Selenoglutathione Diselenide: Unique Redox Reactions in the GPx

Oct 12, 2017 - Selenoglutathione Diselenide: Unique Redox Reactions in the GPx-. Like Catalytic Cycle and Repairing of Disulfide Bonds in Scrambled...
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Article Cite This: Biochemistry XXXX, XXX, XXX-XXX

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Selenoglutathione Diselenide: Unique Redox Reactions in the GPxLike Catalytic Cycle and Repairing of Disulfide Bonds in Scrambled Protein Shingo Shimodaira, Yuki Asano, Kenta Arai, and Michio Iwaoka* Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan S Supporting Information *

ABSTRACT: Selenoglutathione (GSeH) is a selenium analogue of naturally abundant glutathione (GSH). In this study, this water-soluble small tripeptide was synthesized in a high yield (up to 98%) as an oxidized diselenide form, i.e., GSeSeG (1), by liquid-phase peptide synthesis (LPPS). Obtained 1 was applied to the investigation of the glutathione peroxidase (GPx)-like catalytic cycle. The important intermediates, i.e., GSe− and GSeSG, besides GSeO2H were characterized by 77Se NMR spectroscopy. Thiol exchange of GSeSG with various thiols, such as cysteine and dithiothreitol, was found to promote the conversion to GSe− significantly. In addition, disproportionation of GSeSR to 1 and RSSR, which would be initiated by heterolytic cleavage of the Se−S bond and catalyzed by the generated selenolate, was observed. On the basis of these redox behaviors, it was proposed that the heterolytic cleavage of the Se−S bond can be facilitated by the interaction between the Se atom and an amino or aromatic group, which is present at the GPx active site. On the other hand, when a catalytic amount of 1 was reacted with scrambled 4S species of RNase A in the presence of NADPH and glutathione reductase, native protein was efficiently regenerated, suggesting a potential use of 1 to repair misfolded proteins through reduction of the non-native SS bonds.

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In this study, the liquid-phase peptide synthesis (LPPS) method was successfully applied to the high-yield synthesis of GSeSeG (1). Obtained 1 has been used to characterize major intermediates of the GPx-like catalytic cycle by 77Se NMR spectroscopy. In addition, the ability of 1 to repair the nonnative disulfide (SS) bonds in a scrambled protein to the native ones has also been investigated by using bovine pancreatic ribonuclease A (RNase A) as a model protein. Unique redox behaviors of 1 revealed in the study will be informative for controlling redox reactions of various biological processes by using 1 and its analogues.

lutathione (GSH), a water-soluble tripeptide consisting of γ-glutamic acid (γGlu), cysteine (Cys), and glycine (Gly), is abundant in living cells and plays important roles in decomposition of reactive oxygen species (ROS)1,2 and promotion of folding of disulfide-containing proteins. 3 Selenoglutathione (GSeH) is a selenium analogue of GSH, having selenocysteine (Sec) in place of Cys. This small watersoluble selenopeptide is attracting an interest in versatile fields of biological chemistry because it was found in selenium metabolites of plants4 and microbials5,6 and has higher potential than GSH as a redox modulating agent.7,8 Since GSeH should be mostly deprotonated (pKa = 5.24 for SeH of Sec)9 and easily oxidized (E′0 = −407 mV)10 under physiological conditions, it is usually isolated as an oxidized diselenide form (GSeSeG, 1). Several applications of 1 have already been attempted in the literature. For example, use of 1 as an oxidant and a catalyst in oxidative protein folding study,10−13 a radical scavenger,14,15 and a ligand to heavy metal ions16,17 was reported. It is also known that 1 can be reduced to GSeH by nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of glutathione reductase (GR).10,18 We previously reported that GSeSeG (1) has an antioxidant capacity like glutathione peroxidase (GPx), a well-known selenoenzyme catalyzing the reduction of ROS with GSH.18 The stable intermediates, i.e., GSeH, GSeSG, and GSeO2H, were observed in monitoring the reactions by HPLC and mass spectroscopy. However, details of the GPx-like catalytic cycle could not be analyzed because the yield of 1 by solid-phase peptide synthesis (SPPS) was low. © XXXX American Chemical Society

