Different Oxidation Pathways of 2-Selenouracil and 2-Thiouracil

International Journal of Molecular Sciences

Article Diﬀerent Oxidation Pathways of 2-Selenouracil and 2-Thiouracil, Natural Components of Transfer RNA

1, 2, 1 Katarzyna Kulik † , Klaudia Sadowska † , Ewelina Wielgus , Barbara Pacholczyk-Sienicka 2 , Elzbieta Sochacka 2 and Barbara Nawrot 1,* 1 Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland; [email protected] (K.K.); [email protected] (E.W.) 2 Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; [email protected] (K.S.); [email protected] (B.P.-S.); [email protected] (E.S.) * Correspondence: [email protected]; Tel.: +48-42-6803248 These authors contributed equally to this work. † Received: 28 July 2020; Accepted: 13 August 2020; Published: 19 August 2020

Abstract: Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and tuning of protein biosynthesis in all three domains of life. Both sulfur and selenium chalcogens functionally operate as key elements of biological molecules involved in the protection of cells against oxidative damage. In this work, 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura) were treated with hydrogen peroxide at 1:0.5, 1:1, and 1:10 molar ratios and at selected pH values ranging from 5 to 8. It was found that Se2Ura was more prone to oxidation than its sulfur analog, and if reacted with H2O2 at a 1:1 or lower molar ratio, it predominantly produced diselenide Ura-Se-Se-Ura, which spontaneously transformed to a previously unknown Se-containing two-ring compound. Its deselenation furnished the major reaction product, a structure not related to any known biological species. Under the same conditions, only a small amount of S2Ura was oxidized to form Ura-SO2H and uracil (Ura). In contrast, 10-fold excess hydrogen peroxide converted Se2Ura and S2Ura into corresponding Ura-SeOnH and Ura-SOnH intermediates, which decomposed with the release of selenium and sulfur oxide(s) to yield Ura as either a predominant or exclusive product, respectively. Our results confirmed significantly different oxidation pathways of 2-selenouracil and 2-thiouracil.

Keywords: 2-thiouridine; 2-selenouridine; 2-thiouracil; 2-selenouracil; oxidative stress; modiﬁed nucleoside; tRNA

1. Introduction Sulfur and selenium elements are natural components of living organisms in all three domains of life. Among them, the most commonly known are cysteine (Cys) and selenocysteine (Sec) building blocks of proteins and 2-thiouridine (S2U) and 2-selenouridine (Se2U) present in transfer RNAs (tRNAs). Both elements functionally operate as key elements of the enzymes involved in the protection of cells against oxidative damage. These elements are active redox components involved in thiol/disulfide exchange, reactive oxygen species (ROS) metabolism, and redox homeostasis [1–8]. Although selenium and sulfur have similar chemical properties [9], they differ significantly with respect to their polarizability and redox properties [10]. For example, due to weak Se–O π-bonding, Se-oxides are more easily reduced than S-oxides. This so-called “selenium paradox” [11] and other experimental data have allowed the suggestion that selenium-bearing compounds are superior ROS scavengers due to their unique ability to react with oxidizing species in a reversible manner, in contrast to their sulfur analogs [12].

Int. J. Mol. Sci. 2020, 21, 5956; doi:10.3390/ijms21175956 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5956 2 of 18

Unfortunately, limited data are available in the literature on the susceptibility of S2U/Se2U to oxidation. In the course of our research on the functional properties of chalcogen-containing nucleotides present in transfer RNAs, we have shown that in the presence of hydrogen peroxide at high concentrations or in the presence of oxone®, 2-thiouridine (S2U) either as a nucleoside or in an RNA chain undergoes efficient oxidative desulfuration, furnishing uridine (U) and/or 4-pyrimidinone ribonucleoside (H2U) [13,14]. The ratio of products depends on buffer pH, the concentration of reagents, and the electronic properties of a substituent at the C5 position of the nucleobase [15,16]. The reaction proceeds through sulfur-oxidized intermediates (bearing a sulfenic, sulfinic, or sulfonic moiety) and is catalyzed in vitro by cytochrome C [17]. The S2U-RNA H2U-RNA transformation → leads to the loss of U-A base pairing in RNA duplexes [14]. Although this kind of tRNA damage has not been confirmed in cells and is still a subject of research, one can consider that it may impair tRNA function during protein synthesis due to the disruption of codon-anticodon interactions. Recently, Hondal’s group investigated the oxidative transformation of 2-thio-and 2-selenouracils substituted at position 5 with a nonnative substituent (introduced to enhance nucleobase solubility in aqueous media), which is a carboxyl group directly attached to C5 (c5S2Ura and c5Se2Ura), and demonstrated that 5-carboxy-2-thiouracil in the presence of a 1:1 molar ratio of H2O2 underwent irreversible desulfuration, leading to 5-carboxyuracil. In contrast, the 2-seleno-analog, under the same conditions, was converted into the corresponding diselenide and seleninic acid, which, in a reducing environment, could be converted back to the starting 5-carboxy-2-selenouracil [18]. This result allowed them to conclude that selenium-containing biomolecules are resistant to permanent oxidation, and this is the main reason for naturally occurring selenium in both 2-selenouridine- and Sec-containing proteins. This conclusion complementary to the latest data on the role of seleno modifications in tRNA ensuring the fidelity of the reading of synonymous 30-G ending codons [19], prompted us to perform more detailed studies on the oxidation of 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura), especially in light of the recently discovered “oxidative desulfuration” of S2U, leading predominantly to 4-pyrimidinone derivatives [13–17]. In work presented here, we treated 2-selenouracil (Se2Ura, 1a) and 2-thiouracil (S2Ura, 1b) with hydrogen peroxide under different conditions (at different concentrations and various pH values) and identified intermediates and final products at several time points using 1H NMR spectroscopy and ultra-performance liquid chromatography coupled with high-resolution mass spectrometry and photodiode array detection (UPLC-PDA-ESI( )-HRMS). The obtained data allowed us to propose − possible transformation paths for selenium- and sulfur-containing uracils and to characterize their redox properties.

2. Results

2.1. General Approach for Analysis of the Course of Oxidation of 2-Selenouracil (1a) and 2-Thiouracil (1b) and Identification of the Reaction Products To obtain data on a sequence of events during oxidation of 2-selenouracil (1a, Se2Ura) and 2-thiouracil (1b, S2Ura), 10 mM solutions in phosphate buffer at pH 5.0, 7.4, or 8.0 or in water were reacted at room temperature (r.t.) with 5, 10, or 100 mM hydrogen peroxide (1:0.5, 1:1, and 1:10 molar ratio, respectively). The reaction progress (from 1 min to 24 h) and structural data on the intermediates/products were gathered from 1H NMR and UPLC-PDA-ESI( )-HRMS measurements. − The relative content of the detected compounds was calculated using integrations of 1H NMR signals for non-exchangeable H5 and H6 protons (δ range 8.8–5.2 ppm). The UPLC-ESI( )-MS retention times − (Rt, min), m/z values of the ions, which correspond to the deprotonated molecules, λmax in UV/VIS spectra (nm), and 1H NMR chemical shifts (δ, ppm) for H5 and H6 protons and coupling constants JH6-H5 for all identified compounds are presented in Table1. Spectral data for all identified compounds are given in the Supplementary Materials (Figures S1–S20). Int. J. Mol. Sci. 2020, 21, 5956 3 of 18

Table 1. UPLC-PDA-ESI ( )-HRMS and 1H NMR data of 1a and 1b and their oxidation products 2–10. − The UPLC retention time (Rt, min) and m/z data (atomic mass unit to its formal charge ratio) for [M-H]− 1 in negative mode, the maximum wavelength λmax in UV/VIS spectra (nm), and H NMR chemical shifts (δ, ppm) of H5 and H6 protons and their coupling constant JH6-H5 for all identiﬁed compounds are given.

UPLC-PDA-ESI( )-HRMS UV 1H NMR − Compound Elemental Rt m/z [M-H]− H6 H5 λmax JH6-H5 Composition [min] Calcd Found [ppm] [ppm]

1a Se2Ura C4H4N2OSe 2.00 174.9411 174.9415 306 7.61 6.21 7.7 1b S2Ura C4H4N2OS 2.14 126.9966 126.9977 271 7.59 6.11 7.5 2a Ura-Se-Se-Ura C8H6N4O2Se2 3.61 348.8743 348.8748 275 7.88 6.26 6.6 2b Ura-S-S-Ura C8H6N4O2S2 3.35 252.9854 252.9853 228/274 7.88 6.22 6.4 3a Ura-Se-Se-Se-Ura C8H6N4O2Se3 4.17 428.7908 428.7909 238 - - - 3b Ura-S-S-S-Ura C8H6N4O2S3 4.11 284.9575 284.9576 - - - - 4a n = 1 Ura-SeOH C4H4N2O2Se 1.99 190.9360 190.9367 - - - - 4a n = 2 Ura-SeO2HC4H4N2O3Se 1.21 206.9309 206.9307 230/267 8.09 6.48 6.6 4b n = 1 Ura-SOH C4H4N2O2S 1.39 142.9915 142.9921 309 - - - 4b n = 2 Ura-SO2HC4H4N2O3S 1.32 158.9864 158.9865 228/275 8.07 6.49 6.8 4b n = 3 Ura-SO3HC4H4N2O4S 1.11 174.9814 174.9820 232/270 8.02 6.44 6.3 5 Ura C4H4N2O2 1.72 111.0194 111.0190 258 7.55 5.83 7.6 6 4HP C4H4N2O 1.72 95.0245 95.0239 223 8.02 6.55 7.0 7a C8H6N4O2Se 3.71 268.9578 268.9584 224/275 - - - 7b C8H6N4O2S 3.64 221.0133 221.0135 - - - - 8.09 6.42 8.0 8 C H N O 1.52 205.0362 205.0362 267 8 6 4 3 7.85 5.98 6.2 8.45 6.62 7.5 9 C12H8N6O4 1.89 299.0529 299.0534 247 8.01 6.35 6.2 7.87 6.05 7.9 10 C4H4N2O4S2 1.90 206.9534 206.9539 277 7.82 5.96 7.0

