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

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.


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][2][3][4][5][6][7][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]. Table 1. UPLC-PDA-ESI (−)-HRMS and 1 H 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] − in negative mode, the maximum wavelength λ max in UV/VIS spectra (nm), and 1  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 1  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 (∆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 1 H NMR spectrum. The N1 a -C2 b 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 Figure 2c.  Table 1. The time course of the formation/disappearance of each product was assessed by 1 H NMR integration data. (c) The pH-dependent UV spectra of 8 in the pH range from 3 to 10. The inorganic selenous acid was identified by UPLC-ESI(−)-HRMS, and its  Table 1. The time course of the formation/disappearance of each product was assessed by 1 H NMR integration data. (c) The pH-dependent UV spectra of 8 in the pH range from 3 to 10. The inorganic selenous acid was identified by UPLC-ESI(−)-HRMS, and its  Table 1. The time course of the formation/disappearance of each product was assessed by 1 H NMR integration data. (c) The pH-dependent UV spectra of 8 in the pH range from 3 to 10. The inorganic selenous acid was identified by UPLC-ESI(−)-HRMS, and its retention time was determined based on extracted ion chromatograms (EICs) for the ion corresponding to its deprotonated molecule (m/z 128.909).
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 1 H 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 Figure 2c. The structure of 8 was confirmed by NMR analysis in the 2D HMBC (Heteronuclear Multiple Bond Coherence) spectrum, where the correlation between the H6 a proton (δ 7.85 ppm) and the C2 b carbon (δ 156.10 ppm) indicated a covalent bond between N1 a and C2 b . Moreover, the same carbon atom C2 b also had a three-bond correlation with proton H6 b at 8.10 ppm (see Figure 4a). The 1 H and 13 C 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 1 H 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 λ max 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 N1 a -C2 b and N1 b -C2 c covalent bonds. The structure was also confirmed by the 2D HMBC spectrum shown in Figure 4b, where three bond correlations between C2 a and H6 a (δ 7.90 ppm) and H6 b (δ 8.46 ppm) were found. Similarly, correlations between C2 c (δ 156.79 ppm) and H6 b (δ 8.46 ppm) and H6 c (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 (Figure 2a), 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-SeO 2 H, 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 H 2 SeO 3 was observed in UPLC-HRMS analysis.   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 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.
(a) (b) 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 (Figure 2a), 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.
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 1 H 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 H 2 SeO 3 was released. The reaction of 1a (10 mM) was carried out with 0.5 equivalents (5 mM) of H 2 O 2 in phosphate buffer at pH 7.4 and monitored by 1 H NMR (see Figure 5 and Figure S24). Interestingly, in this case 1a was almost immediately converted to diselenide 2a as a sole product (see Figure 5), and only a small amount of 4a (n = 2, Ura-SeO 2 H) 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 1 H NMR signals indicated the formation of 8 (57%), 9 (26%), and 5 (6%), as well as restored 1a (11%) (Scheme 2).  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 1 H 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-SeO2H) and the two-ring derivative 8 (11%, from the rearrangement of diselenide 2a) (Scheme 3). Interestingly, no three-ring product was Figure 5. 1 H NMR analysis (H5 and H6 signals) of the reaction mixtures for the oxidation of Se2Ura (1a, 10 mM) with H 2 O 2 (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. 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 1 H 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 1 H NMR (see Figures 5 and S24). Interestingly, in this case 1a was almost immediately converted to diselenide 2a as a sole product (see Figure 5), 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 1 H NMR signals indicated the formation of 8 (57%), 9 (26%), and 5 (6%), as well as restored 1a (11%) (Scheme 2).   1 H 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.