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MATERIALS AND METHODS General. 1H (500 MHz), 13C (125.8 MHz), and 77Se (95.4 MHz) NMR spectra were recorded at 298 K in CDCl3 or D2O. The chemical shifts (δ) are expressed in ppm against solvent peaks as internal references for 1H and 13C NMR. For 77Se NMR, diphenyl diselenide in CDCl3 was used as an external standard. The coupling constants (J) are reported in Hz. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) spectra were measured by using α-cyano-4-hydroxycinnamic acid as a matrix. Optical rotation ([α]D) was measured in CHCl3 or H2O as a solvent. Reactions were monitored by thin-layer chromatography Received: August 4, 2017 Revised: September 26, 2017

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DOI: 10.1021/acs.biochem.7b00751 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Synthesis of N-(tert-Butoxycarbonyl)-O1-tert-butyl-Lglutamyl-L-selenocysteinyl-glycine tert-Butyl Ester Diselenide (7). To a solution of Boc-γ-Glu(α-OtBu)-Sec(MPM)Gly-OtBu (6) (0.2683 g, 0.39 mmol) in a mixture solution of MeOH, H2O, and AcOH (8:1:1) (7.5 mL) was added I2 (0.1488 g, 0.59 mmol). After stirring for 1 h, water was added to the resulting brown solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water (×2), 10% sodium thiosulfate, saturated sodium hydrogen bicarbonate, and brine, dried over magnesium sulfate, and evaporated. The obtained yellow oil was purified by silica gel column chromatography (hexane−ethyl acetate, 1:1) to give 7 as a yellow oil (0.2031 g, 92%). Spectral data for 7: 1H NMR (CDCl3) δ 8.43 (s, 1H, Gly-NH), 7.05 (d, 2H, J = 8.8, GluNH), 5.39 (m, 1H, Sec-Hα), 5.24 (d, 1H, J = 8.0, Sec-NH), 4.23 (m, 1H, Glu-Hα), 4.00 (dd, 1H, J = 18.0, 6.0, Sec-Hβ), 3.79 (dd, 1H, J = 18.0, 4.8, Sec-Hβ), 3.23 (m, 2H, Gly-CH2), 2.31 (m, 2H, Glu-Hβ), 2.16 (m, 1H, Glu-Hγ), 1.85 (m, 1H, Glu-Hγ), 1.42 (s, 18H, tBu), 1.40 (s, 9H, tBu); 13C NMR (CDCl3) δ 172.4, 171.5, 170.9, 155.8, 82.1, 81.7,79.9, 77.3, 53.7, 53.4, 42.0, 35.6, 32.5, 29.0, 28.3, 28.1, 28.0; 77Se NMR (CDCl3) δ 340.1; MALDI-TOF-MS (m/z) found: 1155.33, calcd for [M + Na]+: 1155.38. Synthesis of Selenoglutathione Diselenide (1). A solution of Boc-γ-Glu(α-OtBu)-Sec-Gly-OtBu diselenide (7) (0.2552 g, 0.23 mmol) in 5% H2O/TFA (6 mL) was stirred for 1 h. TFA was then removed by N2 stream. The deprotected peptide thus obtained was precipitated with diethyl ether and washed with diethyl ether (×3). The product was dissolved in 50% MeCN/H2O containing 0.1% TFA, and lyophilized to give 1·2TFA as a yellow solid (0.2128 g, quant). An alternative method: A solution of Boc-γ-Glu(α-OtBu)Sec(MPM)-Gly-OtBu (6) (0.2605 g, 0.38 mmol) in 20% TA/ TFA (4 mL) was stirred overnight at 37 °C. TFA was then removed by N2 stream. The deprotected peptide thus obtained was precipitated with diethyl ether and washed with diethyl ether (×3). The product was dissolved in 50% MeCN/H2O containing 0.1% TFA and lyophilized to give 1·2TFA as a yellow solid (0.1805 g, quant). Spectral data for 1: 1H NMR (D2O) δ 4.54 (dd, 1H, J = 9.2, 5.0, Sec-Hα), 3.87 (t, 1H, J = 6.5, Glu-Hα), 3.85 (s, 2H, Gly-CH2), 3.23 (dd, 1H, J = 13.2, 5.0, Sec-Hβ), 3.00 (dd, 1H, J = 13.1, 9.2, Sec-Hβ), 2.41 (m, 2H, Glu-Hβ), 2.06 (m, 2H, Glu-Hγ); 13C NMR (D2O) δ 174.3, 172.89, 172.7, 171.9, 54.0, 52.5, 41.2, 31.0, 29.3, 25.5; 77Se NMR (D2O) δ 292.9; MALDI-TOF-MS (m/z) found: 709.01, calcd for [M + H]+: 709.05; [α]D −59.3° (c = 1.00 in H2O). Synthesis of N-(tert-Butoxycarbonyl)-O1-tert-butyl-Lglutamyl-Se-(acetamide)-L-selenocysteinyl-glycine tertButyl Ester (8). To a solution of Boc-γ-Glu(α-OtBu)-Sec-GlyOtBu diselenide (7) (0.2270 g, 0.20 mmol) in THF−H2O (2:1) (6 mL) cooled on an ice bath was added NaBH4 (0.0199 g, 0.53 mmol). After stirring 5 min on an ice bath, the mixture was added with iodoacetamide (0.1425 g, 0.77 mmol) and stirred for 1 h. The resulting pale yellow solution was extracted with ethyl acetate. The combined organic layer was washed with water (×2) and brine, dried over magnesium sulfate, and evaporated. The obtained pale yellow oil was purified by GPC (chloroform) to give 8 as a colorless oil (0.2337 g, 93%). Spectral data for 8: 1H NMR (CDCl3) δ 7.70 (d, 1H, J = 7.2, AM-NH), 7.51 (t, 1H, J = 5.4, Gly-NH), 7.06 (s, 1H, AM-NH), 6.45 (s, 1H, Glu-NH), 5.49 (d, 1H, J = 7.7, Sec-NH), 4.74 (m, 1H, Sec-Hα), 4.10 (m, 1H, Glu-Hα), 3.86 (d, 2H, J = 5.4, GlyCH2), 3.22 (s, 2H, AM-CH2), 3.13 (dd, 1H, J = 13.4, 6.1, Sec-