2.2. Analysis of the Oxidation Course of Se2Ura (1a)

2.2.1. The Reaction of Se2Ura and H2O2 at a 1:1 Molar Ratio, pH 7.4 Preliminary experiments showed that to obtain reliable data on possibly all intermediates, the oxidation process should be performed at a pH close to neutral, so pH 7.4 was selected. A set of 1H NMR spectra (Figure1) was acquired in the 24-h time course and compared with the spectrum of substrate 1a. At similar time points, the UPLC-PDA-ESI( )-HRMS data (Figure2a) were collected. − Dynamic changes were observed in the first stage of the reaction course (t < 60 min, see Figure2b), since over the first 1–2 min, the resonances and MS signals characteristic of 1a (δ 7.61 ppm and 6.21 ppm; m/z 174.9415) disappeared almost completely, while a new compound appeared, which was identified as diselenide 2a (Ura-Se-Se-Ura) (δ 7.88 ppm and 6.26 ppm; m/z 348.8748) (Scheme1). Later (from 5 to 30 min), the diselenide content gradually decreased, and after ca. 2 h, 2a disappeared almost completely (see Figure2a,b). Simultaneously, a new, stable product gradually appeared, for which two pairs of resonance signals were observed, i.e., δ 8.09 and 7.85 ppm for two different H6 protons and δ 6.42 and 5.98 ppm for two H5 protons. The m/z 205.0362 value in the mass spectrum for this product was lower than that for diselenide 2a (m/z 348.8748) but higher than that for 1a (m/z 174.9415). Detailed analysis (UPLC-ESI( )-MS/MS fragmentation, Figure3c) revealed that a selenium-free, two-ring − compound 8 (Scheme1) was formed, which, after 24 h, constituted 87% of the reaction products. The traces of compound 7a (m/z 268.9584, Figure3a), which might be a selenium-bearing precursor of 8, were observed in the UPLC-HRMS measurement (∆m/z 63.9222 between the ions related to the deprotonated molecules of 7a and 8 conformed to the Se O replacement) but not in the 1H → NMR spectrum. The N1a-C2b covalent bonding between two rings was confirmed by the presence of fragmentation ions at m/z 163.0305 and 134.0355 for 8 and at m/z 225.9521 and 162.0300 for 7a in the collision-induced dissociation (CID) spectra. The pH-dependent UV measurements of 8 in the pH range of 3–10 are shown in Figure2c. Int.Int. J. Mol.J. Mol. Sci. Sci.2020 2020, 21, 21,, 5956 x FOR PEER REVIEW 4 of4 18 of 18 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 18

Figure 1. 1H NMR analysis of the reaction mixtures for the oxidation of Se2Ura (1a, 10 mM) with H2O2

(10 mM)1 in1 67 mM phosphate buffer at pH 7.4 and room temperature. 2 2 FigureFigure 1. 1.H H NMR NMR analysis analysis ofof thethe reactionreaction mixturesmixtures for the oxidation of of Se2Ura Se2Ura ( (1a1a, ,10 10 mM) mM) with with H HO2O 2 (10(10 mM) mM) in in 67 67 mM mM phosphate phosphate bu bufferﬀer at at pH pH 7.4 7.4 andand roomroom temperature.temperature.

(b) (b)

(a) (c)

FigureFigure 2. 2.(a ()a) UPLC-PDA UPLC-PDA(a chromatographicchromatographic) analysis analysis and and (b (b) )time time course course of of the the formation formation(c) of products of products for the oxidation reaction of Se2Ura (1a, 10 mM) with H2O2 (10 mM) in 67 mM phosphate buffer at forFigure the oxidation 2. (a) UPLC-PDA reaction ofchromatographic Se2Ura (1a, 10 analysis mM) with and H (2bO) time2 (10 course mM) in of 67 the mM formation phosphate of products buﬀer at pH 7.4 and r.t. Them m/zz values are given in Table 1. The time course of the formation/disappearance pHfor 7.4 the and oxidation r.t. The reaction/ values of areSe2Ura given (1a in, 10 Table mM)1. Thewith time H2O course2 (10 mM) of thein 67 formation mM phosphate/disappearance buffer atof of each product was assessed 1by 1H NMR integration data. (c) The pH-dependent UV spectra of 8 in eachpH product7.4 and wasr.t. The assessed m/z values by H are NMR given integration in Table 1. data. The time (c) The course pH-dependent of the formation/disappearance UV spectra of 8 in the the pH range from 3 to 10. The inorganic selenous acid was identified by UPLC-ESI(−)-HRMS, and its pH range from 3 to 10. The inorganic1 selenous acid was identiﬁed by UPLC-ESI( )-HRMS, and its of each product was assessed by H NMR integration data. (c) The pH-dependent UV− spectra of 8 in retentionthe pH range time was from determined 3 to 10. The based inorganic on extracted selenous ion acid chromatograms was identified by (EICs) UPLC-ESI( for the− ion)-HRMS, corresponding and its to its deprotonated molecule (m/z 128.909).

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 18

2.2. Analysis of the Oxidation Course of Se2Ura (1a)

2.2.1. The Reaction of Se2Ura and H2O2 at a 1:1 Molar Ratio, pH 7.4 Preliminary experiments showed that to obtain reliable data on possibly all intermediates, the oxidation process should be performed at a pH close to neutral, so pH 7.4 was selected. A set of 1H NMR spectra (Figure 1) was acquired in the 24-h time course and compared with the spectrum of substrate 1a. At similar time points, the UPLC-PDA-ESI(−)-HRMS data (Figure 2a) were collected. Dynamic changes were observed in the first stage of the reaction course (t < 60 min, see Figure 2b), since over the first 1–2 min, the resonances and MS signals characteristic of 1a (δ 7.61 ppm and 6.21 ppm; m/z 174.9415) disappeared almost completely, while a new compound appeared, which was identified as diselenide 2a (Ura-Se-Se-Ura) (δ 7.88 ppm and 6.26 ppm; m/z 348.8748) (Scheme 1). Later (from 5 to 30 min), the diselenide content gradually decreased, and after ca. 2 h, 2a disappeared almost completely (see Figure 2a,b). Simultaneously, a new, stable product gradually appeared, for which two pairs of resonance signals were observed, i.e., δ 8.09 and 7.85 ppm for two different H6 protons and δ 6.42 and 5.98 ppm for two H5 protons. The m/z 205.0362 value in the mass spectrum for this product was lower than that for diselenide 2a (m/z 348.8748) but higher than that for 1a (m/z 174.9415). Detailed analysis (UPLC-ESI(−)-MS/MS fragmentation, Figure 3c) revealed that a selenium-free, two-ring compound 8 (Scheme 1) was formed, which, after 24 h, constituted 87% of the reaction products. The traces of compound 7a (m/z 268.9584, Figure 3a), which might be a selenium-bearing precursor of 8, were observed in the UPLC-HRMS measurement (Δm/z 63.9222 between the ions related to the deprotonated molecules of 7a and 8 conformed to the Se → O replacement) but not in the 1H NMR spectrum. The N1a-C2b covalent bonding between two rings was confirmed by the presence of fragmentation ions at m/z 163.0305 and 134.0355 for 8 and at m/z Int.225.9521 J. Mol. Sci.and2020 162.0300, 21, 5956 for 7a in the collision-induced dissociation (CID) spectra. The pH-dependent5 of 18 UV measurements of 8 in the pH range of 3–10 are shown in Figure 2c.

1a 2a 5 8 9 pH 8.0 14% 6% 48% 17% pH 5.0 0% 54% 46% 0% H2O pH 6.5 → 2.5 0% 63% 37% 0%

SchemeScheme 1.1.Oxidation Oxidation products products formed formed in in the the reaction reaction of 1aof (2-selenouracil,1a (2-selenouracil, 10 mM) 10 mM) with with H2O 2H(102O2 mM) (10 inmM) phosphate in phosphate buﬀer buffer at pH at 7.4. pH The 7.4. contents The contents of products of products found found after 24after h at 24 other h at pHother values pH values are given are belowgiven thebelow structures. the structures. The minor The productsminor products were identiﬁed were identified in the reaction in the reaction mixture timemixture course time but course were notbut presentwere not after present 24 h. after 24 h.

The structure of 8 was confirmed by NMR analysis in the 2D HMBC (Heteronuclear Multiple Bond Coherence) spectrum, where the correlation between the H6a proton (δ 7.85 ppm) and the C2b carbon (δ 156.10 ppm) indicated a covalent bond between N1 and C2 . Moreover, the same carbon a b 1 atom C2b also had a three-bond correlation with proton H6b at 8.10 ppm (see Figure4a). The H and 13C NMR spectra recorded for 8 are shown in Figures S9 and S10. Another minor product (ca. 5%), identified in the reaction mixture after 24 h, was found to be three-ring derivative 9 (see Scheme1). Its structure was confirmed by UPLC-HRMS analysis (m/z 299.0534) and 1H NMR resonances for three different H5 atoms and three different H6 atoms (Figures1 and2a, Table1). The UV spectra over a broad pH range (see Figure S13) were almost identical, and only one band with λmax 257 nm at pH 3 shifted to 245 nm at pH 10. The collision-induced dissociation (CID) spectrum of 9 (Figure3d) confirmed the presence of the three six-membered rings a, b, and c connected by N1a-C2b and N1b-C2c covalent bonds. The structure was also confirmed by the 2D HMBC spectrum shown in Figure4b, where three bond correlations between C2 a and H6a (δ 7.90 ppm) and H6b (δ 8.46 ppm) were found. Similarly, correlations between C2c (δ 156.79 ppm) and H6b (δ 8.46 ppm) and H6c (δ 8.02 ppm) were identified. Our approach also allowed the identification of triselenide 3a (Ura-Se-Se-Se-Ura, m/z 428.7909) with its highest abundance (ca. 5%) after 10 min and final disappearance after 2 h. Very weak signals for 1a were noted after ca. 60 min (Figure2a), indicating its partial restoration; however, its content after 24 h was virtually negligible. At an early time point (5 min), small amounts of seleninic acid 4a (n = 2, Ura-SeO2H, m/z 206.9307) appeared, for which resonances at δ 8.09 ppm and δ 6.48 ppm were noted. A minute amount of very reactive selenenic acid, Ura-SeOH (4a, n = 1, m/z 190.9367), was observed at a rather late stage of ca. 60 min, so its appearance might be attributed to the disproportionation of an intermediate compound(s). After 24 h, 8% uracil (5) was identified, and the residual amount of inorganic H2SeO3 was observed in UPLC-HRMS analysis. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 18

retention time was determined based on extracted ion chromatograms (EICs) for the ion Int.corresponding J. Mol. Sci. 2020 to, 21 its, 5956deprotonated molecule (m/z 128.909). 6 of 18

(a)

(b)

(c)

(d)

FigureFigure 3. UPLC/ESI(-)-MS/MS 3. UPLC/ESI(-)-MS/ MSmass mass spectra spectra of the of thedeprotonated deprotonated molecule molecule of 7a of 7a(a),( a7b), 7b(b),(b 8), (8c),(c and), and 9 9 (d()d recorded) recorded with with collision collision ener energygy ramping ramping from from 15 15 to to 35 35 eV. eV.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 18

The structure of 8 was confirmed by NMR analysis in the 2D HMBC (Heteronuclear Multiple Bond Coherence) spectrum, where the correlation between the H6a proton (δ 7.85 ppm) and the C2b carbon (δ 156.10 ppm) indicated a covalent bond between N1a and C2b. Moreover, the same carbon atom C2b also had a three-bond correlation with proton H6b at 8.10 ppm (see Figure 4a). The 1H and 13C NMR spectra recorded for 8 are shown in Figures S9 and S10. Another minor product (ca. 5%), identified in the reaction mixture after 24 h, was found to be three-ring derivative 9 (see Scheme 1). Its structure was confirmed by UPLC-HRMS analysis (m/z 299.0534) and 1H NMR resonances for three different H5 atoms and three different H6 atoms (Figures 1 and 2a, Table 1). The UV spectra over a broad pH range (see Figure S13) were almost identical, and only one band with λmax 257 nm at pH 3 shifted to 245 nm at pH 10. The collision-induced dissociation (CID) spectrum of 9 (Figure 3d) confirmed the presence of the three six-membered rings a, b, and c connected by N1a-C2b and N1b-C2c covalent bonds. The structure was also confirmed by the 2D HMBC spectrum shown in Figure 4b, where three bond correlations between C2a and H6a (δ 7.90 Int.ppm) J. Mol. and Sci. H62020b (,δ21 8.46, 5956 ppm) were found. Similarly, correlations between C2c (δ 156.79 ppm) and7 ofH6 18b (δ 8.46 ppm) and H6c (δ 8.02 ppm) were identified.