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 1 H 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-SeO2H) and the two-ring derivative 8 (11%, from the rearrangement of diselenide 2a) (Scheme 3). Interestingly, no three-ring product was detected. Only traces of 7a, as well as inorganic H2SeO3 and H2SeO4, were detected by UPLC-HRMS ( Figure S26).   The reaction of S2Ura (1b, 10 mM) with hydrogen peroxide at a 1:1 molar ratio (Scheme 4, 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-SO 2 H) (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-SO 3 H, 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 1 H NMR spectra, as well as the time course of the oxidation reaction ( Figure S28), are included in the Supplementary Materials. The reaction of S2Ura (1b, 10 mM) with hydrogen peroxide at a 1:1 molar ratio (Scheme 4, 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, 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 1 H NMR spectra, as well as the time course of the oxidation reaction ( Figure S28), are included in the Supplementary Materials.  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 1     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, Figure S29  by 1 H 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
Generally, our investigations have shown that 2-selenouracil (1a) and 2-thiouracil (1b) have significantly different redox properties (Scheme 6, the red path is preferable for selenium components, and the blue path is preferable for sulfur components). Compound 1a is extremely prone to H 2 O 2 -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 H 2 O 2 , where 2a is almost exclusively formed ( Figure 5 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-SeO 2 H (see Scheme 7). 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" (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 [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-SeI 2 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 N3 a of one R6Se2Ura (a) ring and the C2 b 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 N3 a -C2 b bond, leading to the uracil molecule being substituted with an alkyl group at position 6.
In contrast to the cited two-ring compound R6Se2Ura(N3 a )-(C2 b )R6Ura, the two-ring compound 8 reported here has a spanning bond between the C2 b and N1 a atoms, as documented by 2D NMR studies (Figure 4a). If the amount of oxidant is stoichiometrically limiting (see data for a 1:0.5 molar ratio of reactants at pH 7.4), or the reaction occurs under more basic conditions (pH 8), derivative 8 could be transformed to three-ring product 9 with a yield up to 26% of the total content. At first, we have supposed that compound 9 is a product of the condensation of 8 with 1a, with the departure of hydrogen selenide as a second product (according to the mechanism proposed in Figure S30). If so, this reaction is expected to run between 8 and 1a without any additional reagent. However, as shown by the 1 H NMR analysis of 8 in the mixture with an excess of 1a (0.4:1 molar ratio), only signals of traces of compound 9 are noted after 24 h. However, the prolonged incubation time of this mixture up to 7 days allowed to increase the content of 9 up to ca 14%, while the content of 1a clearly decreased to 32% (Fig. S30). Interestingly, when the same reaction was tested (8 and 1a, 0.4:1 molar ratio) but with the addition of 0.5 eq. of hydrogen peroxide over 1a, the formation of 9 after 1 h was observed, to reach finally the yield of ca. 14% after 24 h (see Figure S31). Thus, we conclude that compound 9 is mostly a product of the condensation of 8 and 2a, as shown in Scheme 8b, in which 2-selenouracil 1a and selenium are released, Thus, the reconstituted 2-selenouracil, present as a final product in the reaction, shown in Scheme 2, may originate from the diselenide reacting with compound 8.
In general, we can summarize that 2-selenouracil (1a) in the presence of a stoichiometrically limiting amount of the oxidant is preferably transformed to diselenide 2a, which spontaneously rearranges to the two-ring product 8, which, in turn, reacts with the remaining diselenide 2a to yield 9 (red path, Scheme 6).
Generally, our investigations have shown that 2-selenouracil (1a) and 2-thiouracil (1b) have significantly different redox properties (Scheme 6, 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 (Figures 5 and 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. 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.
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 Scheme 7). 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" (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 [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.
In contrast to the cited two-ring compound R6Se2Ura(N3a)-(C2b)R6Ura, the two-ring compound 8 reported here has a spanning bond between the C2b and N1a atoms, as documented by 2D NMR studies (Figure 4a). If the amount of oxidant is stoichiometrically limiting (see data for a 1:0.5 molar ratio of reactants at pH 7.4), or the reaction occurs under more basic conditions (pH 8), derivative 8 could be transformed to three-ring product 9 with a yield up to 26% of the total content. At first, we have supposed that compound 9 is a product of the condensation of 8 with 1a, with the departure of hydrogen selenide as a second product (according to the mechanism proposed in Figure S30). If so, this reaction is expected to run between 8 and 1a without any additional reagent. However, as shown by the 1 H NMR analysis of 8 in the mixture with an excess of 1a (0.4:1 molar ratio), only signals of traces of compound 9 are noted after 24 h. However, the prolonged incubation time of this mixture up to 7 days allowed to increase the content of 9 up to ca 14%, while the content of 1a clearly decreased to 32% (Fig. S30). Interestingly, when the same reaction was tested (8 and 1a, 0.4:1 molar ratio) but with the addition of 0.5 eq. of hydrogen peroxide over 1a, the formation of 9 after 1 h was observed, to reach finally the yield of ca. 14% after 24 h (see Figure S31). Thus, we conclude that compound 9 is Scheme 7. A possible scheme of hydrolysis of diselenides under basic conditions [22]. reaction, shown in Scheme 2, may originate from the diselenide reacting with compound 8.