(TLC). Gel permeation chromatography (GPC) was performed with an isocratic HPLC system using CHCl3 as an eluent. All chemicals were used as purchased without further purification. Synthesis of N-(9-Fluolenylmethoxycarbonyl)-Se-(pmethoxyphenylmethyl)-L-selenocysteinyl-glycine tertButyl Ester (4). To a solution of Fmoc-Gly-OtBu (0.2259 g, 0.64 mmol) in DMF (5.4 mL) was added Et2NH (0.6 mL). After stirring for 5 min at r.t., the reaction mixture was evaporated to remove excess Et2NH. Subsequently, FmocSec(MPM)-OH (2) (0.2017 g, 0.40 mmol), HOBt (0.0959 g, 0.63 mmol), EDCI (0.1168 g, 0.61 mmol), and DMF (2 mL) were added to the residue, and the mixture was stirred for 2 h. The resulting pale yellow solution was extracted with a mixture of hexane−ethyl acetate (7:3). The combined organic layer was washed with water (×2), saturated sodium hydrogen bicarbonate, 1 M HCl, and brine, dried over magnesium sulfate, and evaporated. The obtained pale yellow oil was purified by silica gel column chromatography (hexane−ethyl acetate, 7:3) to give 4 as a colorless oil (0.2420 g, 98%). Spectral data for 4: 1H NMR (CDCl3) δ 7.76 (d, 2H, J = 8.0, Fmoc-Ar), 7.59 (m, 2H, Fmoc-Ar), 7.40 (m, 2H, Fmoc-Ar), 7.32 (m, 2H, Fmoc-Ar), 7.22 (d, 2H, J = 8.5, MPM-Ar), 6.82 (d, 2H, J = 8.7, MPM-Ar), 6.64 (bs, 1H, Gly-NH), 5.59 (d, 1H, J = 5.4, Sec-NH), 4.41 (m, 3H, Fmoc-CH2, Sec-Hα), 4.23 (t, 1H, J = 6.9, Fmoc-CH), 3.92 (m, 2H, Gly-CH2), 3.78 (s, 2H, MPM-CH2), 3.736(s, 3H, p-OMe), 2.96 (d, 1H, J = 8.2, SecHβ), 2.78 (dd, 1H, J = 12.1, 6.8, Sec-Hβ), 1.47 (s, 9H, tBu); 13 C NMR (CDCl3) δ 170.3, 168.4, 158.6, 143.7, 141.3, 130.1, 127.8, 127.1, 125.1, 120.0, 114.1, 82.5, 67.2, 55.2, 47.1, 42.2, 28.1, 27.5, 25.7; 77Se NMR (CDCl3) δ 220.4; MALDI-TOFMS (m/z) found: 646.94, calcd for [M + Na]+: 647.16; [α]D −3.4° (c = 1.00 in CHCl3). Synthesis of N-(tert-Butoxycarbonyl)-O1-tert-butyl-Lglutamyl-Se-(p-methoxyphenylmethyl)-L-selenocysteinyl-glycine tert-Butyl Ester (6). To a solution of FmocSec(MPM)-Gly-OtBu (4) (0.2420 g, 0.39 mmol) in 20% DCM/DMF (5.4 mL) was added Et2NH (0.6 mL). After stirring for 5 min at r.t., the reaction mixture was evaporated to remove excess Et2NH. Subsequently, Boc-Glu-OtBu (5) (0.1795 g, 0.59 mmol), HOBt (0.1355 g, 0.88 mmol), EDCI (0.1690 g, 0.88 mmol), and DMF (2 mL) were added to the residue, and the mixture was stirred for 2 h. The resulting pale yellow solution was extracted with hexane−ethyl acetate (6:4). The combined organic layer was washed with water (×2), saturated sodium hydrogen bicarbonate, 1 M HCl, and brine, dried over magnesium sulfate, and evaporated. The obtained pale yellow oil was purified by silica gel column chromatography (hexane−ethyl acetate, 55:45) to give 6 as a colorless oil (0.2702 g, quant). Spectral data for 6: 1H NMR (CDCl3) δ 7.22 (d, 2H, J = 8.7, MPM-Ar), 6.88 (t, 1H, J = 5.1, Gly-NH), 6.82 (d, 2H, J = 8.7, MPM-Ar), 6.74 (d, 1H, J = 7.1, Glu-NH), 5.29 (d, 1H, J = 7.9, Sec-NH), 4.60 (q, 1H, J = 6.8, Sec-Hα), 4.21 (m, 1H, Glu-Hα), 3.89 (m, 2H, Gly-CH2), 3.78 (s, 2H, MPM-CH2), 3.77 (s, 3H, p-OMe), 2.93 (dd, 1H, J = 13.1, 6.2, Sec-Hβ), 2.78 (dd, 1H, J = 13.0, 6.4, Sec-Hβ), 2.26 (m, 2H, Glu-Hβ) 2.14 (m, 1H, Glu-Hγ), 1.86 (m, 1H, Glu-Hγ), 1.44 (s, 9H, tBu), 1.44 (s, 9H, tBu), 1.42 (s, 9H, tBu); 13C NMR (CDCl3) δ 172.3, 171.4, 107.5, 168.5, 158.5, 155.8, 131.0, 130.0, 114.0, 82.3, 82.3, 80.0, 77.3, 55.3, 53.4, 52.7, 42.1, 32.4, 29.0, 28.3, 28.0, 28.0, 27.7, 25.1; 77Se NMR (CDCl3) δ 225.8; MALDI-TOF-MS (m/z) found: 709.91, calcd for [M + Na]+: 710.25; [α]D −8.6° (c = 1.00 in CHCl3). B