(a) (b)

1 13 Figure 4. The 1H/H/13C-HMBCC-HMBC spectra spectra (in (in D D22O)O) showing showing the the correlations correlations between between the hydrogen atoms and the carbon atoms in 8 (a) and 9 ((b).).

InOur an approach analogous also reaction allowed carried the identification out in phosphate of triselenide buffer at pH3a (Ura-Se-Se-Se-Ura, 8.0, the stability of m/z diselenide 428.7909)2a decreasedwith its highest remarkably abundance since (ca. after 5%) 1 min,after the10 min efficient and formationfinal disappearance of 8 was observed after 2 h. (seeVery NMR weak analysis signals Figurefor 1a were S21). noted Compared after ca. to that60 min detected (Figure for 2a), the indicating reaction at its pH partial 7.4, more restoration; seleninic however, acid 4a ( itsn = content2) was detectedafter 24 h in was the virtually reaction mixturenegligible. after At 24 an h. early The formationtime point of (5 the min), three-ring small amounts product 9 ofwas seleninic also observed. acid 4a After(n = 2,24 Ura-SeO h, the2 reactionH, m/z 206.9307) mixture appeared, consisted offor compounds which resonances8 (48%), at 9δ (17%),8.09 ppm5 (6%), and δ4a 6.48(n = ppm2) (15%), were andnoted. reconstituted A minute amount1a (14%) of (Scheme very re1active). selenenic acid, Ura-SeOH (4a, n = 1, m/z 190.9367), was observedFinally, at thea rather reactions late were stage performed of ca. 60 under min, slightlyso its appearance acidic conditions might (in be phosphate attributed bu toffer the at 1 pHdisproportionation 5.0 and in deionized of an waterintermediate at pH 6.5). compound(s). The relevant AfterH 24 NMR h, 8% spectra uracil (Figure(5) was S22)identified, indicated and that the afterresidual 2 min amount at pH of 5.0, inorganic diselenide H2SeO2a was3 was not observed detected, in andUPLC-HRMS the two-ring analysis. products 8 (46%) and uracil (5) (54%)In an were analogous highly reaction abundant. carried Interestingly, out in phosphate neither three-ringbuffer at pH compound 8.0, the stability9 nor restored of diselenide1a were 2a found.decreased For remarkably the reaction since carried after out 1 min, in the the water, efficient the formation reaction course of 8 was was observed quite similar, (see NMR and analysis8 and 5 wereFigure obtained S21). Compared in 37% and to that 63% detected yield, respectively for the reaction (Figure at S23).pH 7.4, This more change seleninic in the acid product 4a (n ratio = 2) was notdetected unexpected in the sincereaction the mixture reacting mixtureafter 24 becameh. The formation more acidic of (to the pH three-ring 1.0–2.5) asproduct inorganic 9 was H2SeO also3 wasobserved. released. After 24 h, the reaction mixture consisted of compounds 8 (48%), 9 (17%), 5 (6%), 4a (n = 2) (15%), and reconstituted 1a (14%) (Scheme 1). 2.2.2. The Reaction of Se2Ura and H2O2 at a 1:0.5 Molar Ratio and pH 7.4

The reaction of 1a (10 mM) was carried out with 0.5 equivalents (5 mM) of H2O2 in phosphate buﬀer at pH 7.4 and monitored by 1H NMR (see Figure5 and Figure S24). Interestingly, in this case 1a was almost immediately converted to diselenide 2a as a sole product (see Figure5), and only a small amount of 4a (n = 2, Ura-SeO2H) was observed after longer reaction times. Over the next 60 min, the formation of the two-ring and three-ring derivatives 8 and 9 was observed. After 24 h, the integration of 1H NMR signals indicated the formation of 8 (57%), 9 (26%), and 5 (6%), as well as restored 1a (11%) (Scheme2). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 18

Finally, the reactions were performed under slightly acidic conditions (in phosphate buffer at pH 5.0 and in deionized water at pH 6.5). The relevant 1H NMR spectra (Figure S22) indicated that after 2 min at pH 5.0, diselenide 2a was not detected, and the two-ring products 8 (46%) and uracil (5) (54%) were highly abundant. Interestingly, neither three-ring compound 9 nor restored 1a were found. For the reaction carried out in the water, the reaction course was quite similar, and 8 and 5 were obtained in 37% and 63% yield, respectively (Figure S23). This change in the product ratio was not unexpected since the reacting mixture became more acidic (to pH 1.0–2.5) as inorganic H2SeO3 was released.

2.2.2. The Reaction of Se2Ura and H2O2 at a 1:0.5 Molar Ratio and pH 7.4

O O O O O O NH NH O NH NH 5 mM H2O2 NH NH N SeSe N N O + NH N O + + N Se phosphate buffer pH 7.4 N O N Se H r.t., 24h HN HN N N N N H H

O O O 1a 2a 8 (57%) 9 (26%) 5 (6%) 1a (11%) Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 18

Int. J. Mol.Scheme Sci. 2020 2. Oxidation, 21, 5956 products formed in the reaction of 2-selenouracil (10 mM) with H2O2 (5 mM) in8 of 18 Finally,phosphate the buffer reactions at pH 7.4.were performed under slightly acidic conditions (in phosphate buffer at pH 5.0 and in deionized water at pH 6.5). The relevant 1H NMR spectra (Figure S22) indicated that after 2 min at pH 5.0, diselenide 2a was not detected, and the two-ring products 8 (46%) and uracil (5) (54%) were highly abundant. Interestingly, neither three-ring compound 9 nor restored 1a were found. For the reaction carried out in the water, the reaction course was quite similar, and 8 and 5 were obtained in 37% and 63% yield, respectively (Figure S23). This change in the product ratio was not unexpected since the reacting mixture became more acidic (to pH 1.0–2.5) as inorganic H2SeO3 was released.

2.2.2. The Reaction of Se2Ura and H2O2 at a 1:0.5 Molar Ratio and pH 7.4

The reaction of 1a (10 mM) was carried out with 0.5 equivalents (5 mM) of H2O2 in phosphate buffer at pH 7.4 and monitored by 1H NMR (see Figures 5 and S24). Interestingly, in this case 1a was almost immediately1 converted to diselenide 2a as a sole product (see Figure 5), and only a small Figure 5.5. 1H NMR analysis (H5 and H6 signals) of the reactionreaction mixtures for thethe oxidationoxidation ofof Se2UraSe2Ura amount(1a, of 10 4a mM) (n with= 2, Ura-SeO H O (52 mM)H) was in 67 observed mM phosphate after longer buﬀer reaction at pH 7.4, times. r.t, taken Over after the 2 next min (upper60 min, the (1a, 10 mM) with H22O2 (5 mM) in 67 mM phosphate buffer at pH 7.4, r.t, taken after 2 min (upper formation of the two-ring and three-ring derivatives 8 and 9 was observed. After 24 h, the integration spectrum)spectrum) andand withwith referencereference 1a1a,, lowerlower spectrum.spectrum. The ﬁgurefigure presentspresents thethe completecomplete transformationtransformation ofof of 1H NMR signals indicated the formation of 8 (57%), 9 (26%), and 5 (6%), as well as restored 1a Se2UraSe2Ura toto Ura-Se-Se-Ura Ura-Se-Se-Ura with with 0.5 0.5 eq. eq. of hydrogenof hydrog peroxide,en peroxide, through through two consecutive two consecutive reactions: reactions: Se2Ura (11%) (SchemeUra-SeOH 2). and Se2Ura + Ura-SeOH Ura-Se-Se-Ura. →Se2Ura → Ura-SeOH and Se2Ura + Ura-SeOH→ → Ura-Se-Se-Ura.

O O O 2.2.3.O The Reaction of Se2Ura and H2O2 at a 1:10 Molar Ratio and pH 7.4 O O NH NH O NH NH 5 mM H2O2 NH NH A 10-fold molar excess of H2ON2 towardsSeSe N Se2Ura (1a) NcausedO + almostNH immediateN O + disappearance+ of N Se phosphate buffer pH 7.4 N O N Se 1a (FiguresH S25 andr.t., 24h S26) and formationHN of the seleninicHN acid N derivativeN N 4aN (n = H2, Ura-SeO2HH) and diselenide 2a in yields of ca. 16% and 75%, Orespectively.O The 1H NMR spectrumO recorded after 5 min indicated1a an increased amount of 4a 2a(n = 2) at the expense 8 (57%) of diselenide 9 (26%) 2a . After 5 (6%) 24 h, the 1a (11%) mixture consisted of uracil (5, 89%, from the decomposition of Ura-SeO2H) and the two-ring derivative 8 (11%, Scheme 2. Oxidation products formed inin thethe reactionreaction ofof 2-selenouracil2-selenouracil (10(10 mM)mM) withwith HH22OO22 (5 mM) in fromphosphate the rearrangement bubufferﬀer atat pHpH 7.4.of7.4. diselenide 2a) (Scheme 3). Interestingly, no three-ring product was

2.2.3. The Reaction of Se2Ura and H2O2 at a 1:10 Molar Ratio and pH 7.4

A 10-fold molar excess of H2O2 towards Se2Ura (1a) caused almost immediate disappearance of 1a (Figures S25 and S26) and formation of the seleninic acid derivative 4a (n = 2, Ura-SeO2H) and diselenide 2a in yields of ca. 16% and 75%, respectively. The 1H NMR spectrum recorded after 5 min indicated an increased amount of 4a (n = 2) at the expense of diselenide 2a. After 24 h, the mixture consisted of uracil (5, 89%, from the decomposition of Ura-SeO H) and the two-ring derivative 8 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 8 of 18 (11%, from the rearrangement of diselenide 2a) (Scheme3). Interestingly, no three-ring product was detected. Only traces of 7a, as well asas inorganicinorganic HH22SeO33 and H2SeOSeO44,, were were detected detected by by UPLC-HRMS UPLC-HRMS (Figure S26).

Figure 5. 1H NMR analysis (H5 and H6 signals) of the reaction mixtures for the oxidation of Se2Ura (1a, 10 mM) with H2O2 (5 mM) in 67 mM phosphate buffer at pH 7.4, r.t, taken after 2 min (upper spectrum) and with reference 1a, lower spectrum. The figure presents the complete transformation of Se2Ura to Ura-Se-Se-Ura with 0.5 eq. of hydrogen peroxide, through two consecutive reactions: Se2Ura → Ura-SeOH and Se2Ura + Ura-SeOH → Ura-Se-Se-Ura.