In general, we can summarize that 2-selenouracil (1a) in the presence of a stoichiometrically limiting amount of the oxidant is preferably transformed to diselenide 2a, which spontaneously rearranges to the two-ring product 8, which, in turn, reacts with the remaining diselenide 2a to yield 9 (red path, Scheme 6).
This type of rearrangement has not been 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. In our case, in contrast, the main component is the two-ring product 8. This inconsistency may originate from the use of Se2Ura bearing a carboxyl substituent in position C5, which exerts an electron-withdrawing effect (both by induction and resonance). This effect is evidenced by the change in the pKa value from 7.18 for Se2Ura (1a) to 7.11 for c5Se2Ura. A similar effect has been observed for 2-thiouracils, where the pKa value changes from 7.75 for S2Ura (1b) to 7.68 for c5S2Ura [27]. The route leading to diselenide 2a and its rearrangement to 7a, 8, and 9 is less favorable if the oxidant is present in high excess (see Figures S25 and S26). In these conditions, the prevalent formation of seleninic acid 4a (n = 2, Ura-SeO2H) is noted, which, in aqueous solutions, undergoes water-assisted hydrolysis, resulting in the formation of uracil (5) (89%) accompanied by elimination of selenium (IV) oxide, which reacts with water to form H2SeO3 (blue path, Scheme 6). Besides, it 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][29][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. This type of rearrangement has not been 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. In our case, in contrast, the main component is the two-ring product 8. This inconsistency may originate from the use of Se2Ura bearing a carboxyl substituent in position C5, which exerts an electron-withdrawing effect (both by induction and resonance). This effect is evidenced by the change in the pKa value from 7.18 for Se2Ura (1a) to 7.11 for c5Se2Ura. A similar effect has been observed for 2-thiouracils, where the pKa value changes from 7.75 for S2Ura (1b) to 7.68 for c5S2Ura [27].
The route leading to diselenide 2a and its rearrangement to 7a, 8, and 9 is less favorable if the oxidant is present in high excess (see Figures S25 and S26). In these conditions, the prevalent formation of seleninic acid 4a (n = 2, Ura-SeO 2 H) is noted, which, in aqueous solutions, undergoes water-assisted hydrolysis, resulting in the formation of uracil (5) (89%) accompanied by elimination of selenium (IV) oxide, which reacts with water to form H 2 SeO 3 (blue path, Scheme 6). Besides, it 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][29][30]. These compounds are hydrolyzed to seleninic acid (RSeO 2 H) 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.
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 H 2 O 2 after 18 h led to a product resonating at δ 1273 ppm in 77 Se NMR, which was assigned as seleninic acid c5Ura-SeO 2 H. In our reaction carried out in phosphate buffer at pH 7.4, seleninic acid Ura-SeO 2 H (4a, n = 2) is present only in the first several minutes, while inorganic seleninic acid H 2 SeO 3 is present after 1 h (see Figure 2a). In addition, similar chemical shifts are known to be associated with Na 2 SeO 3 (1263 ppm) and H 2 SeO 3 (1300 ppm) [32].
Unlike 2-selenouracil (1a), 2-thiouracil (1b) is less prone to oxidation with H 2 O 2 . The reaction is remarkably slower, and a 10-fold molar excess of oxidant leads to predominant conversion to uracil (5) (Scheme 5). With equal or stoichiometrically limiting amounts of H 2 O 2 , ca. 40% of 1b remains intact. The identified intermediates include sulfenic acid 4b (n = 1, Ura-SOH), sulfinic acid 4b (n = 2, Ura-SO 2 H), and sulfonic acid 4b (n = 3, Ura-SO 3 H) (Scheme 6, blue). Notably, among those three acid forms, sulfinic acid (Ura-SO 2 H) is the most abundant, as shown by 1 H NMR analysis (having the highest signal integration and the longest time, the signals remained in the reaction mixture). Similar to sulfonic acid (Ura-SO 3 H), 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][14][15][16][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. and HMBC NMR spectra were recorded on a Bruker Avance 700 MHz spectrometer. The NMR spectra for 1 H and 13 C 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).
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-Spray TM , (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, 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.

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.

1 H-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, pH 5.0) or deionized water (using D 2 O). The first 1 H NMR spectrum was acquired to establish the initial point. Compounds 1a or 1b were treated with 1 or 10 equivalents of H 2 O 2 . The reactions were monitored by 1 H NMR, and the spectra were acquired after 1 or 2 min, 5 min, 20 min, 30 min, 60 min, 120 min, and 24 h.

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 H 2 O 2 . 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.