DOI: 10.1021/acs.biochem.7b00751 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry Scheme 1. Synthesis of GSeSeG (1) and GSeAM (9)a

a

(a) H-Gly-OtBu (3), EDCI, HOBt, DMF (98%), (b) Et2NH, 20% DCM/DMF, then Boc-Glu-OtBu (5), EDCI, HOBt, DMF (quant), (c) I2, MeOH/H2O/AcOH (8:1:1) (92%), (d) 5% H2O/TFA (quant), (e) 20% TA/TFA (quant), (f) NaBH4, THF/H2O (2:1), then IAM (93%), (g) 5% H2O/TFA (98%).

mL)21 to quench the refolding reaction after a certain period of time (1−24 h). The obtained sample solution was desalted by gel filtration chromatography using a Sephadex G-25 resin and then analyzed by HPLC according to the method as described previously.19,20 The separated intermediates were unambiguously assigned to R, 1S, 2S, 3S, 4S, des[40−95], des[65−72], or N by comparing the retention times to those reported in the literature.

Hβ), 2.95 (dd, 1H, J = 13.3, 5.5, Sec-Hβ), 2.33 (m, 2H, GluHβ), 2.10 (m, 1H, Glu-Hγ), 1.88 (m, 1H, Glu-Hγ), 1.41 (s, 9H, tBu), 1.40 (s, 9H, tBu), 1.38 (s, 9H, tBu); 13C NMR (CDCl3) δ 174.2, 172.9, 171.6, 170.9, 168.7, 155.8, 82.2, 79.9, 77.3, 58.1, 53.6, 53.0, 42.1, 32.2, 28.3, 28.0, 28.0; 77Se NMR (CDCl3) δ 189.2 (major), 185.0 (minor); MALDI-TOF-MS (m/z) found: 646.94, calcd for [M + Na]+: 647.22. Synthesis of γ-L-Glutamyl-Se-(acetamide)-L-selenocysteinyl-glycine (9). A solution of Boc-γ-Glu(α-OtBu)-Sec(AM)-Gly-OtBu (8) (0.1282 g, 0.21 mmol) in 5% H2O/TFA (2 mL) was stirred for 2 h at r.t. TFA was then removed by N2 stream. The deprotected peptide was precipitated with diethyl ether and washed with diethyl ether (×3). The product was dissolved in 50% MeCN/H2O containing 0.1% TFA and lyophilized to give 9 as a white solid (0.1058 g, 98%). Spectral data for 9: 1H NMR (D2O) δ 4.47 (dd, 1H, J = 8.5, 5.3, SecHα), 3.92 (t, 1H, J = 6.6, Glu-Hα), 3.84 (d, 2H, J = 0.7, GlyCH2), 3.12 (d, 2H, J = 1.4, AM-CH2), 3.00 (dd, 1H, J = 13.3, 5.3, Sec-Hβ), 2.85 (dd, 1H, J = 13.3, 8.3, Sec-Hβ), 2.42 (m, 2H, Glu-Hβ), 2.07 (m, 2H, Glu-Hγ); 13C NMR (D2O) δ 176.5, 174.2, 172.9, 172.8, 171.6, 53.4, 52.2, 41.1, 30.9, 25.4, 25.3, 24.9; 77Se NMR (D2O) δ 189.1; MALDI- TOF-MS (m/z) found: 412.88, calcd for [M + H]+: 413.06. Refolding of Scrambled RNase A (4S). 