2.2.3.Scheme The Reaction 3. OxidationOxidation of Se2Ura products and of H2-selenouracil 2O2 at a 1:10 (10 Molar mM) Ratio carried and outout withpHwith 7.4 HH22 O2 (100(100 mM) mM) in in phosphate phosphate bufferbuﬀer at pH 7.4. A 10-fold molar excess of H2O2 towards Se2Ura (1a) caused almost immediate disappearance of 1a (Figures S25 and S26) and formation of the seleninic acid derivative 4a (n = 2, Ura-SeO2H) and 2.3. Analysis Analysis of the Oxidation Course of S2Ura ( 1b)) diselenide 2a in yields of ca. 16% and 75%, respectively. The 1H NMR spectrum recorded after 5 min 2.3.1.indicated The an Reaction increased of S2Ura amount and of H 4a2O 2(nat = a2) 1:1 at Molarthe expense Ratio andof diselenide pH 7.4. 2a. After 24 h, the mixture 2.3.1. The Reaction of S2Ura and H2O2 at a 1:1 Molar Ratio and pH 7.4. consistedThe reactionof uracil ( of5, 89%, S2Ura from (1b ,the 10 decomposition mM) with hydrogen of Ura-SeO peroxide2H) and at the a 1:1two-ring molar derivative ratio (Scheme 8 (11%,4, The reaction of S2Ura (1b, 10 mM) with hydrogen peroxide at a 1:1 molar ratio (Scheme 4, Figuresfrom the6 and rearrangement7a) was much of slower diselenide thanthat 2a) of(Scheme the selenium 3). Interestingly, analog 1a, andno afterthree-ring 24 h, ca.product 45% of was the Figures 6 and 7a) was much slower than that of the selenium analog 1a, and after 24 h, ca. 45% of the substrate remained unchanged. Formation of sulﬁnic acid 4b (n = 2, Ura-SO2H) (δ 8.07 and 6.49 ppm, substrate remained unchanged. Formation of sulfinic acid 4b (n = 2, Ura-SO2H) (δ 8.07 and 6.49 ppm, m/z 158.9865) was noted immediately after mixing the reactants. Its maximum concentration occurred after approximately 2 h and decreased to approximately 34% after 24 h. In contrast to the oxidation of 1a, where at the early time points, diselenide 2a was the main intermediate, disulfide 2b (δ 7.88 and 6.22 ppm, m/z 252.9853) was present in a small amount only (0.4%), and it was stable until the end of the experiment. At consecutive time points, other low-abundance compounds were identified: sulfonic acid 4b (n = 3, Ura-SO3H, δ 8.02 and 6.44 ppm, m/z 174.9820, approx. 5%), sulfenic acid 4b (n = 1, Ura-SOH, m/z 142.9921), 4-pyrimidinone (6, δ 8.02 and 6.55 ppm, m/z 95.0239), trisulfide 3b (m/z 284.9576), and the 2-thiouracil member of the Bunte salt family 10 (δ 7.82 and 5.96, m/z 206.9539, see Figure S27), as well as the two-ring derivatives 7b (m/z 221.0135, see Figure 3b) and 8 (m/z 205.0362). After 24 h, the reaction mixture contained three major products (Scheme 4): substrate 1b (37%), sulfinic acid 4b (n = 2, 34%), and uracil (5, 17%). The spectroscopic and chromatographic data for the identified compounds are given in Table 1, and 1H NMR spectra, as well as the time course of the oxidation reaction (Figure S28), are included in the Supplementary Materials.

Scheme 4. The identified products of a reaction of 2-thiouracil (10 mM) with H2O2 (10 mM) in phosphate buffer at pH 7.4.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 18 detected. Only traces of 7a, as well as inorganic H2SeO3 and H2SeO4, were detected by UPLC-HRMS (Figure S26).

Scheme 3. Oxidation products of 2-selenouracil (10 mM) carried out with H2O2 (100 mM) in phosphate buffer at pH 7.4.

2.3. Analysis of the Oxidation Course of S2Ura (1b)

2.3.1. The Reaction of S2Ura and H2O2 at a 1:1 Molar Ratio and pH 7.4.

Int. J. Mol.The Sci.reaction2020, 21 ,of 5956 S2Ura (1b, 10 mM) with hydrogen peroxide at a 1:1 molar ratio (Scheme9 of 184, Figures 6 and 7a) was much slower than that of the selenium analog 1a, and after 24 h, ca. 45% of the substrate remained unchanged. Formation of sulfinic acid 4b (n = 2, Ura-SO2H) (δ 8.07 and 6.49 ppm, mm/z/z 158.9865) 158.9865) waswas notednoted immediatelyimmediately afterafter mixingmixing thethe reactants.reactants. Its maximum concentration occurred afterafter approximatelyapproximately 22 hh andand decreaseddecreased toto approximatelyapproximately 34%34% afterafter 2424 h.h. InIn contrastcontrast toto thethe oxidationoxidation ofof 1a1a,, wherewhere atat thethe earlyearly timetime points,points, diselenidediselenide 2a2a waswas thethe mainmain intermediate,intermediate, disulfidedisulfide 2b ((δδ 7.887.88 andand 6.226.22 ppm,ppm, mm/z/z 252.9853)252.9853) waswas presentpresent inin aa smallsmall amountamount onlyonly (0.4%),(0.4%), andand itit waswas stablestable untiluntil thethe endend ofof the experiment. At consecutive time points points,, other low-abundance compounds were identified: identified: δ sulfonicsulfonic acid 4b (n = 3,3, Ura-SO Ura-SO3H,3H, δ 8.028.02 and and 6.44 6.44 ppm, ppm, m/zm/ z174.9820,174.9820, approx. approx. 5%), 5%), sulfenic sulfenic acid acid 4b4b (n (=n 1,= Ura-SOH,1, Ura-SOH, m/zm 142.9921),/z 142.9921), 4-pyrimidinone 4-pyrimidinone (6, δ ( 68.02, δ 8.02 and and6.55 6.55ppm, ppm, m/z 95.0239),m/z 95.0239), trisulfide trisulfide 3b (m/z3b (284.9576),m/z 284.9576), and andthe 2-thiouracil the 2-thiouracil member member of the of Bunte the Bunte salt family salt family 10 (δ10 7.82(δ 7.82 and and5.96, 5.96, m/z m206.9539,/z 206.9539, see seeFigure Figure S27), S27), as well as well as the as the two-ring two-ring derivatives derivatives 7b7b (m/z(m /221.0135,z 221.0135, see see Figure Figure 3b)3b) and and 88 ((m/zm/z 205.0362).205.0362). AfterAfter 2424 h,h, thethe reactionreaction mixturemixture containedcontained threethree majormajor productsproducts (Scheme(Scheme4 ):4): substratesubstrate 1b (37%), sulfinicsulfinic acidacid 4b4b ((nn == 2, 34%), and uracil (5,, 17%).17%). The spectroscopic and chromatographic datadata forfor thethe 1 identifiedidentified compoundscompounds areare givengiven inin TableTable1 1,, and and 1H NMRNMR spectra,spectra, as wellwell asas thethe timetime coursecourse ofof thethe oxidationoxidation reactionreaction (Figure(Figure S28),S28), areare includedincluded inin thethe SupplementarySupplementary Materials.Materials.

SchemeScheme 4.4. The identified identiﬁed products of a reactionreaction ofof 2-thiouracil2-thiouracil (10(10 mM)mM) withwith HH22OO22 (10(10 mM)mM) inin Int. J. phosphateMol.phosphate Sci. 2020 bubuffer, 21ﬀ,er x FOR atat pHpH PEER 7.4.7.4. REVIEW 9 of 18

11 Figure 6.6. H NMRNMR spectraspectra collected collected for for the the reaction reaction of S2Uraof S2Ura (1b (,1b 10, mM)10 mM) with with H2O H2 2(10O2 mM)(10 mM) in 67 in mM 67 phosphatemM phosphate buﬀer buffer at pH at 7.4 pH and 7.4 r.t. and at the r.t. time at the points time indicated points indicated on the left-hand on the sideleft-hand of each side spectrum. of each spectrum.

2.3.2. The Reaction of S2Ura and H2O2 at a 1:10 Molar Ratio and pH 7.4 The reaction of 1b with a 10-fold molar excess of hydrogen peroxide was relatively fast, and the substrate disappeared after 2 h (Scheme 5, Figures S29 and 7b). Initially, mainly sulfinic acid intermediate 4b (n = 2; Ura-SO2H) (m/z 158.9865) was observed, but after 30 min, its content decreased, while the content of uracil increased. A minute amount of disulfide 2b (m/z 252.9853) was detected by 1H NMR. The content of sulfonic acid 4b (n = 3; m/z 174.9820) increased over the first hour of the reaction course. Traces of sulfenic acid 4b (n = 1; m/z 142.9921), 4-pyrimidinone (6, m/z 95.0239), and Bunte salt 10 (m/z 206.9539), as well as two-ring compound 7b (m/z 221.0135), were identified in UPLC-HRMS analysis. After 24 h, only the resonance signals of uracil 5 were present in the NMR spectrum, but traces of sulfinic and sulfonic acids were still detected in the UPLC-MS chromatogram (Figure 7b).

O O O NH NH 100 mM H2O2 NH

N S phosphate buffer pH 7.4 N SO H N O H r.t., 24h 2 H

1b 4b, n=2 100% (5)

Scheme 5. Oxidation products of 2-thiouracil (10 mM) carried out with H2O2 (100 mM) in phosphate buffer at pH 7.4.

Int. J. Mol. Sci. 2020, 21, 5956 10 of 18 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 18

(a) (b)

FigureFigure 7. UPLC-PDA 7. UPLC-PDA chromatographic chromatographic analysis analysis of the re ofaction the reaction mixtures mixtures of oxidation of oxidation of S2Ura (1b of,S2Ura 10 (1b,

mM)10 with mM) H with2O2 (10 H2 mM)O2 (10 (a) mM) or with (a) H or2O with2 (100 H mM)2O2 ((100b) in mM) 67 mM (b )phosphate in 67 mM buffer phosphate at pH 7.4, buff r.t.er atMass pH 7.4, r.t. spectraMass of spectra all compounds of all compounds identified identified here are shown here are in the shown Supplementary in the Supplementary Materials. The Materials. m/z data The arem /z data givenare in given Table in 1. Table Inorganic1. Inorganic sulfurous sulfurous and sulfuric and acids sulfuric were acids identified were identifiedby UPLC-ESI by UPLC-ESI(−)-HRMS, (and)-HRMS, − theirand retention their retention times were times determined were determined based on based extracted on extracted ion chromatograms ion chromatograms (EICs) for (EICs) the ions for the ions correspondingcorresponding to their to their deprotonated deprotonated molecules molecules (m/z 80.9646 (m/z 80.9646 and 96.9596, and 96.9596, respectively). respectively).

