometry with the TS energy closer to the reactant complexwhere the open conformer is present. The relevant structures of this mechanism are shown in Scheme 3.

Discussion
The PES scan performed at ZORA-OPBE-D3(BJ)/ZORA-def2-TZVP level of theory across the dihedral angle ψ (C-Se-Se-C *, Scheme 1) reveals two minima corresponding to approximately −80 and +80 degrees (Figure 1). The spikes in the curve correspond to switches in the orientation of the phenyl rings converting the open/closed conformations. Solvation brings minimal changes, and the two minima are just slightly shifted, while the phenyl rings orientation in correspondence of these minima is maintained as in the gas phase. The actual detoxifying of MeHg + is the selenol metabolite obtained from the reduction of (PhSe)2 by two electrons transfer process (Scheme 2, Figure 3, breaking the Se-Se bond. The orientation of the phenyl rings is expected to modulate the equilibrium shift, either in favor or against the formation of selenol, hence modifying indirectly the detoxification process. Hydride transfer process are strongly affected by solvent [16]. The crystallographic structure retrieved from the Cambridge Structural Database (CSD) [33,34] is in the closed state. At ZORA-CAM-B3LYP/zora-def2-TZVP ( Figure 3

Conclusions
In this work, we have used both experimental and theoretical approach to identify the reactive state of (PhSe)2. We ascribe the experimental absorption of (PhSe)2 at 239 nm to a dominant open state with a π → π * electron transfer character. The reaction with MeHg + was observed to be preceded by a reduction reaction. A hydride transfer from NaBH4 to (PhSe)2 open state was postulated and confirmed by a NEB profile: a reactant-like transition state (TS) geometry is present leading to selenol as the product. This study provides new insight into the study of (PhSe)2 use as a methylmercury detoxificant agent.

Conflicts of Interest:
The authors declare no conflict of interest.