4S of RNase A, which is fully oxidized species but has non-native SS bonds along the peptide chain, was prepared as described previously.19,20 The 100 mM Tris-HCl/1 mM EDTA buffer solution at pH 7.5 deoxygenated by bubbling nitrogen gas for 1.5 h was used for the refolding experiment. A powder (∼2 mg) of 4S was dissolved in 10 mM HCl (100 μL). This stock solution was diluted 30-fold with the Tris buffer solution. The concentration of 4S was then adjusted to 30 μM based on the molar extinction coefficient (ε275 = 8600 M−1 cm−1). The prepared solution of 4S (300 μL) was mixed with 0.6 to 60 μM 1 in the buffer solution (150 μL). To the resulting solution was then added a mixture solution containing 1.2 mM NADPH and glutathione reductase (2.4 unit) in the buffer solution (150 μL) to initiate the refolding reaction. The sample solution was incubated in a dry thermo bath regulated at 25.0 (±0.2 °C) and was added with aqueous solution of AEMTS (300 μL, 7 mg/

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RESULTS AND DISCUSSION Synthesis of GSeSeG (1) by LPPS. Two methods, i.e., LPPS and SPPS, were previously applied for the synthesis of 1. Soda and co-workers22 obtained 0.13 g of 1 as an ammonium salt starting from N-(p-methoxybenzyloxycarbonyl)-Se-(p-methoxyphenylmethyl)-L-selenocysteine (Z(OMe)-Sec(MPM)OH) by LPPS and characterized 1 by 1H NMR, UV, and circular dichroism (CD). However, since the yield was low (9%), LPPS was not further investigated after Soda’s report. In 2007, Hilvert and co-workers.10 reported SPPS of 1 (33% yield) by using N-(9-fluorenylmethoxycarbonyl)-Se-(p-methoxyphenylmethyl)-L-selenocysteine (Fmoc-Sec(MPM)-OH, 2), which was activated to the corresponding pentafluorophenyl (Pfp) ester when coupled to Gly on the resin. SPPS of 1 without preactivation of 2 to the Pfp ester was also possible, but the yield (9%) was low.18 Thus, in the previous synthesis, the yield of 1 was not satisfactory for further investigation of the redox reactions in a practical scale. To obtain a large amount of 1, LPPS would be more appropriate than SPPS because 1 is just a tripeptide and Sec derivatives, such as 2, are not easily available: In SPPS, excess amounts of amino acid derivatives are required to complete the coupling reaction. Therefore, we revisited LPPS in this study. Coupling between Sec and Gly Derivatives (2 and 3). The coupling condition between Fmoc-Sec(MPM)-OH (2) and HGly-OtBu (3) was examined based on Kisfaludy’s method.23 When Gly derivative 3 was reacted with preactivated 2, i.e., Fmoc-Sec(MPM)-OPfp, in the presence of Et3N at r.t. for 15 min, dipeptide Fmoc-Sec(MPM)-Gly-OtBu (4) was obtained in a yield of 93%. The yield could be increased up to 98% by optimization of the reaction conditions as follows (Scheme 1). C