1 2.3.2.Table The 1. UPLC-PDA-ESI Reaction of S2Ura (−)-HRMS and Hand2O 2Hat NMR a 1:10 data Molar of 1a Ratioand 1b and and pHtheir 7.4 oxidation products 2– 10. The UPLC retention time (Rt, min) and m/z data (atomic mass unit to its formal charge ratio) for The reaction of 1b with a 10-fold molar excess of hydrogen peroxide was relatively fast, and the [M-H]- in negative mode, the maximum wavelength λmax in UV/VIS spectra (nm), and 1H NMR substrate disappeared after 2 h (Scheme5, Figure S29 and Figure7b). Initially, mainly sulfinic acid chemical shifts (δ, ppm) of H5 and H6 protons and their coupling constant JH6-H5 for all identified intermediatecompounds 4bare (given.n = 2; Ura-SO2H) (m/z 158.9865) was observed, but after 30 min, its content decreased, while the content of uracil increased. A minute amount of disulfide 2b (m/z 252.9853) was detected 1 1 UPLC-PDA-ESI(−)-HRMS UV H NMR by HCompound NMR. The contentElemental of sulfonic Rt acid 4b (nm/z= [M-H]3; m-/ z 174.9820) increasedH6 overH5 the first hour of λmax JH6-H5 the reaction course. TracesComposition of sulfenic [min] acid 4bCalcd(n = 1; Foundm/z 142.9921), 4-pyrimidinone[ppm] [ppm] (6, m/z 95.0239), and1a Bunte saltSe2Ura10 (m/z 206.9539),C4H4N2OSe as well2.00 as two-ring174.9411 compound174.9415 3067b (m/z 221.0135),7.61 6.21 were identified7.7 in UPLC-HRMS1b S2Ura analysis. C After4H4N2OS 24 h, only2.14 the126.9966 resonance 126.9977 signals of271 uracil 7.595 were 6.11 present 7.5 in the NMR 2a Ura-Se-Se-Ura C8H6N4O2Se2 3.61 348.8743 348.8748 275 7.88 6.26 6.6 spectrum,2b Ura-S-S-Ura but traces of sulfinicC8H6N4O2S and2 sulfonic3.35 252.9854 acids were 252.9853 still detected228/274 in the7.88 UPLC-MS 6.22 chromatogram6.4 (Figure7b).Ura-Se-Se-Se- 3a C8H6N4O2Se3 4.17 428.7908 428.7909 238 - - - Ura 3b Ura-S-S-S-Ura C8H6N4O2S3 4.11 284.9575 284.9576 - - - - 4a n = 1 Ura-SeOH C4H4N2O2Se 1.99 190.9360 190.9367 - - - - 4a n = 2 Ura-SeO2H C4H4N2O3Se 1.21 206.9309 206.9307 230/267 8.09 6.48 6.6 4b n = 1 Ura-SOH C4H4N2O2S 1.39 142.9915 142.9921 309 - - - 4b n = 2 Ura-SO2H C4H4N2O3S 1.32 158.9864 158.9865 228/275 8.07 6.49 6.8 4b n = 3 Ura-SO3H C4H4N2O4S 1.11 174.9814 174.9820 232/270 8.02 6.44 6.3 5 Ura C4H4N2O2 1.72 111.0194 111.0190 258 7.55 5.83 7.6

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 18

Figure 6. 1H NMR spectra collected for the reaction of S2Ura (1b, 10 mM) with H2O2 (10 mM) in 67 mM phosphate buffer at pH 7.4 and r.t. at the time points indicated on the left-hand side of each spectrum.

2.3.2. The Reaction of S2Ura and H2O2 at a 1:10 Molar Ratio and pH 7.4 The reaction of 1b with a 10-fold molar excess of hydrogen peroxide was relatively fast, and the substrate disappeared after 2 h (Scheme 5, Figures S29 and 7b). Initially, mainly sulfinic acid intermediate 4b (n = 2; Ura-SO2H) (m/z 158.9865) was observed, but after 30 min, its content decreased, while the content of uracil increased. A minute amount of disulfide 2b (m/z 252.9853) was detected by 1H NMR. The content of sulfonic acid 4b (n = 3; m/z 174.9820) increased over the first hour of the reaction course. Traces of sulfenic acid 4b (n = 1; m/z 142.9921), 4-pyrimidinone (6, m/z 95.0239), and Bunte salt 10 (m/z 206.9539), as well as two-ring compound 7b (m/z 221.0135), were identified in UPLC-HRMS analysis. After 24 h, only the resonance signals of uracil 5 were present in the NMR Int.spectrum, J. Mol. Sci. but2020 traces, 21, 5956 of sulfinic and sulfonic acids were still detected in the UPLC-MS chromatogram11 of 18 (Figure 7b).

O O O NH NH 100 mM H2O2 NH

N S phosphate buffer pH 7.4 N SO H N O H r.t., 24h 2 H

1b 4b, n=2 100% (5)

Scheme 5.5. Oxidation products ofof 2-thiouracil2-thiouracil (10(10 mM)mM) carriedcarried outout withwith HH22OO22 (100 mM) inin phosphatephosphate bubufferﬀer atat pHpH 7.4.7.4.

3. Discussion 2-Selenouracil (1a, Se2Ura) and 2-thiouracil (1b, S2Ura) are relatively rare nucleobases present in transfer RNAs (http://modomics.genesilico.pl[ 20], http://mods.rna.albany.edu[ 21]). Within presumably acceptable simplification (no specific substituent at position C5), these compounds may be used as models in preliminary investigations of the processes occurring with tRNA under oxidative stress conditions. Both compounds are sufficiently soluble in aqueous solutions for 1H NMR spectroscopy (in D2O) and advanced high-resolution LC/MS analysis. Generally, our investigations have shown that 2-selenouracil (1a) and 2-thiouracil (1b) have significantly different redox properties (Scheme6, the red path is preferable for selenium components, and the blue path is preferable for sulfur components). Compound 1a is extremely prone to H2O2-assisted oxidation, plausibly resulting in the formation of selenenic acid 4a (n = 1, Ura-SeOH). This very reactive compound is not identified in the first few minutes of the reaction (it is detected later on in a relatively complex reaction mixture) since, probably, it reacts rapidly with 1a, producing diselenide 2a. This path is in agreement with the results of the reaction carried out with 0.5 equivalents of H2O2, where 2a is almost exclusively formed (Figure5 and Figure S24) from 1a and 4a (n = 1). According to our assumption, this pathway of oxidation includes the two following steps: (i) Ura-SeH Ura-SeOH and (ii) Ura-SeH + Ura-SeOH Ura-Se-Se-Ura. → → On the other hand, according to the literature data [22], diselenide R-Se-Se-R under alkaline conditions may hydrolyze to R-SeH and selenenic acid R-SeOH, and the latter, as an extremely unstable derivative, may disproportionate to produce the R-SeH substrate and seleninic acid R-SeO2H (see Scheme7). This mechanism may explain the partial reconstitution of Ura-SeH ( 1a) from diselenide 2a (without a reducing environment). We also demonstrate that the route for reconstitution of 1a from 2a is limited due to the consumption/depletion of diselenide 2a by its rapid intramolecular rearrangement, leading to the selenium-containing intermediate 7a called “Jaffe’s base” (Scheme1)[ 23]. In aqueous environments, this compound easily loses the selenium atom to form stable compound 8. The relevant mechanism is similar to that proposed for the formation of “Jaffe’s base” during the oxidation of ethylene thiourea (Scheme8a) [ 24]. This reaction, obviously, does not take place on the level of tRNA, when Se2Ura is built in the nucleoside moiety. However, one cannot exclude the possibility that the Se2U-tRNA is metabolized (nucleolytically degraded [25]) to Se2Ura, which, in oxidative stress, might be transformed into two- and three-ring products described herein. There are some reports suggesting that 2-selenouracil derivatives substituted at the C6 position with alkyl groups react with iodine to form corresponding R6Ura-SeI2 complexes (where R = methyl, ethyl, n-propyl, or iso-propyl), which may further react with the starting selenouracil R6Se2Ura to form the two-ring products R6Se2Ura-R6Ura, which are deprived of the selenium atom in ring b [26]. As shown by crystallographic analysis, these products have a covalent bond between N3a of one R6Se2Ura (a) ring and the C2b atom of the second R6Se2Ura molecule (b). Ultimately, these compounds spontaneously transform into four-ring derivatives in which the two-ring components are linked with a diselenide bridge. Alternatively, in aqueous solutions, these compounds undergo deselenation and cleavage of the N3a-C2b bond, leading to the uracil molecule being substituted with an alkyl group at position 6. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 18

6 4HP C4H4N2O 1.72 95.0245 95.0239 223 8.02 6.55 7.0 7a C8H6N4O2Se 3.71 268.9578 268.9584 224/275 - - - 7b C8H6N4O2S 3.64 221.0133 221.0135 - - - - Int. J. Mol. Sci. 2020, 21, 5956 8.09 6.42 128.0 of 18 8 C8H6N4O3 1.52 205.0362 205.0362 267 7.85 5.98 6.2 8.45 6.62 7.5

In9 contrast to the cited two-ringC12H8N6O4 compound1.89 R6Se2Ura(N3299.0529 299.0534a)-(C2 b)R6Ura,247 the8.01 two-ring 6.35 compound6.2 8 reported here has a spanning bond between the C2b and N1a atoms, as documented7.87 6.05 by 2D NMR7.9 studies10 (Figure4a). If the amountC4H4N2O of4S2 oxidant 1.90 is stoichiometrically206.9534 206.9539 limiting277 (see 7.82data for5.96 a 1:0.5 molar 7.0 ratio of reactants at pH 7.4), or the reaction occurs under more basic conditions (pH 8), derivative 83.could Discussion be transformed to three-ring product 9 with a yield up to 26% of the total content. At first, we have2-Selenouracil supposed that (1a, compoundSe2Ura) and9 is 2-thiouracil a product of(1b the, S2Ura) condensation are relatively of 8 with rare1a nucleobases, with the departure present ofin hydrogentransfer selenideRNAs (http://modomics.genesilico.pl as a second product (according [20], to the http://mods.rna.albany.edu mechanism proposed in Figure [21]). S30). Within If so, thispresumably reaction isacceptable expected simplification to run between (no8 and specific1a without substituent any additionalat position reagent. C5), these However, compounds as shown may 1 bybe theusedH as NMR models analysis in preliminary of 8 in the mixtureinvestigations with an of excess the processes of 1a (0.4:1 occurring molar ratio), with only tRNA signals under of tracesoxidative of compound stress conditions.9 are noted Both after compounds 24 h. However, are sufficiently the prolonged soluble incubation in aqueous time solutions of this for mixture 1H NMR up tospectroscopy 7 days allowed (in D to2O) increase and advanced the content high-resolution of 9 up to ca LC/MS 14%, while analysis. the content of 1a clearly decreased to 32%Generally, (Fig. S30). our Interestingly, investigations when have the shown same reactionthat 2-selenouracil was tested ((81aand) and1a ,2-thiouracil 0.4:1 molar ( ratio)1b) have but withsignificantly the addition different of 0.5 eq.redox of hydrogenproperties peroxide (Scheme over 6, 1athe, the red formation path is of preferable9 after 1 h wasfor observed,selenium tocomponents, reach finally and the the yield blue of ca.path 14% is preferable after 24 h (seefor Figuresulfur S31).components). Thus, we Compound conclude that 1a compoundis extremely9 isprone mostly to H a2 productO2-assisted of the oxidation, condensation plausibly of 8 resultingand 2a, as in shownthe formation in Scheme of selenenic8b, in which acid 2-selenouracil 4a (n = 1, Ura- 1aSeOH).and selenium This very are reactive released, compound Thus, the is reconstituted not identified 2-selenouracil, in the first few present minutes as a of final the product reaction in (it the is reaction,detected shownlater on in in Scheme a relatively2, may complex originate reaction from the mixture) diselenide since, reacting probably, with it compound reacts rapidly8. with 1a, producingIn general, diselenide we can 2a. summarize This path is that in agreement 2-selenouracil with ( 1athe) inresults the presence of the reaction of a stoichiometrically carried out with limiting0.5 equivalents amount of ofH2 theO2, where oxidant 2a is is preferablyalmost exclusiv transformedely formed to (Figures diselenide 5 and2a, S24) which from spontaneously 1a and 4a (n rearranges= 1). According to the to two-ring our assumption, product 8, this which, pathway in turn, of reacts oxidation with theincludes remaining the two diselenide following2a tosteps: yield (i)9 (redUra-SeH path, → Scheme Ura-SeOH6). and (ii) Ura-SeH + Ura-SeOH → Ura-Se-Se-Ura.