DOI: 10.1021/acs.biochem.7b00751 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry First, 3 was prepared from Fmoc-Gly-OtBu by using Et2NH as a base in DMF: Et2NH (bp 56 °C) was employed because it could be more easily removed from the reaction solution than widely used piperidine (bp 106 °C). After evaporation of excess Et2NH, obtained 3 was reacted with 2 (without preactivation) in the presence of 1.5 equiv of EDCI and 1.5 equiv of HOBt at r.t. for 2 h to yield dipeptide 4 (98%) after purification by silica gel column chromatography. The same yield was achieved by using 2 equiv of DIC instead of EDCI, although a longer reaction time (6 h) was necessary. Thus, direct coupling between 2 and 3 without preactivation was found to be more efficient and convenient than the coupling using the Pfp ester of 2. Coupling between 4 and Glu Derivative (5). The next step was the coupling of obtained dipeptide 4 with glutamic acid derivative Boc-Glu-OtBu (5). In this step, the deprotection condition of the Fmoc group of 4 was needed to be optimized because Besse and Moroder24 reported that dehydroalanine and β-piperidinoalanine derivatives can be produced by elimination of the selenium moiety in highly polar solvents, such as DMF and NMP, when a Sec-containing peptide is treated with piperidine. Therefore, deprotection of the Fmoc group of 4 was carried out by using Et2NH in 20% DCM/ DMF. The subsequent reaction with preactivated 5, i.e., BocGlu(OPfp)-OtBu, afforded tripeptide 6 in up to 94% yield. On the other hand, direct coupling of deprotected 4 with 5 quantitatively yielded 6 under the conditions using 1.5 equiv of 5, 2.25 equiv of EDCI, and 2.25 equiv of HOBt after stirring at r.t. for 2 h. Deprotection of 6 to GSeSeG (1). The deprotection was examined by two methods. In the first attempt, 6 was treated with 1.5 equiv of I2 in MeOH:H2O:AcOH (8:1:1) to obtain diselenide 7 in a yield of 92%, and obtained 7 was subsequently treated with 5% H2O/TFA for 2 h. Through this stepwise deprotection, 1 was obtained in >90% yield. In the other method, all protecting groups were deprotected in one-pot by treatment of 6 with a mixture of thioanisole (TA) and TFA according to the literature method.25 Diselenide 1 was obtained as a TFA salt (1·2TFA) quantitatively after stirring in 20% TA/ TFA at 37 °C for overnight. According to Scheme 1, 1 could be obtained from Sec derivative 2 in three steps (paths a, b, and e) in an overall yield of up to 98%. Redox Reactions of GSeSeG (1). Since a semigram scale of 1 was obtained by LPPS as described above, the GPx-like catalytic cycle of selenoglutathione was reinvestigated by 77Se NMR spectroscopy. The sample solution was prepared by dissolving 1 in D2O (64 mM), the 77Se NMR resonance of which was observed at δ 293. In the following experiments, the redox reactions of 1 were monitored by a shift of the 77Se NMR signal but in some cases other spectroscopic methods, such as 1 H NMR and MS, were supplementarily utilized. Reduction of GSeSeG (1). The attempt to reduce the diselenide bond of 1 by NADPH with GR in a NMR sample tube failed probably because the solvent and concentration conditions (i.e., [1] = 64 mM in D2O) were very different from those applied in the literature (i.e., [1] = 0.05 mM in a pH 7.4 buffer solution).18 Therefore, other reduction conditions were investigated. When dithiothreitol (DTTred) was reacted with 1, no signal change was observed in the 77Se NMR spectrum, whereas a new broad signal (full width at half-maximum ∼500 Hz) appeared at δ − 210 when the solution pH was adjusted to 10 by using aqueous NaOH (Figure 1). The observed line broadening is probably due to rapid proton or deuterium