Scheme 6. Proposed transformation pathways for Se2Ura (1a) and S2Ura (1b) under oxidizing conditions. The red path is preferable for 2-selenouracil 1a, and the blue path is preferable for 2-thiouracil 1b. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 18 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 18

Scheme 6. Proposed transformation pathways for Se2Ura (1a) and S2Ura (1b) under oxidizing mostly a product of the condensation of 8 and 2a, as shown in Scheme 8b, in which 2-selenouracil 1a conditions. The red path is preferable for 2-selenouracil 1a, and the blue path is preferable for 2- and selenium are released, Thus, the reconstituted 2-selenouracil, present as a final product in the thiouracil 1b. reaction, shown in Scheme 2, may originate from the diselenide reacting with compound 8. In general,On wethe canother summari hand, accordingze that 2-selenouracil to the literature (1a) inda tathe [22], presence diselenide of a stoichiometricallyR-Se-Se-R under alkaline limitingconditions amount of may the oxidanthydrolyze is preferablyto R-SeH andtransformed selenenic toacid diselenide R-SeOH, 2a and, which the latter,spontaneously as an extremely rearrangesunstable to the derivative,two-ring product may disproportio 8, which, in nateturn, toreacts produce with the remainingR-SeH substrate diselenide and 2a seleninic to yield acid R- 9 (red path, Scheme 6). Int. J. Mol.SeO Sci.22020H (see, 21, 5956Scheme 7). This mechanism may explain the partial reconstitution of Ura-SeH13 of 18(1a) from Thisdiselenide type of rearrangement 2a (without a reducinghas not been environment). reported earlier for the oxidation experiments carried out with c5Se2Ura [18]. In this cited work, the oxidation of c5Se2Ura furnishes oxidized forms of c5Se2Ura and uracil.1 In our2 case, in contrast, the main1 component2 is the two-ring product 8. This 3 R Se Se R + 3H O 3R SeOH + 3 R SeH inconsistency may originate from the2 use of Se2Ura bearing a carboxyl substituent in position C5, 1 1 1 which exerts an electron-withdrawing2R SeOH effect (botR hSeO by 2inductionH + R SeHand resonance). This effect is evidenced by the change1 in the p1Ka value from 7.181 for Se2Ura1 (1a) to 7.11 for c5Se2Ura. A similar R SeOH + R SeH R Se Se R + H O effect has been observed for 2-thiouracils, where the pKa value changes2 from 7.75 for S2Ura (1b) to 7.68 for c5S2Ura [27].1 2 2 1 1 1 3 R Se Se R + 2H2O 3 R SeH + R Se Se R + R SeO2H

Scheme 7. A possible scheme of hydrolysis of diselenides under basic conditions [22]. Scheme 7. A possible scheme of hydrolysis of diselenides under basic conditions [22].

We also demonstrate that the route for reconstitution of 1a from 2a is limited due to the consumption/depletion of diselenide 2a by its rapid intramolecular rearrangement, leading to the selenium-containing intermediate 7a called “Jaffe’s base” (Scheme 1) [23]. In aqueous environments, this compound easily loses the selenium atom to form stable compound 8. The relevant mechanism is similar to that proposed for the formation of “Jaffe’s base” during the oxidation of ethylene thiourea (Scheme 8a) [24]. This reaction, obviously, does not take place on the level of tRNA, when Se2Ura is

built in the nucleoside moiety. However, one cannot exclude the possibility that the Se2U-tRNA is metabolized (nucleolytically degraded (a[25])) to Se2Ura, which, in oxidative stress, might be transformed into two- and three-ring products described herein. There are some reports suggesting that 2-selenouracil derivatives substituted at the C6 position with alkyl groups react with iodine to form corresponding R6Ura-SeI2 complexes (where R = methyl, ethyl, n-propyl, or iso-propyl), which may further react with the starting selenouracil R6Se2Ura to form the two-ring products R6Se2Ura-R6Ura, which are deprived of the selenium atom in ring b [26]. As shown by crystallographic analysis, these products have a covalent bond between N3a of one R6Se2Ura (a) ring and the C2b atom of the second R6Se2Ura molecule (b). Ultimately, these compounds spontaneously transform into four-ring derivatives in which the two-ring components are linked with a diselenide bridge. Alternatively, in aqueous solutions, these compounds undergo deselenation and cleavage of the N3a-C2b( bond,b) leading to the uracil molecule being substituted with an alkyl group at position 6. SchemeScheme 8. 8. TheThe proposed proposed mechanism mechanism of of formation formation of of two-ring two-ring compounds compounds 7a7a andand 88 ((aa)) and and three-ring three-ring In contrast to the cited two-ring compound R6Se2Ura(N3a)-(C2b)R6Ura, the two-ring compound compoundcompound 99 ((bb).). 8 reported here has a spanning bond between the C2b and N1a atoms, as documented by 2D NMR TheThisstudies route type of leading(Figure rearrangement to4a). diselenide If the has amount not 2a beenand of itsoxidant reported rearrangement is earlier stoichiometrically for to the 7a oxidation, 8, and limiting 9experiments is less (see favorable data carried for ifa 1:0.5the out molar oxidantwith c5Se2Ura ratiois present of [ 18reactants]. in In thishigh at cited pHexcess 7.4), work, (see or the the Figures oxidation reaction S25 ofoccu and c5Se2Urars S26).under furnishesIn more these basic oxidizedconditions, conditions forms the (pH of prevalent c5Se2Ura 8), derivative 8 could be transformed to three-ring product 9 with a yield up to 26% of the total content. At first, we formationand uracil. of In seleninic our case, acid in contrast, 4a (n = the2, Ura-SeO main component2H) is noted, is the which, two-ring in aqueous product solutions,8. This inconsistency undergoes water-assistedmay originatehave supposed fromhydrolysis, the that use resulting compound of Se2Ura in bearingthe 9 is formation a product a carboxyl ofof uracilthe substituent condensation (5) (89%) in accompanied position of 8 with C5, 1a which ,by with elimination exertsthe departure an of hydrogen selenide as a second product (according to the mechanism proposed in Figure S30). If so, ofelectron-withdrawing selenium (IV) oxide, e ffwhichect (both reacts by inductionwith water and to form resonance). H2SeO This3 (blue eff path,ect is evidencedScheme 6). by Besides, the change it is worthin the pmentioningKthisa value reaction from that, is 7.18expected according for Se2Ura to runto published( between1a) to 7.11 8 data and for c5Se2Ura., 1ain withoutthe presence A any similar additional of eexcessffect has rehydrogenagent. been observedHowever, peroxide, for as shown 1 the2-thiouracils, diselenidesby the where Hare NMR further the panalysisK oxidizeda value of changes8 to in selenoseleninate the frommixture 7.75 with for derivatives S2Ura an excess (1b) (-Se(O)-Se-), toof 7.681a (0.4:1 for c5S2Ura molar which ratio), [are27]. unstable only signals of traces of compound 9 are noted after 24 h. However, the prolonged incubation time of this mixture underThe basic route conditions leading [28–30]. to diselenide These 2acompoundand its rearrangements are hydrolyzed to to7a ,seleninic8, and 9 acidis less (RSeO favorable2H) and if selenolthe oxidant (RSeH),up to is 7 days present while, allowed in in acidic high to increase excessenvironmen (see the Figurescontentts, selenenic of S25 9 up and acid to S26).ca (RSeOH) 14%, In while these is formed the conditions, content [31]. of Our the 1a prevalentclearlyefforts decreasedto to 32% (Fig. S30). Interestingly, when the same reaction was tested (8 and 1a, 0.4:1 molar ratio) but confirmformation such of seleninictransformation acid 4a pathways(n = 2, Ura-SeO have been2H) unsuccessful, is noted, which, probably in aqueous due to solutions, the low stability undergoes of thewater-assisted selenoseleninatewith the hydrolysis, addition derivative. of resulting 0.5 eq. of in hydrogen the formation peroxide of uracil over (1a5), (89%) the formation accompanied of 9 after by elimination 1 h was observed, of seleniumto reach (IV) finally oxide, the which yield reacts of ca. with 14% water after 24 to h form (see HFigure2SeO3 S31).(blue Thus, path, we Scheme conclude6). Besides, that compound it is 9 is worth mentioning that, according to published data, in the presence of excess hydrogen peroxide,

the diselenides are further oxidized to selenoseleninate derivatives (-Se(O)-Se-), which are unstable under basic conditions [28–30]. These compounds are hydrolyzed to seleninic acid (RSeO2H) and selenol (RSeH), while, in acidic environments, selenenic acid (RSeOH) is formed [31]. Our eﬀorts to conﬁrm such transformation pathways have been unsuccessful, probably due to the low stability of the selenoseleninate derivative. Int. J. Mol. Sci. 2020, 21, 5956 14 of 18

It is noteworthy that, in the Hondal team’s work [18], treatment of 5-carboxy-2-selenouracil (c5Se2Ura, 100 mM) with 1 molar equivalent of H2O2 after 18 h led to a product resonating at 77 δ 1273 ppm in Se NMR, which was assigned as seleninic acid c5Ura-SeO2H. In our reaction carried out in phosphate buffer at pH 7.4, seleninic acid Ura-SeO2H(4a, n = 2) is present only in the first several minutes, while inorganic seleninic acid H2SeO3 is present after 1 h (see Figure2a). In addition, similar chemical shifts are known to be associated with Na2SeO3 (1263 ppm) and H2SeO3 (1300 ppm) [32]. Unlike 2-selenouracil (1a), 2-thiouracil (1b) is less prone to oxidation with H2O2. The reaction is remarkably slower, and a 10-fold molar excess of oxidant leads to predominant conversion to uracil (5) (Scheme5). With equal or stoichiometrically limiting amounts of H 2O2, ca. 40% of 1b remains intact. The identified intermediates include sulfenic acid 4b (n = 1, Ura-SOH), sulfinic acid 4b (n = 2, Ura-SO2H), and sulfonic acid 4b (n = 3, Ura-SO3H) (Scheme6, blue). Notably, among those three 1 acid forms, sulfinic acid (Ura-SO2H) is the most abundant, as shown by H NMR analysis (having the highest signal integration and the longest time, the signals remained in the reaction mixture). Similar to sulfonic acid (Ura-SO3H), this compound can eliminate sulfur oxide upon nucleophilic substitution of a water molecule at the C2 position of the nucleobase ring. Although the small amount of 4-pyrimidinone (6) is identified, this process is marginal in comparison to the previously reported data for 2-thiouridines [13–17]. Traces of disulfide 1b, trisulfide 1c, or bicyclic compounds 7b and 8 (detected by UPLC-HRMS) indicate that while the responses of S2Ura and Se2Ura to hydrogen peroxide are common, their different redox properties are decisive for the preferred paths and the final content of oxidized products.

4. Materials and Methods

4.1. Methods and Instrumentation

4.1.1. NMR Spectroscopy 1H, 13C, COSY (COrrelation SpectroscopY), HMQC (Heteronuclear Multiple Quantum Correlation) and HMBC NMR spectra were recorded on a Bruker Avance 700 MHz spectrometer. The NMR spectra for 1H and 13C were recorded at 700 MHz and 176 MHz, respectively. Chemical shifts (δ) are reported in ppm, and the signal multiplicities are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Coupling constants are reported in hertz (Hz).

4.1.2. Ultra-Performance Liquid Chromatography Coupled with a High-Resolution Mass Spectrometry and Photodiode Array Detection (UPLC-PDA-ESI( )-HRMS) − The identiﬁcation of the reaction products was carried out using an ACQUITY UPLC I-Class chromatography system equipped with a photodiode array detector with a binary solvent manager (Waters Corp., Milford, MA, USA ) coupled with an SYNAPT G2-Si mass spectrometer equipped with an electrospray source and quadrupole-Time-of-Flight mass analyzer (Waters Corp., Milford, MA, USA). An Acquity HSS T3 1.8 µm column (100 2.1 mm) (Waters Corp., Milford, MA) thermostated at × 30 ◦C was used for the chromatographic separation of the analyte. A gradient program was employed with the mobile phase combining solvent A (10 mM CH3COONH4) and solvent B (50% CH3CN in 10 mM CH3COONH4) as follows: 10% B (0–1.0 min), 10–95% B (1.0–3.5 min), 95–99% B (3.5–4.0 min), 95–10% B (4.0–4.1 min), and 10–10% B (4.1–6 min). The ﬂow rate was 0.2 mL/min, and the injection volume was 1 µL. For mass spectrometric detection, the electrospray source was operated in a negative, high-resolution mode at a 50,000 FWHM resolving power of the TOF analyzer. To ensure accurate mass measurements, data were collected in centroid mode, and mass was corrected during acquisition using leucine encephalin solution as an external reference, Lock-SprayTM, (Waters Corp., Milford, MA, USA), which generated reference ion at m/z 554.2615 ([M-H]) in negative ESI mode. The optimized source parameters were: capillary voltage 3 kV, cone voltage 20 V, source temperature 90 ◦C, Int. J. Mol. Sci. 2020, 21, 5956 15 of 18

desolvation gas (nitrogen) ﬂow rate 600 L/h with the temperature 350 ◦C, nebulizer gas pressure 6.5 bar. Mass spectrometer conditions were optimized by direct infusion of the standard solution. Mass spectra would be recorded over an m/z range of 100 to 1200. Collision-induced fragmentation experiments were performed using argon as the collision gas. The collision energy was ramped from 15 to 35 eV. The PDA spectra were measured over the wavelength range of 210–400 nm in steps of 1.2 nm. The results of the measurements were processed using the MassLynx 4.1 software (Waters) incorporated with the instrument.

4.1.3. Ultraviolet Spectroscopy Measurements (UV) UV spectra were recorded on a Specord® 50 plus spectrophotometer. Samples were prepared by dilution of 4 µL of stock compounds solution (stock solution-ca 1 mg of compound in 1 mL water) in 996 µL of buﬀer solutions (1 mM HCl at pH 3.0, 67 mM phosphate buﬀer at pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 0.1 mM NaOH at pH 10).

4.2. Experimental Section

4.2.1. Materials All materials, including 2-thiouracil, were purchased from Sigma Aldrich (St. Louis, MO, USA) or TCI Europe n.V., (Zwijndrecht, Belgium).

4.2.2. Synthesis of 2-Selenouracil The synthesis of 2-selenouracil was done according to the published procedure [33], with slight improvement, and is described in Supplementary Materials.

4.2.3. 1H-NMR Analysis of Oxidation Assays of 1a and 1b A 10 mM solution of either 1a or 1b was prepared in 67 mM phosphate buﬀer (pH 7.4, pH 8.0, 1 pH 5.0) or deionized water (using D2O). The ﬁrst H NMR spectrum was acquired to establish the initial point. Compounds 1a or 1b were treated with 1 or 10 equivalents of H2O2. The reactions were monitored by 1H NMR, and the spectra were acquired after 1 or 2 min, 5 min, 20 min, 30 min, 60 min, 120 min, and 24 h.

4.2.4. UPLC-PDA-ESI ( )-HRMS Analysis of the Oxidation Assays of 1a and 1b − The 10 mM solutions of either 1a or 1b were prepared in 67 mM phosphate buffer (pH 7.4) and then were treated with 1 or 10 equivalents of H2O2. The reactions were monitored by UPLC-PDA-ESI ( )-HRMS, and the spectra were acquired after 1, 5, 10, 30, 60, and 120 min and 24 h. − 5. Conclusions By a series of oxidation experiments carried out on 2-seleno- and 2-thiouracil (1a and 1b), we demonstrated that Se2Ura is more prone to oxidation than the sulfur analog. In the first step of oxidation of 1a with H2O2 (1:0.5 molar ratio), diselenide 2a is exclusively formed, which is, as we suggest, the product of the condensation of selenenic acid (Ura-SeOH) and Ura-SeH. Diselenide 2a spontaneously undergoes Jaffe’s rearrangement to the two-ring product 7a, which, upon deselenation, gives 8, the major component of the reaction mixture. If diselenide 2a is still present in the reaction mixture, it reacts with 8 to give the three-ring product 9. These new paths make 2-seleno-uracil unsuitable as a model for the analysis of oxidative stress in transfer RNAs. On the other hand, only excess hydrogen peroxide causes oxidation of S2Ura. This process proceeds via sulfenic (Ura-SOH), sulfinic (Ura-SO2H), and sulfonic (Ura-SO3H) intermediates, and finally, after the elimination of sulfur oxides (SOn, n = 2 or 3), uracil is restored. Thus, we conclude that 2-selenouracil is oxidized more easily than 2-thiouracil, and the dominating oxidation path depends on (i) the concentration of reactants, Int. J. Mol. Sci. 2020, 21, 5956 16 of 18

(ii) excess oxidant, and (iii) the pH of the reaction mixture. Moreover, since the two- and three-ring products may be potential metabolites of degradation/oxidation of selenium-modiﬁed transfer RNAs, it is of interest to elaborate their inﬂuence on cell biology.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/17/ 5956/s1. Chemistry: Synthesis of 2-selenouracil (Se2Ura, 1a); Figure S1. 1H NMR spectrum of 1a; Figure S2. ESI( )-HRMS analysis and UV spectrum of 1a; Figure S3. ESI( )-HRMS analysis and UV spectrum of 2a; Figure S4. ESI(−)-HRMS analysis and UV spectrum of 3a; Figure S5.− ESI( )-HRMS analysis of 4a,(n = 1); Figure S6. ESI(−)-HRMS analysis and UV spectrum of 4a,(n = 2); Figure S7. ESI(− )-HRMS analysis and UV spectrum of 6; − − Figure S8. ESI( )-HRMS analysis and UV spectrum of 7a; Figure S9. NMR analysis of 8: (A) 1H NMR, (B) 13C NMR, (C) COSY,− (D) HMQC; Figure S10. ESI( )-HRMS analysis and UV spectrum of 8; Figure S11. NMR analysis − of 9: (A) 1H NMR, (B) 13C NMR, (C) COSY, (D) HMQC; Figure S12. ESI( )-HRMS analysis and UV spectrum of 9; Figure S13. The pH-dependent UV spectra of 9 in the pH range from− 3 to 10; Figure S14. ESI( )-HRMS analysis and UV spectrum of 2b; Figure S15. ESI( )-HRMS analysis of 3b; Figure S16. ESI( )-HRMS analysis− of 4b,(n = 1); Figure S17. ESI( )-HRMS analysis of 4b−,(n = 2); Figure S18. ESI( )-HRMS analysis− and UV spectrum of 4b,(n = 3); Figure S19. ESI(− )-HRMS analysis of 7b; Figure S20. ESI( )-HRMS− analysis and UV spectrum of 10; 1 − − Figure S21. H NMR analysis of the reaction mixtures for oxidation of Se2Ura (1a, 10 mM) with H2O2 (10 mM) in 67 mM phosphate buffer pH 8.0, at room temperature; FigureS22. 1H NMR analysis of the reaction mixtures for oxidation of Se2Ura (1a, 10 mM) with H2O2 (10 mM) in 67 mM phosphate buffer pH 5.0, at room temperature; 1 Figure S23. H NMR analysis of the reaction mixtures for oxidation of Se2Ura (1a, 10 mM) with H2O2 (10 mM) in water, at room temperature; Figure S24. 1H NMR analysis of the reaction mixtures for oxidation of Se2Ura 1 (1a, 10 mM) with H2O2 (5 mM) in 67 mM phosphate buffer pH 7.4, at room temperature; Figure S25. H NMR analysis of the reaction mixtures for oxidation of Se2Ura (1a, 10 mM) with H2O2 (100 mM) in 67 mM phosphate buffer pH 7.4, at room temperature; Figure S26. UPLC-PDA chromatographic analysis of the reaction mixtures for oxidation of Se2Ura (1a, 10 mM) with H2O2 (100 mM) in 67 mM phosphate buffer pH 7.4, at room temperature; Figure S27. Product ion mass spectrum of 10; Time course of the oxidation of S2Ura (1b) by hydrogen peroxide monitored by 1H NMR spectroscopy and UPLC-PDA-ESI( )-HRMS; Figure S28. The time course of the formation − of products for the oxidation reaction of S2Ura (1b, 10 mM) with H2O2 (10 mM) in 67 mM phosphate buffer 1 pH 7.4, r.t.; Figure S29. H NMR analysis of the reaction mixtures for oxidation of S2Ura (1b, 10 mM) with H2O2 (100 mM) in 67 mM phosphate buffer pH 7.4, at room temperature (r.t.).; Fig. S30. 1H NMR analysis (H5 and H6) of the reaction of 8 (4 mM) with 1a (10 mM) at the 0.4:1 molar ratio, at 67 mM phosphate buffer pH 8, r.t. Traces of compound 9 are seen after 17 and 24 h (signals of H5 and H6 protons are indicated by arrows). After 7 days of incubation the amount of 9 increased to ca. 14%, while the amount of 1a dropped down to ca. 32%, while 8 was 52% and 5 was 2%; Fig. S31. 1H NMR analysis (H5 and H6) of the reaction of 8 (4 mM) and 1a (10 mM) at the 0.4:1 molar ratio, after addition of 0.5 eq. of H2O2 (5 mM), at 67 mM phosphate buffer pH 8, r.t. Signals of compound 9 are seen after 1 h (see signals of H5 and H6 protons indicated by arrows). Author Contributions: Conceptualization, B.N. and E.S.; methodology, K.K., K.S., E.W., and B.P.-S.; investigation, B.N., E.S., K.K., K.S., E.W., and B.P.-S.; writing original draft preparation, K.K., K.S., E.W., and B.P.-S.; writing— review and editing, B.N., E.S., and K.K.; supervision, B.N. and E.S.; funding acquisition, B.N. and E.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The National Science Center in Poland (project UMO-2018/29/B/ST5/02509 to B.N.) and by statutory funds of the Lodz University of Technology and the Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences. Funding for open access charge: The National Science Center in Poland. Acknowledgments: Thanks are directed to Piotr Guga for critical reading of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Gilbert, H.F. Molecular and cellular aspects of thiol-disulfide exchange. In Advances in Enzymology and Related Areas of Molecular Biology; Meister, A., Ed.; John Wiley & Sons: Hoboken, NJ, USA, 1990; Volume 63, pp. 69–172. [CrossRef] 2. Gilbert, H.F. Thiol/disulfide exchange equilibria and disulfide bond stability. Methods Enzymol. 1995, 251, 8–28. [CrossRef][PubMed] 3. Gilbert, H.F. Thiol/disulfide exchange and redox potentials of proteins. In Bioelectrochemistry of Biomacromolecules: Principles and Practice; Lenaz, G., Milazzo, G., Eds.; Birkhäuser Verlag: Basel, Switzerland, 1997; pp. 256–324. [CrossRef] 4. Hondal, R.J.; Marino, S.M.; Gladyshev, V.N.Selenocysteine in thiol/disulfide-like exchange reactions. Antioxid. Redox Signal. 2013, 18, 1675–1689. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 5956 17 of 18