Figure 1. A series of 77Se NMR spectra of 1 in D2O. (a) GSeSeG (1). (b) A 1:3 mixture of GSeSeG and DTTred in D2O containing NaOH (pH 10). (c) After addition of IAM (5 equiv) to (b).

exchange of a selenol group (SeH) under basic conditions.26 Indeed, regeneration of 1 was observed when GSSG was added to the reaction mixture (Figure S1). In addition, when iodoacetamide (IAM) was reacted as an alkylating reagent of selenol, a signal corresponding to GSeAM (9) was observed at δ 189. The same signal was observed for 9, which was synthesized in a different route according to paths f and g in Scheme 1. Thus, it was found that GSeSeG (1) can be reduced to GSeH (or GSe− under basic conditions) by DTTred. However, monothiols, such as glutathione (GSH) and cysteine (CysSH), could not reduce 1. These observations can be reasonably explained by the redox potentials of GSeSeG/GSeH (−407 mV) and DTTox/DTTred (−327 mV), which are significantly lower than those of GSSG/GSH (−256 mV) and CysSSCys/CysSH (−238 mV).10,27 On the other hand, when H2O2 was reacted with GSe−, which was prepared at pH 10 by the reaction of 1 with DTTred, a new signal was observed at δ 1182 in addition to a small signal of 1 (Figure S1). To assign the new signal, oxidation of diselenide 1 with H2O2 was carried out subsequently. Oxidation of GSeSeG (1) by H2O2. When 1 equiv of H2O2 was reacted with 1, the signal of 1 diminished and a new signal was observed at δ 1213 in the 77Se NMR spectrum. The signal of 1 completely disappeared and only one signal was observed at δ 1213 when two more equivalents of H2O2 were added (Figure S2). The same reaction was monitored by 1H NMR (Figure 2) as well. The Hα of the Sec residue was observed at δ 4.51 as a doublet of doublets. By addition of H2O2, the signal was decreased and a new signal appeared at δ 4.83 as triplets. These spectral changes suggested that 1 was oxidized by 3 equiv of H2O2, producing two molecules of GSeO2H. Kamigata28 reported that methaneseleninic acid, 2-methylpropane-1-seleninic acid, 2,2-dimethylpropane-1-seleninic acid, and cyclohexaneseleninic acid show 77Se NMR signals at δ 1294, 1314, 1316, and 1322, respectively. Mugesh29,30 reported the 77Se NMR chemical shift of the seleninic acid that was derived from ebselen to be observed at δ 1122. Thus, the 77Se NMR chemical shift of 1213 observed for GSeO2H reasonably falls in a range of seleninic acid species. However, this assignment may be a little ambiguous as two selenenic acids, which were isolated by applying bulky bowl-shape substituents, showed the 77Se NMR resonance in a similar range (δ 1079 and 1261).31,32 D

DOI: 10.1021/acs.biochem.7b00751 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

observed at δ 367 in the 77Se NMR spectrum (Figure S4). In the previous study using selenocystine, the selenenyl sulfide (CysSeSCys) was observed at δ 349.34 Therefore, the signal of δ 367 could be assigned to the selenenyl sulfide bond of GSeSG. Indeed, according to MS analysis of the 1:3 reaction mixture of GSeO2H and GSH, the signals corresponding to GSeSG and GSSG were observed at m/z 661.00 (calcd for [M + H]+ 661.10) and 613.07 (calcd for [M + H]+ 613.16), respectively (Figure S5). GSeO2 H + 3GSH → GSeSG + GSSG + 2H 2O

(1)

Thus, formation of GSeO2H, not GSeOH, in the reaction mixture of 1 with H2O2 was confirmed. X-ray structure analysis of GPx was indeed performed for this seleninic acid state.35 In the reaction of GSe− with H2O2 at pH 10, we observed a 77 Se NMR resonance at δ 1182 (Figure S1). This signal can now be assigned to GSeO2− based on the observations discussed above. The slight difference of the chemical shift from that of GSeO2H (δ 1213) is likely due to ionization. Interestingly, it was found that when GSeO2H was reacted with 2 equiv of GSH, GSeO2H was completely converted to GSeSG (Figure S4) in the 77Se NMR spectrum. This suggested the possibility of a fast reaction (