5. Yoshida, S.; Kumakura, F.; Komatsu, I.; Arai, K.; Onuma, Y.; Hojo, H.; Singh, B.G.; Priyadarsini, K.I.; Iwaoka, M. Antioxidative glutathione peroxidase activity of selenoglutathione. Angew. Chem. 2011, 50, 2125–2128. [CrossRef][PubMed] 6. Kryukov, G.V.; Castellano, S.; Novoselov, S.V.; Lobanov, A.V.; Zehtab, O.; Guigó, R.; Gladyshev, V.N. Characterization of mammalian selenoproteomes. Science 2003, 300, 1439–1443. [CrossRef] 7. Ramming, T.; Hansen, H.G.; Nagata, K.; Ellgaard, L.; Appenzeller-Herzog, C. Gpx8 peroxidase prevents

leakage of H2O2 from the endoplasmic reticulum. Free Radic. Biol. Med. 2014, 70, 106–116. [CrossRef] 8. Jacob, C.; Giles, G.; Giles, N.M.; Sies, H. Sulfur and selenium: The role of oxidation state in protein structure and function. Angew. Chem. 2003, 42, 4742–4758. [CrossRef] 9. Wessjohann, L.A.; Schneider, A.; Abbas, M.; Brand, W. Selenium in chemistry and biochemistry in comparison to sulfur. Biol. Chem. 2007, 388, 997–1006. [CrossRef] 10. Reich, H.J.; Hondal, R.J. Why Nature chose selenium. ACS Chem. Biol. 2016, 11, 821–841. [CrossRef] 11. Hondal, R.J.; Ruggles, E.L. Differing views of the role of selenium in thioredoxin reductase. Amino Acids 2011, 41, 73–89. [CrossRef] 12. Maroney, M.; Hondal, R.J. Selenium versus sulfur: Reversibility of chemical reactions and resistance to permanent oxidation in proteins and nucleic acids. Free Radic. Biol. Med. 2018, 127, 228–237. [CrossRef] 13. Sochacka, E.; Kraszewska, K.; Sochacki, M.; Sobczak, M.; Janicka, M.; Nawrot, B. The 2-thiouridine unit in the RNA strand is desulfured predominantly to 4-pyrimidinone nucleoside under in vitro oxidative stress conditions. Chem. Commun. 2011, 47, 4914–4916. [CrossRef][PubMed] 14. Sochacka, E.; Szczepanowski, R.H.; Cypryk, M.; Sobczak, M.; Janicka, M.; Kraszewska, K.; Bartos, P.; Chwialkowska, A.; Nawrot, B. 2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes. Nucleic Acids Res. 2015, 11, 2499–2512. [CrossRef] [PubMed] 15. Sochacka, E.; Bartos, P.; Kraszewska, K.; Nawrot, B. Desulfuration of 2-thiouridine with hydrogen peroxide in the physiological pH range 6.6-7.6 is pH-dependent and results in two distinct products. Bioorg. Med. Chem. Lett. 2013, 23, 5803–5805. [CrossRef][PubMed] 16. Bartos, P.; Ebenryter-Olbinska, K.; Sochacka, E.; Nawrot, B. The influence of the C5 substituent on the 2-thiouridine desulfuration pathway and the conformational analysis of the resulting 4-pyrimidinone products. Bioorg. Med. Chem. 2015, 23, 5587–5594. [CrossRef][PubMed] 17. Sierant, M.; Kulik, K.; Sochacka, E.; Szewczyk, R.; Sobczak, M.; Nawrot, B. Cytochrome C catalyzes the hydrogen peroxide-assisted oxidative desulfuration of 2-thiouridines in transfer RNAs. ChemBiochem 2018, 4, 687–695. [CrossRef][PubMed] 18. Payne, N.C.; Geissler, A.; Button, A.; Sasuclark, A.R.; Schroll, A.L.; Ruggles, E.L.; Gladyshev, V.N.; Hondal, R.J. Comparison of the redox chemistry of sulfur- and selenium-containing analogs of uracil. Free Radic. Biol. Med. 2017, 104, 249–261. [CrossRef] 19. Leszczynska, G.; Cypryk, M.; Gostynski, B.; Sadowska, K.; Herman, P.; Bujacz, G.; Lodyga-Chruscinska, E.; Sochacka, E.; Nawrot, B. C5-Substituted 2-selenouridines ensure efficient base pairing with guanosine;

consequences for reading the NNG-30 synonymous mRNA codons. Int. J. Mol. Sci. 2020, 21, 2882. [CrossRef] 20. Boccaletto, P.; Machnicka, M.A.; Purta, E.; Pi ˛atkowski,P.; Bagiński,B.; Wirecki, T.K.; de Crécy-Lagard, V.; Ross, R.; Limbach, P.A.; Kotter, A.; et al. MODOMICS: A database of RNA modification pathways. 2017 update. Nucl. Acids Res. 2018, 46, D303–D307. [CrossRef] 21. Cantara, W.A.; Crain, P.F.; Rozenski, J.; McCloskey, J.A.; Harris, K.A.; Zhang, X.; Vendeix, F.A.P.; Fabris DAgris, P.F. The RNA modification database, RNAMDB: 2011 update. Nucl. Acids Res. 2011, 39, D195–D201. [CrossRef] 22. Rheinboldt, H.; Giesbreoht, E. Unsymmetrische diselenide. Chem. Ber. 1952, 85, 357–368. [CrossRef] 23. Johnson, T.B.; Edens, C.O. Complex formations between iodine and µ-mercapto-dihydroglyoxalines. J. Am. Chem. Soc. 1942, 64, 2706–2708. [CrossRef] 24. Marshall, W.; Singh, J. Oxidative inactivation of ethylenethiourea by hypochlorite in alkaline medium. J. Agric. Food Chem. 1977, 25, 1316–1320. [CrossRef][PubMed] 25. Jackowiak, P.; Nowacka, M.; Strozycki, P.M.; Figlerowicz, M. RNA degradome-its biogenesis and functions. Nucl. Acids Res. 2011, 39, 7361–7370. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 5956 18 of 18

26. Antoniadis, C.D.; Hadjikakou, S.K.; Hadjiliadis, N.; Papakyriakou, A.; Baril, M.; Butler, I.S. Synthesis and structures of Se analogues of the antithyroid drug 6-n-propyl-2-thiouracil and its alkyl derivatives: Formation of dimeric Se-Se compounds and deselenation reactions of charge-transfer adducts of diiodine. Chem. Eur. J. 2006, 12, 6888–6897. [CrossRef][PubMed] 27. Palumbo, A.; d’Ischia, M.; Cioﬃ, F.A. 2-Thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin-dependent enzyme activation and dimerization. FEBS Lett. 2000, 485, 109–112. [CrossRef] 28. Reich, H.J.; Hoeger, C.A.; Willis, J.W.W. Organoselenium chemistry. Characterization of reactive intermediates in the selenoxide syn elimination: Selenenic acids and selenolseleninate esters. J. Am. Chem. Soc. 1982, 104, 2936–2937. [CrossRef] 29. Reich, H.J.; Hoeger, C.A.; Willis, J.W.W. Organoselenium chemistry: A study of intermediates in the fragmentation of aliphatic ketoselenoxides. Characterization of selenoxides, selenenamides and selenolseleninates by 1H-,13C-and 77Se-NMR. Tetrahedron 1985, 41, 4771–4779. [CrossRef] 30. Ayrey, G.; Barnard, D.; Woodbridge, D.T. The oxidation of organoselenium compounds by ozone. J. Chem. Soc. 1962, 2089–2099. [CrossRef] 31. Ishii, A.; Takahashi, T.; Nakayama, J. Hydrolysis of an isolable selenoseleninate under acidic and alkaline conditions. Heteroatom Chem. 2001, 12, 198–203. [CrossRef] 32. Duddeck, H. 77Se NMR spectroscopy and its applications in chemistry. Annu. Reports NMR Spectrosc. 2004, 52, 105–166. [CrossRef] 33. Mautner, H.G. The synthesis and properties of some selenopurines and selenopyrimidines. J. Am. Chem. Soc. 1956, 78, 5292–5294. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).